Rare Neoplastic Diseases

**37**

**Chapter 4**

**Abstract**

Approaches

prognostic factors, treatment

**1. Introduction**

Childhood Langerhans Cell

Factors, and Therapeutic

*Katharina Sterlich and Milen Minkov*

Histiocytosis: Epidemiology,

Clinical Presentations, Prognostic

Childhood LCH is a rare disease, affecting 4–9 per 1,000,000 children below the age of 15 years. It is driven by somatic mutations in the MAPK pathway, arising in myeloid marrow progenitors. Both genders are affected by a slight male preponderance. The clinical spectrum of LCH varies from a single lesion affecting one organ system to severe multisystem disease with dysfunction of vital organs. Likewise, variable and unpredictable is its course, spanning from self-limiting course to progression with lethal outcome. Recognized unfavorable prognostic factors are the involvement of hematopoiesis, liver, and spleen, as well as non-response to systemic treatment. Recent studies suggest that patients carrying the BRAFV600E mutation may have a more severe clinical phenotype and less favorable prognosis. The combination of prednisolone and vinblastine is the standard first-line treatment for disseminated disease. Second-line options used in clinical practice are not well evidenced. Inhibitors of the MAPK pathway are a promising alternative option.

**Keywords:** langerhans cell histiocytosis, epidemiology, manifestations,

cases were described that made the spectrum of this disease clearer.

LCH is a rare disease with a variety of presentations and outcomes. Indeed, for most of its history, it was thought to be several different entities until sufficient

The descriptive approach in medicine in the early 20th century and the extremely heterogeneous clinical presentation of LCH led to the fact that different manifestations of the disease were described as separate syndromes. Thus, the history of the disease began with the description of the Hand-Schüller-Christian syndrome, [1–3], the Letterer-Siwe disease, [4, 5] and the eosinophilic granuloma [6]. In 1953, Dr. L. Lichtenstein in a critical review of the literature introduced a unifying concept, stating that the conditions previously designated eosinophilic

#### **Chapter 4**

## Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic Factors, and Therapeutic Approaches

*Katharina Sterlich and Milen Minkov*

### **Abstract**

Childhood LCH is a rare disease, affecting 4–9 per 1,000,000 children below the age of 15 years. It is driven by somatic mutations in the MAPK pathway, arising in myeloid marrow progenitors. Both genders are affected by a slight male preponderance. The clinical spectrum of LCH varies from a single lesion affecting one organ system to severe multisystem disease with dysfunction of vital organs. Likewise, variable and unpredictable is its course, spanning from self-limiting course to progression with lethal outcome. Recognized unfavorable prognostic factors are the involvement of hematopoiesis, liver, and spleen, as well as non-response to systemic treatment. Recent studies suggest that patients carrying the BRAFV600E mutation may have a more severe clinical phenotype and less favorable prognosis. The combination of prednisolone and vinblastine is the standard first-line treatment for disseminated disease. Second-line options used in clinical practice are not well evidenced. Inhibitors of the MAPK pathway are a promising alternative option.

**Keywords:** langerhans cell histiocytosis, epidemiology, manifestations, prognostic factors, treatment

#### **1. Introduction**

LCH is a rare disease with a variety of presentations and outcomes. Indeed, for most of its history, it was thought to be several different entities until sufficient cases were described that made the spectrum of this disease clearer.

The descriptive approach in medicine in the early 20th century and the extremely heterogeneous clinical presentation of LCH led to the fact that different manifestations of the disease were described as separate syndromes. Thus, the history of the disease began with the description of the Hand-Schüller-Christian syndrome, [1–3], the Letterer-Siwe disease, [4, 5] and the eosinophilic granuloma [6].

In 1953, Dr. L. Lichtenstein in a critical review of the literature introduced a unifying concept, stating that the conditions previously designated eosinophilic granuloma of bone, Letterer-Siwe disease and Hand-Schüller-Christian disease, are interrelated manifestations of a single disease [7]. The name "histiocytosis X" was suggested to underscore the unknown origin of the disease.

In 1973, Dr. Christian Nezelof, a French pediatric pathologist, proposed the Langerhans cells as the origin of histiocytosis X [8]. His hypothesis was based on morphologic similarities (e.g. Birbeck granule, a cytoplasmic pentalaminar structure with a tennis racket shape) between normal Langerhans cells and the abnormal cells in histiocytosis X. Since then the disease is referred to as Langerhans cell histiocytosis.

#### **2. Epidemiology**

The estimated incidence of LCH is 4–9 children younger than 15 years per million [9–12]. The peak incidence of childhood LCH is between 0 and 4 years [13]. There is a relation between age at manifestation and disease extent, younger children have more disseminated disease [14]. All large epidemiologic studies report a male predominance in the range of 1.2–1.5 [9–12]. The causes and risk factors for developing LCH are unclear [15]. However, the unique patterns of presentation, ranging from localized bone lesions with spontaneous regression to disseminated forms with involvement of multiple organs, suggest a complex pathogenesis. Familial clustering, particularly the observation of increased incidence in monozygotic twins, have suggested the presence of a germline predisposition at least for a proportion of cases [16, 17]. In addition, populationbased studies have shown differences in the incidence of disseminated LCH by race and ethnic group; a higher incidence has been reported for Hispanics and a lower incidence for blacks [18]. Studies have also shown a correlation with maternal and neonatal infections, [15, 19, 20] lack of childhood vaccinations, [15, 20] family history of thyroid disease, [15] in vitro fertilization, [21] and feeding problems and transfusions during infancy [19]. Finally, lower socioeconomic conditions have been associated with an increased incidence of disseminated LCH [18].

#### **3. Clinical presentation**

The clinical presentations of LCH range from incidentally detected asymptomatic bone lesions to severe multisystem disease manifesting with disseminated rash, fever, failure to thrive, enlarged liver and spleen and transfusion-dependent cytopenia (**Figure 1**). The disease can present with insidious nonspecific manifestations such as fever, impaired appetite, anxiety, and sleep disturbances, particularly in infants. Virtually all organ systems can be affected either individually (single system LCH; SS-LCH) or in different combinations (multisystem LCH; MS-LCH). Hence, LCH can mimic a large spectrum of diseases (**Table 1**). Frequent, though unspecific manifestations are: bone pain, soft tissue swelling ("bumps") in the head and neck area, persistent polymorphic skin eruptions, mucous membrane ulcerations, respiratory symptoms (cough, shortness of breath, chest pain), enlargement of the liver, spleen and lymph nodes, growth failure, polyuria with polydipsia or, rarely, neurological symptoms.

The organs mostly affected are bone (80%), skin (33%) and pituitary (25%). The hematopoietic system, spleen, liver and lungs are affected in up to 15%, lymph nodes in 5–10% and the central nervous system without the pituitary in 2–4% of the patients [22].

**39**

**3.1 Skeleton**

**Figure 1.**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

Flat bones and particularly the cranial bones are most commonly affected. Other common locations in decreasing order are the long bones of the extremities, the vertebral bodies and the pelvic bones. The proximal bones of the extremities are more frequently involved than the distal ones. The bones of the hands and feet are usually sparred. The lesions are characteristically located in the diaphysis or

*Spectrum of clinical manifestations. Bone lesions in the skull (A) with irregular punched out appearance, vertebra (B) with partial collapse of the vertebral body, and the tibia (C) with periosteal reaction and bone deformity; Cutaneous manifestations: purpuric rash with erythema and maceration of the inguinal folds resembling diaper dermatitis (D), characteristic confluent maculopapular rash with purpuric appearance and crusting on the trunk (E), and papulonodular reddish-brown rash in a newborn (F); Right-sided proptosis caused by a lesion of the orbital bones (G); Jawbone lesion presenting as a massive soft-tissue swelling (H) and the respective MRI findings (I); J - Thickening of the pituitary infundibulum, manifesting as central diabetes insipidus; Characteristic non-granulomatous lesions of the cerebellum (K) and the basal ganglia (L); Massive hepatosplenomegaly and skin rash in severe multisystem LCH (M); Pulmonary LCH with bullae visible on* 

Vertebral lesions typically localize in the vertebral body, vertebral arch, transverse or spinous process and present with pain, kyphoscoliosis, or neurological deficits due to compression of the spinal cord [23]. While vertebra plana in LCH are

Unilateral or bilateral lesions in the temporal bone range from unspecific opacification of the mastoid cells with minimal bone destruction, to extensive osseous destruction and intracranial soft tissue infiltration. The most common symptoms are recurrent or persistent otitis, mucopurulent otorrhea, swelling of the mastoid, and eczema or polyps of the ear canal. In rare cases of inner ear involvement, hear-

Lesions of the orbital bones in LCH are mostly unilateral and have exclusively extraconal location, typically affecting the roof and the lateral wall [24]. They manifest with lid swelling (with or without inflammatory appearance), palpable mass, or proptosis. The differentials of orbital involvement include acute infections, inflammatory pseudotumor, hemangioma, rhabdomyosarcoma, retinoblastoma, metastatic neuroblastoma, lymphoma, and optic glioma. However, other histiocytic disorders, such as juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-

metaphysis, but the epiphysis can be affected as well.

*radiography (N) and combination of nodules and cysts on CT scan (O).*

rare, LCH is the leading cause of vertebra plana in children.

ing loss, dizziness or paralysis of the facial nerve also occur.

Dorfman disease can present with orbital involvement as well.

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

#### **Figure 1.**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

histiocytosis.

LCH [18].

**3. Clinical presentation**

rarely, neurological symptoms.

**2. Epidemiology**

granuloma of bone, Letterer-Siwe disease and Hand-Schüller-Christian disease, are interrelated manifestations of a single disease [7]. The name "histiocytosis X"

In 1973, Dr. Christian Nezelof, a French pediatric pathologist, proposed the Langerhans cells as the origin of histiocytosis X [8]. His hypothesis was based on morphologic similarities (e.g. Birbeck granule, a cytoplasmic pentalaminar structure with a tennis racket shape) between normal Langerhans cells and the abnormal cells in histiocytosis X. Since then the disease is referred to as Langerhans cell

The estimated incidence of LCH is 4–9 children younger than 15 years per million [9–12]. The peak incidence of childhood LCH is between 0 and 4 years [13]. There is a relation between age at manifestation and disease extent, younger children have more disseminated disease [14]. All large epidemiologic studies report a male predominance in the range of 1.2–1.5 [9–12]. The causes and risk factors for developing LCH are unclear [15]. However, the unique patterns of presentation, ranging from localized bone lesions with spontaneous regression to disseminated forms with involvement of multiple organs, suggest a complex pathogenesis. Familial clustering, particularly the observation of increased incidence in monozygotic twins, have suggested the presence of a germline predisposition at least for a proportion of cases [16, 17]. In addition, populationbased studies have shown differences in the incidence of disseminated LCH by race and ethnic group; a higher incidence has been reported for Hispanics and a lower incidence for blacks [18]. Studies have also shown a correlation with maternal and neonatal infections, [15, 19, 20] lack of childhood vaccinations, [15, 20] family history of thyroid disease, [15] in vitro fertilization, [21] and feeding problems and transfusions during infancy [19]. Finally, lower socioeconomic conditions have been associated with an increased incidence of disseminated

The clinical presentations of LCH range from incidentally detected asymptomatic bone lesions to severe multisystem disease manifesting with disseminated rash, fever, failure to thrive, enlarged liver and spleen and transfusion-dependent cytopenia (**Figure 1**). The disease can present with insidious nonspecific manifestations such as fever, impaired appetite, anxiety, and sleep disturbances, particularly in infants. Virtually all organ systems can be affected either individually (single system LCH; SS-LCH) or in different combinations (multisystem LCH; MS-LCH). Hence, LCH can mimic a large spectrum of diseases (**Table 1**). Frequent, though unspecific manifestations are: bone pain, soft tissue swelling ("bumps") in the head and neck area, persistent polymorphic skin eruptions, mucous membrane ulcerations, respiratory symptoms (cough, shortness of breath, chest pain), enlargement of the liver, spleen and lymph nodes, growth failure, polyuria with polydipsia or,

The organs mostly affected are bone (80%), skin (33%) and pituitary (25%). The hematopoietic system, spleen, liver and lungs are affected in up to 15%, lymph nodes in 5–10% and the central nervous system without the pituitary in 2–4% of the

was suggested to underscore the unknown origin of the disease.

**38**

patients [22].

*Spectrum of clinical manifestations. Bone lesions in the skull (A) with irregular punched out appearance, vertebra (B) with partial collapse of the vertebral body, and the tibia (C) with periosteal reaction and bone deformity; Cutaneous manifestations: purpuric rash with erythema and maceration of the inguinal folds resembling diaper dermatitis (D), characteristic confluent maculopapular rash with purpuric appearance and crusting on the trunk (E), and papulonodular reddish-brown rash in a newborn (F); Right-sided proptosis caused by a lesion of the orbital bones (G); Jawbone lesion presenting as a massive soft-tissue swelling (H) and the respective MRI findings (I); J - Thickening of the pituitary infundibulum, manifesting as central diabetes insipidus; Characteristic non-granulomatous lesions of the cerebellum (K) and the basal ganglia (L); Massive hepatosplenomegaly and skin rash in severe multisystem LCH (M); Pulmonary LCH with bullae visible on radiography (N) and combination of nodules and cysts on CT scan (O).*

#### **3.1 Skeleton**

Flat bones and particularly the cranial bones are most commonly affected. Other common locations in decreasing order are the long bones of the extremities, the vertebral bodies and the pelvic bones. The proximal bones of the extremities are more frequently involved than the distal ones. The bones of the hands and feet are usually sparred. The lesions are characteristically located in the diaphysis or metaphysis, but the epiphysis can be affected as well.

Vertebral lesions typically localize in the vertebral body, vertebral arch, transverse or spinous process and present with pain, kyphoscoliosis, or neurological deficits due to compression of the spinal cord [23]. While vertebra plana in LCH are rare, LCH is the leading cause of vertebra plana in children.

Unilateral or bilateral lesions in the temporal bone range from unspecific opacification of the mastoid cells with minimal bone destruction, to extensive osseous destruction and intracranial soft tissue infiltration. The most common symptoms are recurrent or persistent otitis, mucopurulent otorrhea, swelling of the mastoid, and eczema or polyps of the ear canal. In rare cases of inner ear involvement, hearing loss, dizziness or paralysis of the facial nerve also occur.

Lesions of the orbital bones in LCH are mostly unilateral and have exclusively extraconal location, typically affecting the roof and the lateral wall [24]. They manifest with lid swelling (with or without inflammatory appearance), palpable mass, or proptosis. The differentials of orbital involvement include acute infections, inflammatory pseudotumor, hemangioma, rhabdomyosarcoma, retinoblastoma, metastatic neuroblastoma, lymphoma, and optic glioma. However, other histiocytic disorders, such as juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease can present with orbital involvement as well.


#### **Table 1.**

*Common differential diagnoses of LCH.*

Jaw involvement can present with gingival hyperplasia or ulceration, extensive dissolution of the jawbone structure with tooth loosening ("floating teeth") or loss.

**41**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

Skin is the second most frequently involved organ system after the skeleton overall. In patients younger than two years, it is even the most frequently involved organ. Cutaneous involvement is typically representative of multisystem disease, as 87–93% also have systemic involvement. Cutaneous lesions may be either circum-

They typically present as pinpoint erythematous or skin-colored papules or pustules. The morphology can mimic a seborrheic dermatitis-like or an eczematous

scabbing, crusting, and/or purpura. In infants, a seborrheic dermatitis-like rash on the scalp often causes LCH to be misdiagnosed as seborrheic dermatitis, while groin involvement can mimic treatment-resistant, recurring diaper dermatitis [25].

Nail involvement is rare, but can present as subungual pustules, hemorrhage, or hyperkeratosis, purpuric striae, purulent discharge, longitudinal grooving, ony-

Enlarged lymph nodes are rarely the only manifestation of a single system LCH. The lymphadenopathy encountered in the setting of multisystem disease is usually

Peripheral blood cytopenia in LCH patients, often referred to as "hematologic dysfunction", is a sign of severe disease and heralds unfavorable prognosis. For decades, the terms "hematopoietic dysfunction" and "bone marrow involvement" were interchangeably used in the literature [26]. Bone marrow studies in LCH patients using immunochemical staining for CD1a or molecular markers (BRAFV600E), have found increased proportion of histiocytes compared to controls, but their numbers did not correlate well with disease extent and severity [26, 27]. Strikingly, the phagocytosis in the bone marrow, which better correlates to disease severity, is carried out by CD1anegative macrophages. Although the exact mechanisms leading to peripheral cytopenia remain uncovered, it is clear that it is not due to marrow infiltration in most cases, and is probably due to increased phagocytosis or inflammatory marrow suppression.

Enlargement of the spleen in LCH occurs exclusively in the setting of MS-LCH. It occurs in 15–30% of the patients mostly coinciding with hematopoietic and liver

Liver involvement occurs exclusively in children with MS-LCH. Patients may present with hepatomegaly only or with functional impairment (elevated liver

Two patterns of liver dysfunction can been seen in children: one with predominant hypoproteinemia/hypoalbuminemia ± mild elevation of transaminases and bilirubin; and a less common cholestatic one, due to progressive sclerosing cholangitis [28]. The former is usually combined with prominent constitutional symptoms, and is characteristically observed in the setting of active LCH, while the latter is usually seen as a disease consequence and often without concomitant activity of LCH elsewhere.

enzymes, hypoproteinemia and hypoalbuminemia) and/or jaundice.

erythematous, skin-colored, or brown petechial rash with or without scale,

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

scribed (nodular) or spread and confluent (rash).

cholysis, paronychia, and pitting [25].

**3.3 Lymph nodes**

mild to moderate.

**3.4 Bone marrow**

**3.5 Spleen**

involvement.

**3.6 Liver**

**3.2 Skin**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

#### **3.2 Skin**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Skin Vesicles and bullae (most

like) Petechia Pruritic rash

Bone Vertebral lesions (vertebra plana)

Lung Respiratory symptoms,

cysts)

Liver Hepatomegaly, jaundice with

reticular lesions (nodules and

direct hyperbilirubinemia Hypoalbuminemia

Polyuria/polydipsia, growth failure, hypothyroidism, hypogonadism

**Affected organ Manifestation/finding Differentials**

Erythema toxicum Herpes simplex Varicella

Seborrheic dermatitis Mastocytosis

Leukemia/Lymphoma Aneurysmal bone cyst Erdheim-Chester disease Ewing sarcoma Osteosarcoma Metabolic bone diseases

Scabies

Mastoiditis Cholesteatoma Soft tissue sarcoma

Dermoid cyst

CRMO

Erdheim–Chester disease Pseudoinflammatory tumor Rhabdomyosarcoma Neuroblastoma

Aneurysmal bone cyst

Fibrous dysplasia Giant cell tumor of bone Atypical mycobacterial infection

Osteogenic sarcoma Ewing's sarcoma

Metabolic diseases Hepatitis

Toxic (Reye syndrome) Neonatal hemochromatosis Chronic inflammatory bowel disease

Renal diabetes insipidus Head trauma

Germ cell tumors of CNS Lymphatic hypophysitis Non-LCH histiocytoses

Sarcoidosis

Bone angiomatosis (Gorham disease)

Mycobacterial or other pulmonary infections

Chronic destructive cholangitis

Diseases obstructing biliary tract Inherited diseases of bilirubin conjugation

Orbit Acute infection (preseptal cellulitis)

Temporal bone Chronic otitis media

Lytic lesions of the long bones Septic osteomyelitis

Juvenile xanthogranuloma Neuroblastoma Infant leukemia Intrauterine infections

Chronic relapsing multifocal osteomyelitis (CRMO)

common in early infancy)

Dermatitis (most frequently scalp, diaper area, or axilla) Nodules ("blueberry muffin"

**40**

or loss.

**Table 1.**

Endocrine glands (pituitary, thyroid)

*Common differential diagnoses of LCH.*

Jaw involvement can present with gingival hyperplasia or ulceration, extensive dissolution of the jawbone structure with tooth loosening ("floating teeth")

Skin is the second most frequently involved organ system after the skeleton overall. In patients younger than two years, it is even the most frequently involved organ. Cutaneous involvement is typically representative of multisystem disease, as 87–93% also have systemic involvement. Cutaneous lesions may be either circumscribed (nodular) or spread and confluent (rash).

They typically present as pinpoint erythematous or skin-colored papules or pustules. The morphology can mimic a seborrheic dermatitis-like or an eczematous erythematous, skin-colored, or brown petechial rash with or without scale, scabbing, crusting, and/or purpura. In infants, a seborrheic dermatitis-like rash on the scalp often causes LCH to be misdiagnosed as seborrheic dermatitis, while groin involvement can mimic treatment-resistant, recurring diaper dermatitis [25].

Nail involvement is rare, but can present as subungual pustules, hemorrhage, or hyperkeratosis, purpuric striae, purulent discharge, longitudinal grooving, onycholysis, paronychia, and pitting [25].

#### **3.3 Lymph nodes**

Enlarged lymph nodes are rarely the only manifestation of a single system LCH. The lymphadenopathy encountered in the setting of multisystem disease is usually mild to moderate.

#### **3.4 Bone marrow**

Peripheral blood cytopenia in LCH patients, often referred to as "hematologic dysfunction", is a sign of severe disease and heralds unfavorable prognosis. For decades, the terms "hematopoietic dysfunction" and "bone marrow involvement" were interchangeably used in the literature [26]. Bone marrow studies in LCH patients using immunochemical staining for CD1a or molecular markers (BRAFV600E), have found increased proportion of histiocytes compared to controls, but their numbers did not correlate well with disease extent and severity [26, 27]. Strikingly, the phagocytosis in the bone marrow, which better correlates to disease severity, is carried out by CD1anegative macrophages. Although the exact mechanisms leading to peripheral cytopenia remain uncovered, it is clear that it is not due to marrow infiltration in most cases, and is probably due to increased phagocytosis or inflammatory marrow suppression.

#### **3.5 Spleen**

Enlargement of the spleen in LCH occurs exclusively in the setting of MS-LCH. It occurs in 15–30% of the patients mostly coinciding with hematopoietic and liver involvement.

#### **3.6 Liver**

Liver involvement occurs exclusively in children with MS-LCH. Patients may present with hepatomegaly only or with functional impairment (elevated liver enzymes, hypoproteinemia and hypoalbuminemia) and/or jaundice.

Two patterns of liver dysfunction can been seen in children: one with predominant hypoproteinemia/hypoalbuminemia ± mild elevation of transaminases and bilirubin; and a less common cholestatic one, due to progressive sclerosing cholangitis [28]. The former is usually combined with prominent constitutional symptoms, and is characteristically observed in the setting of active LCH, while the latter is usually seen as a disease consequence and often without concomitant activity of LCH elsewhere.

#### **3.7 Lungs**

Isolated pulmonary LCH (also known as primary pulmonary LCH) is extremely rare in children, accounting for less than 1% of all pediatric LCH cases. However, pulmonary involvement in the setting of MS-LCH presents at diagnosis in about 25% of cases [29]. The most common clinical symptoms are tachypnea, cyanosis, chest pain and chronic or persistent cough. Characteristic imaging findings are symmetric bilateral reticulonodular opacities ± bullae on radiography and combination of nodules and cysts on CT. Histopathological verification of lung involvement in children with confirmed LCH is required only in case of uncharacteristic or inconsistent imaging findings. Symptom severity and time course can vary. In rare cases, excessive tissue destruction and cyst formation can result in (recurring) life-threatening pneumothorax. Honeycombing with end-stage lung disease is a rare permanent sequela of pediatric LCH.

#### **3.8 Gastrointestinal tract**

Gastrointestinal involvement in LCH (GI-LCH) is infrequent, accounting for about 2–3% of the pediatric series. It usually occurs in the setting of a multisystem LCH, and depending of the affected gut segment, clinically presents with vomiting, abdominal pain, protein-losing enteropathy, bloody and non-bloody diarrhea, malabsorption, and failure to thrive. The prognostic value of gut involvement remains controversial but currently published paper suggests unfavorable impact on survival [30].

#### **3.9 Endocrine system**

Involvement of the hypothalamic–pituitary axis and the resulting central diabetes insipidus (CDI) and dysfunction of the anterior pituitary are a hallmark of LCH. Characteristic findings on MRI are hypothalamic mass, infundibular thickening, and lacking posterior bright spot. CDI manifests with polyuria and polydipsia, and can be the inaugural manifestation of LCH or develop later during disease course. Its prevalence in children with multisystem LCH is between 20 and 35%. Loss of the hormones of the anterior pituitary is less common than CDI. In order of decreasing frequency, pituitary LCH can cause growth hormone (growth failure), thyroid-stimulating hormone (hypothyroidism), adrenocorticotropic hormone (hypocortisolism), luteinizing and follicle-stimulating hormone (hypogonadism) loss. Thyroid involvement is rare, with only 75 cases reported in the literature [25]. It can manifest with gland enlargement due to diffuse or nodular lesions, but the function is mostly preserved.

#### **3.10 Thymus**

Thymus involvement is a rare event with estimated frequency of 1–2% and mostly seen in young children with MS-LCH [31]. Typical imaging findings are enlargement of the gland, cysts and calcifications. Sonography allows for a reliable non-invasive evaluation of the thymus [31].

#### **3.11 Central nervous system (CNS-LCH)**

LCH can affect brain in different ways and result in a variety of manifestations and clinical problems. With respect to risk factors, clinical presentation, imaging findings and the classification of CNS-LCH, the interested readers are referred to two dedicated review papers [32, 33]. For the purposes of clinical management LCH of the brain is divided into granulomatous (tumorous) and non-granulomatous (neurodegenerative) CNS-LCH.

**43**

**Table 5**.

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

Granulomatous (tumorous) lesions of the CNS are defined as space-occupying lesions involving brain structures. Any of the following brain regions may be involved either by isolated lesions or in the context of multisystem disease: hypothalamic– pituitary region (HPR), pineal gland, meninges or choroid plexus [32, 33].

Non-granulomatous (neurodegenerative) lesions encompass two subtypes [32, 33]:

• Radiological neurodegeneration or LCH-associated abnormal CNS imaging (LACI). This term refers to typical signal changes on two consecutive MRI scans performed within an interval of at least 3 months without related clinical

• Clinical neurodegeneration or LCH-associated abnormal CNS symptoms

(LACS). This clinical syndrome is defined as the presence of overt neurological or neuropsychological deficits in the context of consistent radiological findings.

The broad spectrum of clinical manifestations and the variability of disease course and outcome makes prediction of prognosis quite challenging. Attempts to split the disease into categories with distinct prognosis led to elaboration of a number of staging and scoring systems [34–37]. Established prognostic factors in pediatric LCH are disease extent (SS-LCH vs. MC-LCH), involvement of organs crucial for survival (risk organs, e.g. hematopoiesis, liver, spleen) and early response to systemic treatment [38]. SS-LCH has an excellent prognosis, with a survival rate of nearly 100% and a 5-year

recurrence rate of less than 20% [39]. Relapses are usually limited to skeleton and posterior pituitary (diabetes insipidus) and therefore do not affect survival [39, 40]. MS-LCH is a broad category encompassing patients with involvement of two to more than seven organ systems. In 1975, E. Lahey introduced the definition of organ dysfunction [41]. Lahey's definition has been in use for treatment stratification for many decades, and in the 1990s, it was replaced by the definition of "risk organ

Response to an initial 6-week course of systemic therapy has proved to be an additional independent prognostic factor [37, 43–45]. Risk organ involvement at diagnosis and lack of response to 6-weeks of systemic treatment define a subgroup

The French LCH Working Group has developed a disease activity score, which is suitable, for longitudinal objective assessment of disease burden and treatment

The experience from institutional cohorts, registries and clinical trials has unequivocally proven that treatment of LCH has to be tailored to disease extent and severity and to take into account mortality risk. For this purpose, standardized clinical evaluation of each patient at initial diagnosis and relapse is mandatory [22, 48, 49]. The mandatory set of laboratory tests and imaging is presented in **Table 2**. Further investigations to be performed upon specific indications are listed in **Table 3**. Based on the results of the initial evaluation the patients are attributed to one of the disease extent categories of the clinical classification of LCH (**Table 4**). The empirical clinical classification of LCH was developed for the purposes of treatment stratification. The definitions of risk organ involvement are summarized in

of MS-LCH patients with survival of only 20–40% [46].

**5. Pretreatment patient evaluation and stratification**

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

manifestations.

**4. Prognostic factors**

involvement" [34, 35, 37, 42].

success [47].

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

Granulomatous (tumorous) lesions of the CNS are defined as space-occupying lesions involving brain structures. Any of the following brain regions may be involved either by isolated lesions or in the context of multisystem disease: hypothalamic– pituitary region (HPR), pineal gland, meninges or choroid plexus [32, 33].

Non-granulomatous (neurodegenerative) lesions encompass two subtypes [32, 33]:


#### **4. Prognostic factors**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Isolated pulmonary LCH (also known as primary pulmonary LCH) is extremely rare in children, accounting for less than 1% of all pediatric LCH cases. However, pulmonary involvement in the setting of MS-LCH presents at diagnosis in about 25% of cases [29]. The most common clinical symptoms are tachypnea, cyanosis, chest pain and chronic or persistent cough. Characteristic imaging findings are symmetric bilateral reticulonodular opacities ± bullae on radiography and combination of nodules and cysts on CT. Histopathological verification of lung involvement in children with confirmed LCH is required only in case of uncharacteristic or inconsistent imaging findings. Symptom severity and time course can vary. In rare cases, excessive tissue destruction and cyst formation can result in (recurring) life-threatening pneumothorax. Honeycombing

Gastrointestinal involvement in LCH (GI-LCH) is infrequent, accounting for about 2–3% of the pediatric series. It usually occurs in the setting of a multisystem LCH, and depending of the affected gut segment, clinically presents with vomiting, abdominal pain, protein-losing enteropathy, bloody and non-bloody diarrhea, malabsorption, and failure to thrive. The prognostic value of gut involvement remains controversial

Involvement of the hypothalamic–pituitary axis and the resulting central diabetes

insipidus (CDI) and dysfunction of the anterior pituitary are a hallmark of LCH. Characteristic findings on MRI are hypothalamic mass, infundibular thickening, and lacking posterior bright spot. CDI manifests with polyuria and polydipsia, and can be the inaugural manifestation of LCH or develop later during disease course. Its prevalence in children with multisystem LCH is between 20 and 35%. Loss of the hormones of the anterior pituitary is less common than CDI. In order of decreasing frequency, pituitary LCH can cause growth hormone (growth failure), thyroid-stimulating hormone (hypothyroidism), adrenocorticotropic hormone (hypocortisolism), luteinizing and follicle-stimulating hormone (hypogonadism) loss. Thyroid involvement is rare, with only 75 cases reported in the literature [25]. It can manifest with gland enlargement due to diffuse or nodular lesions, but the function is mostly preserved.

Thymus involvement is a rare event with estimated frequency of 1–2% and mostly seen in young children with MS-LCH [31]. Typical imaging findings are enlargement of the gland, cysts and calcifications. Sonography allows for a reliable

LCH can affect brain in different ways and result in a variety of manifestations and clinical problems. With respect to risk factors, clinical presentation, imaging findings and the classification of CNS-LCH, the interested readers are referred to two dedicated review papers [32, 33]. For the purposes of clinical management LCH of the brain is divided into granulomatous (tumorous) and non-granulomatous

with end-stage lung disease is a rare permanent sequela of pediatric LCH.

but currently published paper suggests unfavorable impact on survival [30].

**3.7 Lungs**

**3.8 Gastrointestinal tract**

**3.9 Endocrine system**

**3.10 Thymus**

non-invasive evaluation of the thymus [31].

**3.11 Central nervous system (CNS-LCH)**

(neurodegenerative) CNS-LCH.

**42**

The broad spectrum of clinical manifestations and the variability of disease course and outcome makes prediction of prognosis quite challenging. Attempts to split the disease into categories with distinct prognosis led to elaboration of a number of staging and scoring systems [34–37]. Established prognostic factors in pediatric LCH are disease extent (SS-LCH vs. MC-LCH), involvement of organs crucial for survival (risk organs, e.g. hematopoiesis, liver, spleen) and early response to systemic treatment [38].

SS-LCH has an excellent prognosis, with a survival rate of nearly 100% and a 5-year recurrence rate of less than 20% [39]. Relapses are usually limited to skeleton and posterior pituitary (diabetes insipidus) and therefore do not affect survival [39, 40].

MS-LCH is a broad category encompassing patients with involvement of two to more than seven organ systems. In 1975, E. Lahey introduced the definition of organ dysfunction [41]. Lahey's definition has been in use for treatment stratification for many decades, and in the 1990s, it was replaced by the definition of "risk organ involvement" [34, 35, 37, 42].

Response to an initial 6-week course of systemic therapy has proved to be an additional independent prognostic factor [37, 43–45]. Risk organ involvement at diagnosis and lack of response to 6-weeks of systemic treatment define a subgroup of MS-LCH patients with survival of only 20–40% [46].

The French LCH Working Group has developed a disease activity score, which is suitable, for longitudinal objective assessment of disease burden and treatment success [47].

#### **5. Pretreatment patient evaluation and stratification**

The experience from institutional cohorts, registries and clinical trials has unequivocally proven that treatment of LCH has to be tailored to disease extent and severity and to take into account mortality risk. For this purpose, standardized clinical evaluation of each patient at initial diagnosis and relapse is mandatory [22, 48, 49]. The mandatory set of laboratory tests and imaging is presented in **Table 2**. Further investigations to be performed upon specific indications are listed in **Table 3**. Based on the results of the initial evaluation the patients are attributed to one of the disease extent categories of the clinical classification of LCH (**Table 4**). The empirical clinical classification of LCH was developed for the purposes of treatment stratification. The definitions of risk organ involvement are summarized in **Table 5**.


#### **Table 2.**

*Mandatory baseline evaluation upon initial diagnosis, progression or relapse.*


**45**

**Table 5.**

*\**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

• MRI of head • CT of temporal bone

• Formal hearing assessment

• Endoscopy and biopsy

*In case of verified LCH in other organs, biopsy is indicated ONLY if the pulmonary findings on CT are inconsistent* 

One organ/system involved (uni- or multifocal): • Bone unifocal (single bone) or multifocal (>1 bone)

• Lymph node (not the draining lymph node of another LCH lesion)

• With risk organ involvement (at least one of the following: hematopoietic

• Anemia: hemoglobin <100 g/L (<10 g/dl), infants <90 g/L (<9.0 g/

• dysfunction (i.e. hypoproteinemia <55 g/L, hypoalbuminemia

dl), not due to other causes e.g. iron deficiency • Leukocytopenia: leukocytes <4,0 x109/l (4,000/μL) • Thrombocytopenia: platelets <100 x109/l (100.000/μL)

*Laboratory investigations and imaging recommended upon specific indications.*

• Skin

• Lungs

**Risk organ Involvement criteria**

)

*hypocellularity, myelodysplasia or myelofibrosis may be found.*

*Definition of risk organ involvement.*

• Central nervous system • Other (e.g. thyroid, thymus)

system, liver, or spleen)

• and/or

• and/or

*\*\*Enlargement in cm below the costal margin as assessed by palpation or sonography.*

Two or more organs/systems involved: • Without risk organ involvement

At least 2 of the following:

Spleen • Enlargement >2 cm below costal margin in the midclavicular line\*\* Liver • Enlargement >3 cm below costal margin in the midclavicular line\*\*

<25 g/L, not due to other causes

*Bone marrow infiltration is defined as presence of CD1a positive cells on marrow slides. The clinical significance of marrow CD1a positivity is still unclear. In cases of severe progressive disease, prominent hemophagocytosis, as well as* 

• histopathological findings of active disease

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

**Indication Test**

Aural discharge or suspected hearing impairment/mastoid involvement

Unexplained chronic diarrhea, failure to thrive or evidence of malabsorption

*with LCH or atypical infection is suspected.*

**Disease category Definition**

Single system LCH (SS-LCH)

Multisystem LCH (MS-LCH)

*Clinical classification of LCH.*

Hematopoiesis (with or without bone marrow infiltration\*

**Table 4.**

*\**

**Table 3.**


*with LCH or atypical infection is suspected.*

#### **Table 3.**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

**Test Description** Blood counts • Hemoglobin

Erythrocyte sedimentation rate

Imaging:

**Table 2.**

**Indication Test**

Bi- or pancytopenia, or persistent unexplained single cytopenia

Skeletal survey (radiography, PET/CT, whole-body MRI\*)

Lung involvement (abnormal radiography or symptoms/signs suggestive for lung involvement)

atypical infection\*

excluded)

Abnormal lung CT AND findings not characteristic for LCH or suspicion for

Suspected craniofacial bone lesions including maxilla (mandible

Suspected endocrine abnormality (i.e. short stature, growth failure, polyuria, polydipsia, hypothalamic syndromes, precocious or delayed puberty) and/or imaging abnormality

of hypothalamus/ pituitary

Risk organ involvement • HLA typing

Visual or neurological abnormalities • MRI of head

• Bone marrow aspirate & trephine biopsy to also exclude causes

\* Marrow signal alterations detected by MRI need confirmation. Only bone lesions confirmed by x-ray, CT, PET/CT, or biopsy count for

> liver involvement and the result will alter treatment i.e. to differentiate active LCH from sclerosing cholangitis

• Low dose multi-detector volume-CT is preferable to high-

• Bronchoalveolar lavage (BAL), >5% CD1a-positive cells in BAL

• CT could be considered in addition, if needed for better view

• Endocrine assessment (including water deprivation test and

• MRI of brain (focussed on hypothalamic–pituitary region)

other than LCH Liver dysfunction • Liver biopsy only recommended if there is clinically significant

• White blood cell and differential count

Blood chemistry • Total protein, albumin, bilirubin, ALT (SGPT), AST (SGOT), alkaline

• BUN, creatinine, electrolytes

Urinalysis • Specific gravity and osmolality in an early morning urine sample

• Platelet count

• Ferritin

Coagulation studies • PT, APTT/PTT, fibrinogen

Abdominal ultrasound Size and structure of liver and spleen? Chest radiography Reticulo-nodular opacifications, bullae?

*Mandatory baseline evaluation upon initial diagnosis, progression or relapse.*

stratification.

phosphatase, γGT

• MRI of head

of skeletal lesions Suspected vertebral lesions • MRI of spine (to exclude spinal cord compression and evaluate soft tissue masses)

• Neurology assessment

• Neuropsychometric assessment

dynamic tests of the anterior pituitary)

resolution CT of the lungs

• Lung function test (if age appropriate)

fluid is diagnostic in non-smokers • Lung biopsy (if BAL not diagnostic)

**44**

*Laboratory investigations and imaging recommended upon specific indications.*


#### **Table 4.**

*Clinical classification of LCH.*


*hypocellularity, myelodysplasia or myelofibrosis may be found.*

*\*\*Enlargement in cm below the costal margin as assessed by palpation or sonography.*

#### **Table 5.**

*Definition of risk organ involvement.*

#### **6. Treatment**

Patients with single skeletal lesions usually do not need systemic treatment, except for large symptomatic lesions or lesions in weight-bearing bones, which are not easily accessible for surgical treatment. Treatment of isolated cutaneous LCH is controversial, but if topical treatments fail, systemic treatment needs consideration in infants.

Multifocal skeletal disease and MS-LCH indicate systemic treatment.

#### **6.1 Approach to localized (single-system single site) LCH**

Randomized prospective trials for the treatment of localized LCH are not available. Therefore, current treatment recommendations for localized LCH based on experience gained from retrospective cohorts and non-randomized controlled trials [39, 50].

According to existing clinical experience, the majority of patients with localized LCH (mostly confined to skeleton) do not need systemic treatment. Established treatment options range from expectant attitude, through surgery or topical drug application, to systemic therapy in selected cases. Decisive for the treatment choice in unifocal skeletal LCH is the location (weight-bearing bones or imminent compression of adjacent structures), the size, the surgical accessibility, the presence of considerable adjacent soft-tissue mass, pain or functional impairment, and the risk of permanent consequences.

A best practice based treatment approach to SS-LCH is depicted on **Figure 2**.

#### *6.1.1 Wait and watch*

A "wait and see" approach is justified in small asymptomatic osseous or cutaneous lesions in view of the high likelihood for spontaneous healing.

#### *6.1.2 Surgery*

Surgical procedures such as biopsy, curettage, or resection are used to treat solitary bone lesions, solitary affected lymph nodes, or solitary circumscribed nodular skin lesions. A biopsy is necessary to confirm the diagnosis and at the same time represents a healing stimulus. Clinical experience showed that radical surgery is not necessary and usually not useful in localized LCH [22, 51]. Wide surgical resection

**47**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

is particularly harmful in skull vault, jawbone lesions, as it impedes bone remodeling, and causes permanent defects, which are unlikely in non-resected lesions.

An intralesional application of crystalline methylprednisolone (100-150 mg) in symptomatic bone lesion can quickly bring about a reduction in symptoms and

Because of its potential to induce secondary malignancies, radiotherapy at a low dose (6–10 Gy) is nowadays limited to specific indications (for example, imminent

compression of vital structures (e.g. the spinal cord or the optic nerve).

In case of large, symptomatic lesions, which are not easily accessible and bear high likelihood for pathologic fractures and permanent consequences, mild systemic treatment of short duration (3–6 months) using the same regimen as in

disseminated LCH, may be the preferable option for local disease control.

**6.2 Treatment of disseminated (multifocal skeletal and multisystem) LCH**

Multifocal skeletal and multisystem LCH (earlier unified under the term disseminated LCH) have been traditionally considered an indication for systemic treatment. While there is a general agreement on the indication of systemic therapy for patients with MS-LCH, the value of systemic therapy for multifocal skeletal SS-LCH is less well documented and still needs evaluation in controlled prospective trials [46, 50, 54, 55]. A number of individual drugs, drug combinations and regimens have been tested in LCH since the 1960s. Most trials before the era of international cooperation have pooled patients with varying clinical presentation, course, and prognosis to gain meaningful numbers [56]. Methodological weaknesses and inappropriate sample size lead to contradicting results, and most of the

The current standard of care foots on evidence of the consecutive clinical trials of the Histiocyte Society [42, 44, 57]. The cumulative evidence of the empirical

• The standard front-line therapy for patients with MS-LCH treated outside of controlled clinical trials should consist of a 6–12 weeks of initial therapy (oral steroids and weekly vinblastine injections), followed by pulses of prednisolone/vinblastine every 3 weeks, for a total treatment duration of 12 months.

• Patients with risk organ involvement (particularly those with bi-, pancytopenia and liver dysfunction), who do not respond to 6 weeks of standard treatment have particularly dismal prognosis (survival less than 50%). This small subgroup categorized as "very high risk" deserves treatment intensification. To date only few options have shown promising results in the treatment of severe progressive LCH in small series and pilot trials [58–63]. Their applicability is limited by either high toxicity (cladribine + cytarabine), limited availability of matched donors (hematopoietic stem cell transplantation), or the high relapse

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

early trials are of historic importance only.

trials LCH I-III can be summarized as follows:

rate (MAPK inhibitors when used as single drugs).

*6.1.3 Topical steroids*

facilitated cure [52, 53].

*6.1.4 Radiation therapy*

*6.1.5 Systemic therapy*

**Figure 2.** *Treatment approach to single system LCH.*

is particularly harmful in skull vault, jawbone lesions, as it impedes bone remodeling, and causes permanent defects, which are unlikely in non-resected lesions.

#### *6.1.3 Topical steroids*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Patients with single skeletal lesions usually do not need systemic treatment, except for large symptomatic lesions or lesions in weight-bearing bones, which are not easily accessible for surgical treatment. Treatment of isolated cutaneous LCH is controversial, but if topical treatments fail, systemic treatment needs consideration

Randomized prospective trials for the treatment of localized LCH are not available. Therefore, current treatment recommendations for localized LCH based on experience gained from retrospective cohorts and non-randomized controlled trials

According to existing clinical experience, the majority of patients with localized LCH (mostly confined to skeleton) do not need systemic treatment. Established treatment options range from expectant attitude, through surgery or topical drug application, to systemic therapy in selected cases. Decisive for the treatment choice in unifocal skeletal LCH is the location (weight-bearing bones or imminent compression of adjacent structures), the size, the surgical accessibility, the presence of considerable adjacent soft-tissue mass, pain or functional impairment, and the risk

A best practice based treatment approach to SS-LCH is depicted on **Figure 2**.

A "wait and see" approach is justified in small asymptomatic osseous or cutane-

Surgical procedures such as biopsy, curettage, or resection are used to treat solitary bone lesions, solitary affected lymph nodes, or solitary circumscribed nodular skin lesions. A biopsy is necessary to confirm the diagnosis and at the same time represents a healing stimulus. Clinical experience showed that radical surgery is not necessary and usually not useful in localized LCH [22, 51]. Wide surgical resection

ous lesions in view of the high likelihood for spontaneous healing.

Multifocal skeletal disease and MS-LCH indicate systemic treatment.

**6.1 Approach to localized (single-system single site) LCH**

**6. Treatment**

in infants.

[39, 50].

of permanent consequences.

*6.1.1 Wait and watch*

*6.1.2 Surgery*

**46**

**Figure 2.**

*Treatment approach to single system LCH.*

An intralesional application of crystalline methylprednisolone (100-150 mg) in symptomatic bone lesion can quickly bring about a reduction in symptoms and facilitated cure [52, 53].

#### *6.1.4 Radiation therapy*

Because of its potential to induce secondary malignancies, radiotherapy at a low dose (6–10 Gy) is nowadays limited to specific indications (for example, imminent compression of vital structures (e.g. the spinal cord or the optic nerve).

#### *6.1.5 Systemic therapy*

In case of large, symptomatic lesions, which are not easily accessible and bear high likelihood for pathologic fractures and permanent consequences, mild systemic treatment of short duration (3–6 months) using the same regimen as in disseminated LCH, may be the preferable option for local disease control.

#### **6.2 Treatment of disseminated (multifocal skeletal and multisystem) LCH**

Multifocal skeletal and multisystem LCH (earlier unified under the term disseminated LCH) have been traditionally considered an indication for systemic treatment. While there is a general agreement on the indication of systemic therapy for patients with MS-LCH, the value of systemic therapy for multifocal skeletal SS-LCH is less well documented and still needs evaluation in controlled prospective trials [46, 50, 54, 55]. A number of individual drugs, drug combinations and regimens have been tested in LCH since the 1960s. Most trials before the era of international cooperation have pooled patients with varying clinical presentation, course, and prognosis to gain meaningful numbers [56]. Methodological weaknesses and inappropriate sample size lead to contradicting results, and most of the early trials are of historic importance only.

The current standard of care foots on evidence of the consecutive clinical trials of the Histiocyte Society [42, 44, 57]. The cumulative evidence of the empirical trials LCH I-III can be summarized as follows:


A currently ongoing international trial of the Histiocyte Society (LCH-IV International Collaborative Treatment Protocol for Children and Adolescents with Langerhans Cell Histiocytosis; NCT02205762) with a complex design (5 interventional and 2 observational strata) is looking for improvement of relapse-free survival and quality of life by targeting still unsolved clinical issues [56, 64].

#### *6.2.1 Front-line treatment*

The combination of prednisolone plus vinblastine is the most extensively studied first-line therapy in pediatric-onset LCH [42, 57, 65–68]. The major advantages are its extensively documented activity, its favorable toxicity profile and good tolerability in children, and its moderate costs, which make this treatment applicable even in countries with limited health-care resources [56]. In 'high-risk' patients of the LCH-III trial, the prednisolone plus vinblastine combination has induced response in risk organs in 70% of the patients after 6–12 weeks of treatment, and resulted in an overall 5-year survival of 84%, and a reactivation-free survival of 73% [57].

This regimen is the current standard frontline therapy for pediatric patients with multifocal and multisystem LCH treated outside of clinical trials **(Figure 3A** and **B)**. It consists of 6–12 weeks of initial therapy (oral steroids and weekly vinblastine injections), followed by a continuation therapy given to total treatment duration of 12 months. The continuation therapy consists of prednisolone (day 1–5)/vinblastine (day 1) pulses given every 3 weeks.

#### *6.2.2 Second-line treatment options for non-risk LCH relapses*

The role of systemic treatment and the most appropriate drugs and regimens for patients with non-risk LCH who fail frontline therapy, is less clear. In the majority of those cases, LCH is confined to skeleton, skin and pituitary, and does not influence survival [40, 69, 70]. Similarly, most relapses of LCH are confined to non-risk organs and are not life-threatening. Relapses of LCH, however, are associated with an increased risk of permanent consequences [40, 69, 70]. The belief that control of the disease will prevent subsequent relapses and, thus, related permanent consequences, prompts physicians to use systemic chemotherapy for 'low-risk' multisystem LCH.

Temporary disease control in patients with low-risk disease, particularly in those, who have a relapse after complete disease resolution, is achievable both by repetition of the front-line regimen, or by application of a number of other single drugs or drug combinations [40, 50, 64, 69–71]. Remarkably, none of the available options can prevent further relapses and permanent consequences in all patients. Therefore, second-line treatment of non-risk LCH should be preferably offered within controlled trials. Future trials seeking effective treatment for 'low-risk' LCH should focus on appropriate end-points such as quality of life, risk for and severity of permanent consequences, instead on control of active lesions or remission rates [38].

**49**

**Figure 3.**

*Standard treatment of disseminated LCH.*

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

Such trials are only possible within the frame of a large-scale cooperation and require implementation of innovative study designs and appropriate statistical methods.

• Patients with relapse months or years after stopping prednisone and vinblas-

cytosine arabinoside [72]. This regimen, modified for prednisolone duration,

• Cytosine-arabinoside 100 mg/m2/das for 5 days every 28 days has been used with success both in patients with extracranial non-risk LCH and in CNS-LCH [51, 73].

• 2-Cholorodeoxyadenosine (2-CdA, Cladribine®, Leustatin®) at 5 mg/m2/day for 5 days per course has also been shown to be effective therapy for recurrent low-risk LCH (multifocal bone and low-risk multisystem LCH) with acceptable toxicity [71]. Use of 2-CdA should be limited to a maximum of six cycles to avoid cumulative toxicity and potentially long-lasting or irreversible cytopenias.

tine can benefit from re-induction of the first-line regimen [39].

• An alternative treatment regimen employs vincristine, prednisone, and

from the clinical practice, as well as, justifiable toxicity:

is being prospectively tested in the LCH-IV trial.

For treatment outside of clinical trials, the following drugs and regimens seem to be reasonable choices, based on existing evidence for activity in LCH or experience

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

*6.2.1 Front-line treatment*

(day 1) pulses given every 3 weeks.

*6.2.2 Second-line treatment options for non-risk LCH relapses*

• A standard of care for patients who fail front-line therapy (suboptimal

• The same is true for some specific or rare clinical scenarios, i.e. isolated

A currently ongoing international trial of the Histiocyte Society (LCH-IV International Collaborative Treatment Protocol for Children and Adolescents with Langerhans Cell Histiocytosis; NCT02205762) with a complex design (5 interventional and 2 observational strata) is looking for improvement of relapse-free survival and quality of life by targeting still unsolved clinical issues [56, 64].

response, disease progression or relapse) but the disease is not life-threatening (low risk LCH), remains to be established. Controlled prospective trials with appropriate endpoints (prevention of subsequent relapses and permanent consequences, as well as, improvement of quality of life) are still lacking.

destructive pulmonary LCH, sclerosing cholangitis, LCH reactivation presenting with isolated diabetes insipidus, CNS-LCH of neurodegenerative type.

The combination of prednisolone plus vinblastine is the most extensively studied first-line therapy in pediatric-onset LCH [42, 57, 65–68]. The major advantages are its extensively documented activity, its favorable toxicity profile and good tolerability in children, and its moderate costs, which make this treatment applicable even in countries with limited health-care resources [56]. In 'high-risk' patients of the LCH-III trial, the prednisolone plus vinblastine combination has induced response in risk organs in 70% of the patients after 6–12 weeks of treatment, and resulted in an overall 5-year survival of 84%, and a reactivation-free survival of 73% [57].

This regimen is the current standard frontline therapy for pediatric patients with multifocal and multisystem LCH treated outside of clinical trials **(Figure 3A** and **B)**. It consists of 6–12 weeks of initial therapy (oral steroids and weekly vinblastine injections), followed by a continuation therapy given to total treatment duration of 12 months. The continuation therapy consists of prednisolone (day 1–5)/vinblastine

The role of systemic treatment and the most appropriate drugs and regimens for patients with non-risk LCH who fail frontline therapy, is less clear. In the majority of those cases, LCH is confined to skeleton, skin and pituitary, and does not influence survival [40, 69, 70]. Similarly, most relapses of LCH are confined to non-risk organs and are not life-threatening. Relapses of LCH, however, are associated with an increased risk of permanent consequences [40, 69, 70]. The belief that control of the disease will prevent subsequent relapses and, thus, related permanent consequences, prompts physicians to use systemic chemotherapy for 'low-risk'

Temporary disease control in patients with low-risk disease, particularly in those, who have a relapse after complete disease resolution, is achievable both by repetition of the front-line regimen, or by application of a number of other single drugs or drug combinations [40, 50, 64, 69–71]. Remarkably, none of the available options can prevent further relapses and permanent consequences in all patients. Therefore, second-line treatment of non-risk LCH should be preferably offered within controlled trials. Future trials seeking effective treatment for 'low-risk' LCH should focus on appropriate end-points such as quality of life, risk for and severity of permanent consequences, instead on control of active lesions or remission rates [38].

**48**

multisystem LCH.

#### **Figure 3.**

*Standard treatment of disseminated LCH.*

Such trials are only possible within the frame of a large-scale cooperation and require implementation of innovative study designs and appropriate statistical methods.

For treatment outside of clinical trials, the following drugs and regimens seem to be reasonable choices, based on existing evidence for activity in LCH or experience from the clinical practice, as well as, justifiable toxicity:


The choice of an individual drug or regimen requires consideration of comorbidities, previous treatments, cumulative toxicities and known individual intolerances and side effects. The decision remains on discretion of the treating physician, as the level of published evidence is not sufficient for a clear recommendation of a particular regimen or for a ranked list of preference.

#### *6.2.3 Established salvage therapies for severe progressing multisystem LCH (very high risk LCH)*

Two prospective trials have confirmed the curative potential of the combination of 2-CdA and Ara-C in patients with severe refractory to front-line systemic therapy MS-LCH [58, 59]. Unfortunately, this regimen is highly myelotoxic and associated with treatment-related mortality even if applied in experienced centers [59].

Allogeneic hematopoietic stem cell transplantation is another treatment option for very high-risk multisystem LCH with curative rate comparable to those achieved with the combination of 2-CdA and Ara-C [63, 78]. However, the most optimal conditioning regimen remains to be defined [78].

#### *6.2.4 Toward rational treatment of LCH*

The mitogen-activated protein kinase (MAPK) signalling pathway plays a key role in the regulation of gene expression, cellular growth and survival. A number of activating mutations affecting this pathway result in overactive downstream extracellular-signal-regulated kinase (ERK), which proves to be the ultimate driving event in LCH. Both specific inhibition of the mutated RAF and MEK kinases, as well as, downstream ERK inhibition (**Figure 4**) are undoubtedly appealing treatment options [49, 60, 61, 79].

The clinical experience available to date confirmed at least two essential expectations to BRAF inhibitors, namely in vivo activity and rapid clinical effect [80–83]. In patients with severe life-threatening LCH rapid clinical response is of particular importance. Currently published pediatric series show impressive rapid response to vemurafenib and prove that BRAF inhibitors can induce remission in patients with the most severe form of the disease [60, 61, 84, 85]. The clinical remission is sustainable as far as the treatment is given. However, most patients experience disease relapse shortly after treatment discontinuation. Hence, it is currently unclear whether treatment with a single inhibitor can eradicate the disease.

The European experience with vemurafenib in children with severe MS-LCH has shown that a daily dose of 20 mg/kg (2 x 10 mg/kg) is both well tolerated and clinically effective [60].

**51**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

downstream inhibition for patients without known mutations, defining appropriate pediatric dosages, and establishing optimal treatment duration and drug combina-

Treatment of the disease form referred to as a "non-granulomatous" or "neurodegenerative" CNS-LCH remains frustrating. The two currently recommended treatment options, monthly cytarabine pulses and/or monthly intravenous immunoglobulins have limited effect on disease course, mostly slowing down the process and achieving some improvement in anecdotal cases [33, 86–88]. A pilot study testing retinoic acid could achieve stabilization of the neurologic manifestations

A recently published paper has shed light on the underlying mechanism of the neurodegenerative CNS-LCH with possible therapeutic implications [90]. The authors could reproduce "neurodegenerative" LCH in a mouse model, by introducing the BRAFV600E mutation in the early erythro-myeloid progenitors, which give rise to the microglia. Moreover, in that model the neurodegeneration was preventable by BRAF inhibition. Human data are still limited and indicate that treatment with MAPK inhibitors can be effective if started in advance of irreversible brain

Apart of organ transplantation, effective treatments are still not available for the most severe disease-related complications of LCH, such as sclerosing cholangitis,

The current standard of care for pediatric onset LCH has been developed through laborious empirical trials over four decades. Future optimization of the

**6.3 Treatment of LCH of the central nervous system (CNS-LCH)**

**6.4 Treatment of other life-threatening complications of LCH**

and end-stage lung disease (honeycombing).

**7. Current challenges and future directions**

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

tion for a definitive cure.

*MAPK pathway and relevant inhibition options.*

only [89].

**Figure 4.**

damage [61, 91].

The major tasks to be addressed in controlled prospective trials are therefore: finding the most effective and least toxic specific inhibitors, establishing *Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

dose, but reduced to 3 days can be given.

particular regimen or for a ranked list of preference.

conditioning regimen remains to be defined [78].

*6.2.4 Toward rational treatment of LCH*

ment options [49, 60, 61, 79].

clinically effective [60].

skeletal LCH in adults.

*high risk LCH)*

• Clofarabine is a proven effective therapy for patients with multiple relapses of low-risk or high-risk organs [51, 62]. In LCH, it is usually applied at a dose of 25 mg/m2/day for 5 days every 28 days for six cycles. Depending on hematopoietic toxicity or the need for longer treatment, (further) cycles at the same daily

• Bisphosphonate therapy has reported effects in treating recurrent skeletal LCH [74–77]. The regimen most commonly used in children consist of six doses of pamidronate at 1 mg/kg, given at 4-week intervals. Other bisphosphonates, such as zoledronate and oral alendronate, have also been successful in treating

The choice of an individual drug or regimen requires consideration of comorbidities, previous treatments, cumulative toxicities and known individual intolerances and side effects. The decision remains on discretion of the treating physician, as the level of published evidence is not sufficient for a clear recommendation of a

Two prospective trials have confirmed the curative potential of the combination of 2-CdA and Ara-C in patients with severe refractory to front-line systemic therapy MS-LCH [58, 59]. Unfortunately, this regimen is highly myelotoxic and associated with treatment-related mortality even if applied in experienced centers [59].

Allogeneic hematopoietic stem cell transplantation is another treatment option for very high-risk multisystem LCH with curative rate comparable to those achieved with the combination of 2-CdA and Ara-C [63, 78]. However, the most optimal

The mitogen-activated protein kinase (MAPK) signalling pathway plays a key role in the regulation of gene expression, cellular growth and survival. A number of activating mutations affecting this pathway result in overactive downstream extracellular-signal-regulated kinase (ERK), which proves to be the ultimate driving event in LCH. Both specific inhibition of the mutated RAF and MEK kinases, as well as, downstream ERK inhibition (**Figure 4**) are undoubtedly appealing treat-

The clinical experience available to date confirmed at least two essential expectations to BRAF inhibitors, namely in vivo activity and rapid clinical effect [80–83]. In patients with severe life-threatening LCH rapid clinical response is of particular importance. Currently published pediatric series show impressive rapid response to vemurafenib and prove that BRAF inhibitors can induce remission in patients with the most severe form of the disease [60, 61, 84, 85]. The clinical remission is sustainable as far as the treatment is given. However, most patients experience disease relapse shortly after treatment discontinuation. Hence, it is currently unclear

The European experience with vemurafenib in children with severe MS-LCH has shown that a daily dose of 20 mg/kg (2 x 10 mg/kg) is both well tolerated and

The major tasks to be addressed in controlled prospective trials are therefore: finding the most effective and least toxic specific inhibitors, establishing

whether treatment with a single inhibitor can eradicate the disease.

*6.2.3 Established salvage therapies for severe progressing multisystem LCH (very* 

**50**

downstream inhibition for patients without known mutations, defining appropriate pediatric dosages, and establishing optimal treatment duration and drug combination for a definitive cure.

#### **6.3 Treatment of LCH of the central nervous system (CNS-LCH)**

Treatment of the disease form referred to as a "non-granulomatous" or "neurodegenerative" CNS-LCH remains frustrating. The two currently recommended treatment options, monthly cytarabine pulses and/or monthly intravenous immunoglobulins have limited effect on disease course, mostly slowing down the process and achieving some improvement in anecdotal cases [33, 86–88]. A pilot study testing retinoic acid could achieve stabilization of the neurologic manifestations only [89].

A recently published paper has shed light on the underlying mechanism of the neurodegenerative CNS-LCH with possible therapeutic implications [90]. The authors could reproduce "neurodegenerative" LCH in a mouse model, by introducing the BRAFV600E mutation in the early erythro-myeloid progenitors, which give rise to the microglia. Moreover, in that model the neurodegeneration was preventable by BRAF inhibition. Human data are still limited and indicate that treatment with MAPK inhibitors can be effective if started in advance of irreversible brain damage [61, 91].

#### **6.4 Treatment of other life-threatening complications of LCH**

Apart of organ transplantation, effective treatments are still not available for the most severe disease-related complications of LCH, such as sclerosing cholangitis, and end-stage lung disease (honeycombing).

#### **7. Current challenges and future directions**

The current standard of care for pediatric onset LCH has been developed through laborious empirical trials over four decades. Future optimization of the treatment approach to MS-LCH and development of targeted drugs should be guided by biology insights. In the absence of this knowledge, the clinical needs have to be met by optimization of available treatments. The ongoing LCH –IV trial (ClinicalTrials.gov Identifier: NCT02205762) is designed to address the still remaining problems and unmet patient needs [46, 50]. The most urgent need is eliminating mortality (15–20%) among patients with risk MS-LCH. A two-step stratification based on risk organ involvement at diagnosis and lack of response to standard initial treatment (e.g. at week 6) allows for an early identification of patients who are at risk to die [50]. The combination of 2-CdA and Ara-C has proved to be curative, albeit too toxic. On the other hand, BRAF inhibitors provide rapid control of organ dysfunction, but alone are obviously unable to eradicate the mutated clone. A combination of the empiric and the targeted treatment options may be able to achieve ultimate cure, as this has been the case in other malignancies (e.g. Ph + acute lymphoblastic leukemia).

With all regimens used to date high relapse rates remain another unsolved problem in MS-LCH. Historical controls and preliminary data of the LCH-III trial have shown that treatment duration of 12 months significantly reduces relapse rates compared to 6 months of treatment [46, 57]. The question whether further prolongation of the total treatment duration will result in further reduction of the relapse risk is under investigation in the ongoing LCH-IV trial. A "2x2" factorial design will allow for additional evaluation of the role of oral 6-MP in the continuation treatment of MS-LCH.

There is an urgent need to address optimal treatment of some special disease presentations (i.e. new-onset central diabetes insipidus and non-granulomatous CNS-LCH. The potential of BRAF and MEK inhibitors is still insufficiently explored for these particular indications, but a mouse model delivers a rationale and awakes expectations [90].

Whatever new drugs and regimens appear appropriate testing in LCH, the design of the future prospective studies has to take into account the extreme clinical diversity and unpredictable natural course of MS-LCH, in order to avoid wrong conclusions and therapeutic strays [50, 56].

#### **Acknowledgements**

The authors are indebted to the medical student Martina Minkov for her assistance in the preparation of the figures.

Sigmund Freud University, Vienna, Austria provided financial support for the publication fees.

**53**

**Author details**

Katharina Sterlich1

and Milen Minkov1,2\*

2 Faculty of Medicine, Sigmund Freud University, Vienna, Austria

\*Address all correspondence to: milen.minkov@gesundheitsverbund.at

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Clinic Floridsdorf, Vienna Health Network, Austria

provided the original work is properly cited.

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

#### **Conflict of interest**

The authors declare no conflict of interest.

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

#### **Author details**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

(e.g. Ph + acute lymphoblastic leukemia).

conclusions and therapeutic strays [50, 56].

assistance in the preparation of the figures.

The authors declare no conflict of interest.

ment of MS-LCH.

awakes expectations [90].

**Acknowledgements**

publication fees.

**Conflict of interest**

treatment approach to MS-LCH and development of targeted drugs should be guided by biology insights. In the absence of this knowledge, the clinical needs have to be met by optimization of available treatments. The ongoing LCH –IV trial (ClinicalTrials.gov Identifier: NCT02205762) is designed to address the still remaining problems and unmet patient needs [46, 50]. The most urgent need is eliminating mortality (15–20%) among patients with risk MS-LCH. A two-step stratification based on risk organ involvement at diagnosis and lack of response to standard initial treatment (e.g. at week 6) allows for an early identification of patients who are at risk to die [50]. The combination of 2-CdA and Ara-C has proved to be curative, albeit too toxic. On the other hand, BRAF inhibitors provide rapid control of organ dysfunction, but alone are obviously unable to eradicate the mutated clone. A combination of the empiric and the targeted treatment options may be able to achieve ultimate cure, as this has been the case in other malignancies

With all regimens used to date high relapse rates remain another unsolved problem in MS-LCH. Historical controls and preliminary data of the LCH-III trial have shown that treatment duration of 12 months significantly reduces relapse rates compared to 6 months of treatment [46, 57]. The question whether further prolongation of the total treatment duration will result in further reduction of the relapse risk is under investigation in the ongoing LCH-IV trial. A "2x2" factorial design will allow for additional evaluation of the role of oral 6-MP in the continuation treat-

There is an urgent need to address optimal treatment of some special disease presentations (i.e. new-onset central diabetes insipidus and non-granulomatous CNS-LCH. The potential of BRAF and MEK inhibitors is still insufficiently

explored for these particular indications, but a mouse model delivers a rationale and

Whatever new drugs and regimens appear appropriate testing in LCH, the design of the future prospective studies has to take into account the extreme clinical diversity and unpredictable natural course of MS-LCH, in order to avoid wrong

The authors are indebted to the medical student Martina Minkov for her

Sigmund Freud University, Vienna, Austria provided financial support for the

**52**

Katharina Sterlich1 and Milen Minkov1,2\*

1 Clinic Floridsdorf, Vienna Health Network, Austria

2 Faculty of Medicine, Sigmund Freud University, Vienna, Austria

\*Address all correspondence to: milen.minkov@gesundheitsverbund.at

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**54**

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1999;107(4):883-888.

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histiocytosis. Hematol Oncol Clin North

Current therapy for Langerhans cell histiocytosis. Hematol Oncol Clin North

[56] Minkov M, Rodriguez-Galindo C.

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Munzer M, Landman Parker J, Ouache M, et al. Multi-centre pilot study of 2-chlorodeoxyadenosine and cytosine arabinoside combined chemotherapy in refractory Langerhans cell histiocytosis with haematological

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How I treat Langerhans cell

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*Rare Diseases - Diagnostic and Therapeutic Odyssey*

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[53] Egeler RM, Thompson RC, Jr., Voute PA, Nesbit ME, Jr. Intralesional infiltration of corticosteroids in localized Langerhans' cell histiocytosis. J Pediatr Orthop. 1992;12(6):811-814.

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[58] Bernard F, Thomas C, Bertrand Y, Munzer M, Landman Parker J, Ouache M, et al. Multi-centre pilot study of 2-chlorodeoxyadenosine and cytosine arabinoside combined chemotherapy in refractory Langerhans cell histiocytosis with haematological

dysfunction. Eur J Cancer. 2005;41(17):2682-2689.

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section of the Swiss Work Group for Clinical Cancer Research (SAKK)]. Schweiz Med Wochenschr. 1980;110(23):912-915.

[66] Gadner H, Heitger A, Grois N, Gatterer-Menz I, Ladisch S. Treatment strategy for disseminated Langerhans cell histiocytosis. DAL HX-83 Study Group. Med Pediatr Oncol. 1994;23(2):72-80.

[67] Lahey ME. Histiocytosis X--comparison of three treatment regimens. J Pediatr. 1975;87(2):179-183.

[68] Rigaud C, Barkaoui MA, Thomas C, Bertrand Y, Lambilliotte A, Miron J, et al. Langerhans cell histiocytosis: therapeutic strategy and outcome in a 30-year nationwide cohort of 1478 patients under 18 years of age. Br J Haematol. 2016;174(6):887-898.

[69] Morimoto A, Kobayashi R, Maeda M, Asami K, Bessho F, Imashuku S. Impact of reactivation on the sequelae of multi-system Langerhans cell histiocytosis patients. Pediatr Blood Cancer. 2008;50(4):931- 932; author reply 2.

[70] Pollono D, Rey G, Latella A, Rosso D, Chantada G, Braier J. Reactivation and risk of sequelae in Langerhans cell histiocytosis. Pediatr Blood Cancer. 2007;48(7):696-699.

[71] Weitzman S, Braier J, Donadieu J, Egeler RM, Grois N, Ladisch S, et al. 2'-Chlorodeoxyadenosine (2-CdA) as salvage therapy for Langerhans cell histiocytosis (LCH). results of the LCH-S-98 protocol of the histiocyte society. Pediatr Blood Cancer. 2009;53(7):1271-1276.

[72] Egeler RM, de Kraker J, Voute PA. Cytosine-arabinoside, vincristine, and prednisolone in the treatment of children with disseminated Langerhans cell histiocytosis with organ dysfunction: experience at a

single institution. Med Pediatr Oncol. 1993;21(4):265-270.

[73] Simko SJ, McClain KL, Allen CE. Up-front therapy for LCH: is it time to test an alternative to vinblastine/ prednisone? Br J Haematol. 2015;169(2):299-301.

[74] Arzoo K, Sadeghi S, Pullarkat V. Pamidronate for bone pain from osteolytic lesions in Langerhans' cell histiocytosis. N Engl J Med. 2001;345(3):225.

[75] Brown RE. More on pamidronate in Langerhans'-cell histiocytosis. N Engl J Med. 2001;345(20):1503.

[76] Farran RP, Zaretski E, Egeler RM. Treatment of Langerhans cell histiocytosis with pamidronate. J Pediatr Hematol Oncol. 2001;23(1):54-56.

[77] Morimoto A, Shioda Y, Imamura T, Kanegane H, Sato T, Kudo K, et al. Nationwide survey of bisphosphonate therapy for children with reactivated Langerhans cell histiocytosis in Japan. Pediatr Blood Cancer. 2011;56(1):110-115.

[78] Veys PA, Nanduri V, Baker KS, He W, Bandini G, Biondi A, et al. Haematopoietic stem cell transplantation for refractory Langerhans cell histiocytosis: outcome by intensity of conditioning. Br J Haematol. 2015;169(5):711-718.

[79] Chakraborty R, Hampton OA, Shen X, Simko SJ, Shih A, Abhyankar H, et al. Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis. Blood. 2014;124(19):3007-3015.

[80] Charles J, Beani JC, Fiandrino G, Busser B. Major response to vemurafenib in patient with severe cutaneous Langerhans cell histiocytosis harboring BRAF V600E mutation. J Am Acad Dermatol. 2014;71(3):e97–e99.

**59**

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic…*

[88] Imashuku S, Okazaki NA, Nakayama M, Fujita N, Fukuyama T, Koike K, et al. Treatment of neurodegenerative CNS disease in Langerhans cell histiocytosis with a combination of intravenous immunoglobulin and chemotherapy. Pediatr Blood Cancer.

[89] Idbaih A, Donadieu J, Barthez MA, Geissmann F, Bertrand Y, Hermine O,

"degenerative-like" neuro-langerhans cell histiocytosis: a prospective pilot study. Pediatr Blood Cancer.

[90] Mass E, Jacome-Galarza CE, Blank T, Lazarov T, Durham BH, Ozkaya N, et al. A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease. Nature.

2017;549(7672):389-393.

[91] Diamond EL, Durham BH, Ulaner GA, Drill E, Buthorn J, Ki M, et al. Efficacy of MEK inhibition in patients with histiocytic neoplasms. Nature. 2019;567(7749):521-524.

et al. Retinoic acid therapy in

2008;50(2):308-311.

2004;43(1):55-58.

*DOI: http://dx.doi.org/10.5772/intechopen.96543*

Charlotte F, et al. Dramatic efficacy of vemurafenib in both multisystemic and refractory Erdheim-Chester disease and Langerhans cell histiocytosis harboring the BRAF V600E mutation. Blood.

[81] Haroche J, Cohen-Aubart F, Emile JF, Arnaud L, Maksud P,

[82] Haroche J, Cohen-Aubart F, Emile JF, Maksud P, Drier A, Toledano D, et al. Reproducible and sustained efficacy of targeted therapy with vemurafenib in patients with BRAF(V600E)-mutated Erdheim-Chester disease. J Clin Oncol.

[83] Hyman DM, Puzanov I, Subbiah V,

Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations. N Engl J Med. 2015;373(8):726-736.

[84] Heritier S, Jehanne M, Leverger G, Emile JF, Alvarez JC, Haroche J, et al. Vemurafenib Use in an Infant for High-Risk Langerhans Cell Histiocytosis. JAMA Oncol. 2015;1(6):836-838.

[85] Kolenova A, Schwentner R, Jug G, Simonitsch-Klupp I, Kornauth C, Plank L, et al. Targeted inhibition of the MAPK pathway: emerging salvage option for progressive life-threatening multisystem LCH. Blood Advances.

[86] Allen CE, Flores R, Rauch R, Dauser R, Murray JC, Puccetti D, et al. Neurodegenerative central nervous system Langerhans cell histiocytosis and coincident hydrocephalus treated with vincristine/cytosine arabinoside. Pediatr Blood Cancer. 2010;54(3):416-423.

[87] Imashuku S, Fujita N, Shioda Y, Noma H, Seto S, Minato T, et al. Follow-up of pediatric patients treated by IVIG for Langerhans cell histiocytosis (LCH)-related neurodegenerative CNS disease. Int J Hematol. 2015;101(2):191-197.

Faris JE, Chau I, Blay JY, et al.

2013;121(9):1495-1500.

2015;33(5):411-418.

2017;1(6):352-356.

*Childhood Langerhans Cell Histiocytosis: Epidemiology, Clinical Presentations, Prognostic… DOI: http://dx.doi.org/10.5772/intechopen.96543*

[81] Haroche J, Cohen-Aubart F, Emile JF, Arnaud L, Maksud P, Charlotte F, et al. Dramatic efficacy of vemurafenib in both multisystemic and refractory Erdheim-Chester disease and Langerhans cell histiocytosis harboring the BRAF V600E mutation. Blood. 2013;121(9):1495-1500.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

single institution. Med Pediatr Oncol.

[73] Simko SJ, McClain KL, Allen CE. Up-front therapy for LCH: is it time to test an alternative to vinblastine/

[74] Arzoo K, Sadeghi S, Pullarkat V. Pamidronate for bone pain from osteolytic lesions in Langerhans' cell histiocytosis. N Engl J Med.

[75] Brown RE. More on pamidronate in Langerhans'-cell histiocytosis. N Engl J

[76] Farran RP, Zaretski E, Egeler RM.

histiocytosis with pamidronate. J Pediatr Hematol Oncol. 2001;23(1):54-56.

[77] Morimoto A, Shioda Y, Imamura T, Kanegane H, Sato T, Kudo K, et al. Nationwide survey of bisphosphonate therapy for children with reactivated Langerhans cell histiocytosis in Japan. Pediatr Blood Cancer. 2011;56(1):110-115.

[78] Veys PA, Nanduri V, Baker KS, He W, Bandini G, Biondi A, et al. Haematopoietic stem cell transplantation for refractory

Langerhans cell histiocytosis: outcome by intensity of conditioning. Br J Haematol. 2015;169(5):711-718.

[79] Chakraborty R, Hampton OA,

Abhyankar H, et al. Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis. Blood. 2014;124(19):3007-3015.

[80] Charles J, Beani JC, Fiandrino G,

Busser B. Major response to vemurafenib in patient with severe cutaneous Langerhans cell histiocytosis harboring BRAF V600E mutation. J Am Acad Dermatol. 2014;71(3):e97–e99.

Shen X, Simko SJ, Shih A,

prednisone? Br J Haematol. 2015;169(2):299-301.

1993;21(4):265-270.

2001;345(3):225.

Med. 2001;345(20):1503.

Treatment of Langerhans cell

section of the Swiss Work Group for Clinical Cancer Research

1980;110(23):912-915.

1994;23(2):72-80.

(SAKK)]. Schweiz Med Wochenschr.

[66] Gadner H, Heitger A, Grois N, Gatterer-Menz I, Ladisch S. Treatment strategy for disseminated Langerhans

cell histiocytosis. DAL HX-83 Study Group. Med Pediatr Oncol.

[67] Lahey ME. Histiocytosis X--comparison of three treatment regimens. J Pediatr. 1975;87(2):179-183.

[69] Morimoto A, Kobayashi R, Maeda M, Asami K, Bessho F, Imashuku S. Impact of reactivation on the sequelae of multi-system Langerhans cell histiocytosis patients. Pediatr Blood Cancer. 2008;50(4):931-

[70] Pollono D, Rey G, Latella A, Rosso D, Chantada G, Braier J. Reactivation and risk of sequelae in Langerhans cell histiocytosis. Pediatr Blood Cancer. 2007;48(7):696-699.

[71] Weitzman S, Braier J, Donadieu J, Egeler RM, Grois N, Ladisch S, et al. 2'-Chlorodeoxyadenosine (2-CdA) as salvage therapy for Langerhans cell histiocytosis (LCH). results of the LCH-S-98 protocol of the histiocyte society. Pediatr Blood Cancer.

[72] Egeler RM, de Kraker J, Voute PA. Cytosine-arabinoside, vincristine, and prednisolone in the treatment of children with disseminated Langerhans cell histiocytosis with organ dysfunction: experience at a

932; author reply 2.

2009;53(7):1271-1276.

[68] Rigaud C, Barkaoui MA, Thomas C, Bertrand Y, Lambilliotte A, Miron J, et al. Langerhans cell histiocytosis: therapeutic strategy and outcome in a 30-year nationwide cohort of 1478 patients under 18 years of age. Br J Haematol. 2016;174(6):887-898.

**58**

[82] Haroche J, Cohen-Aubart F, Emile JF, Maksud P, Drier A, Toledano D, et al. Reproducible and sustained efficacy of targeted therapy with vemurafenib in patients with BRAF(V600E)-mutated Erdheim-Chester disease. J Clin Oncol. 2015;33(5):411-418.

[83] Hyman DM, Puzanov I, Subbiah V, Faris JE, Chau I, Blay JY, et al. Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations. N Engl J Med. 2015;373(8):726-736.

[84] Heritier S, Jehanne M, Leverger G, Emile JF, Alvarez JC, Haroche J, et al. Vemurafenib Use in an Infant for High-Risk Langerhans Cell Histiocytosis. JAMA Oncol. 2015;1(6):836-838.

[85] Kolenova A, Schwentner R, Jug G, Simonitsch-Klupp I, Kornauth C, Plank L, et al. Targeted inhibition of the MAPK pathway: emerging salvage option for progressive life-threatening multisystem LCH. Blood Advances. 2017;1(6):352-356.

[86] Allen CE, Flores R, Rauch R, Dauser R, Murray JC, Puccetti D, et al. Neurodegenerative central nervous system Langerhans cell histiocytosis and coincident hydrocephalus treated with vincristine/cytosine arabinoside. Pediatr Blood Cancer. 2010;54(3):416-423.

[87] Imashuku S, Fujita N, Shioda Y, Noma H, Seto S, Minato T, et al. Follow-up of pediatric patients treated by IVIG for Langerhans cell histiocytosis (LCH)-related neurodegenerative CNS disease. Int J Hematol. 2015;101(2):191-197.

[88] Imashuku S, Okazaki NA, Nakayama M, Fujita N, Fukuyama T, Koike K, et al. Treatment of neurodegenerative CNS disease in Langerhans cell histiocytosis with a combination of intravenous immunoglobulin and chemotherapy. Pediatr Blood Cancer. 2008;50(2):308-311.

[89] Idbaih A, Donadieu J, Barthez MA, Geissmann F, Bertrand Y, Hermine O, et al. Retinoic acid therapy in "degenerative-like" neuro-langerhans cell histiocytosis: a prospective pilot study. Pediatr Blood Cancer. 2004;43(1):55-58.

[90] Mass E, Jacome-Galarza CE, Blank T, Lazarov T, Durham BH, Ozkaya N, et al. A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease. Nature. 2017;549(7672):389-393.

[91] Diamond EL, Durham BH, Ulaner GA, Drill E, Buthorn J, Ki M, et al. Efficacy of MEK inhibition in patients with histiocytic neoplasms. Nature. 2019;567(7749):521-524.

**61**

**Chapter 5**

**Abstract**

innovative screening strategies.

**1. Introduction**

The Immune System of

Mesothelioma Patients:

Novel Immunotherapies

*Fabio Nicolini and Massimiliano Mazza*

A Window of Opportunity for

The interplay between the immune system and the pleural mesothelium is crucial both for the development of malignant pleural mesothelioma (MPM) and for the response of MPM patients to therapy. MPM is heavily infiltrated by several immune cell types which affect the progression of the disease. The presence of organized tertiary lymphoid structures (TLSs) witness the attempt to fight the disease *in situ* by adaptive immunity which is often suppressed by tumor expressed

factors. In rare patients physiological, pharmacological or vaccine-induced immune response is efficient, rendering their plasma a valuable resource of antitumor immune cells and molecules. Of particular interest are human antibodies targeting antigens at the tumor cell surface. Here we review current knowledge regarding MPM immune infiltration, MPM immunotherapy and the harnessing of this response to identify novel biologics as biomarkers and therapeutics through

**Keywords:** Malignant pleural mesothelioma (MPM), Immunotherapy, Fully human

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm principally due to asbestos exposure with a poor prognosis and a median overall survival (OS) of only 14 months [1]. Heavy asbestos utilization during earlier decades in Europe is the cause of actual disease incidence [2] and, despite many countries have banned asbestos use in recent years, a peak of MPM incidence is expected for 2020s due to a long latency and delayed disease onset [1, 3–5]. On the contrary, other countries that still make use asbestos are very likely to observe a substantial increase of asbestosrelated disease and MPM in the future. BRCA1 associated protein-1 (*BAP1*) protein is an important player in DNA repair mechanisms, cell cycle control, carcinogenesis and apoptosis and almost 60% of MPM patients have BAP1 mutation [6–13]. BAP1 mutational status determines the insurgence of MPM [9, 10, 12–15], and influences the response to chemotherapy [16] and patient' s clinical outcome [17]. When other gene alterations are coupled to BAP1 mutation, synthetic lethality approaches could be evaluated as therapeutic options [18, 19]. Other frequent mutations are in the

antibody, Tertiary lymphoid structure (TLS), BCR repertoire

#### **Chapter 5**

## The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel Immunotherapies

*Fabio Nicolini and Massimiliano Mazza*

#### **Abstract**

The interplay between the immune system and the pleural mesothelium is crucial both for the development of malignant pleural mesothelioma (MPM) and for the response of MPM patients to therapy. MPM is heavily infiltrated by several immune cell types which affect the progression of the disease. The presence of organized tertiary lymphoid structures (TLSs) witness the attempt to fight the disease *in situ* by adaptive immunity which is often suppressed by tumor expressed factors. In rare patients physiological, pharmacological or vaccine-induced immune response is efficient, rendering their plasma a valuable resource of antitumor immune cells and molecules. Of particular interest are human antibodies targeting antigens at the tumor cell surface. Here we review current knowledge regarding MPM immune infiltration, MPM immunotherapy and the harnessing of this response to identify novel biologics as biomarkers and therapeutics through innovative screening strategies.

**Keywords:** Malignant pleural mesothelioma (MPM), Immunotherapy, Fully human antibody, Tertiary lymphoid structure (TLS), BCR repertoire

#### **1. Introduction**

Malignant pleural mesothelioma (MPM) is an aggressive neoplasm principally due to asbestos exposure with a poor prognosis and a median overall survival (OS) of only 14 months [1]. Heavy asbestos utilization during earlier decades in Europe is the cause of actual disease incidence [2] and, despite many countries have banned asbestos use in recent years, a peak of MPM incidence is expected for 2020s due to a long latency and delayed disease onset [1, 3–5]. On the contrary, other countries that still make use asbestos are very likely to observe a substantial increase of asbestosrelated disease and MPM in the future. BRCA1 associated protein-1 (*BAP1*) protein is an important player in DNA repair mechanisms, cell cycle control, carcinogenesis and apoptosis and almost 60% of MPM patients have BAP1 mutation [6–13]. BAP1 mutational status determines the insurgence of MPM [9, 10, 12–15], and influences the response to chemotherapy [16] and patient' s clinical outcome [17]. When other gene alterations are coupled to BAP1 mutation, synthetic lethality approaches could be evaluated as therapeutic options [18, 19]. Other frequent mutations are in the

genes NF2, LATS2, TP53, SETD2 and TERT promoter as recently reported and are associated with different histotypes of MPM with epithelioid, biphasic and sarcomatoid features [20]. MPM is characterized by a lack of early and specific symptomatology and few reliable biomarkers and screening tools are available causing a late prognosis. As we recently reviewed [21], current therapies in clinical practice consist of surgery, radiotherapy and chemotherapy and innovative therapeutic approaches are being explored. From this survey emerged that new therapeutic modalities and prognostic biomarkers are urgently needed in order to grant a fair chance of survival to all MPM patients. Here we describe the interplay of the immune system and MPM at the tumor tissue level and envision strategies to take advantage of it and derive novel fully human MPM-targeting antibodies to be used as biomarkers and for the design of novel immunotherapies.

#### **2. Inflammatory response and carcinogenesis in MPM**

MPM's development is intertwined with the inflammatory response provoked by asbestos exposure. Asbestos fibers and fluid enter the pleural space where they reach the outer pulmonary parenchyma inducing an inflammatory response [22]. Later steps see macrophage infiltration guided by the presence of the chemokine CCL2 generated by mesothelial cells in response to asbestos fibers contact. Reactive oxygen species (ROS) and nitrogen species are produced by macrophages that, together with already present nitrogen and oxygen species generated from iron particles associated with the fibers, create reactive and dangerous free radicals responsible for mutagenic events and genomic instability [23–25].

Normally, cells which suffer genotoxic DNA damage undergo PARP-dependent apoptosis. Despite that, an *in vitro* study [26] demonstrated that damaged human mesothelial cells could be rescued and skip apoptosis by TNF-alfa produced by macrophages and by other intracellular pathways activated in mesothelial cells, such as NFkB [26–28]. Conversely, TNF-alfa receptor knock-out mice are protected from fibroproliferative lesions when exposed to asbestos fibers [29]. In summary, among innate immune system players, macrophages contribute to genomic alterations as well as survival of mesothelial cells in a context of inflammatory response to asbestos fibers.

#### **3. Immune cell infiltrate in MPM**

#### **3.1 Tumor-Associated Macrophages**

Tumor-Associated Macrophages (TAMs) are the most abundant cells infiltrating the pleural effusions [30–33] and are associated with poor prognosis [32, 34, 35]. *In vitro* and *in vivo* experiments support TAMs as potential targets for MPM treatment. Chemokines released by mesothelioma cells such as CCL4, CCL5, CXCL12 and, in particular, CCL2, are chemoattractants for monocytes [36–38]. CCL2 concentration is particularly high in malignant pleural effusions with respect to benign lesions or lung adenocarcinoma pleural effusions [39, 40] and affects CCR2-expressing monocyte trafficking in MPM [41]. When recruited to MPM lesions, monocytes and macrophages switch to immunosuppressive cells under the influence of growth factors such as M-CSF, IL-34, MCSF [41, 42] and cytokines such as IL-10 and TGF-β released by MPM cells. Those cytokines act both on monocyte and macrophage development and activation but also exert autocrine feedback loop functions on MPM cells [42, 43]. Also, the macrophage checkpoint marker and "do not eat

**63**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

the number of tumor nodules, metastases and tissue invasiveness [46].

me" signal CD47 is found to be highly expressed in the majority of patients with epithelioid mesothelioma [44]. In mesothelioma, TAMs show an immunosuppressive phenotype, characterized by CD14midCD16hi expression, reduced phagocytic activity and increased IL-10 production [45]. In addition, in vitro co-culture of TAMs with MPM cells boosts tumor proliferation and concomitantly reduces sensitivity to chemotherapy treatment [41]. Pro-tumoral activity of TAMs is also evident in mesothelioma mouse models where the removal of macrophages reduces

Granulocytes and neutrophils are also present in MPM microenvironment and recruited by CXCR2 or CXCL5 and CXCL1 chemokines, respectively [36, 47]. Also, polarization and phenotype of granulocytes are affected by growth factors from the mesothelioma secretome which increases their expression of CD11b, CD15 and CD66b markers. These cells function as Myeloid-Derived Suppressor Cells (MDSC) and negatively affect T-cell proliferation via the production and release of ROS [48]. Also, the presence of consistent neutrophilic infiltrate as well as high numbers of neutrophils in the peripheral blood is associated with poor prognosis in epithelioid mesothelioma [49, 50]. However, MDSC targeting in MPM is still debated and

lower numbers compared to macrophages [32, 51–53]. T-regulatory cells (Tregs) are also present in MPM tissue but are less abundant compared to other solid tumors [54]. Principal chemokines present in mesothelioma secretome involved in T-cell trafficking are CXCL12, CXCL10 and CCL5. CXCR3, the receptor of CXCL10 chemokine, is upregulated in mouse models of MPM [47]. CCL5 concentration is high in MPM patients' peripheral blood with respect to asbestos workers and healthy individuals [55] while its receptor CCR5 is expressed on T-cell infiltrating pleural effusions [56]. As discussed in the following chapters, T-cells activation and programming is determined by the presence of neo antigenic stimuli [57, 58] and immune checkpoint expression [59, 60] in specialized immune structures organized

**4. Tertiary lymphoid structures in solid tumors and MPM: where the** 

(DCs) present antigens to T-cells in a major histocompatibility complex (MHC) dependent way acting an efficient adaptive response against cancer, requiring the migration of DCs from the tumor site to the SLOs [61]. Consequently, B-cells are also activated in the SLOs by CD4+ T-cells, begin to proliferate and form a secondary follicle that will be converted to a germinal center (GC). This process induces T and B-lymphocyte proliferation and differentiation into effector T-cells and memory B-cells (MBCs), respectively, that migrate into the tumor contributing to cancer cells elimination, unless unfavorable/antagonizing events or exhaustive signals are in place. However, studies on the role of the immune system in tumors revealed that anti-tumor mechanisms can take place also at the tumor site within organized lymphoid aggregates similar to SLOs [62] called tertiary lymphoid structures (TLSs) [63].

Secondary lymphoid organs (SLOs) are lymphoid regions wherein dendritic cells

T-lymphocytes are present in MPM microenvironment but in

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

**3.2 Myeloid-Derived Suppressor Cells**

controversial and requires further investigations.

**3.3 T-lymphocytes**

and CD8+

**anti-tumor response begins**

CD4<sup>+</sup>

*in situ*.

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

me" signal CD47 is found to be highly expressed in the majority of patients with epithelioid mesothelioma [44]. In mesothelioma, TAMs show an immunosuppressive phenotype, characterized by CD14midCD16hi expression, reduced phagocytic activity and increased IL-10 production [45]. In addition, in vitro co-culture of TAMs with MPM cells boosts tumor proliferation and concomitantly reduces sensitivity to chemotherapy treatment [41]. Pro-tumoral activity of TAMs is also evident in mesothelioma mouse models where the removal of macrophages reduces the number of tumor nodules, metastases and tissue invasiveness [46].

#### **3.2 Myeloid-Derived Suppressor Cells**

Granulocytes and neutrophils are also present in MPM microenvironment and recruited by CXCR2 or CXCL5 and CXCL1 chemokines, respectively [36, 47]. Also, polarization and phenotype of granulocytes are affected by growth factors from the mesothelioma secretome which increases their expression of CD11b, CD15 and CD66b markers. These cells function as Myeloid-Derived Suppressor Cells (MDSC) and negatively affect T-cell proliferation via the production and release of ROS [48]. Also, the presence of consistent neutrophilic infiltrate as well as high numbers of neutrophils in the peripheral blood is associated with poor prognosis in epithelioid mesothelioma [49, 50]. However, MDSC targeting in MPM is still debated and controversial and requires further investigations.

#### **3.3 T-lymphocytes**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

design of novel immunotherapies.

**2. Inflammatory response and carcinogenesis in MPM**

responsible for mutagenic events and genomic instability [23–25].

genes NF2, LATS2, TP53, SETD2 and TERT promoter as recently reported and are associated with different histotypes of MPM with epithelioid, biphasic and sarcomatoid features [20]. MPM is characterized by a lack of early and specific symptomatology and few reliable biomarkers and screening tools are available causing a late prognosis. As we recently reviewed [21], current therapies in clinical practice consist of surgery, radiotherapy and chemotherapy and innovative therapeutic approaches are being explored. From this survey emerged that new therapeutic modalities and prognostic biomarkers are urgently needed in order to grant a fair chance of survival to all MPM patients. Here we describe the interplay of the immune system and MPM at the tumor tissue level and envision strategies to take advantage of it and derive novel fully human MPM-targeting antibodies to be used as biomarkers and for the

MPM's development is intertwined with the inflammatory response provoked by asbestos exposure. Asbestos fibers and fluid enter the pleural space where they reach the outer pulmonary parenchyma inducing an inflammatory response [22]. Later steps see macrophage infiltration guided by the presence of the chemokine CCL2 generated by mesothelial cells in response to asbestos fibers contact. Reactive oxygen species (ROS) and nitrogen species are produced by macrophages that, together with already present nitrogen and oxygen species generated from iron particles associated with the fibers, create reactive and dangerous free radicals

Normally, cells which suffer genotoxic DNA damage undergo PARP-dependent apoptosis. Despite that, an *in vitro* study [26] demonstrated that damaged human mesothelial cells could be rescued and skip apoptosis by TNF-alfa produced by macrophages and by other intracellular pathways activated in mesothelial cells, such as NFkB [26–28]. Conversely, TNF-alfa receptor knock-out mice are protected from fibroproliferative lesions when exposed to asbestos fibers [29]. In summary, among innate immune system players, macrophages contribute to genomic alterations as well as survival of mesothelial cells in a context of inflammatory response

Tumor-Associated Macrophages (TAMs) are the most abundant cells infiltrating the pleural effusions [30–33] and are associated with poor prognosis [32, 34, 35]. *In vitro* and *in vivo* experiments support TAMs as potential targets for MPM treatment. Chemokines released by mesothelioma cells such as CCL4, CCL5, CXCL12 and, in particular, CCL2, are chemoattractants for monocytes [36–38]. CCL2 concentration is particularly high in malignant pleural effusions with respect to benign lesions or lung adenocarcinoma pleural effusions [39, 40] and affects CCR2-expressing monocyte trafficking in MPM [41]. When recruited to MPM lesions, monocytes and macrophages switch to immunosuppressive cells under the influence of growth factors such as M-CSF, IL-34, MCSF [41, 42] and cytokines such as IL-10 and TGF-β released by MPM cells. Those cytokines act both on monocyte and macrophage development and activation but also exert autocrine feedback loop functions on MPM cells [42, 43]. Also, the macrophage checkpoint marker and "do not eat

**62**

to asbestos fibers.

**3. Immune cell infiltrate in MPM**

**3.1 Tumor-Associated Macrophages**

CD4<sup>+</sup> and CD8+ T-lymphocytes are present in MPM microenvironment but in lower numbers compared to macrophages [32, 51–53]. T-regulatory cells (Tregs) are also present in MPM tissue but are less abundant compared to other solid tumors [54]. Principal chemokines present in mesothelioma secretome involved in T-cell trafficking are CXCL12, CXCL10 and CCL5. CXCR3, the receptor of CXCL10 chemokine, is upregulated in mouse models of MPM [47]. CCL5 concentration is high in MPM patients' peripheral blood with respect to asbestos workers and healthy individuals [55] while its receptor CCR5 is expressed on T-cell infiltrating pleural effusions [56]. As discussed in the following chapters, T-cells activation and programming is determined by the presence of neo antigenic stimuli [57, 58] and immune checkpoint expression [59, 60] in specialized immune structures organized *in situ*.

#### **4. Tertiary lymphoid structures in solid tumors and MPM: where the anti-tumor response begins**

Secondary lymphoid organs (SLOs) are lymphoid regions wherein dendritic cells (DCs) present antigens to T-cells in a major histocompatibility complex (MHC) dependent way acting an efficient adaptive response against cancer, requiring the migration of DCs from the tumor site to the SLOs [61]. Consequently, B-cells are also activated in the SLOs by CD4+ T-cells, begin to proliferate and form a secondary follicle that will be converted to a germinal center (GC). This process induces T and B-lymphocyte proliferation and differentiation into effector T-cells and memory B-cells (MBCs), respectively, that migrate into the tumor contributing to cancer cells elimination, unless unfavorable/antagonizing events or exhaustive signals are in place. However, studies on the role of the immune system in tumors revealed that anti-tumor mechanisms can take place also at the tumor site within organized lymphoid aggregates similar to SLOs [62] called tertiary lymphoid structures (TLSs) [63].

TLSs are also present in the stroma, at the invasive margin and/or in the core of different tumor types [63, 64]. TLSs are composed of a T-cell-rich zone together with mature DCs but also by B-cell rich-GC surrounded by plasma cells (PCs). Inside TLSs tumor antigens are presented to T-cells by DCs. and both T- and B- cells are activated, begin to proliferate and to differentiate to effector memory T helper (TH) cells, effector memory cytotoxic T-cells, MBCs or antibody-producing PCs [53, 65–69]. High numbers of CD8+ and CD4+ T-cells in tumors determine TLS density [70] and evidence indicates a positive correlation of TLS density on OS and disease-free survival in lung cancer [66, 70–72], colorectal cancer [73, 74], pancreatic cancer [75, 76], oral squamous cell carcinoma [77] and invasive breast cancer [65, 78–80].

Importantly, its prognostic value is independent of tumor–node–metastasis (TNM) staging in most malignancies suggesting TLS can induce a systemic longlasting anti-tumor response. High endothelial venules (HEVs) similar to those that allow entry of lymphocytes into SLOs could be detected near TLSs [65]. In this context HEVs allow lymphocytes to enter into tumors. Therefore, therapeutic approaches that enhance HEV formation would be beneficial to improve antitumor immune responses. Tregs negatively regulate HEV formation and their absence in cancer murine models promotes T-cell activation and tumor infiltration, favoring the eradication of the lesions [81, 82]. Also other immunosuppressive cell types, such as MDSCs, regulatory B-cell (Bregs) and cytokines, like TGFβ and IL-10, play a part in the development of an immunosuppressive tumor microenvironment (TME).

Tumor-resident Tregs co-express high levels of CTLA-4, OX-40 and GITR compared to effector T-cells and In murine models of MPM, the combination of anti-OX-40 and anti-CTLA-4 antibodies has synergistic effect on CTLA-4+ , OX-40<sup>+</sup> tumor resident T-regs and contributing to a clear tumor regression when compared to single-antibody treatment [83]. Coherently with this point, combined anti-angiogenic and anti-PD-L1 therapies favor HEV and TLS formation in murine models of breast cancer and neuroendocrine pancreatic tumors [84] suggesting that a powerful anti-tumor systemic response by ICIs is sustained, if not triggered, by the presence of TLSs *in situ*. TLS heterogeneity among human cancers has been analyzed via a pan-cancer gene expression analysis of TME cellular composition on The Cancer Genome Atlas (TCGA) data and MPM, as well as lung adenocarcinoma and lung squamous cell carcinoma, display high expression of a 12-chemokine gene signature associated with TLS presence [85] suggesting TLSs are frequent, but also heterogeneous [86].

Seventy percent of MPM cases contain lymphoid aggregates and about 30% of them contain GCs [31]. These aggregates are functionally similar to TLSs, in which T- and B- lymphocytes are apart in two adjacent regions surrounded by HEV, as already shown for ovarian and prostate cancer [87, 88]. Intratumoral CD8+ T-lymphocytes in high numbers are an independent good prognostic marker for MPM patients [68]. Additionally, structural inter- or intra-chromosomal rearrangements and single nucleotide variants have been recently reported from mate-pair and RNA sequencing-based analyses on mesothelioma specimens predicting the expression of potentially-targetable neoantigens [58]. Moreover, some of these neoantigens bind patient-specific MHC and specific tumor-infiltrating T-cell clones are expanded as observed through TCR repertoire analysis [58]. Indeed, TCR diversity and mutation/neoantigen load are inversely correlated, but both active and suppressive TME immune components, such as Treg and CD8+ T-cells, were present and equally balanced suggesting a scenario where activated anti-tumor CD8+ T-cells are counteracted by pro-tumoral immune suppressive molecules and Treg cells [57] or activated CD8+ T and CD4+ T-helper cells displaying phenotypic markers of exhaustion like PD-1, TIM-3 and LAG3 [59].

**65**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

**5. The importance of B-cell infiltration in solid tumors and MPM**

B-cell follicles in TLS from non-small cell lung cancer and ovarian cancers contain bona fide Ki67+ GC B-cells expressing the activation-induced deaminase (AID) gene, that codes a critical enzyme in somatic hypermutation and class switch recombination processes typical of immunoreceptor genes, as well as, of BCL-6, the transcription factor involved in the late stage of B-cell maturation [66, 89]. Additionally, the presence of CD38+ CD138+ PCs around the follicle is highly suggestive of antibody production *in situ* [90]. Indeed, micro-dissected follicles subjected to BCR repertoire analysis revealed clonal amplification compared to peripheral B-cells, supporting the idea that locally presented antigens can elicit specific B-cell responses

Additionally, PCs isolated from dense aggregates in tumor stroma [90], produce anti-tumor antibodies of the immunoglobulin G (IgG) isotype *in vivo* whose mechanism of action has not been yet determined. One possibility is that anti-tumor IgGs produced locally increase antigen presentation by DCs and/or directly promote the activity of specific subsets of CD4+ T-cells endowed with Fcγ receptors (FcγRs) [95]. The presence of IgG deposits in TLS, the spatial organization of TLSs that may favor DC priming by locally produced IgGs and the observation that tumor-derived immune complexes increase the expression of the co-stimulatory molecule CD86 on DCs *in vivo* [87] suggest that these mechanisms take place. In favor of the latter are the results of a meta-analysis in a large set of human cancers showing that the prognostic effect of T-cells is generally stronger when tumor-infiltrating B-cells or PCs are present, supporting the hypothesis that a coordination between cellular and humoral adaptive immune responses is crucial for effective anti-tumor adaptive

The role of B-cells and the association of B-cell rich TLSs with survival and anti-PD-1 immunotherapy response in sarcoma and melanoma have been recently established [97, 98]. Interestingly B-cells are the strongest prognostic factor even in the context of low CD8+ T-cells [97] in sarcoma and class-switched MBCs are specifically enriched in melanoma ICI-treated responders [99]. In murine models of MPM treated with immunotherapy, the presence of B-cells is essential for good prognosis, indicating that antibodies are generated and contribute significantly and essentially to the therapeutic effect [100]. Consistently, B-lymphocyte infiltration in MPM tissue positively correlates with prognosis [38] although variable in its extent [101]. Moreover clinical [52] and preclinical data on B-lymphocytes contribution to MPM prognosis suggest that they elicit an adaptive cytotoxic immune response rather than acting directly as antigen presenting cells (APCs) [100, 102]. In this respect MPM and other solid tumors share many similarities and provide a solid opportunity to develop novel immunotherapies via the identification of MPM

Immune checkpoint (IC) proteins, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1) and PD-L1, are regulators of the immune system that preserve homeostasis and hinder autoimmunity in physiological conditions [103]. ICs overexpression in MPM keeps anti-tumor immune response in check contributing to the creation of a local immunosuppressive TME [31, 104]. IC inhibitors (ICIs), i.e. antibodies targeting ICs, are used as immunomodulatory agents to interfere with the CTLA-4/B7.1/2 or PD-1/PD-L1 axes thereby

helping to overcome tumor-immune escape [95, 105, 106].

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

in several malignancies [87, 89, 91–94].

responses [96].

targeting molecules in patients.

**6. Immunotherapy in MPM**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

#### **5. The importance of B-cell infiltration in solid tumors and MPM**

B-cell follicles in TLS from non-small cell lung cancer and ovarian cancers contain bona fide Ki67+ GC B-cells expressing the activation-induced deaminase (AID) gene, that codes a critical enzyme in somatic hypermutation and class switch recombination processes typical of immunoreceptor genes, as well as, of BCL-6, the transcription factor involved in the late stage of B-cell maturation [66, 89]. Additionally, the presence of CD38+ CD138+ PCs around the follicle is highly suggestive of antibody production *in situ* [90]. Indeed, micro-dissected follicles subjected to BCR repertoire analysis revealed clonal amplification compared to peripheral B-cells, supporting the idea that locally presented antigens can elicit specific B-cell responses in several malignancies [87, 89, 91–94].

Additionally, PCs isolated from dense aggregates in tumor stroma [90], produce anti-tumor antibodies of the immunoglobulin G (IgG) isotype *in vivo* whose mechanism of action has not been yet determined. One possibility is that anti-tumor IgGs produced locally increase antigen presentation by DCs and/or directly promote the activity of specific subsets of CD4+ T-cells endowed with Fcγ receptors (FcγRs) [95]. The presence of IgG deposits in TLS, the spatial organization of TLSs that may favor DC priming by locally produced IgGs and the observation that tumor-derived immune complexes increase the expression of the co-stimulatory molecule CD86 on DCs *in vivo* [87] suggest that these mechanisms take place. In favor of the latter are the results of a meta-analysis in a large set of human cancers showing that the prognostic effect of T-cells is generally stronger when tumor-infiltrating B-cells or PCs are present, supporting the hypothesis that a coordination between cellular and humoral adaptive immune responses is crucial for effective anti-tumor adaptive responses [96].

The role of B-cells and the association of B-cell rich TLSs with survival and anti-PD-1 immunotherapy response in sarcoma and melanoma have been recently established [97, 98]. Interestingly B-cells are the strongest prognostic factor even in the context of low CD8+ T-cells [97] in sarcoma and class-switched MBCs are specifically enriched in melanoma ICI-treated responders [99]. In murine models of MPM treated with immunotherapy, the presence of B-cells is essential for good prognosis, indicating that antibodies are generated and contribute significantly and essentially to the therapeutic effect [100]. Consistently, B-lymphocyte infiltration in MPM tissue positively correlates with prognosis [38] although variable in its extent [101]. Moreover clinical [52] and preclinical data on B-lymphocytes contribution to MPM prognosis suggest that they elicit an adaptive cytotoxic immune response rather than acting directly as antigen presenting cells (APCs) [100, 102]. In this respect MPM and other solid tumors share many similarities and provide a solid opportunity to develop novel immunotherapies via the identification of MPM targeting molecules in patients.

#### **6. Immunotherapy in MPM**

Immune checkpoint (IC) proteins, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1) and PD-L1, are regulators of the immune system that preserve homeostasis and hinder autoimmunity in physiological conditions [103]. ICs overexpression in MPM keeps anti-tumor immune response in check contributing to the creation of a local immunosuppressive TME [31, 104]. IC inhibitors (ICIs), i.e. antibodies targeting ICs, are used as immunomodulatory agents to interfere with the CTLA-4/B7.1/2 or PD-1/PD-L1 axes thereby helping to overcome tumor-immune escape [95, 105, 106].

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

microenvironment (TME).

heterogeneous [86].

exhaustion like PD-1, TIM-3 and LAG3 [59].

OX-40<sup>+</sup>

TLSs are also present in the stroma, at the invasive margin and/or in the core of different tumor types [63, 64]. TLSs are composed of a T-cell-rich zone together with mature DCs but also by B-cell rich-GC surrounded by plasma cells (PCs). Inside TLSs tumor antigens are presented to T-cells by DCs. and both T- and B- cells are activated, begin to proliferate and to differentiate to effector memory T helper (TH) cells, effector memory cytotoxic T-cells, MBCs or antibody-producing PCs [53, 65–69]. High numbers of CD8+ and CD4+ T-cells in tumors determine TLS density [70] and evidence indicates a positive correlation of TLS density on OS and disease-free survival in lung cancer [66, 70–72], colorectal cancer [73, 74], pancreatic cancer [75, 76], oral

Importantly, its prognostic value is independent of tumor–node–metastasis (TNM) staging in most malignancies suggesting TLS can induce a systemic longlasting anti-tumor response. High endothelial venules (HEVs) similar to those that allow entry of lymphocytes into SLOs could be detected near TLSs [65]. In this context HEVs allow lymphocytes to enter into tumors. Therefore, therapeutic approaches that enhance HEV formation would be beneficial to improve antitumor immune responses. Tregs negatively regulate HEV formation and their absence in cancer murine models promotes T-cell activation and tumor infiltration, favoring the eradication of the lesions [81, 82]. Also other immunosuppressive cell types, such as MDSCs, regulatory B-cell (Bregs) and cytokines, like TGFβ and IL-10, play a part in the development of an immunosuppressive tumor

Tumor-resident Tregs co-express high levels of CTLA-4, OX-40 and GITR compared to effector T-cells and In murine models of MPM, the combination of anti-OX-40 and anti-CTLA-4 antibodies has synergistic effect on CTLA-4+

Seventy percent of MPM cases contain lymphoid aggregates and about 30% of them contain GCs [31]. These aggregates are functionally similar to TLSs, in which T- and B- lymphocytes are apart in two adjacent regions surrounded by HEV, as already shown for ovarian and prostate cancer [87, 88]. Intratumoral CD8+ T-lymphocytes in high numbers are an independent good prognostic marker for MPM patients [68]. Additionally, structural inter- or intra-chromosomal rearrangements and single nucleotide variants have been recently reported from mate-pair and RNA sequencing-based analyses on mesothelioma specimens predicting the expression of potentially-targetable neoantigens [58]. Moreover, some of these neoantigens bind patient-specific MHC and specific tumor-infiltrating T-cell clones are expanded as observed through TCR repertoire analysis [58]. Indeed, TCR diversity and mutation/neoantigen load are inversely correlated, but both active and suppressive TME immune components, such as Treg and CD8+ T-cells, were present and equally balanced suggesting a scenario where activated anti-tumor CD8+ T-cells are counteracted by pro-tumoral immune suppressive molecules and Treg cells [57] or activated CD8+ T and CD4+ T-helper cells displaying phenotypic markers of

 tumor resident T-regs and contributing to a clear tumor regression when compared to single-antibody treatment [83]. Coherently with this point, combined anti-angiogenic and anti-PD-L1 therapies favor HEV and TLS formation in murine models of breast cancer and neuroendocrine pancreatic tumors [84] suggesting that a powerful anti-tumor systemic response by ICIs is sustained, if not triggered, by the presence of TLSs *in situ*. TLS heterogeneity among human cancers has been analyzed via a pan-cancer gene expression analysis of TME cellular composition on The Cancer Genome Atlas (TCGA) data and MPM, as well as lung adenocarcinoma and lung squamous cell carcinoma, display high expression of a 12-chemokine gene signature associated with TLS presence [85] suggesting TLSs are frequent, but also

,

squamous cell carcinoma [77] and invasive breast cancer [65, 78–80].

**64**

Recently, PD-L1 expression in MPM has been assessed on tissue microarrays using two different FDA-approved antibodies and 22–27% of cases were positive for PD-L1 (1% cut off) [107]. PD-L1 is expressed in a high proportion of biphasic and sarcomatoid MPM cases and its positivity >1% is associated with a significant 10-months reduction in median OS compared to PD-L1 negative tumors [108, 109]. Similarly, high PD-L1 expression (>50%) in epithelioid MPM patients correlates with shorter PFS (6.7 vs. 9.9 months) [108]. Despite its prognostic value [59, 60, 110], PD-L1 expression is not a valid predictive marker of response to anti-PD-L1 therapies for several tumor types [111, 112], including MPM [113]. Anti-PD-1/PD-L1 therapies were tested in different trials in MPM patients [114–121]. Combination of pembrolizumab with PPC in first-line treatment compared to pembrolizumab or PPC alone, is currently being evaluated in the phase III trial NCT0278417, while nivolumab is being investigated in the randomized phase III trial CONFIRM (NCT03063450) in comparison with placebo [119]. Durvalumab activity, a PD-L1 inhibitor, in combination with first-line CCP was tested in the DREAM study (ANZ clinical trial registry number: ACTRN12616001170415). This combination resulted in an ORR of 61% using mRECIST and 53% using iRECIST criteria and in a 6 months PFS of 71% (mRECIST). On the basis of these observations a randomized phase 3 trial will be started [122].

ICs expression is controlled at different stages of T-lymphocyte activation and variable in tumor cells. For these reasons, a combination strategy employing two different ICIs in addition to chemotherapy has been proposed to achieve a synergistic effect by overcoming immune-resistance observed in some MPM patients. Encouraging results observed for different ICIs in combination [113, 123, 124] prompted the investigation of the nivolumab plus ipilimumab combination in comparison to standard PPC alone as first-line option in the phase III clinical trial Checkmate-743 (NCT02899299). Checkmate-743 has clearly demonstrated the benefit of nivolumab in combination with ipilimumab in first line mesothelioma treatment and based on those results obtained approval from FDA from October 2020. On 22 April 2021, the Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion recommending a change to the terms of the marketing authorization for the medicinal product Opdivo (nivolumab) in combination with ipilimumab for the first line treatment of adult patients with unresectable malignant pleural mesothelioma in Europe as well.

At present, efficacy and safety of adoptive T-cell therapies, in particular chimeric antigen receptor-transduced T-cells (CAR-T), in MPM and other solid tumors are under investigation [125, 126]. CAR-T-cells directed against mesothelin (MSLN), a glycoprotein expressed on MPM and other solid tumor cells, with a limited presence on normal tissues [127], represent a promising therapeutic option [128, 129]. Recently, Adusumilli and colleagues reported the outcome of a phase I clinical trial, NCT02414269, [130, 131] on MPM patients with pleural metastases from lung or breast cancer treated with anti-MSLN CAR-T-cells. Of note, the inclusion of anti-PD-1 therapy was crucial to elicit clinical efficacy and avoid T-cell exhaustion since no patient had an objective response before pembrolizumab addition showing the importance of conditioning the immune suppressive features of the TME also in this therapeutic setting.

Pembrolizumab plus anti-MSLN CAR-T-cell combination showed the best clinical outcome with an ORR of 63% (10/16) and a DCR of 75% (12/16). No evidence of on-target, off-tumor or therapy related toxicities higher than grade 1 was observed. Although applied to a limited number of patients so far, CAR-T therapies against MPM have shown really impressive results highlighting the different efficacy for advanced cell therapies compared to small molecule drugs or antibodies. Recently,

**67**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

a comprehensive review about immunotherapy in MPM has been published [132]. However, the limited availability of therapeutic targetable antigens hinders the efficacy of CAR based strategies for MPM patients. More targets are needed for

ICIs effectiveness in MPM treated patients highlight the presence of potentially active immune cells *in situ that* if properly unleashed can elicit anti-tumor responses. However, to achieve this goal, TME must be modified in order to abolish/ interfere with specific immune suppressive cues. Interestingly, Barsky and colleagues recently reported a case of a man with MPM treated with a combination of palliative radiation and immune-gene therapy (GM-CSF) [133]. The outcome of this treatment combination was outstanding, resulting in a so-called "abscopal

The abscopal effect is observed when a localized radiation induces an antitumor response at distant sites. RT can trigger an immunogenic cell death (ICD) [134, 135] and can stimulate antigen-specific, adaptive immunity [136]. ICD sets the stage for anti-tumor immune responses which include the release of tumor antigens by irradiated tumor cells, the cross-presentation of tumor-derived antigens to T-cells by antigen-presenting cells (APCs), and the migration of effector T-cells from the lymph nodes to distant tumor sites, suggesting that irradiated tumors can act as an *in situ* vaccine if appropriate conditions are in place [137–139]. The overall incidence of the abscopal effect of RT alone is low with 46 clinical cases reported from 1969 [139]. Those poor numbers witness the insufficiency of RT alone to overcome the immune resistance of malignant tumors. Immunotherapy can lower host immune tolerance towards tumors, therefore the combination of RT and immunotherapy can amplify the anti-tumor immune response, a hypothesis currently under investigation in the trial NCT02959463 where adjuvant pembrolizumab after RT in lung-intact MPM patients is tested. In a murine model of MPM, the abscopal effect can be induced by local RT and enhanced by immune suppressive CTLA-4 blockade as infiltrated T-cells, both in primary and secondary tumor sites, are predominantly cytotoxic CD8+ T-cells while Tregs are reduced [140]. Those observations corroborate the idea that a systemic tumor response can be unleashed by a local treatment thereby modifying

**8. The quest for specificity in malignant mesothelioma: how can we fill** 

Adoptive cell therapies in combination with ICIs are showing promising results for MPM patients. Their specificity or preference of targeting is granted almost exclusively by the use of antibodies or their derived fragments that are directed to tumor specific/associated antigens. First attempts of therapy using murine monoclonal antibodies (mAbs) in cancer patients failed due to neutralizing antibodies generation and to mismatch with components of the human immune system. These results highlighted the importance of using human or human compatible/tolerable biomolecules and prompted the design of novel screening platforms to find them. Antigen unbiased screening methods (**Figure 1**) can be used to this end to test *a priori* the targeting ability of antibodies to cells postponing the identification of

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

**7. Making a hot tumor microenvironment**

MPM treatment in the future.

effect".

the features of the TME.

antigens to lead candidates only.

**this gap?**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

a comprehensive review about immunotherapy in MPM has been published [132]. However, the limited availability of therapeutic targetable antigens hinders the efficacy of CAR based strategies for MPM patients. More targets are needed for MPM treatment in the future.

#### **7. Making a hot tumor microenvironment**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

be started [122].

Recently, PD-L1 expression in MPM has been assessed on tissue microarrays using two different FDA-approved antibodies and 22–27% of cases were positive for PD-L1 (1% cut off) [107]. PD-L1 is expressed in a high proportion of biphasic and sarcomatoid MPM cases and its positivity >1% is associated with a significant 10-months reduction in median OS compared to PD-L1 negative tumors [108, 109]. Similarly, high PD-L1 expression (>50%) in epithelioid MPM patients correlates with shorter PFS (6.7 vs. 9.9 months) [108]. Despite its prognostic value [59, 60, 110], PD-L1 expression is not a valid predictive marker of response to anti-PD-L1 therapies for several tumor types [111, 112], including MPM [113]. Anti-PD-1/PD-L1 therapies were tested in different trials in MPM patients [114–121]. Combination of pembrolizumab with PPC in first-line treatment compared to pembrolizumab or PPC alone, is currently being evaluated in the phase III trial NCT0278417, while nivolumab is being investigated in the randomized phase III trial CONFIRM (NCT03063450) in comparison with placebo [119]. Durvalumab activity, a PD-L1 inhibitor, in combination with first-line CCP was tested in the DREAM study (ANZ clinical trial registry number: ACTRN12616001170415). This combination resulted in an ORR of 61% using mRECIST and 53% using iRECIST criteria and in a 6 months PFS of 71% (mRECIST). On the basis of these observations a randomized phase 3 trial will

ICs expression is controlled at different stages of T-lymphocyte activation and variable in tumor cells. For these reasons, a combination strategy employing two different ICIs in addition to chemotherapy has been proposed to achieve a synergistic effect by overcoming immune-resistance observed in some MPM patients. Encouraging results observed for different ICIs in combination [113, 123, 124] prompted the investigation of the nivolumab plus ipilimumab combination in comparison to standard PPC alone as first-line option in the phase III clinical trial Checkmate-743 (NCT02899299). Checkmate-743 has clearly demonstrated the benefit of nivolumab in combination with ipilimumab in first line mesothelioma treatment and based on those results obtained approval from FDA from October 2020. On 22 April 2021, the Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion recommending a change to the terms of the marketing authorization for the medicinal product Opdivo (nivolumab) in combination with ipilimumab for the first line treatment of adult patients with unresect-

At present, efficacy and safety of adoptive T-cell therapies, in particular chimeric antigen receptor-transduced T-cells (CAR-T), in MPM and other solid tumors are under investigation [125, 126]. CAR-T-cells directed against mesothelin (MSLN), a glycoprotein expressed on MPM and other solid tumor cells, with a limited presence on normal tissues [127], represent a promising therapeutic option [128, 129]. Recently, Adusumilli and colleagues reported the outcome of a phase I clinical trial, NCT02414269, [130, 131] on MPM patients with pleural metastases from lung or breast cancer treated with anti-MSLN CAR-T-cells. Of note, the inclusion of anti-PD-1 therapy was crucial to elicit clinical efficacy and avoid T-cell exhaustion since no patient had an objective response before pembrolizumab addition showing the importance of conditioning the immune suppressive features of

Pembrolizumab plus anti-MSLN CAR-T-cell combination showed the best clinical outcome with an ORR of 63% (10/16) and a DCR of 75% (12/16). No evidence of on-target, off-tumor or therapy related toxicities higher than grade 1 was observed. Although applied to a limited number of patients so far, CAR-T therapies against MPM have shown really impressive results highlighting the different efficacy for advanced cell therapies compared to small molecule drugs or antibodies. Recently,

able malignant pleural mesothelioma in Europe as well.

the TME also in this therapeutic setting.

**66**

ICIs effectiveness in MPM treated patients highlight the presence of potentially active immune cells *in situ that* if properly unleashed can elicit anti-tumor responses. However, to achieve this goal, TME must be modified in order to abolish/ interfere with specific immune suppressive cues. Interestingly, Barsky and colleagues recently reported a case of a man with MPM treated with a combination of palliative radiation and immune-gene therapy (GM-CSF) [133]. The outcome of this treatment combination was outstanding, resulting in a so-called "abscopal effect".

The abscopal effect is observed when a localized radiation induces an antitumor response at distant sites. RT can trigger an immunogenic cell death (ICD) [134, 135] and can stimulate antigen-specific, adaptive immunity [136]. ICD sets the stage for anti-tumor immune responses which include the release of tumor antigens by irradiated tumor cells, the cross-presentation of tumor-derived antigens to T-cells by antigen-presenting cells (APCs), and the migration of effector T-cells from the lymph nodes to distant tumor sites, suggesting that irradiated tumors can act as an *in situ* vaccine if appropriate conditions are in place [137–139]. The overall incidence of the abscopal effect of RT alone is low with 46 clinical cases reported from 1969 [139]. Those poor numbers witness the insufficiency of RT alone to overcome the immune resistance of malignant tumors. Immunotherapy can lower host immune tolerance towards tumors, therefore the combination of RT and immunotherapy can amplify the anti-tumor immune response, a hypothesis currently under investigation in the trial NCT02959463 where adjuvant pembrolizumab after RT in lung-intact MPM patients is tested. In a murine model of MPM, the abscopal effect can be induced by local RT and enhanced by immune suppressive CTLA-4 blockade as infiltrated T-cells, both in primary and secondary tumor sites, are predominantly cytotoxic CD8+ T-cells while Tregs are reduced [140]. Those observations corroborate the idea that a systemic tumor response can be unleashed by a local treatment thereby modifying the features of the TME.

#### **8. The quest for specificity in malignant mesothelioma: how can we fill this gap?**

Adoptive cell therapies in combination with ICIs are showing promising results for MPM patients. Their specificity or preference of targeting is granted almost exclusively by the use of antibodies or their derived fragments that are directed to tumor specific/associated antigens. First attempts of therapy using murine monoclonal antibodies (mAbs) in cancer patients failed due to neutralizing antibodies generation and to mismatch with components of the human immune system. These results highlighted the importance of using human or human compatible/tolerable biomolecules and prompted the design of novel screening platforms to find them. Antigen unbiased screening methods (**Figure 1**) can be used to this end to test *a priori* the targeting ability of antibodies to cells postponing the identification of antigens to lead candidates only.

#### **Figure 1.**

*Schematic representation of 4 antigen-unbiased screening strategies to obtain fully human tumor targeting antibodies. Panel A: Patient derived scFv phage display libraries can be generated from MPM patient peripheral blood B cells. Those libraries are used to screen for novel specificities. Phage displayed scFvs undergo selection through consecutive rounds of panning on tumor cells to enrich for specific binders. NGS analysis allows the prediction of scFv sequences enriched on tumor cells. Panel B:* de novo *formed sequences, like those codifying the BCRs of infiltrated immune cells in tumor tissue can be retrieved using specific bioinformatic tools. Combinations of different heavy (VH) and light (VL) variable chains are used to generate candidate antibodies to be screened on cell or tumor tissues. Panel C: Analysis of BCR repertoire could be performed from memory B-cells from MPM patients. Enriched or de novo formed sequences could be monitored before and after a specific treatment in order to identify specific clones. Panel D: Memory B-cells from MPM patients can be immortalized through EBV infection and the immunoglobulins released in the medium of clonal cell cultures are tested on tumor cells by FACS or ELISA assays.*

**69**

ing errors.

retrospective patients' cohorts.

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

As explained above, patients develop an immune response against MPM that, if unleashed, can be very effective. The presence of TLSs and the development of oligoclonal families of B-cells inside or at the border of MPM tissue are positive prognostic features and constitute a window of opportunity to capture human therapeutic antibodies. Now the next question is: how can we exploit this powerful reservoir of biologics to isolate or design targeting drugs? In other words: what

Bulk RNA-Seq data from tumor tissue contain a hitherto overlooked picture of tumor and its ecosystem. Typically, data are analyzed to assess the expression of known transcripts, while *de novo* formed sequences, like those generated by T- and B-cells in the assembly and generation of their specific receptors, are usually disregarded since they fall off from the comparison with the reference transcriptome. However, these sequences can be retrieved from raw data and employed to extract the sequence of TCRs and BCRs from tumor tissue infiltrated immune cells using specific bioinformatic tools. One of them is MiXCR [141], a universal tool which takes raw sequencing data, including RNA-seq, as input and profiles TCR and BCR repertoires. As a reference, it uses a built-in library of V, D, J and C gene sequences from the human or mouse genome. MixCR output provides a list of clonotypes derived by assembling identical and homologous reads, corrected for sequenc-

V'DJer is another software that can process RNA-seq data for this purpose [142]. It can be run on BCR light and heavy chain data and employs unmapped paired end short reads aligning them against a reference transcriptome. Then, V'DJer detects VDJ rearrangements, generates contigs and quantifies the ones that represent the most abundant portions of the BCR repertoire. When the expression levels of BCR are low, there is an option to increase sensitivity of the algorithm at the cost of increasing the demand for computational resources. V'DJer has been used, for example, to retrieve antibodies from RNA sequencing data of melanoma patients from TCGA repository [142, 143]. At present, TCGA contains expression analyses of 87 MPM patients (TCGA-MESO) that could be used for this purpose. In addition, RNA can be obtained from FFPE samples containing TLSs in prospective and

It is possible to infer the sequence of resident B-cell clones by applying bioinformatic tools to RNA-Seq or by sequencing amplicons for immunoglobulin chains using specific sets of degenerate universal primers from whole tissue DNA or RNA/cDNA. The latter approach is implemented by the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). In contrast to profiling using bulk RNA-Seq data, it is more precise since the experimental design is optimized to identify the BCR repertoire through the ImmunoSeq Analyzer software which is specific for this purpose. Its starting material can be both genomic DNA (gDNA) and cDNA: in order to assess clonal expansion of B-cells in tissues, gDNA is the best solution since each cell contains the same copy number, while mRNA transcripts can be very different among cells, depending on cellular activation and even the retrotranscription procedure can add other confounding factors. However, cDNA is a better choice if the goal is to find the most abundantly produced antibodies in situ, since there is a difference in the mRNA expression between activated and naive B-cells. Finally, independently of the method employed for their derivation,

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

**9. From today's patients the future cures for MPM**

technologies are available to take up this challenge?

**10. BCR repertoire from sequencing data**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

#### **9. From today's patients the future cures for MPM**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

**68**

*tested on tumor cells by FACS or ELISA assays.*

**Figure 1.**

*Schematic representation of 4 antigen-unbiased screening strategies to obtain fully human tumor targeting antibodies. Panel A: Patient derived scFv phage display libraries can be generated from MPM patient peripheral blood B cells. Those libraries are used to screen for novel specificities. Phage displayed scFvs undergo selection through consecutive rounds of panning on tumor cells to enrich for specific binders. NGS analysis allows the prediction of scFv sequences enriched on tumor cells. Panel B:* de novo *formed sequences, like those codifying the BCRs of infiltrated immune cells in tumor tissue can be retrieved using specific bioinformatic tools. Combinations of different heavy (VH) and light (VL) variable chains are used to generate candidate antibodies to be screened on cell or tumor tissues. Panel C: Analysis of BCR repertoire could be performed from memory B-cells from MPM patients. Enriched or de novo formed sequences could be monitored before and after a specific treatment in order to identify specific clones. Panel D: Memory B-cells from MPM patients can be immortalized through EBV infection and the immunoglobulins released in the medium of clonal cell cultures are* 

As explained above, patients develop an immune response against MPM that, if unleashed, can be very effective. The presence of TLSs and the development of oligoclonal families of B-cells inside or at the border of MPM tissue are positive prognostic features and constitute a window of opportunity to capture human therapeutic antibodies. Now the next question is: how can we exploit this powerful reservoir of biologics to isolate or design targeting drugs? In other words: what technologies are available to take up this challenge?

#### **10. BCR repertoire from sequencing data**

Bulk RNA-Seq data from tumor tissue contain a hitherto overlooked picture of tumor and its ecosystem. Typically, data are analyzed to assess the expression of known transcripts, while *de novo* formed sequences, like those generated by T- and B-cells in the assembly and generation of their specific receptors, are usually disregarded since they fall off from the comparison with the reference transcriptome. However, these sequences can be retrieved from raw data and employed to extract the sequence of TCRs and BCRs from tumor tissue infiltrated immune cells using specific bioinformatic tools. One of them is MiXCR [141], a universal tool which takes raw sequencing data, including RNA-seq, as input and profiles TCR and BCR repertoires. As a reference, it uses a built-in library of V, D, J and C gene sequences from the human or mouse genome. MixCR output provides a list of clonotypes derived by assembling identical and homologous reads, corrected for sequencing errors.

V'DJer is another software that can process RNA-seq data for this purpose [142]. It can be run on BCR light and heavy chain data and employs unmapped paired end short reads aligning them against a reference transcriptome. Then, V'DJer detects VDJ rearrangements, generates contigs and quantifies the ones that represent the most abundant portions of the BCR repertoire. When the expression levels of BCR are low, there is an option to increase sensitivity of the algorithm at the cost of increasing the demand for computational resources. V'DJer has been used, for example, to retrieve antibodies from RNA sequencing data of melanoma patients from TCGA repository [142, 143]. At present, TCGA contains expression analyses of 87 MPM patients (TCGA-MESO) that could be used for this purpose. In addition, RNA can be obtained from FFPE samples containing TLSs in prospective and retrospective patients' cohorts.

It is possible to infer the sequence of resident B-cell clones by applying bioinformatic tools to RNA-Seq or by sequencing amplicons for immunoglobulin chains using specific sets of degenerate universal primers from whole tissue DNA or RNA/cDNA. The latter approach is implemented by the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). In contrast to profiling using bulk RNA-Seq data, it is more precise since the experimental design is optimized to identify the BCR repertoire through the ImmunoSeq Analyzer software which is specific for this purpose. Its starting material can be both genomic DNA (gDNA) and cDNA: in order to assess clonal expansion of B-cells in tissues, gDNA is the best solution since each cell contains the same copy number, while mRNA transcripts can be very different among cells, depending on cellular activation and even the retrotranscription procedure can add other confounding factors. However, cDNA is a better choice if the goal is to find the most abundantly produced antibodies in situ, since there is a difference in the mRNA expression between activated and naive B-cells. Finally, independently of the method employed for their derivation, identified immunoglobulin heavy and light chain sequences can be assembled to produce candidate antibodies and test them for MPM target cells binding.

#### **11. Memory B-cell receptor repertoire in MPM patients**

A second powerful approach to obtain human antibodies targeting MPM cancer cells exploits directly the immune system of patients. Individuals exposed to viral agents, parasites and tumors develop an adaptive response against non-self and neoantigens. Anti-cancer treatments such as vaccines and ICIs elicit impressive clinical responses (reviewed in [95]) and an immunological memory in subgroups of cancer patients ("elite responders") has been reported. MPM is not characterized by high mutational burden [15] an important determinant of the response to checkpoint blockade.

The efficacy of the anti-PD-1 pembrolizumab was shown by Alley and colleagues in KEYNOTE-028 [116]. In addition, ipilimumab in combination with anti-TGFβ and anti-CD25 antibodies of syngeneic MPM in BALB/c animals resulted in: i) disease eradication in treated mice; ii) elevated levels of tumor-specific IgG antibodies in healed animals; iii) failure to regrow tumors in cured mice when re-challenged with the same tumor; iv) importantly, no response in the absence of B-cells, suggesting that antibodies generated upon treatment contribute significantly to the curative effect [100]. Besides that, CD20+ B-cells infiltration in MPM tumor tissue is a positive prognostic factor as previously discussed [38].

Therefore, the immune system of elite responders can be mined to isolate MBCs producing targeting antibodies. MBCs derive mostly from affinity matured and somatically hypermutated B-cells arising in the germinal centers [144] and constitute a reservoir of high-affinity antibody producers. These features make the MBC pool very attractive so biotech and pharma companies invest in the design of screening platforms to exploit it. For example, Oncoresponse, a company that developed a proprietary, clinically validated human-antibody discovery platform in partnership with MD Anderson Cancer Center follows this paradigm and identifies therapeutically relevant antibodies from patients showing elite response against cancer after immunotherapy. MBCs are easily accessible from the peripheral blood of donors and are suitable for viral immortalization to generate lymphoblastoid cultures for high throughput screens. MBC immortalization is usually performed by infection of peripheral MBCs by Epstein Barr Virus (EBV) [145] or by BCL-6/BCL-XL expressing vectors [146]. Those procedures generate cells that express BCR on the membrane and release their antibody into culture medium at the same time. BCR presence is exploited to isolate cells binding to labeled soluble antigens by cell sorting [146] so that subsequently immunoglobulin sequences from isolated cells can be cloned into expression vectors for large-scale antibody production. Companies like Humabs and AIMM therapeutics exploit those strategies to raise antibodies against specific targets. However, the same technology can be used to isolate targeting antibodies in an antigen unbiased manner as shown for melanoma via cell-based screenings of EBV immortalized B-cells [147]. In addition, human plasmablasts and MBCs can be cultured for a limited time using specific cytokines [147–152].

Importantly, these approaches to retrieve targeting antibodies do not rely on a prior knowledge of the target. Target identification in this case is postponed, initially drawing on the demonstration of efficacy and specificity towards MPM cancer cells. MBCs receptor repertoire can be obtained also from peripheral blood or draining lymph node purified MBCs by RNA-Seq mining for *de novo* formed or highly enriched variants after treatment in elite responders [142]. Advantages and

**71**

are summarized in **Table 1**.

**libraries**

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

**Advantages Disadvantages**

• Affinity maturation step is

often needed • Reformatting in IgG format, if needed • Binding to normal human tissues to establish specific-

ity *a posteriori*

required

• Possible downsampling • Cloning and production of candidate antibodies is

• VH and VL pairs are not known (unless single cell sequencing is used) • Requires a test of binding specificity to normal human tissues *a posteriori*

• Requires cloning and production of the antibodies • Possible downsampling due to low quality or limited sample material

• VH and VL pairs cannot be

• Requires a test of binding specificity to normal human tissues *a posteriori*

• Requires a test of binding specificity to normal human tissues *a posteriori*

• Requires a BSL2 area • Identification of the antigens can be technically

challenging

known

• Cheap instrumentation • Used with any cell type • Established technology • Fastest strategy to lead candidates

• NGS driven selection of

• Antibodies are derived from affinity matured human immunoglobulins

• Availability of large number of FFPE samples • Applicable to retrospective

• Applicable to any RNA-Seq

• Easy availability of elite responder samples (blood/

• Established protocols • Isolation of *in vivo* high-affinity matured and human compatible immunoglobulins • Basic technical expertise on viral manipulation

case series

dataset

PBMCs)

• Availability of blood samples from elite responders

candidates

drawbacks of the different screening strategies for fully human antibody selection

A useful strategy to select human antibody fragments (Fabs and scFvs) against specific antigens or cells is phage display (reviewed in [153]). The importance of phage display has been restated in 2018 by the award of Nobel Prize in Chemistry

**12. Phage display screening using patient-derived scFv antibody** 

*Advantages and drawbacks of antigen-unbiased screens to obtain fully human antibodies.*

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

**display**

Antigen on cell surface

Antigen on cell surface

Antigen on cell surface

Antigen on cell surface

**Approach Antigen** 

**Phage-display technology with patient derived scFv antibody libraries**

**BCR repertoire from the peripheral blood of elite responders pre and post therapy**

**Bioinformatic analysis of BCR repertoire in tumor** 

**tissue**

**MBC** 

**Table 1.**

**immortalization**


*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

**Table 1.**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

checkpoint blockade.

identified immunoglobulin heavy and light chain sequences can be assembled to produce candidate antibodies and test them for MPM target cells binding.

A second powerful approach to obtain human antibodies targeting MPM cancer cells exploits directly the immune system of patients. Individuals exposed to viral agents, parasites and tumors develop an adaptive response against non-self and neoantigens. Anti-cancer treatments such as vaccines and ICIs elicit impressive clinical responses (reviewed in [95]) and an immunological memory in subgroups of cancer patients ("elite responders") has been reported. MPM is not characterized by high mutational burden [15] an important determinant of the response to

The efficacy of the anti-PD-1 pembrolizumab was shown by Alley and colleagues in KEYNOTE-028 [116]. In addition, ipilimumab in combination with anti-TGFβ and anti-CD25 antibodies of syngeneic MPM in BALB/c animals resulted in: i) disease eradication in treated mice; ii) elevated levels of tumor-specific IgG antibodies in healed animals; iii) failure to regrow tumors in cured mice when re-challenged with the same tumor; iv) importantly, no response in the absence of B-cells, suggesting that antibodies generated upon treatment contribute significantly to the curative effect [100]. Besides that, CD20+ B-cells infiltration in MPM

Therefore, the immune system of elite responders can be mined to isolate MBCs producing targeting antibodies. MBCs derive mostly from affinity matured and somatically hypermutated B-cells arising in the germinal centers [144] and constitute a reservoir of high-affinity antibody producers. These features make the MBC pool very attractive so biotech and pharma companies invest in the design of screening platforms to exploit it. For example, Oncoresponse, a company that developed a proprietary, clinically validated human-antibody discovery platform in partnership with MD Anderson Cancer Center follows this paradigm and identifies therapeutically relevant antibodies from patients showing elite response against cancer after immunotherapy. MBCs are easily accessible from the peripheral blood of donors and are suitable for viral immortalization to generate lymphoblastoid cultures for high throughput screens. MBC immortalization is usually performed by infection of peripheral MBCs by Epstein Barr Virus (EBV) [145] or by BCL-6/BCL-XL expressing vectors [146]. Those procedures generate cells that express BCR on the membrane and release their antibody into culture medium at the same time. BCR presence is exploited to isolate cells binding to labeled soluble antigens by cell sorting [146] so that subsequently immunoglobulin sequences from isolated cells can be cloned into expression vectors for large-scale antibody production. Companies like Humabs and AIMM therapeutics exploit those strategies to raise antibodies against specific targets. However, the same technology can be used to isolate targeting antibodies in an antigen unbiased manner as shown for melanoma via cell-based screenings of EBV immortalized B-cells [147]. In addition, human plasmablasts and MBCs can be cultured for a limited time using

Importantly, these approaches to retrieve targeting antibodies do not rely on a prior knowledge of the target. Target identification in this case is postponed, initially drawing on the demonstration of efficacy and specificity towards MPM cancer cells. MBCs receptor repertoire can be obtained also from peripheral blood or draining lymph node purified MBCs by RNA-Seq mining for *de novo* formed or highly enriched variants after treatment in elite responders [142]. Advantages and

tumor tissue is a positive prognostic factor as previously discussed [38].

**11. Memory B-cell receptor repertoire in MPM patients**

**70**

specific cytokines [147–152].

*Advantages and drawbacks of antigen-unbiased screens to obtain fully human antibodies.*

drawbacks of the different screening strategies for fully human antibody selection are summarized in **Table 1**.

#### **12. Phage display screening using patient-derived scFv antibody libraries**

A useful strategy to select human antibody fragments (Fabs and scFvs) against specific antigens or cells is phage display (reviewed in [153]). The importance of phage display has been restated in 2018 by the award of Nobel Prize in Chemistry

to George P. Smith and Sir Gregory P. Winter"for the phage display of peptides and antibodies". Phage display has allowed the production of clinically relevant antibodies (reviewed in [154]). The presence of BCRs in TME, SLOs and in the peripheral blood of MPM and other tumor patients allows for the generation of patient derived scFv phage display libraries [155] that can be used to screen for novel specificities. Phage displayed scFvs undergo selection through consecutive rounds of panning on tumor cells to enrich for specific binders (**Figure 1**). Identified antibodies can be reformatted to fully human antibodies or used as fragments or building blocks for CAR constructs.

Importantly those antibodies will derive from the permutation of original VH and VL sequences of the B-cell repertoire during library preparation while for EBV immortalized cells VH and VL pairs will be the original ones as in the patient. Classically single bacterial clones were selected and grown to produce antibodies or phages displaying specific antibodies in order to test individually their targeting of a cell of interest. Nowadays, next generation sequencing provides an efficient, quantitative and quick analytical tool to assess the evolution of complexity of phage antibody-display libraries during consecutive biopanning enrichment stages. Phage clonal evolution during screening can be studied and used to identify putative candidate antibodies and promote their cloning and production for further testing their binding to cells [156].

An unbiased phage display approach has been used to identify tumor-targeting scFvs for both sarcomatoid and epithelioid MPM. In this study, 95 mesotheliomatargeting scFvs were identified and 21 candidates were characterized for binding by FACS and IHC and for their *in vitro* internalization capacity by MPM cells with the goal to deliver conjugated anti-tumor drugs directly inside tumor cells [157]. Further analyses identified MCAM/CD146 as one of the antigens. CD146 had been previously described as a marker in advanced melanoma [158] and other tumors [159, 160], it is expressed in all MPM histotypes and by a limited spectrum of normal human adult tissues [161]. The clinical utility of MCAM/CD146 detection in pleural effusion fluid and peripheral blood samples as a diagnostic and prognostic marker for MPM [162] is under evaluation. The generation of a phage antibodydisplay library from the entire antibody genes repertoire of a cancer patient has been also attempted. Rare cancer targeting antibodies have been identified by this strategy [163]. However, the immunodominance phenomenon typical of certain cancers [153, 164, 165] has hindered a wider use of this strategy in early attempts.

#### **13. Conclusions**

Despite amazing efforts made by the scientific community and the therapeutic options developed over the last decades, the discovery of a curative treatment for MPM is still elusive and constitutes an unmet clinical need. To-date, the most promising therapeutic approaches comprise immunotherapies and CAR-based therapies that have shown impressive although preliminary clinical results. The field needs to bet on and implement these novel approaches towards novel targets and antigens to cope with tumor heterogeneity and to provide effective treatments to be used in combination. The most innovative screening technologies for the generation of fully human antibodies are in place and combine elements from fields of science that started far apart and came together to serve the purpose. These include protein engineering, next-generation sequencing (NGS), virology and cell biology providing an opportunity to find novel and unknown therapeutic targets for MPM and cancer in general. Based on these premises, we believe that a future breakthrough in MPM management will come from the design of novel

**73**

**Author details**

Fabio Nicolini\* and Massimiliano Mazza\*

provided the original work is properly cited.

massimiliano.mazza@irst.emr.it

lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy

\*Address all correspondence to: fabio.nicolini@irst.emr.it and

Immunotherapy, Cell Therapy and Biobank (ITCB), IRCCS Istituto Romagnolo per

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel…*

ATMPs engineered to target antigens that are still unknown but that can be identi-

We thank Alicja M. Gruszka for her help in proofreading and editing this

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

fied via unbiased screening strategies.

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

manuscript.

*The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

ATMPs engineered to target antigens that are still unknown but that can be identified via unbiased screening strategies.

### **Acknowledgements**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

CAR constructs.

their binding to cells [156].

**13. Conclusions**

to George P. Smith and Sir Gregory P. Winter"for the phage display of peptides and antibodies". Phage display has allowed the production of clinically relevant antibodies (reviewed in [154]). The presence of BCRs in TME, SLOs and in the peripheral blood of MPM and other tumor patients allows for the generation of patient derived scFv phage display libraries [155] that can be used to screen for novel specificities. Phage displayed scFvs undergo selection through consecutive rounds of panning on tumor cells to enrich for specific binders (**Figure 1**). Identified antibodies can be reformatted to fully human antibodies or used as fragments or building blocks for

Importantly those antibodies will derive from the permutation of original VH and VL sequences of the B-cell repertoire during library preparation while for EBV immortalized cells VH and VL pairs will be the original ones as in the patient. Classically single bacterial clones were selected and grown to produce antibodies or phages displaying specific antibodies in order to test individually their targeting of a cell of interest. Nowadays, next generation sequencing provides an efficient, quantitative and quick analytical tool to assess the evolution of complexity of phage antibody-display libraries during consecutive biopanning enrichment stages. Phage clonal evolution during screening can be studied and used to identify putative candidate antibodies and promote their cloning and production for further testing

An unbiased phage display approach has been used to identify tumor-targeting scFvs for both sarcomatoid and epithelioid MPM. In this study, 95 mesotheliomatargeting scFvs were identified and 21 candidates were characterized for binding by FACS and IHC and for their *in vitro* internalization capacity by MPM cells with the goal to deliver conjugated anti-tumor drugs directly inside tumor cells [157]. Further analyses identified MCAM/CD146 as one of the antigens. CD146 had been previously described as a marker in advanced melanoma [158] and other tumors [159, 160], it is expressed in all MPM histotypes and by a limited spectrum of normal human adult tissues [161]. The clinical utility of MCAM/CD146 detection in pleural effusion fluid and peripheral blood samples as a diagnostic and prognostic marker for MPM [162] is under evaluation. The generation of a phage antibodydisplay library from the entire antibody genes repertoire of a cancer patient has been also attempted. Rare cancer targeting antibodies have been identified by this strategy [163]. However, the immunodominance phenomenon typical of certain cancers [153, 164, 165] has hindered a wider use of this strategy in early attempts.

Despite amazing efforts made by the scientific community and the therapeutic

options developed over the last decades, the discovery of a curative treatment for MPM is still elusive and constitutes an unmet clinical need. To-date, the most promising therapeutic approaches comprise immunotherapies and CAR-based therapies that have shown impressive although preliminary clinical results. The field needs to bet on and implement these novel approaches towards novel targets and antigens to cope with tumor heterogeneity and to provide effective treatments to be used in combination. The most innovative screening technologies for the generation of fully human antibodies are in place and combine elements from fields of science that started far apart and came together to serve the purpose. These include protein engineering, next-generation sequencing (NGS), virology and cell biology providing an opportunity to find novel and unknown therapeutic targets for MPM and cancer in general. Based on these premises, we believe that a future breakthrough in MPM management will come from the design of novel

**72**

We thank Alicja M. Gruszka for her help in proofreading and editing this manuscript.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Fabio Nicolini\* and Massimiliano Mazza\* Immunotherapy, Cell Therapy and Biobank (ITCB), IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori", Meldola, Italy

\*Address all correspondence to: fabio.nicolini@irst.emr.it and massimiliano.mazza@irst.emr.it

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

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[22] Donaldson K, Murphy FA, Duffin R, Poland CA. Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and

mesothelioma. Part Fibre Toxicol. BioMed Central; 2010 Mar 22;7(1):5-17.

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[25] Hei TK, Piao CQ, He ZY, Vannais D, Waldren CA. Chrysotile fiber is a strong mutagen in mammalian cells. Cancer Res. Cancer Res; 1992 Nov 15;52(22):6305-9.

[26] Yang H, Bocchetta M, Kroczynska B, Elmishad AG, Chen Y, Liu Z, et al. TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestosinduced oncogenesis. Proc Natl Acad Sci USA. National Academy of Sciences; 2006 Jul 5;103(27):10397-402.

[27] Yang H, Rivera Z, Jube S, Nasu M, Bertino P, Goparaju C, et al. Programmed necrosis induced by asbestos in human mesothelial cells causes high-mobility group box 1 protein release and resultant inflammation. Proc Natl Acad Sci USA. National Academy of Sciences; 2010 Jul 13;107(28):12611-6.

[28] Padmore T, Stark C, Turkevich LA, Champion JA. Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):58-67.

[29] Liu JY, Brass DM, Hoyle GW, Brody AR. TNF-alpha receptor

**74**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

[9] Affar EB, Carbone M. BAP1 regulates different mechanisms of cell death. Cell Death Dis. Nature Publishing Group;

2018 Nov 19;9(12):1151-3.

Oct;20(10):1181-92.

[10] Zhang Y, Shi J, Liu X, Feng L, Gong Z, Koppula P, et al. BAP1 links metabolic regulation of ferroptosis to tumour suppression. Nat Cell Biol. Nature Publishing Group; 2018

[11] Dey A, Seshasayee D, Noubade R, French DM, Liu J, Chaurushiya MS, et al. Loss of the tumor suppressor BAP1 causes myeloid transformation. Science.

[12] Bononi A, Giorgi C, Patergnani S, Larson D, Verbruggen K, Tanji M, et al. BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation. Nature. Nature Publishing Group; 2017 Jun 22;546(7659):549-53.

2012 Sep 21;337(6101):1541-6.

[13] Bononi A, Yang H, Giorgi C, Patergnani S, Pellegrini L, Su M, et al. Germline BAP1 mutations induce a Warburg effect. Cell Death Differ. 2017

[14] Betti M, Casalone E, Ferrante D, Aspesi A, Morleo G, Biasi A, et al. Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural

mesothelioma. Cancer Letters. 2017 Oct

[15] Bueno R, Stawiski EW, Goldstein LD, Durinck S, De Rienzo A, Modrusan Z, et al. Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations. Nat Genet. Nature Publishing Group; 2016

[16] Hassan R, Morrow B, Thomas A, Walsh T, Lee MK, Gulsuner S, et al.

Oct;24(10):1694-704.

1;405:38-45.

Apr;48(4):407-16.

[1] Carbone M, Adusumilli PS, Alexander HR Jr, Baas P, Bardelli F, Bononi A, et al. Mesothelioma: Scientific clues for prevention, diagnosis, and therapy. CA A Cancer J

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Clin. 2019 Jul 30;69(5):402-29.

2014 Nov 1;92(11):790-7.

1999 Feb;79(3-4):666-72.

2013 Jan;19(1):1-10.

Wales. Lung Cancer. 2015

Jun;88(3):344-8.

2017 Jun;6(3):270-8.

2014 Jan 7;111(1):285-90.

2013 Mar;13(3):153-9.

[8] Carbone M, Yang H, Pass HI, Krausz T, Testa JR, Gaudino G. BAP1 and cancer. Nature Publishing Group.

[2] Kameda T, Takahashi K, Kim R, Jiang Y, Movahed M, Park E-K, et al. Asbestos: use, bans and disease burden in Europe. Bull World Health Organ.

[3] Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer.

[4] Henley SJ, Larson TC, Wu M,

[5] Beckett P, Edwards J, Fennell D, Hubbard R, Woolhouse I, Peake MD. Demographics, management and survival of patients with malignant pleural mesothelioma in the National Lung Cancer Audit in England and

[6] Cheung M, Testa JR. BAP1, a tumor suppressor gene driving malignant mesothelioma. Transl Lung Cancer Res.

[7] Yu H, Pak H, Hammond-Martel I, Ghram M, Rodrigue A, Daou S, et al. Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair. Proc Natl Acad Sci USA.

Antao VCS, Lewis M, Pinheiro GA, et al. Mesothelioma incidence in 50 states and the District of Columbia, United States, 2003-2008. Int J Occup Environ Health.

knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am J Pathol. 1998 Dec;153(6):1839-47.

[30] Lievense LA, Cornelissen R, Bezemer K, Kaijen-Lambers MEH, Hegmans JPJJ, Aerts JGJV. Pleural Effusion of Patients with Malignant Mesothelioma Induces Macrophage-Mediated T Cell Suppression. J Thorac Oncol. 2016 Oct;11(10):1755-64.

[31] Marcq E, Siozopoulou V, De Waele J, van Audenaerde J, Zwaenepoel K, Santermans E, et al. Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma. Oncoimmunology. Taylor & Francis; 2017 Jan 20;6(1):1-10.

[32] Burt BM, Rodig SJ, Tilleman TR, Elbardissi AW, Bueno R, Sugarbaker DJ. Circulating and tumor-infiltrating myeloid cells predict survival in human pleural mesothelioma. Cancer. John Wiley & Sons, Ltd; 2011 Nov 15;117(22): 5234-44.

[33] Awad MM, Jones RE, Liu H, Lizotte PH, Ivanova EV, Kulkarni M, et al. Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors. Cancer Immunology Research. American Association for Cancer Research; 2016 Dec;4(12):1038-48.

[34] Cornelissen R, Hegmans JPJJ, Maat APWM, Kaijen-Lambers MEH, Bezemer K, Hendriks RW, et al. Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma. Am J Respir Crit Care Med. 2016 May;193(9):1023-31.

[35] Tanrikulu AC, Abakay A, Komek H, Abakay O. Prognostic value of the lymphocyte-to-monocyte ratio and

other inflammatory markers in malignant pleural mesothelioma. Environ Health Prev Med. BioMed Central; 2016 Sep;21(5):304-11.

[36] Hegmans JPJJ, Hemmes A, Hammad H, Boon L, Hoogsteden HC, Lambrecht BN. Mesothelioma environment comprises cytokines and T-regulatory cells that suppress immune responses. Eur Respir J. European Respiratory Society; 2006 Jun;27(6): 1086-95.

[37] Li T, Li H, Wang Y, Harvard C, Tan J-L, Au A, et al. The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma. J Pathol. John Wiley & Sons, Ltd; 2011 Mar;223(4):519-30.

[38] Ujiie H, Kadota K, Nitadori J-I, Aerts JG, Woo KM, Sima CS, et al. The tumoral and stromal immune microenvironment in malignant pleural mesothelioma: A comprehensive analysis reveals prognostic immune markers. Oncoimmunology. Taylor & Francis; 2015 Jun;4(6):e1009285.

[39] Blanquart C, Gueugnon F, Nguyen J-M, Roulois D, Cellerin L, Sagan C, et al. CCL2, galectin-3, and SMRP combination improves the diagnosis of mesothelioma in pleural effusions. J Thorac Oncol. 2012 May;7(5):883-9.

[40] Gueugnon F, Leclercq S, Blanquart C, Sagan C, Cellerin L, Padieu M, et al. Identification of novel markers for the diagnosis of malignant pleural mesothelioma. Am J Pathol. 2011 Mar;178(3):1033-42.

[41] Chéné A-L, d'Almeida S, Blondy T, Tabiasco J, Deshayes S, Fonteneau J-F, et al. Pleural Effusions from Patients with Mesothelioma Induce Recruitment of Monocytes and Their Differentiation into M2 Macrophages. J Thorac Oncol. 2016 Oct;11(10):1765-73.

[42] Cioce M, Canino C, Goparaju C, Yang H, Carbone M, Pass HI. Autocrine

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al. High blood neutrophil-to-

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[51] Marcq E, Siozopoulou V, De Waele J, van Audenaerde J, Zwaenepoel K, Santermans E, et al. Prognostic and predictive aspects of the tumor immune

microenvironment and immune checkpoints in malignant pleural mesothelioma. Oncoimmunology. Taylor & Francis; 2017 Jan 20;6(1):1-10.

[52] Chee SJ, Lopez M, Mellows T, Gankande S, Moutasim KA, Harris S, et al. Evaluating the effect of immune cells

on the outcome of patients with mesothelioma. Nature Publishing Group; 2017 Aug 17;117(9):1341-8.

Cardiovasc Surg. 2008 Apr;135(4):823-9.

Mar;4(3):342-6.

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5805-13.

1341-8.

lymphocyte ratio is an indicator of poor prognosis in malignant mesothelioma patients undergoing systemic therapy. Clin Cancer Res. American Association for Cancer Research; 2010 Dec 1;16(23):

*DOI: http://dx.doi.org/10.5772/intechopen.98617*

CSF-1R signaling drives mesothelioma chemoresistance via AKT activation. Cell Death Dis. Nature Publishing Group; 2014 Apr 10;5(4):e1167-7.

[43] Fujii M, Toyoda T, Nakanishi H, Yatabe Y, Sato A, Matsudaira Y, et al. TGF-β synergizes with defects in the Hippo pathway to stimulate human malignant mesothelioma growth. J Exp Med. 2012 Mar 12;209(3):479-94.

[44] Schürch CM, Forster S, Brühl F, Yang SH, Felley Bosco E, Hewer E. The "don't eat me" signal CD47 is a novel diagnostic biomarker and potential therapeutic target for diffuse malignant mesothelioma. Oncoimmunology. Taylor & Francis; 2017;7(1):e1373235.

[45] Izzi V, Chiurchiù V, D'Aquilio F, Palumbo C, Tresoldi I, Modesti A, et al. Differential effects of malignant

mesothelioma cells on THP-1 monocytes and macrophages. Int J Oncol. Int J Oncol; 2009 Feb;34(2):543-50.

[46] Miselis NR, Wu ZJ, Van Rooijen N, Kane AB. Targeting tumor-associated macrophages in an orthotopic murine

model of diffuse malignant mesothelioma. Molecular Cancer Therapeutics. American Association for Cancer Research; 2008 Apr;7(4):788-99.

[47] Rehrauer H, Wu L, Blum W, Pecze L, Henzi T, Serre-Beinier V, et al. How asbestos drives the tissue towards tumors: YAP activation, macrophage and mesothelial precursor recruitment, RNA editing, and somatic mutations. Oncogene. Nature Publishing Group;

2018 May;37(20):2645-59.

[48] Khanna S, Graef S, Mussai F, Thomas A, Wali N, Yenidunya BG, et al. Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clin Cancer Res. American Association for Cancer Research; 2018 Jun 15;24(12):2859-72.

[49] Kao SCH, Pavlakis N, Harvie R, Vardy JL, Boyer MJ, van Zandwijk N, et *The Immune System of Mesothelioma Patients: A Window of Opportunity for Novel… DOI: http://dx.doi.org/10.5772/intechopen.98617*

CSF-1R signaling drives mesothelioma chemoresistance via AKT activation. Cell Death Dis. Nature Publishing Group; 2014 Apr 10;5(4):e1167-7.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

other inflammatory markers in malignant pleural mesothelioma. Environ Health Prev Med. BioMed Central; 2016 Sep;21(5):304-11.

[36] Hegmans JPJJ, Hemmes A, Hammad H, Boon L, Hoogsteden HC,

Lambrecht BN. Mesothelioma

1086-95.

Mar;223(4):519-30.

environment comprises cytokines and T-regulatory cells that suppress immune responses. Eur Respir J. European Respiratory Society; 2006 Jun;27(6):

[37] Li T, Li H, Wang Y, Harvard C, Tan J-L, Au A, et al. The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma. J Pathol. John Wiley & Sons, Ltd; 2011

[38] Ujiie H, Kadota K, Nitadori J-I, Aerts JG, Woo KM, Sima CS, et al. The

microenvironment in malignant pleural mesothelioma: A comprehensive analysis reveals prognostic immune markers. Oncoimmunology. Taylor & Francis; 2015 Jun;4(6):e1009285.

[39] Blanquart C, Gueugnon F, Nguyen J-M, Roulois D, Cellerin L, Sagan C, et al. CCL2, galectin-3, and SMRP combination improves the diagnosis of mesothelioma in pleural effusions. J Thorac Oncol. 2012 May;7(5):883-9.

[41] Chéné A-L, d'Almeida S, Blondy T, Tabiasco J, Deshayes S, Fonteneau J-F, et al. Pleural Effusions from Patients with Mesothelioma Induce Recruitment of Monocytes and Their Differentiation into M2 Macrophages. J Thorac Oncol.

[42] Cioce M, Canino C, Goparaju C, Yang H, Carbone M, Pass HI. Autocrine

tumoral and stromal immune

[40] Gueugnon F, Leclercq S, Blanquart C, Sagan C, Cellerin L, Padieu M, et al. Identification of novel markers for the diagnosis of malignant pleural mesothelioma. Am J Pathol. 2011

Mar;178(3):1033-42.

2016 Oct;11(10):1765-73.

knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. Am J Pathol. 1998

[30] Lievense LA, Cornelissen R, Bezemer K, Kaijen-Lambers MEH, Hegmans JPJJ, Aerts JGJV. Pleural Effusion of Patients with Malignant Mesothelioma Induces Macrophage-Mediated T Cell Suppression. J Thorac Oncol. 2016 Oct;11(10):1755-64.

[31] Marcq E, Siozopoulou V, De Waele J, van Audenaerde J, Zwaenepoel K, Santermans E, et al. Prognostic and predictive aspects of the tumor immune

microenvironment and immune checkpoints in malignant pleural mesothelioma. Oncoimmunology. Taylor & Francis; 2017 Jan 20;6(1):1-10.

[32] Burt BM, Rodig SJ, Tilleman TR, Elbardissi AW, Bueno R, Sugarbaker DJ. Circulating and tumor-infiltrating myeloid cells predict survival in human pleural mesothelioma. Cancer. John Wiley & Sons, Ltd; 2011 Nov 15;117(22):

[33] Awad MM, Jones RE, Liu H, Lizotte PH, Ivanova EV, Kulkarni M, et al. Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are

Counterbalanced by Distinct Immunosuppressive Factors. Cancer Immunology Research. American Association for Cancer Research; 2016

[34] Cornelissen R, Hegmans JPJJ, Maat APWM, Kaijen-Lambers MEH, Bezemer K, Hendriks RW, et al. Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma. Am J Respir Crit Care Med. 2016 May;193(9):1023-31.

[35] Tanrikulu AC, Abakay A, Komek H, Abakay O. Prognostic value of the lymphocyte-to-monocyte ratio and

Dec;4(12):1038-48.

5234-44.

Dec;153(6):1839-47.

**76**

[43] Fujii M, Toyoda T, Nakanishi H, Yatabe Y, Sato A, Matsudaira Y, et al. TGF-β synergizes with defects in the Hippo pathway to stimulate human malignant mesothelioma growth. J Exp Med. 2012 Mar 12;209(3):479-94.

[44] Schürch CM, Forster S, Brühl F, Yang SH, Felley Bosco E, Hewer E. The "don't eat me" signal CD47 is a novel diagnostic biomarker and potential therapeutic target for diffuse malignant mesothelioma. Oncoimmunology. Taylor & Francis; 2017;7(1):e1373235.

[45] Izzi V, Chiurchiù V, D'Aquilio F, Palumbo C, Tresoldi I, Modesti A, et al. Differential effects of malignant mesothelioma cells on THP-1 monocytes and macrophages. Int J Oncol. Int J Oncol; 2009 Feb;34(2):543-50.

[46] Miselis NR, Wu ZJ, Van Rooijen N, Kane AB. Targeting tumor-associated macrophages in an orthotopic murine model of diffuse malignant mesothelioma. Molecular Cancer Therapeutics. American Association for Cancer Research; 2008 Apr;7(4):788-99.

[47] Rehrauer H, Wu L, Blum W, Pecze L, Henzi T, Serre-Beinier V, et al. How asbestos drives the tissue towards tumors: YAP activation, macrophage and mesothelial precursor recruitment, RNA editing, and somatic mutations. Oncogene. Nature Publishing Group; 2018 May;37(20):2645-59.

[48] Khanna S, Graef S, Mussai F, Thomas A, Wali N, Yenidunya BG, et al. Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients. Clin Cancer Res. American Association for Cancer Research; 2018 Jun 15;24(12):2859-72.

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[140] Wu L, Wu MO, la Maza De L, Yun Z, Yu J, Zhao Y, et al. Targeting the inhibitory receptor CTLA-4 on T cells increased abscopal effects in murine mesothelioma model. Oncotarget. 2015

May 20;6(14):12468-80.

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Mitrophanov I, Shugay M, Mamedov IZ, Putintseva EV, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat Methods. Nature Publishing Group; 2015 May;12(5):

[142] Mose LE, Selitsky SR, Bixby LM, Marron DL, Iglesia MD, Serody JS, et al. Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V'DJer.

[143] Selitsky SR, Mose LE, Smith CC, Chai S, Hoadley KA, Dittmer DP, et al.

Bioinformatics. 2016 Dec 15;32(24):3729-34.

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[137] Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from

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[152] Walker LM, Phogat SK, Chan-Hui P-Y, Wagner D, Phung P, Goss JL, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009 Oct 9;326(5950):285-9.

[153] Dantas-Barbosa C, de Macedo Brigido M, Maranhao AQ. Antibody phage display libraries: contributions to oncology. Int J Mol Sci. Molecular Diversity Preservation International; 2012;13(5):5420-40.

[154] Alfaleh MA, Alsaab HO, Mahmoud AB, Alkayyal AA, Jones ML, Mahler SM, et al. Phage Display Derived Monoclonal Antibodies: From Bench to Bedside. Front Immunol. Frontiers; 2020;11:1986.

[155] Rothe A, Klimka A, Tur MK, Pfitzner T, Huhn M, Sasse S, et al. Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines. Int J Mol Med. Spandidos Publications; 2004 Oct;14(4):729-35.

[156] Rouet R, Jackson KJL, Langley DB, Christ D. Next-Generation Sequencing of Antibody Display Repertoires. Front Immunol. 2018 Feb 2;9:1315.

[157] An F, Drummond DC, Wilson S, Kirpotin DB, Nishimura SL,

Broaddus VC, et al. Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies. Molecular Cancer Therapeutics. 2008 Mar 1;7(3):569-78.

[158] Lei X, Guan C-W, Song Y, Wang H. The multifaceted role of CD146/MCAM in the promotion of melanoma progression. Cancer Cell International. BioMed Central; 2015;15(1):3-11.

[159] de Kruijff I, Timmermans A, Bakker den M, Trapman-Jansen A, Foekens R, Meijer-Van Gelder M, et al. The Prevalence of CD146 Expression in Breast Cancer Subtypes and Its Relation to Outcome. Cancers. Multidisciplinary Digital Publishing Institute; 2018 May;10(5):134.

[160] Olajuyin AM, Olajuyin AK, Wang Z, Zhao X, Zhang X. CD146 T cells in lung cancer: its function, detection, and clinical implications as a biomarker and therapeutic target. Cancer Cell International. BioMed Central; 2019 Sep 25;:1-13.

[161] Bidlingmaier S, He J, Wang Y, An F, Feng J, Barbone D, et al. Identification of MCAM/CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma. Cancer Res. American Association for Cancer Research; 2009 Feb 15;69(4):1570-7.

[162] Beije N, Kraan J, Bakker den MA, Maat APWM, van der Leest C, Cornelissen R, et al. Improved diagnosis and prognostication of patients with pleural malignant mesothelioma using biomarkers in pleural effusions and peripheral blood samples - a short report. Cell Oncol (Dordr). Springer Netherlands; 2017 Oct;40(5):511-9.

[163] Barbas CF, Kang AS, Lerner RA, Benkovic SJ. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci USA. National Academy of Sciences; 1991 Sep 15;88(18):7978-82.

[164] Cai X, Garen A. Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc Natl Acad Sci USA. 1995 Jul 3;92(14):6537-41.

[165] Wortzel RD, Urban JL, Philipps C, Fitch FW, Schreiber H. Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones. JI. 1983 May;130(5):2461-6.

**87**

Section 4

Rare Skin Diseases

Section 4

## Rare Skin Diseases

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

gene III site. Proc Natl Acad Sci USA. National Academy of Sciences; 1991 Sep

[164] Cai X, Garen A. Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc Natl Acad Sci USA. 1995 Jul 3;92(14):6537-41.

[165] Wortzel RD, Urban JL, Philipps C, Fitch FW, Schreiber H. Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones. JI. 1983

15;88(18):7978-82.

May;130(5):2461-6.

**86**

**89**

**1. Introduction**

**Chapter 6**

**Abstract**

Therapy Development for

*Christina Guttmann-Gruber, Iris Gratz and Ulrich Koller*

Although rare genodermatoses such as Epidermolysis bullosa have received more attention over the last years, no approved treatment options targeting causal mutations are currently available. Still, such diseases can be devastating, in some cases even associated with life-threatening secondary manifestations. Therefore, developing treatments that target disease-associated complications along with causal therapies remains the focus of current research efforts, in order to increase patient's quality of life and potentially their life expectancy. Epidermolysis bullosa is a genodermatosis that is caused by mutations in either one of 16 genes, predominantly encoding structural components of the skin and mucosal epithelia that are crucial to give these barrier organs physical and mechanical resilience to stress. The genetic heterogeneity of the disease is recapitulated in the high variability of phenotypic expressivity observed, ranging from minor and localized blistering to generalized erosions and wound chronification, rendering certain subtypes a systemic disease that is complicated by a plethora of secondary manifestations. During the last decades, several studies have focused on developing treatments for EB patients and significant progress has been made, as reflected by numerous publications, patents, and registered trials available. Overall, strategies range from causal to symptom-relieving approaches, and include gene, RNA and cell therapies, as well as drug developments based on biologics and small molecules. In this chapter, we highlight the most recent and promising approaches that are currently being investigated in order to provide

effective treatments for patients with epidermolysis bullosa in the future.

wound healing, pruritus, fibrosis, squamous cell carcinoma

**Keywords:** Epidermolysis bullosa, genodermatoses, gene therapy, drug development,

Epidermolysis bullosa (EB) refers to a group of rare genodermatoses typically characterized by vulnerability of the skin to friction or trauma, leading to blistering and wounding to various extents, *i.e.* from localized and mild to generalized and severe, depending on the respective subtype of the disease. EB subtypes are classified according to the mutated gene, the resulting product of which is either functionally impaired or absent, and the level at which tissue cleavage occurs. While involvement in some subtypes are restricted to the skin, for others, extracutaneous organs (*i.e.* mucosa and eyes) may be involved, along with a multitude of secondary manifestations that significantly impair patients' life quality (QoL) and may

Epidermolysis Bullosa

*Josefina Piñón Hofbauer, Verena Wally,* 

#### **Chapter 6**

## Therapy Development for Epidermolysis Bullosa

*Josefina Piñón Hofbauer, Verena Wally, Christina Guttmann-Gruber, Iris Gratz and Ulrich Koller*

#### **Abstract**

Although rare genodermatoses such as Epidermolysis bullosa have received more attention over the last years, no approved treatment options targeting causal mutations are currently available. Still, such diseases can be devastating, in some cases even associated with life-threatening secondary manifestations. Therefore, developing treatments that target disease-associated complications along with causal therapies remains the focus of current research efforts, in order to increase patient's quality of life and potentially their life expectancy. Epidermolysis bullosa is a genodermatosis that is caused by mutations in either one of 16 genes, predominantly encoding structural components of the skin and mucosal epithelia that are crucial to give these barrier organs physical and mechanical resilience to stress. The genetic heterogeneity of the disease is recapitulated in the high variability of phenotypic expressivity observed, ranging from minor and localized blistering to generalized erosions and wound chronification, rendering certain subtypes a systemic disease that is complicated by a plethora of secondary manifestations. During the last decades, several studies have focused on developing treatments for EB patients and significant progress has been made, as reflected by numerous publications, patents, and registered trials available. Overall, strategies range from causal to symptom-relieving approaches, and include gene, RNA and cell therapies, as well as drug developments based on biologics and small molecules. In this chapter, we highlight the most recent and promising approaches that are currently being investigated in order to provide effective treatments for patients with epidermolysis bullosa in the future.

**Keywords:** Epidermolysis bullosa, genodermatoses, gene therapy, drug development, wound healing, pruritus, fibrosis, squamous cell carcinoma

#### **1. Introduction**

Epidermolysis bullosa (EB) refers to a group of rare genodermatoses typically characterized by vulnerability of the skin to friction or trauma, leading to blistering and wounding to various extents, *i.e.* from localized and mild to generalized and severe, depending on the respective subtype of the disease. EB subtypes are classified according to the mutated gene, the resulting product of which is either functionally impaired or absent, and the level at which tissue cleavage occurs. While involvement in some subtypes are restricted to the skin, for others, extracutaneous organs (*i.e.* mucosa and eyes) may be involved, along with a multitude of secondary manifestations that significantly impair patients' life quality (QoL) and may

even be life-threatening. Among these is the development of an aggressive form of cutaneous squamous cell carcinoma (cSCC) in patients with the severe dystrophic subtype of the disease [1–3].

Worldwide approximately 500,000 people suffer from EB, which can be classified into four major groups [1]. Mainly dominantly inherited mutations within genes encoding keratin 5, 14 and plectin lead to EB simplex (EBS), associated with intraepidermal blister formation (**Figure 1**). Mutations within genes, encoding laminin-332, type XVII collagen or integrin-α6β4, are the main causes of the junctional form of EB (JEB), characterized by tissue separation within the lamina lucida of the basement membrane zone (BMZ). The severe dystrophic variant of EB (DEB) is caused exclusively by mutations within the gene *COL7A1*, encoding type VII collagen, resulting in tissue separation within the sub-lamina densa (**Figure 1**). Kindler syndrome is caused by recessive mutations within the *KIND1* gene leading to a complete loss of encoded Kindlin-1 and blistering in multiple skin layers [1].

Despite advances in our understanding of the spectrum of pathologies associated with the different subtypes of EB, a systemic cure is still out of reach. However, the increasing number of trials that are being conducted reflects significant progress in clinical research, including strategies to correct and/or modulate the aberrant molecules and mechanisms underlying this devastating disease. [4–7] The remarkable differences between EB-types and numerous subtypes, renders the development of therapies a complex challenge, as both inter-subtype and inter-individual differences require the development of more personalized treatments.

Major complications in EB range from itch and pain, to a predisposition to wound chronification and tumor development, with molecular contributors deriving from various sources including different tissue-associated cell types, matrix components, as well as inflammatory events. The majority of these comorbidities, in and of themselves, are not unique to EB, so that the repurposing of clinically approved treatments against such symptoms represents an attractive path for a more rapid market approval of these compounds for EB. Furthermore, new

#### **Figure 1.**

*Therapeutic targets in EB and strategies in development against them. EB is a rare hereditary skin fragility disorder characterized by blistering and wounding. In severe subtypes of the disease, wounds degenerate into tumors. The different therapeutic strategies to target important aspects of the disease are depicted.*

**91**

**Table 1.**

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

deficiencies associated with the disease.

**2.1 Causal therapies for epidermolysis bullosa**

*2.1.1 Gene replacement therapies for epidermolysis bullosa*

**2. Therapy development for EB**

**EB subtype Therapeutic** 

**goal**

Read-through therapy

Protein therapy

Read-through therapy

Cell therapy Allogeneic fibroblast injection

*Overview on causal therapies in epidermolysis bullosa and their current clinical status.*

research (**Table 1**).

*Recessive dystrophic EB*

evidence arising in the study of these common conditions can be leveraged to direct therapy development in EB. Nevertheless, rigorous evaluation for safety in this specific, vulnerable patient group is warranted for any candidate therapeutic. This is especially true for cancer therapies which are associated with significant cellular toxicity and often have the adverse effect of exacerbating the wound healing

The development of causal therapies has always been a main focus of EB

**Therapeutic strategy**

SMaRT *KRT14,* 

*Junctional EB* Causal therapy cDNA replacement *LAMB3* clinical [14–17]

*EB simplex* Causal therapy Gene editing *KRT5, KRT14* pre-

**Targeted gene/protein**

*PLEC1*

PTC read-through *LAMB3* clinical [7]

Gene editing *LAMB3* pre-

Causal therapy cDNA replacement *COL7A1* clinical [19–23]

AON *COL7A1* pre-

Gene editing *COL7A1* pre-

SMaRT *COL7A1* pre-

vollagen

Type VII collagen

PTC read-through *COL7A1* clinical [43]

Protein replacement Type VII

**Status Ref**

[8, 9]

[10–13]

[18]

[24–27]

[24, 28–36]

[10, 37–40]

[41]

clinical

clinical

clinical

clinical

clinical

clinical [42]

preclinical

preclinical

Currently, gene replacement strategies that exploit viral vectors to introduce full-length wild type cDNA copies of the affected gene into the skin cells of patients [14], have advanced the furthest in clinical trials. However, this strategy relies on genomic integration of the transgene to achieve long-term restoration of gene

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

subtype of the disease [1–3].

even be life-threatening. Among these is the development of an aggressive form of cutaneous squamous cell carcinoma (cSCC) in patients with the severe dystrophic

Worldwide approximately 500,000 people suffer from EB, which can be classified into four major groups [1]. Mainly dominantly inherited mutations within genes encoding keratin 5, 14 and plectin lead to EB simplex (EBS), associated with intraepidermal blister formation (**Figure 1**). Mutations within genes, encoding laminin-332, type XVII collagen or integrin-α6β4, are the main causes of the junctional form of EB (JEB), characterized by tissue separation within the lamina lucida of the basement membrane zone (BMZ). The severe dystrophic variant of EB (DEB) is caused exclusively by mutations within the gene *COL7A1*, encoding type VII collagen, resulting in tissue separation within the sub-lamina densa (**Figure 1**). Kindler syndrome is caused by recessive mutations within the *KIND1* gene leading to a complete loss of encoded Kindlin-1 and blistering in multiple skin layers [1]. Despite advances in our understanding of the spectrum of pathologies associated with the different subtypes of EB, a systemic cure is still out of reach. However, the increasing number of trials that are being conducted reflects significant progress in clinical research, including strategies to correct and/or modulate the aberrant molecules and mechanisms underlying this devastating disease. [4–7] The remarkable differences between EB-types and numerous subtypes, renders the development of therapies a complex challenge, as both inter-subtype and inter-individual

differences require the development of more personalized treatments.

Major complications in EB range from itch and pain, to a predisposition to wound chronification and tumor development, with molecular contributors deriving from various sources including different tissue-associated cell types, matrix components, as well as inflammatory events. The majority of these comorbidities, in and of themselves, are not unique to EB, so that the repurposing of clinically approved treatments against such symptoms represents an attractive path for a more rapid market approval of these compounds for EB. Furthermore, new

*Therapeutic targets in EB and strategies in development against them. EB is a rare hereditary skin fragility disorder characterized by blistering and wounding. In severe subtypes of the disease, wounds degenerate into tumors. The different therapeutic strategies to target important aspects of the disease are depicted.*

**90**

**Figure 1.**

evidence arising in the study of these common conditions can be leveraged to direct therapy development in EB. Nevertheless, rigorous evaluation for safety in this specific, vulnerable patient group is warranted for any candidate therapeutic. This is especially true for cancer therapies which are associated with significant cellular toxicity and often have the adverse effect of exacerbating the wound healing deficiencies associated with the disease.

#### **2. Therapy development for EB**

#### **2.1 Causal therapies for epidermolysis bullosa**

#### *2.1.1 Gene replacement therapies for epidermolysis bullosa*

The development of causal therapies has always been a main focus of EB research (**Table 1**).

Currently, gene replacement strategies that exploit viral vectors to introduce full-length wild type cDNA copies of the affected gene into the skin cells of patients [14], have advanced the furthest in clinical trials. However, this strategy relies on genomic integration of the transgene to achieve long-term restoration of gene


#### **Table 1.**

*Overview on causal therapies in epidermolysis bullosa and their current clinical status.*

function, which bears a low risk of genomic toxicity due to insertional mutagenesis that can result in tumor development as shown for for X-linked severe combined immunodeficiency (X-SCID) [44]. However, no such deleterious events have been observed thus far for EB [14–17, 19]. In general, cutaneous gene therapies have the advantage that grafted skin areas are easy to monitor, with developing tumors easily detected and promptly excised. Until now, transplantation of genetically corrected skin grafts represents the most auspicious approach, due to the limited number of viral vectors suitable for *in vivo* targeting. Poor transcutaneous delivery of the vector and the size of the transgene represent further limitations [45, 46].

To date, the most successful application of gene replacement therapy has been achieved in junctional EB (JEB) patients carrying mutations in the *LAMB3* gene [14, 16, 17]. In all three cases, epidermal stem cells isolated from skin biopsies and expanded *in vitro*, were treated with a Moloney leukemia virus (MLV)-derived retroviral vector expressing the full-length cDNA of *LAMB3*. Treated cells were then expanded into epidermal sheets, which were transplanted back onto the patient. To this day, the first patient treated in 2006 still retains the transgenic epidermis, which at 6.5 years follow-up, appeared normal, blister-free, and showed accurate localized expression of laminin-332 within the skin, and no adverse effects reported thus far [15]. More recently, the same procedure was applied to treat an eight-year old JEB patient with life-threatening skin loss due to a bacterial infection. Over the course of successive treatments, up to ~80% of the patient's skin was surgically replaced by genetically corrected skin, demonstrating the life-saving potential of this therapeutic strategy for genodermatoses. Genetic analyses of the skin grafts clearly demonstrated that the transgenic epidermis was sustained by a defined number of epidermal stem cells with long-term regenerative potential [17].

Despite these successes, attempts to apply the same strategy in recessive dystrophic EB (RDEB) demonstrated no long-lasting effects. While long-term *COL7A1* expression could be attained following treatment, variable clinical outcomes were observed, with persistent type VII collagen expression detected in only two out of seven treated patients at two years post transplantation. [19, 20]

For most patients, improved wound healing and an accurate deposition of type VII collagen within the regenerated skin could be detected. However, expression of the transgene significantly decreased over time [19]. Possible reasons for this include the size of the *COL7A1* cDNA (~9 kb), the random nature of viral integrations, or post-transcriptional deregulation via aberrant splicing [21, 47]. In this respect, targeting *LAMB3* via a gene replacement therapy bears a big advantage over *COL7A1*, as the phenotype of JEB is associated with a significant depletion of epidermal stem cells [48]. Here, the YAP/TAZ mechano-sensing pathway plays a major role in sustaining holoclones downstream of Laminin-332 signaling. Holoclones represent stem cells with the greatest reproductive capacity and are essential for epidermal regeneration. Thus laminin-332 gene therapy likely rescues YAP activity, enabling the maintenance of the pool of epidermal stem cells [48]. Thus, while these clinical trials in JEB and DEB demonstrate the potential of gene replacement-based therapies in EB, they also reveal the varying therapeutic efficiencies that can be expected among the different EB types, potentially depending on the biology of the respective matrix protein affected.

In contrast to targeting patient keratinocytes, a combined gene and cell therapy approach using patient autologous fibroblasts was recently evaluated in a phase I, open-label, single-center clinical trial in four RDEB patients [22]. Based on previous preclinical data [23], patient fibroblasts were first modified *ex vivo* to carry a full-length codon-optimized *COL7A1* cDNA using a self-inactivating lentiviral vector. The gene-modified autologous fibroblasts were then injected intradermally back into the patients. The treatment was well-tolerated and no serious side effects

**93**

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*2.1.2 Gene editing strategies for epidermolysis bullosa*

a dominant negative mutation within *COL7A1* [30].

for the patient.

were observed. Mean fluorescence intensity of type VII collagen staining in treated skin was 1.26-fold to 26.10-fold increased over non-injected skin, with enhanced expression sustained for up to 12 months in 2 out of the 4 patients [22]. This study demonstrated the potential of a combined gene & cell therapy approach for the treatment of RDEB, although more clinical data is required to evaluate the benefit

Gene editing platforms based on programmable nucleases have advanced at a rapid pace, such that the correction of any gene is now, at least in theory, conceivable. At their core, designer nucleases consist of DNA endonucleases, such as zinc-finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9), which are guided to specific DNA loci of interest, where they generate double-strand breaks (DSBs) and trigger the activation of DNA repair mechanisms [49, 50]. The most frequent repair pathway, termed non-homologous end joining pathway (NHEJ), relies on the introduction of small insertions and deletions (indels) at the DSB site [51], which can be leveraged for the inactivation of genes carrying dominant negative missense mutations [8, 52], or for

the reframing of genes bearing pathogenic frameshift mutations [28, 29].

While not a genetic correction *per se*, gene disruption can be a suitable way of targeting dominant alleles via causing the coding sequence to run into a premature termination codon (PTC) during translation. Ribosomal stalling and activation of the nonsense-mediated mRNA decay pathway triggers the degradation of the edited mutant transcripts [53]. We have exploited this strategy to disrupt a dominant negative *KRT5* mutation whilst leaving the wild type allele intact [8]. This targeting strategy should be applicable to a broad number of EBS patients. Similarly, for DDEB, Shinkuma et al. applied an allele-specific gene disruption approach to target

The same strategy can also be used to reframe mutant mRNA and was recently shown by several groups to be a promising editing approach in RDEB [28, 29, 31, 32]. Leveraging recently developed algorithms to accurately predict end-joining (EJ) repair outcomes following CRISPR/Cas9-mediated cleavage, we were able to achieve significant restoration of *COL7A1* function after targeting a pathogenic frameshift mutation within exon 73 of *COL7A1* [29]. Sequence composition around the Cas9 binding site predicted a single adenine sense-strand insertion at the *COL7A1* target locus as the dominant EJ repair outcome, which would restore the reading frame of the message while introducing a single amino acid change in the protein. Indeed, we detected this precise nucleotide modification in 17% of all next generation sequencing (NGS)-analyzed *COL7A1* alleles, following a single RNP treatment. In all gene-edited cells analyzed, > 70% exhibited restored functional protein expression, underscoring the potential of end-joining based DNA repair strategies for restoring gene function in EB [29]. Of course, the holy grail of gene therapy has been to achieve a traceless repair of the disease-causing mutation. With current editing technologies, this is now attainable by invoking the high-fidelity homology directed repair (HDR) pathway. By providing an exogenous HDR donor sequence, which bears homology to the target region, the exchange of whole gene regions or individual nucleotides can be achieved [18, 24, 49, 50]. However, in comparison to EJ-based targeting strategies, gene editing efficiency is generally reduced, as homologous recombination is only active during the late S/G2 phase of the cell cycle [54]. Nevertheless, in EB, HDR-based gene repair strategies have been successfully applied to EBS [9] and

#### *Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

function, which bears a low risk of genomic toxicity due to insertional mutagenesis that can result in tumor development as shown for for X-linked severe combined immunodeficiency (X-SCID) [44]. However, no such deleterious events have been observed thus far for EB [14–17, 19]. In general, cutaneous gene therapies have the advantage that grafted skin areas are easy to monitor, with developing tumors easily detected and promptly excised. Until now, transplantation of genetically corrected skin grafts represents the most auspicious approach, due to the limited number of viral vectors suitable for *in vivo* targeting. Poor transcutaneous delivery of the vec-

To date, the most successful application of gene replacement therapy has been achieved in junctional EB (JEB) patients carrying mutations in the *LAMB3* gene [14, 16, 17]. In all three cases, epidermal stem cells isolated from skin biopsies and expanded *in vitro*, were treated with a Moloney leukemia virus (MLV)-derived retroviral vector expressing the full-length cDNA of *LAMB3*. Treated cells were then expanded into epidermal sheets, which were transplanted back onto the patient. To this day, the first patient treated in 2006 still retains the transgenic epidermis, which at 6.5 years follow-up, appeared normal, blister-free, and showed accurate localized expression of laminin-332 within the skin, and no adverse effects reported thus far [15]. More recently, the same procedure was applied to treat an eight-year old JEB patient with life-threatening skin loss due to a bacterial infection. Over the course of successive treatments, up to ~80% of the patient's skin was surgically replaced by genetically corrected skin, demonstrating the life-saving potential of this therapeutic strategy for genodermatoses. Genetic analyses of the skin grafts clearly demonstrated that the transgenic epidermis was sustained by a defined number of epidermal stem cells with long-term regenerative potential [17].

Despite these successes, attempts to apply the same strategy in recessive dystrophic EB (RDEB) demonstrated no long-lasting effects. While long-term *COL7A1* expression could be attained following treatment, variable clinical outcomes were observed, with persistent type VII collagen expression detected in only two out of

For most patients, improved wound healing and an accurate deposition of type VII collagen within the regenerated skin could be detected. However, expression of the transgene significantly decreased over time [19]. Possible reasons for this include the size of the *COL7A1* cDNA (~9 kb), the random nature of viral integrations, or post-transcriptional deregulation via aberrant splicing [21, 47]. In this respect, targeting *LAMB3* via a gene replacement therapy bears a big advantage over *COL7A1*, as the phenotype of JEB is associated with a significant depletion of epidermal stem cells [48]. Here, the YAP/TAZ mechano-sensing pathway plays a major role in sustaining holoclones downstream of Laminin-332 signaling. Holoclones represent stem cells with the greatest reproductive capacity and are essential for epidermal regeneration. Thus laminin-332 gene therapy likely rescues YAP activity, enabling the maintenance of the pool of epidermal stem cells [48]. Thus, while these clinical trials in JEB and DEB demonstrate the potential of gene replacement-based therapies in EB, they also reveal the varying therapeutic efficiencies that can be expected among the different EB types, potentially depending

In contrast to targeting patient keratinocytes, a combined gene and cell therapy approach using patient autologous fibroblasts was recently evaluated in a phase I, open-label, single-center clinical trial in four RDEB patients [22]. Based on previous preclinical data [23], patient fibroblasts were first modified *ex vivo* to carry a full-length codon-optimized *COL7A1* cDNA using a self-inactivating lentiviral vector. The gene-modified autologous fibroblasts were then injected intradermally back into the patients. The treatment was well-tolerated and no serious side effects

seven treated patients at two years post transplantation. [19, 20]

on the biology of the respective matrix protein affected.

tor and the size of the transgene represent further limitations [45, 46].

**92**

were observed. Mean fluorescence intensity of type VII collagen staining in treated skin was 1.26-fold to 26.10-fold increased over non-injected skin, with enhanced expression sustained for up to 12 months in 2 out of the 4 patients [22]. This study demonstrated the potential of a combined gene & cell therapy approach for the treatment of RDEB, although more clinical data is required to evaluate the benefit for the patient.

#### *2.1.2 Gene editing strategies for epidermolysis bullosa*

Gene editing platforms based on programmable nucleases have advanced at a rapid pace, such that the correction of any gene is now, at least in theory, conceivable. At their core, designer nucleases consist of DNA endonucleases, such as zinc-finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9), which are guided to specific DNA loci of interest, where they generate double-strand breaks (DSBs) and trigger the activation of DNA repair mechanisms [49, 50]. The most frequent repair pathway, termed non-homologous end joining pathway (NHEJ), relies on the introduction of small insertions and deletions (indels) at the DSB site [51], which can be leveraged for the inactivation of genes carrying dominant negative missense mutations [8, 52], or for the reframing of genes bearing pathogenic frameshift mutations [28, 29].

While not a genetic correction *per se*, gene disruption can be a suitable way of targeting dominant alleles via causing the coding sequence to run into a premature termination codon (PTC) during translation. Ribosomal stalling and activation of the nonsense-mediated mRNA decay pathway triggers the degradation of the edited mutant transcripts [53]. We have exploited this strategy to disrupt a dominant negative *KRT5* mutation whilst leaving the wild type allele intact [8]. This targeting strategy should be applicable to a broad number of EBS patients. Similarly, for DDEB, Shinkuma et al. applied an allele-specific gene disruption approach to target a dominant negative mutation within *COL7A1* [30].

The same strategy can also be used to reframe mutant mRNA and was recently shown by several groups to be a promising editing approach in RDEB [28, 29, 31, 32]. Leveraging recently developed algorithms to accurately predict end-joining (EJ) repair outcomes following CRISPR/Cas9-mediated cleavage, we were able to achieve significant restoration of *COL7A1* function after targeting a pathogenic frameshift mutation within exon 73 of *COL7A1* [29]. Sequence composition around the Cas9 binding site predicted a single adenine sense-strand insertion at the *COL7A1* target locus as the dominant EJ repair outcome, which would restore the reading frame of the message while introducing a single amino acid change in the protein. Indeed, we detected this precise nucleotide modification in 17% of all next generation sequencing (NGS)-analyzed *COL7A1* alleles, following a single RNP treatment. In all gene-edited cells analyzed, > 70% exhibited restored functional protein expression, underscoring the potential of end-joining based DNA repair strategies for restoring gene function in EB [29].

Of course, the holy grail of gene therapy has been to achieve a traceless repair of the disease-causing mutation. With current editing technologies, this is now attainable by invoking the high-fidelity homology directed repair (HDR) pathway. By providing an exogenous HDR donor sequence, which bears homology to the target region, the exchange of whole gene regions or individual nucleotides can be achieved [18, 24, 49, 50]. However, in comparison to EJ-based targeting strategies, gene editing efficiency is generally reduced, as homologous recombination is only active during the late S/G2 phase of the cell cycle [54]. Nevertheless, in EB, HDR-based gene repair strategies have been successfully applied to EBS [9] and

RDEB cells [24, 33, 35] *in vitro*. The focus of therapy development currently lies on improving safety and efficiency, which are both prerequisites for any future *in vivo* application of this strategy. Towards this end, to circumvent the known off-target activity of wild type *Streptococcus pyogenes* Cas9 (SpCas9), we utilized a mutant Cas9D10A nuclease, which predominantly induces single-strand nicks instead of double-strand breaks within the DNA [55, 56]. Indeed, we found that targeting of mutant *KRT14* or *COL7A1* alleles with two guided nickases in a double-nicking configuration was safer and more efficient than the use of wild type spCas9 [9, 24]. Additional efficiency can be gained by optimizing both, the format and the delivery of the gene-editing molecules. Currently, electroporation of *COL7A1*-specific RNPs together with single-stranded oligonucleotide HDR templates have resulted in the highest repair efficiencies [34].

The application of HDR-based approaches to the correction of patient-derived induced pluripotent stem cells (iPSCs) further increases the range of therapeutic options for patients, especially as the isolation of epidermal holoclones can be a limiting factor [14, 34]. Pre-clinical studies using corrected iPSCs, that were then differentiated into keratinocytes and fibroblasts, and used to generate three-dimensional skin equivalents (HSEs) on the backs of immunodeficient mice, showed normal type VII collagen expression and restored anchoring fibrils [34]. Alternative strategies, not based on homology-directed repair, comprise base editing, which has proven to be a suitable option for correcting pathogenic mutations in RDEB [36]. The most recent genome editing tool, prime editing, can be used to directly write new genetic information into a selected genomic locus using a Cas9 nickase fused to an engineered reverse transcriptase (RT) domain [57]. Via a prime editing guide RNA (pegRNA), which specifies the target site and represents the RT template encoding the desired edit, the prime editor is directed to the target locus. Here, the Cas9 nickase makes a single strand DNA break, that induces the hybridization of the nicked genomic strand to the complementary primer binding site (PBS) sequence, located within the pegRNA. A subsequent reverse transcription of the RT template, carrying the desired edit, followed by a cellular DNA repair mechanism lead to the insertion of the respective genetic modification at the target site [57]. Prime editing potentially improves safety, efficiency and applicability, and its application to EB will undoubtedly be confirmed in appropriate disease models in the near future.

#### *2.1.3 RNA-based therapies for epidermolysis bullosa*

A promising RNA-based strategy for the restoration of functional protein expression in EB is based on the use of antisense oligonucleotides (AON) for the specific knockdown of genes or their modification via splicing interference [58]. AONs are generally short fragments of modified DNA or RNA which, in the case of splicing modulation, hybridize to splicing elements (*e.g.* splice acceptor site or enhancers) within an in-frame target exon during pre-mRNA splicing, thereby masking it from the splicing machinery and resulting in its exclusion from the mature transcript. Thus, a truncated protein carrying an in-frame deletion of one or more exons is translated from the new transcript. In the last decade, AON-mediated splicing modulation has been successfully applied in DEB keratinocytes to skip inframe *COL7A1* exon 70 [25], exon 73 [24, 26], exon 80 [26], and exon 105 [27] that carried dominant or recessive disease-causing mutations. The resulting proteins, though shorter, retained functionality. Indeed, *COL7A1* is amenable to AON-based exon skipping strategies because of the numerous short in-frame exons that encode its collagenous domains [24]. Beyond proof-of-concept, formulation of an exon 73-specific AON, named QR-313, within a carbomer-composed gel for topical treatment of wounds in DEB, led to enhanced type VII collagen levels in human RDEB

**95**

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

skin [24], and is currently being evaluated for safety and efficacy in clinical trials (NCT03605069). However, not all exons are suitable targets for exon skipping. Furthermore, amino acid sequences potentially vital for protein function may be deleted. Thus, AON-based gene repair approaches need to be carefully evaluated for each targeted gene, exon and even mutation, prior to their clinical application [27]. Another RNA-based strategy for mRNA correction, namely RNA *trans*-splicing (also termed spliceosome-mediated RNA *trans*-splicing, SMaRT), has emerged as an attractive therapeutic option for the correction of EB-associated mutations on the RNA level [10–12]. Here, the endogenous splicing machinery is exploited to selectively exchange mutation-bearing regions of the target pre-mRNA, with the corresponding wild type sequence from an exogenously provided RNA *trans*-splicing molecule (RTM) [59]. The engineered RTM contains a binding domain to direct its hybridization to the target pre-mRNA, as well as splicing elements required for efficient splicing, in addition to the wild-type coding sequence to be restored [59]. This approach has been applied to accurately restore gene function in a variety of human genetic diseases, including hemophilia A [60], muscular dystrophy [61, 62] or Alzheimer's disease [63]. In EB, SMaRT-mediated RNA editing was first achieved for the *PLEC* gene [12], but has more recently, been used to correct the EB-associated genes *KRT14* [10, 11, 13] and *COL7A1* [37–40] *in vitro* and in pre-clinical animal models. However, while these studies indicated a potential clinical applicability of SMaRT technology in gene therapy for EB, the efficiency of this RNA-based method needs to

be significantly improved if it is to move forward towards clinical translation.

when compared to standard of care after 2 and 12 weeks of treatment [42].

Protein replacement strategies were recently applied in preclinical studies for RDEB. The local or intravenous injection of recombinant type VII collagen led to its homing to the dermal-epidermal junction and promoted wound healing [41]. Another therapeutic option for RDEB is the administration of type VII collagen-expressing allogeneic fibroblasts into healing RDEB wounds. Particularly when injected intradermally allogeneic fibroblast therapy resulted in a significant decrease in wound area

Premature stop codon (PTC) read-through strategies rely on agents that allow for the incorporation of a random amino acid at the PTC position in the mRNA. Depending on the importance of the original amino acid to protein function, as well as impact of the introduced amino acid to *e.g.* protein folding, stability, and posttranslational processing, PTC read-through therapies can result in the synthesis of a functional full-length protein. This strategy has been shown to be feasible in RDEB-derived cells [64, 65]. Cogan et al. treated RDEB keratinocyte cell lines and RDEB fibroblasts carrying PTC mutations, with the aminoglycosides geneticin, gentamicin and paromomycin. Full-length type VII collagen was accurately synthesized and secreted in a dose-dependent and sustained manner, highlighting the therapeutic potential of PTC read-through approaches for RDEB patients [65]. This resulted in a clinical trial to assess safety and efficacy of topical and intradermal gentamicin treatment in 5 RDEB patients with nonsense mutations application led to induced type VII collagen expression and anchoring fibril generation at the dermal-epidermal junction of treated skin areas, with improved wound closure and reduced blistering [43]. However, the toxicity associated with long-term aminoglycoside use currently hinders their widespread clinical application for these purposes. Alternatively, the FDA-approved anti-inflammatory drug amlexanox has

*2.1.4 Protein- and cell-based therapies for epidermolysis bullosa*

*2.1.5 Read-through strategies for epidermolysis bullosa*

#### *Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

highest repair efficiencies [34].

*2.1.3 RNA-based therapies for epidermolysis bullosa*

RDEB cells [24, 33, 35] *in vitro*. The focus of therapy development currently lies on improving safety and efficiency, which are both prerequisites for any future *in vivo* application of this strategy. Towards this end, to circumvent the known off-target activity of wild type *Streptococcus pyogenes* Cas9 (SpCas9), we utilized a mutant Cas9D10A nuclease, which predominantly induces single-strand nicks instead of double-strand breaks within the DNA [55, 56]. Indeed, we found that targeting of mutant *KRT14* or *COL7A1* alleles with two guided nickases in a double-nicking configuration was safer and more efficient than the use of wild type spCas9 [9, 24]. Additional efficiency can be gained by optimizing both, the format and the delivery of the gene-editing molecules. Currently, electroporation of *COL7A1*-specific RNPs together with single-stranded oligonucleotide HDR templates have resulted in the

The application of HDR-based approaches to the correction of patient-derived induced pluripotent stem cells (iPSCs) further increases the range of therapeutic options for patients, especially as the isolation of epidermal holoclones can be a limiting factor [14, 34]. Pre-clinical studies using corrected iPSCs, that were then differentiated into keratinocytes and fibroblasts, and used to generate three-dimensional skin equivalents (HSEs) on the backs of immunodeficient mice, showed normal type VII collagen expression and restored anchoring fibrils [34]. Alternative strategies, not based on homology-directed repair, comprise base editing, which has proven to be a suitable option for correcting pathogenic mutations in RDEB [36]. The most recent genome editing tool, prime editing, can be used to directly write new genetic information into a selected genomic locus using a Cas9 nickase fused to an engineered reverse transcriptase (RT) domain [57]. Via a prime editing guide RNA (pegRNA), which specifies the target site and represents the RT template encoding the desired edit, the prime editor is directed to the target locus. Here, the Cas9 nickase makes a single strand DNA break, that induces the hybridization of the nicked genomic strand to the complementary primer binding site (PBS) sequence, located within the pegRNA. A subsequent reverse transcription of the RT template, carrying the desired edit, followed by a cellular DNA repair mechanism lead to the insertion of the respective genetic modification at the target site [57]. Prime editing potentially improves safety, efficiency and applicability, and its application to EB will undoubtedly be confirmed in appropriate disease models in the near future.

A promising RNA-based strategy for the restoration of functional protein expression in EB is based on the use of antisense oligonucleotides (AON) for the specific knockdown of genes or their modification via splicing interference [58]. AONs are generally short fragments of modified DNA or RNA which, in the case of splicing modulation, hybridize to splicing elements (*e.g.* splice acceptor site or enhancers) within an in-frame target exon during pre-mRNA splicing, thereby masking it from the splicing machinery and resulting in its exclusion from the mature transcript. Thus, a truncated protein carrying an in-frame deletion of one or more exons is translated from the new transcript. In the last decade, AON-mediated splicing modulation has been successfully applied in DEB keratinocytes to skip inframe *COL7A1* exon 70 [25], exon 73 [24, 26], exon 80 [26], and exon 105 [27] that carried dominant or recessive disease-causing mutations. The resulting proteins, though shorter, retained functionality. Indeed, *COL7A1* is amenable to AON-based exon skipping strategies because of the numerous short in-frame exons that encode its collagenous domains [24]. Beyond proof-of-concept, formulation of an exon 73-specific AON, named QR-313, within a carbomer-composed gel for topical treatment of wounds in DEB, led to enhanced type VII collagen levels in human RDEB

**94**

skin [24], and is currently being evaluated for safety and efficacy in clinical trials (NCT03605069). However, not all exons are suitable targets for exon skipping. Furthermore, amino acid sequences potentially vital for protein function may be deleted. Thus, AON-based gene repair approaches need to be carefully evaluated for each targeted gene, exon and even mutation, prior to their clinical application [27].

Another RNA-based strategy for mRNA correction, namely RNA *trans*-splicing (also termed spliceosome-mediated RNA *trans*-splicing, SMaRT), has emerged as an attractive therapeutic option for the correction of EB-associated mutations on the RNA level [10–12]. Here, the endogenous splicing machinery is exploited to selectively exchange mutation-bearing regions of the target pre-mRNA, with the corresponding wild type sequence from an exogenously provided RNA *trans*-splicing molecule (RTM) [59]. The engineered RTM contains a binding domain to direct its hybridization to the target pre-mRNA, as well as splicing elements required for efficient splicing, in addition to the wild-type coding sequence to be restored [59]. This approach has been applied to accurately restore gene function in a variety of human genetic diseases, including hemophilia A [60], muscular dystrophy [61, 62] or Alzheimer's disease [63]. In EB, SMaRT-mediated RNA editing was first achieved for the *PLEC* gene [12], but has more recently, been used to correct the EB-associated genes *KRT14* [10, 11, 13] and *COL7A1* [37–40] *in vitro* and in pre-clinical animal models. However, while these studies indicated a potential clinical applicability of SMaRT technology in gene therapy for EB, the efficiency of this RNA-based method needs to be significantly improved if it is to move forward towards clinical translation.

#### *2.1.4 Protein- and cell-based therapies for epidermolysis bullosa*

Protein replacement strategies were recently applied in preclinical studies for RDEB. The local or intravenous injection of recombinant type VII collagen led to its homing to the dermal-epidermal junction and promoted wound healing [41]. Another therapeutic option for RDEB is the administration of type VII collagen-expressing allogeneic fibroblasts into healing RDEB wounds. Particularly when injected intradermally allogeneic fibroblast therapy resulted in a significant decrease in wound area when compared to standard of care after 2 and 12 weeks of treatment [42].

#### *2.1.5 Read-through strategies for epidermolysis bullosa*

Premature stop codon (PTC) read-through strategies rely on agents that allow for the incorporation of a random amino acid at the PTC position in the mRNA. Depending on the importance of the original amino acid to protein function, as well as impact of the introduced amino acid to *e.g.* protein folding, stability, and posttranslational processing, PTC read-through therapies can result in the synthesis of a functional full-length protein. This strategy has been shown to be feasible in RDEB-derived cells [64, 65]. Cogan et al. treated RDEB keratinocyte cell lines and RDEB fibroblasts carrying PTC mutations, with the aminoglycosides geneticin, gentamicin and paromomycin. Full-length type VII collagen was accurately synthesized and secreted in a dose-dependent and sustained manner, highlighting the therapeutic potential of PTC read-through approaches for RDEB patients [65]. This resulted in a clinical trial to assess safety and efficacy of topical and intradermal gentamicin treatment in 5 RDEB patients with nonsense mutations application led to induced type VII collagen expression and anchoring fibril generation at the dermal-epidermal junction of treated skin areas, with improved wound closure and reduced blistering [43]. However, the toxicity associated with long-term aminoglycoside use currently hinders their widespread clinical application for these purposes. Alternatively, the FDA-approved anti-inflammatory drug amlexanox has

been demonstrated to induce full-length collagen type VII expression *in vitro* in 8 out of 12 different RDEB PTC alleles tested. Furthermore, read-through synthesis correlated with the phosphorylation of the RNA helicase UPF-1, suggesting that inhibition of nonsense mediated decay of the PTC-containing mRNA contributed to its mechanism of action [64]. However, increased read-through translation alone is insufficient to achieve proper function, and additionally accurate deposition of the protein at the basement membrane zone likely needs to be confirmed for each PTC mutation. Similar to the mentioned RDEB studies, the aminoglycoside gentamicin was recently applied to JEB keratinocytes carrying various nonsense mutations within the *LAMB3* gene [66]. As a result, the authors achieved PTC read-through leading to the synthesis and secretion of the respective laminin chain protein as well as the restoration of laminin-332 assembly [66]. Nevertheless, future studies are required to address current issues concerning read-through-based approaches such as the toxicity and bioavailability of applied compounds and their interactions within treated cells and organisms.

In summary, numerous strategies to target the genetic cause of EB, as well as ameliorate disease-associated complications, are under intensive investigation. These act at various levels, from genes and gene products, to cellular pathways, tissue processes, and systemic events. While each strategy has distinct strengths and challenges, they all share the overarching aim of significatnly improving the QoL of patients (**Figure 2**).

#### *2.1.6 Immunological aspects of causal therapy*

Immunological tolerance to self-antigens result from central and peripheral tolerance mechanisms. Central tolerance in the thymus results in either negative selection of self-reactive T cells or development of self-specific suppressive regulatory T cells, both of which require expression and presentation of self-antigens to developing thymocytes. Additionally, various peripheral mechanisms of tolerance protect the body from deleterious reactions against self-tissues. These include anatomical sequestration of self-antigens, deletion of peripheral autoreactive lymphocytes, the development of functional unresponsiveness of lymphocytes (anergy) and action of regulatory T cells [67, 68].

A major risk in patients, especially those completely lacking expression of the affected protein, is that this protein is missing from the repertoire of self-antigens presented during central tolerance establishment. As the aim of causal therapies is to restore the missing protein or repair a defective (*e.g.* truncated) one, these include an inherent risk of inducing adverse immune reactivity against the restored wild type protein, at least parts of which would be recognized in patients as foreign. For these reasons, only patients with residual expression of the EB-associated protein have been included in gene therapy trials to date [14, 16, 17, 19]. Despite this, transient circulating antibodies reactive against the gene-correction product, and also tissue-bound antibodies deposited within the graft, were reported in some participants [19, 20]. However, these did not correlate with rejection of the graft. In addition, a phenomenon called epitope spreading could theoretically occur in patients that mount an immune reaction against the gene-correction product, leading to autoimmunity against the endogenous mutated/residual protein present throughout the body's epithelial tissues. This systemic immune reaction would likely manifest as blistering within the graft, as well as worsened blistering distal to the graft. Thus, monitoring immunological parameters (*e.g.* specific antibody formation) and diffuse new-onset blistering is warranted in study participants. While detailed immunological studies are still largely lacking, the results suggest ex vivo gene-replacement therapy to be a safe therapeutic approach in patients who lack pre-existing immune reactivity and express residual protein. However, future

**97**

**Figure 2.**

*MSC therapy).*

trials should aim to also include patients without any residual protein expression, who oftentimes display a more severe phenotype, and where there is likely no preexisting tolerance to prevent adverse immune reactions against the gene-correction

*Causal therapy options for epidermolysis bullosa. Current strategies targeting the genetic cause of EB can be designed to act either on DNA level (cDNA replacement, gene editing), RNA level (AON-mediated exon skipping, SMaRT), RNA/protein level (PTC readthrough) or tissue level (protein replacement fibroblast/*

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

interactions within treated cells and organisms.

*2.1.6 Immunological aspects of causal therapy*

of regulatory T cells [67, 68].

been demonstrated to induce full-length collagen type VII expression *in vitro* in 8 out of 12 different RDEB PTC alleles tested. Furthermore, read-through synthesis correlated with the phosphorylation of the RNA helicase UPF-1, suggesting that inhibition of nonsense mediated decay of the PTC-containing mRNA contributed to its mechanism of action [64]. However, increased read-through translation alone is insufficient to achieve proper function, and additionally accurate deposition of the protein at the basement membrane zone likely needs to be confirmed for each PTC mutation. Similar to the mentioned RDEB studies, the aminoglycoside gentamicin was recently applied to JEB keratinocytes carrying various nonsense mutations within the *LAMB3* gene [66]. As a result, the authors achieved PTC read-through leading to the synthesis and secretion of the respective laminin chain protein as well as the restoration of laminin-332 assembly [66]. Nevertheless, future studies are required to address current issues concerning read-through-based approaches such as the toxicity and bioavailability of applied compounds and their

In summary, numerous strategies to target the genetic cause of EB, as well as ameliorate disease-associated complications, are under intensive investigation. These act at various levels, from genes and gene products, to cellular pathways, tissue processes, and systemic events. While each strategy has distinct strengths and challenges, they all share the overarching aim of significatnly improving the QoL of patients (**Figure 2**).

Immunological tolerance to self-antigens result from central and peripheral tolerance mechanisms. Central tolerance in the thymus results in either negative selection of self-reactive T cells or development of self-specific suppressive regulatory T cells, both of which require expression and presentation of self-antigens to developing thymocytes. Additionally, various peripheral mechanisms of tolerance protect the body from deleterious reactions against self-tissues. These include anatomical sequestration of self-antigens, deletion of peripheral autoreactive lymphocytes, the development of functional unresponsiveness of lymphocytes (anergy) and action

A major risk in patients, especially those completely lacking expression of the affected protein, is that this protein is missing from the repertoire of self-antigens presented during central tolerance establishment. As the aim of causal therapies is to restore the missing protein or repair a defective (*e.g.* truncated) one, these include an inherent risk of inducing adverse immune reactivity against the restored wild type protein, at least parts of which would be recognized in patients as foreign. For these reasons, only patients with residual expression of the EB-associated protein have been included in gene therapy trials to date [14, 16, 17, 19]. Despite this, transient circulating antibodies reactive against the gene-correction product, and also tissue-bound antibodies deposited within the graft, were reported in some participants [19, 20]. However, these did not correlate with rejection of the graft. In addition, a phenomenon called epitope spreading could theoretically occur in patients that mount an immune reaction against the gene-correction product, leading to autoimmunity against the endogenous mutated/residual protein present throughout the body's epithelial tissues. This systemic immune reaction would likely manifest as blistering within the graft, as well as worsened blistering distal to the graft. Thus, monitoring immunological parameters (*e.g.* specific antibody formation) and diffuse new-onset blistering is warranted in study participants. While detailed immunological studies are still largely lacking, the results suggest ex vivo gene-replacement therapy to be a safe therapeutic approach in patients who lack pre-existing immune reactivity and express residual protein. However, future

**96**

#### **Figure 2.**

*Causal therapy options for epidermolysis bullosa. Current strategies targeting the genetic cause of EB can be designed to act either on DNA level (cDNA replacement, gene editing), RNA level (AON-mediated exon skipping, SMaRT), RNA/protein level (PTC readthrough) or tissue level (protein replacement fibroblast/ MSC therapy).*

trials should aim to also include patients without any residual protein expression, who oftentimes display a more severe phenotype, and where there is likely no preexisting tolerance to prevent adverse immune reactions against the gene-correction product. Towards this goal, further research is required to exploit peripheral immune tolerance mechanisms to control the response to neo-antigens in the skin.

#### **2.2 Treating complications of EB**

While gene therapy is the only curative option for EB, strategies to ameliorate symptoms are critically needed to increase patient's QoL and prevent severe complications of the disease until causal therapies are available for all EB patients. The number of preclinical and clinical studies published, including those currently registered, reflects the great effort placed into providing such. Strategies to identify suitable candidates are diverse, but great potential lies in drug repurposing, as this facilitates timely development of potent treatments by leveraging already existing pre-clinical and clinical data. Even so, the methodological challenges inherent to conducting studies in rare disease populations can complicate the clinical evaluation of repositioning such drugs for EB [69].

The active components of drugs generally comprise small molecules or biologics. While low molecular weight small molecules can be derived chemically and exhibit distinct advantages regarding delivery and route of administration, biologics, which are much larger, often interfere very specifically with distinct pathomechanisms and show overall less toxicity. Functionally, small molecules are frequently designed as inhibitors of *e.g.* enzymes, whereas biologics usually have a specific active function (*e.g.* antibodies, enzymes, nucleic acids).

In the context of EB several approaches addressing various complications have been reported, with the majority of primary outcomes measured being improvement of wound healing, reduction of blistering, and mitigation of itch. While some of the evaluated compounds have reached late stage clinical trials, first marketing approvals are still awaited [4–7, 69].

#### *2.2.1 Reduction of blistering*

Across the various EB types, blistering of the skin may be the first clinical manifestation of skin fragility following mechanical friction or trauma. While in some patients blisters heal without scarring, in patients suffering from more severe subtypes these degenerate into wounds and are accompanied by multiple comorbidities. Thus, preventing blistering or accelerating their resolution is a logical primary outcome measure for clinical trials due to its relevance to patients, at least in distinct patient cohorts [7]. Particularly in EBS, where patients only rarely develop wounds, reducing blister numbers will improve patients' QoL substantially. Especially during childhood, EBS patients are prone to developing numerous blisters, which may prevent children from *e.g.* learning to walk, or playing with other children. However, also for dystrophic and junctional EB clinical studies evaluating a treatment's impact on blister numbers have been published [7].

For EBS, uncovering the pathways and molecular mediators underscoring the pathogenic keratin biology in cells, proved instrumental to the development of a topical formulation of the drug diacerein, which has been shown in randomized controlled trials (RCTs) to significantly reduce blister numbers in comparison to patients who received placebo [70, 71]. Moreover, during the patient followup period when no treatment was applied, a delayed recurrence of blisters was observed, pointing towards a long-term stabilization of the skin. Diacerein is a small molecule that interferes with the expression and signaling of the proinflammatory cytokine IL-1ß at various levels. Upregulation of IL-1ß, triggered by the accumulation of mutated keratins, is characteristic for distinct subtypes of EBS. However, the reciprocal effect of IL-1ß to further induce the expression of mutant

**99**

*2.2.3 Pruritus*

an unmet medical need [75].

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*2.2.2 Wound healing*

network and consequently, also the patient's skin [72].

gene products using drugs that induce read-through of PTCs.

keratins was also observed in patient cells. Thus, interfering with this positive feedback loop proved beneficial to stabilizing the keratinocyte's intermediate filament

EB patients develop wounds throughout their lifetime, and their management and daily care routine represent a major burden that is accompanied by substantial discomfort. There is currently no standard treatment for the treatment of nonhealing or severely infected wounds in EB. Defects in wound healing, associated with infections and persistent inflammation are presumed major drivers of wound chronification, which is a major risk factor for the development of particularly aggressive squamous cell carcinomas (SCC) [2, 3]. Thus, means to improve wound healing, and thereby prevent downstream complications that severely decrease QoL and that may even be life-threatening, are urgently needed. This is also reflected by the high number of clinical trials that primarily aim to improve wound healing, either by applying drugs that modulate wound healing associated pathways (*e.g.* modulation of inflammation), or, if feasible, by directly targeting the EB-causing

Promising outcomes were recently reported from a trial using anti-inflammatory/immunomodulatory betulin-rich birch bark extract (Filzuvez, previously Oleogel S-10), wherein 41.3% of patients treated with Oleogel-S10 met the primary endpoint of target wound closure within 45 (± 7) days as compared to 28.9% of patients within the placebo arm. Furthermore, among the EB subtypes evaluated, patients with RDEB appeared to be particularly responsive to treatment (NCT03068780). In the context of wound healing, induction of PTC read-through as a means of triggering re-expression of genes harboring nonsense mutations, is particularly attractive in cases where the drugs being evaluated for these purposes

have known antibacterial and anti-inflammatory activity. Particularly for junctional and dystrophic EB patients, clinical trials investigating the aminoglycoside antibiotic gentamicin are still ongoing [43, 73, 74] (NCT04140786, NCT03526159, NCT04644627, NCT03392909). Additionally for RDEB, the antiinflammatory, anti-allergic immunomodulator Amlexanox, typically used against

mouth ulcers, has emerged as a novel candidate in preclinical studies [64].

Pruritus is a particularly agonizing aspect associated with all subtypes of EB which not only impairs patients' QoL, but also leads to additional skin damage as it provokes scratching. Even though pruritus is not a life-threatening symptom *per se*, its overall influence on well-being is tremendous. In general, itch is a major problem associated with various diseases like atopic dermatitis, psoriasis, and nephrologic conditions. Yet, underlying molecular mechanisms are still not fully understood. Numerous inflammatory mediators have been associated with pathological itch, but despite pruritus being a severe clinical problem, effective treatments still represent

For patients with EB, a handful of studies targeting itch have been published, the most recent being a randomized-controlled trial (RCT) evaluating the neurokinin-1 receptor (NK1R) antagonist Serlopitant in patients with any EB subtype [76]. Serlopitant disrupts Substance P (SP) associated signaling by preventing its binding to NK1R. Expressed on multiple skin cell types, NK1R is thought to play a major role in the transmission of itch signals in the peripheral and central nervous systems [77]. In the RCT above, 14 EB patients received serlopitant or placebo over a period

keratins was also observed in patient cells. Thus, interfering with this positive feedback loop proved beneficial to stabilizing the keratinocyte's intermediate filament network and consequently, also the patient's skin [72].

#### *2.2.2 Wound healing*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

tion of repositioning such drugs for EB [69].

tion (*e.g.* antibodies, enzymes, nucleic acids).

approvals are still awaited [4–7, 69].

*2.2.1 Reduction of blistering*

**2.2 Treating complications of EB**

product. Towards this goal, further research is required to exploit peripheral immune tolerance mechanisms to control the response to neo-antigens in the skin.

While gene therapy is the only curative option for EB, strategies to ameliorate symptoms are critically needed to increase patient's QoL and prevent severe complications of the disease until causal therapies are available for all EB patients. The number of preclinical and clinical studies published, including those currently registered, reflects the great effort placed into providing such. Strategies to identify suitable candidates are diverse, but great potential lies in drug repurposing, as this facilitates timely development of potent treatments by leveraging already existing pre-clinical and clinical data. Even so, the methodological challenges inherent to conducting studies in rare disease populations can complicate the clinical evalua-

The active components of drugs generally comprise small molecules or biologics. While low molecular weight small molecules can be derived chemically and exhibit distinct advantages regarding delivery and route of administration, biologics, which are much larger, often interfere very specifically with distinct pathomechanisms and show overall less toxicity. Functionally, small molecules are frequently designed as inhibitors of *e.g.* enzymes, whereas biologics usually have a specific active func-

In the context of EB several approaches addressing various complications have been reported, with the majority of primary outcomes measured being improvement of wound healing, reduction of blistering, and mitigation of itch. While some of the evaluated compounds have reached late stage clinical trials, first marketing

Across the various EB types, blistering of the skin may be the first clinical manifestation of skin fragility following mechanical friction or trauma. While in some patients blisters heal without scarring, in patients suffering from more severe subtypes these degenerate into wounds and are accompanied by multiple comorbidities. Thus, preventing blistering or accelerating their resolution is a logical primary outcome measure for clinical trials due to its relevance to patients, at least in distinct patient cohorts [7]. Particularly in EBS, where patients only rarely develop wounds, reducing blister numbers will improve patients' QoL substantially. Especially during childhood, EBS patients are prone to developing numerous blisters, which may prevent children from *e.g.* learning to walk, or playing with other children. However, also for dystrophic and junctional EB clinical studies evaluating a treat-

For EBS, uncovering the pathways and molecular mediators underscoring the pathogenic keratin biology in cells, proved instrumental to the development of a topical formulation of the drug diacerein, which has been shown in randomized controlled trials (RCTs) to significantly reduce blister numbers in comparison to patients who received placebo [70, 71]. Moreover, during the patient followup period when no treatment was applied, a delayed recurrence of blisters was observed, pointing towards a long-term stabilization of the skin. Diacerein is a small molecule that interferes with the expression and signaling of the proinflammatory cytokine IL-1ß at various levels. Upregulation of IL-1ß, triggered by the accumulation of mutated keratins, is characteristic for distinct subtypes of EBS. However, the reciprocal effect of IL-1ß to further induce the expression of mutant

ment's impact on blister numbers have been published [7].

**98**

EB patients develop wounds throughout their lifetime, and their management and daily care routine represent a major burden that is accompanied by substantial discomfort. There is currently no standard treatment for the treatment of nonhealing or severely infected wounds in EB. Defects in wound healing, associated with infections and persistent inflammation are presumed major drivers of wound chronification, which is a major risk factor for the development of particularly aggressive squamous cell carcinomas (SCC) [2, 3]. Thus, means to improve wound healing, and thereby prevent downstream complications that severely decrease QoL and that may even be life-threatening, are urgently needed. This is also reflected by the high number of clinical trials that primarily aim to improve wound healing, either by applying drugs that modulate wound healing associated pathways (*e.g.* modulation of inflammation), or, if feasible, by directly targeting the EB-causing gene products using drugs that induce read-through of PTCs.

Promising outcomes were recently reported from a trial using anti-inflammatory/immunomodulatory betulin-rich birch bark extract (Filzuvez, previously Oleogel S-10), wherein 41.3% of patients treated with Oleogel-S10 met the primary endpoint of target wound closure within 45 (± 7) days as compared to 28.9% of patients within the placebo arm. Furthermore, among the EB subtypes evaluated, patients with RDEB appeared to be particularly responsive to treatment (NCT03068780). In the context of wound healing, induction of PTC read-through as a means of triggering re-expression of genes harboring nonsense mutations, is particularly attractive in cases where the drugs being evaluated for these purposes have known antibacterial and anti-inflammatory activity. Particularly for junctional and dystrophic EB patients, clinical trials investigating the aminoglycoside antibiotic gentamicin are still ongoing [43, 73, 74] (NCT04140786, NCT03526159, NCT04644627, NCT03392909). Additionally for RDEB, the antiinflammatory, anti-allergic immunomodulator Amlexanox, typically used against mouth ulcers, has emerged as a novel candidate in preclinical studies [64].

#### *2.2.3 Pruritus*

Pruritus is a particularly agonizing aspect associated with all subtypes of EB which not only impairs patients' QoL, but also leads to additional skin damage as it provokes scratching. Even though pruritus is not a life-threatening symptom *per se*, its overall influence on well-being is tremendous. In general, itch is a major problem associated with various diseases like atopic dermatitis, psoriasis, and nephrologic conditions. Yet, underlying molecular mechanisms are still not fully understood. Numerous inflammatory mediators have been associated with pathological itch, but despite pruritus being a severe clinical problem, effective treatments still represent an unmet medical need [75].

For patients with EB, a handful of studies targeting itch have been published, the most recent being a randomized-controlled trial (RCT) evaluating the neurokinin-1 receptor (NK1R) antagonist Serlopitant in patients with any EB subtype [76]. Serlopitant disrupts Substance P (SP) associated signaling by preventing its binding to NK1R. Expressed on multiple skin cell types, NK1R is thought to play a major role in the transmission of itch signals in the peripheral and central nervous systems [77]. In the RCT above, 14 EB patients received serlopitant or placebo over a period

of 8 weeks, and reduction of pruritus was assessed using a numeric rating scale. Even though results were not statistically significant, patients who had received the investigational drug tended to achieve a ≥ 3 point reduction in itch compared to placebo, and a positive impact on QoL was reported by the patients. However, a larger clinical trial will be needed to provide clear evidence on the efficacy of serlopitant in reducing pruritus in patients with EB [76].

Another agent currently being evaluated against pruritus in patients with EB-pruriginosa is the anti-interleukin-4 receptor alpha (IL-4Rα) monoclonal antibody Dupilumab [78, 79]. Already indicated for atopic dermatitis, Dupilumab inhibits both IL-4 and IL-13 signaling and modulates Th2-mediated immune mechanisms. The promising outcome of both studies, which included a total of three patients, might provide a rationale for larger RCTs in the future that extend to other subtypes of EB.

Interestingly, in a single-patient observational study aimed primarily at investigating the wound healing benefits of a low-dose topical calcipotriol ointment in DEB, a significant reduction of itch was reported as a highly patient-relevant outcome. Calcipotriol is an analogue of the active form of vitamin D3, an important skin homeostasis factor with roles in cell proliferation, differentiation, antimicrobial defense, and immune modulation. Calcipotriol has proven anti-proliferative effects in keratinocytes, which is leveraged for the treatment of plaque psoriasis. For this reason, we investigated a lower concentration with respect to antimicrobial peptide induction in DEB keratinocytes. In addition to the complete closure of a chronic wound within two weeks of treatment, we observed significant improvement in the diversity of the skin microbiota on the treated skin area, with complete clearance of *Staphylococcus aureus* by the end of the treatment [80]. The low dose ointment was evaluated in a small double-blind, placebo-controlled phase II clinical trial (EudraCT: 2016–001967-35), where a significant and steady reduction in pruritus was observed with calcipotriol treatment compared to placebo [81]. These results support conducting a follow-up trial to investigate its impact on itch in patients with EB, particularly given calcipotriol's reported anti-neoplastic effects.

#### *2.2.4 Fibrosis*

Repeated cycles of injury and subsequent persistent inflammation trigger a cascade of events leading to progressive fibrosis, followed by tissue stiffening and increased risk of tumor development in patients with dystrophic EB. Additionally, fibrotic webbing at limb extremities post-wounding ultimately leads to fusion of fingers and toes (called mitten deformities), severely limiting their use. Thus, strategies to support a normal course of wound healing are investigated to avoid or minimize deviations from deposition of a normal skin matrix [82]. A key player in EB-associated fibrosis is TGF-ß, a pro-inflammatory cytokine whose pleiotropic effects are highly contextdependent, and which has been shown to be constitutively expressed in RDEB-skin [83, 84]. While TGF-ß1 promotes wound healing under normal conditions, excessive TGF-ß1 signaling leads to abnormal ECM deposition and scar formation, as confirmed in a type VII collagen hypomorphic mouse model [85, 86]. Thus, modulating the expression of TGF-ß1 was hypothesized to be beneficial in reducing fibrosis. In this context, losartan, an angiotensin II antagonist with anti-fibrotic effects, has been evaluated in preclinical studies, where Nystrom *et al* achieved a significant reduction in fibrosis in collagen VII hypomorphic mice. This approach led to reduced TGF-ß signaling, normalized skin extracellular matrix composition, and delayed progression of mitten deformities [83]. Based on these results, a phase I/II clinical trial to evaluate the safety, tolerability and efficacy of losartan in children and adolescents with RDEB is currently underway (Eudra-CT: 2015–003670-32).

**101**

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

**2.3 EB-associated squamous cell carcinoma**

applied to this vulnerable patient group [91].

when long-term treatment is considered.

*2.3.1 EGFR inhibition*

In a drug repurposing approach, endoglin (CD105), a type III co-receptor for TGF-1, and raloxifene, an estrogen receptor modulator, were tested in a pre-clinical setting for their potential to attenuate RDEB-associated fibrosis. Indeed, both drugs were shown to modulate profibrotic events, rendering them potential candidates for

Cutaneous tumors are a life-threatening complication that arise especially in patients with RDEB. Owing to the repeated cycles of wounding, infection and inflammation, RDEB patients are at especially high risk of developing aggressive squamous cell carcinoma (RDEB-SCC) with high risk-features. The sites of tumor occurrence are predominantly at sites of chronic and long-term wounds [87], especially on the extremities [88], indicating that tumorigenesis is related to the pathology of RDEB. The SCCs tend to arise in early adulthood, with a reported median age of 29 years at time of diagnosis, although the youngest case reported was in a 6-year old patient [89]. In comparison to the general population, RDEB patients have an estimated 70-fold higher risk of developing SCC [90], with cumulative risk rising from 7.5% at age 20 years, to 67.8% at age 35 years, to 90.1% by age 55 [2]. Despite aggressive therapy with multiple treatment modalities, median survival time from time of first diagnosis is 4–5 years [89], making RDEB-SCC the primary cause of premature death in these patients. The first choice of treatment still consists of wide local excision of the tumor, and even amputation of the extremity is sometimes necessary. Radiotherapy and conventional chemotherapeutic approaches have been mostly used palliatively in EB SCC and considering their strong adverse effects (*e.g.* desquamation of the skin upon radiotherapy) should be used carefully when

Genomic analyses combined with transcriptomic profiling of tumors highlight cell endogenous mutagenic processes mediated by APOBEC enzymes, which are associated with an innate defense mechanism against ongoing microbial infection, as a major driver of carcinogenesis in RDEB. These observations indicate that effective wound management, which includes an antimicrobial component, could potentially lower cancer risk in these patients. Genetically, RDEB-SCC closely resembles ultraviolet (UV) light-induced SCC and SCC of the head and neck (HN-SCC), with driver mutations in known cancer-associated genes such as *CASP8, NOTCH1, TP53, FAT1, CDKN2A, HRAS, ARID2, and KMT2B* frequently observed [92]. While these similarities suggest that therapies proven to be efficacious in other SCCs would also be effective in RDEB SCC, careful consideration of the background pathology of EB is needed. The significant overlap in cellular processes associated with wound healing and tumor development (*e.g.* proliferation, migration, vascularisation/ angiogenesis, matrix remodeling) dictates that a delicate balance needs to established wherein tumor inhibition is not interfering with wound healing, especially

Cetuximab, a monoclonal antibody targeting the extracellular domain of epidermal growth factor receptor (EGFR), has been used for therapy against both HN-SCC, as well as advanced unresectable cutaneous UV-SCC [93, 94]. This agent inhibits tumor cell proliferation by blocking receptor tyrosine kinase activity upstream of known survival-, growth-, and migration- signaling cascades mediated by *PI3K/AKT*, *RAS/MAPK*, and *JAK/STAT* [95, 96]. Cetuximab shows significant efficacy against EGFR-positive, wild type RAS tumors, while those bearing RAS

repositioning both compounds for the treatment of patients with EB [86].

#### *Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

in reducing pruritus in patients with EB [76].

other subtypes of EB.

*2.2.4 Fibrosis*

of 8 weeks, and reduction of pruritus was assessed using a numeric rating scale. Even though results were not statistically significant, patients who had received the investigational drug tended to achieve a ≥ 3 point reduction in itch compared to placebo, and a positive impact on QoL was reported by the patients. However, a larger clinical trial will be needed to provide clear evidence on the efficacy of serlopitant

Another agent currently being evaluated against pruritus in patients with EB-pruriginosa is the anti-interleukin-4 receptor alpha (IL-4Rα) monoclonal antibody Dupilumab [78, 79]. Already indicated for atopic dermatitis, Dupilumab inhibits both IL-4 and IL-13 signaling and modulates Th2-mediated immune mechanisms. The promising outcome of both studies, which included a total of three patients, might provide a rationale for larger RCTs in the future that extend to

Interestingly, in a single-patient observational study aimed primarily at investigating the wound healing benefits of a low-dose topical calcipotriol ointment in DEB, a significant reduction of itch was reported as a highly patient-relevant outcome. Calcipotriol is an analogue of the active form of vitamin D3, an important skin homeostasis factor with roles in cell proliferation, differentiation, antimicrobial defense, and immune modulation. Calcipotriol has proven anti-proliferative effects in keratinocytes, which is leveraged for the treatment of plaque psoriasis. For this reason, we investigated a lower concentration with respect to antimicrobial peptide induction in DEB keratinocytes. In addition to the complete closure of a chronic wound within two weeks of treatment, we observed significant improvement in the diversity of the skin microbiota on the treated skin area, with complete clearance of *Staphylococcus aureus* by the end of the treatment [80]. The low dose ointment was evaluated in a small double-blind, placebo-controlled phase II clinical trial (EudraCT: 2016–001967-35), where a significant and steady reduction in pruritus was observed with calcipotriol treatment compared to placebo [81]. These results support conducting a follow-up trial to investigate its impact on itch in patients with EB, particularly given calcipotriol's reported anti-neoplastic effects.

Repeated cycles of injury and subsequent persistent inflammation trigger a cascade of events leading to progressive fibrosis, followed by tissue stiffening and increased risk of tumor development in patients with dystrophic EB. Additionally, fibrotic webbing at limb extremities post-wounding ultimately leads to fusion of fingers and toes (called mitten deformities), severely limiting their use. Thus, strategies to support a normal course of wound healing are investigated to avoid or minimize deviations from deposition of a normal skin matrix [82]. A key player in EB-associated fibrosis is TGF-ß, a pro-inflammatory cytokine whose pleiotropic effects are highly contextdependent, and which has been shown to be constitutively expressed in RDEB-skin [83, 84]. While TGF-ß1 promotes wound healing under normal conditions, excessive TGF-ß1 signaling leads to abnormal ECM deposition and scar formation, as confirmed in a type VII collagen hypomorphic mouse model [85, 86]. Thus, modulating the expression of TGF-ß1 was hypothesized to be beneficial in reducing fibrosis. In this context, losartan, an angiotensin II antagonist with anti-fibrotic effects, has been evaluated in preclinical studies, where Nystrom *et al* achieved a significant reduction in fibrosis in collagen VII hypomorphic mice. This approach led to reduced TGF-ß signaling, normalized skin extracellular matrix composition, and delayed progression of mitten deformities [83]. Based on these results, a phase I/II clinical trial to evaluate the safety, tolerability and efficacy of losartan in children and adolescents with RDEB

**100**

is currently underway (Eudra-CT: 2015–003670-32).

In a drug repurposing approach, endoglin (CD105), a type III co-receptor for TGF-1, and raloxifene, an estrogen receptor modulator, were tested in a pre-clinical setting for their potential to attenuate RDEB-associated fibrosis. Indeed, both drugs were shown to modulate profibrotic events, rendering them potential candidates for repositioning both compounds for the treatment of patients with EB [86].

#### **2.3 EB-associated squamous cell carcinoma**

Cutaneous tumors are a life-threatening complication that arise especially in patients with RDEB. Owing to the repeated cycles of wounding, infection and inflammation, RDEB patients are at especially high risk of developing aggressive squamous cell carcinoma (RDEB-SCC) with high risk-features. The sites of tumor occurrence are predominantly at sites of chronic and long-term wounds [87], especially on the extremities [88], indicating that tumorigenesis is related to the pathology of RDEB. The SCCs tend to arise in early adulthood, with a reported median age of 29 years at time of diagnosis, although the youngest case reported was in a 6-year old patient [89]. In comparison to the general population, RDEB patients have an estimated 70-fold higher risk of developing SCC [90], with cumulative risk rising from 7.5% at age 20 years, to 67.8% at age 35 years, to 90.1% by age 55 [2]. Despite aggressive therapy with multiple treatment modalities, median survival time from time of first diagnosis is 4–5 years [89], making RDEB-SCC the primary cause of premature death in these patients. The first choice of treatment still consists of wide local excision of the tumor, and even amputation of the extremity is sometimes necessary. Radiotherapy and conventional chemotherapeutic approaches have been mostly used palliatively in EB SCC and considering their strong adverse effects (*e.g.* desquamation of the skin upon radiotherapy) should be used carefully when applied to this vulnerable patient group [91].

Genomic analyses combined with transcriptomic profiling of tumors highlight cell endogenous mutagenic processes mediated by APOBEC enzymes, which are associated with an innate defense mechanism against ongoing microbial infection, as a major driver of carcinogenesis in RDEB. These observations indicate that effective wound management, which includes an antimicrobial component, could potentially lower cancer risk in these patients. Genetically, RDEB-SCC closely resembles ultraviolet (UV) light-induced SCC and SCC of the head and neck (HN-SCC), with driver mutations in known cancer-associated genes such as *CASP8, NOTCH1, TP53, FAT1, CDKN2A, HRAS, ARID2, and KMT2B* frequently observed [92]. While these similarities suggest that therapies proven to be efficacious in other SCCs would also be effective in RDEB SCC, careful consideration of the background pathology of EB is needed. The significant overlap in cellular processes associated with wound healing and tumor development (*e.g.* proliferation, migration, vascularisation/ angiogenesis, matrix remodeling) dictates that a delicate balance needs to established wherein tumor inhibition is not interfering with wound healing, especially when long-term treatment is considered.

#### *2.3.1 EGFR inhibition*

Cetuximab, a monoclonal antibody targeting the extracellular domain of epidermal growth factor receptor (EGFR), has been used for therapy against both HN-SCC, as well as advanced unresectable cutaneous UV-SCC [93, 94]. This agent inhibits tumor cell proliferation by blocking receptor tyrosine kinase activity upstream of known survival-, growth-, and migration- signaling cascades mediated by *PI3K/AKT*, *RAS/MAPK*, and *JAK/STAT* [95, 96]. Cetuximab shows significant efficacy against EGFR-positive, wild type RAS tumors, while those bearing RAS

mutations are resistant to treatment [97]. EB-associated SCCs frequently express EGFR, but with noticeable differences in expression level which could impact responsiveness to treatment [98]. To date, only a handful of cases have reported the use of cetuximab in EB patients with advanced cutaneous SCC, with limited beneficial effects on survival [88, 94, 98–100]. Progression-free survival in seven patients reported in the literature ranged from three weeks to nine months. Better outcomes have been observed when cetuximab was given as a first line treatment suggesting that treatment might be more efficacious when administered early. Of note, wound healing deficits and worsening of skin lesions, which negatively impact patient's QoL, were noted in three patients. For one patient, the negative effects on patient wound healing led to a dose reduction of cetuximab and subsequent tumor recurrence soon after [98]. This negative impact of cetuximab in RDEB is likely associated with the important roles of the aforementioned downstream signaling cascades in wound healing processes and exemplifies the challenge of treating tumors in the background of EB. Thus, more targeted strategies, aimed at inhibition of tumoressential pathways downstream of EGFR are already being investigated and are expected to minimize such adverse effects.

#### *2.3.2 JAK1/2 inhibition*

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway mediates cellular responses to a variety of cytokines and growth factors, including IL-6 and EGF, downstream of these ligands binding their cognate receptors. These responses include proliferation, differentiation, migration, survival, and apoptosis, and are dependent on cell- and tissue-type, as well as the context of the signal [101]. In this respect JAK/STAT plays important roles in developmental and homeostatic processes, and is also aberrantly active in numerous cancers, including HN-SCC [102]. Increased levels of phosphorylated STAT3, a downstream effector of JAK, were observed in RDEB-SCC cells over normal keratinocytes, providing rationale for evaluating the effect of the JAK1/2 inhibitor ruxolitinib in a murine xenograft model of human RDEB-SCC [103]. In this preclinical study, ruxolitinib effectively reduced tumor mass, when administered either orally or topically onto tumors, because reduced STAT3 signaling led to decreased cell proliferation. These observations argue that ruxolitinib may be a promising anticancer drug for RDEB-SCC. When ruxolitinib was used with the aim to counteract the fibrotic processes in the skin of type VII collagen hypomorphic mice, a reduction of phosphorylated STAT3 in fibroblasts and SCC *in vitro*, but only limited therapeutic benefit against fibrosis-driven mitten deformities in type VII collagen hypomorphic mice was observed [104]. Additionally, the drug was not well tolerated by the mice and, even more importantly, treatment delayed wound healing, highlighting that caution and rigorous evaluation is warranted before its clinical application in patients.

#### *2.3.3 Polo-like kinase 1 inhibition*

Due to the aggressive and metastasising nature of RDEB-SCC, which is atypical of UV-induced cutaneous SCCs that arise in the general population, gene expression assays were performed to identify differentially regulated genes that might account for this difference in tumor behavior. Among the handful of genes identified was polo-like kinase 1 (PLK1) [105], a serine/threonine protein kinase which was overexpressed in a number of different tumors [106]. Blocking PLK1 leads to mitotic arrest, inhibition of cell proliferation and apoptosis. Notably, cells were more sensitive to PLK1 inhibition when p53 was defective [107]. *TP53* is frequently mutated in RDEB-SCC [92] highlighting PLK1-inhibition as a potential strategy to selectively

**103**

**3. Conclusions**

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*2.3.4 Immune checkpoint blocking*

carcinomas (EudraCT: 2016–002811-16) [6].

In the last decade, the number of clinical trials registered for the evaluation of therapies against various primary and secondary pathologies associated with the various forms of EB has risen dramatically (>70 clinical trials; clinicaltrials.

target tumor cells over normal cells which exhibit normal p53 function. Several small molecules are under investigation for their ability to target PLK1 signaling in cancer, and results from these studies can be leveraged to facilitate the evaluation of these agents also for clinical use in RDEB patients. A study by Atansova et al., identified rigosertib (or ON-01910) among six different small-molecule inhibitors of PLK1 as a strong and selective inducer of apoptosis in RDEB-SCC cells. Its pre-clinical evaluation in a murine xenograft model demonstrated inhibition of tumor growth without obvious toxicity, laying the path for a multi-center phase II clinical trial in RDEB patients with late stage, metastatic, and/or unresectable SCC. [64] (NCT03786237).

Immune checkpoints are molecules that are either able to turn on (co-stimulatory molecules) or turn off (inhibitory molecules) immune signaling, generally referring to the activation of responses in T cells. Tumors have developed mechanisms to exploit these immune checkpoint molecules in order to evade immune surveillance and escape clearance by *e.g*. cytotoxic T cells [108]. Such immune checkpoint molecules include CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD-1 (programmed death-1). The latter is predominantly expressed on T cells, and by binding to its ligands PD-L1 and PD-L2 expressed on tumor cells, induces a negative signal that leads to effector T cell suppression [109]. Blocking these interactions using specific antibodies leads to reactivation of the immune system and improvement of anti-tumor immune responses. Remarkable antitumor effects were achieved with an antibody targeting CTLA-4 (ipilimumab), increasing median overall survival in metastatic melanoma patients [109]. Even better outcomes in survival rate have been observed with pembrolizumab, an anti-PD1 receptor antibody [110]. Furthermore, beneficial evidence in clinical trials using anti-PD1 treatment in advanced HN-SCC [111], and also locally advanced/ metastatic SCC [112] support PD-1 blocking as well in RDEB SCC. In this respect, 2 reports can be found in the literature describing the use of anti-PD1 blocking antibodies in RDEB-SCC patients. The first case described the use of pembrolizumab (PD-1 antibody) as second line therapy after cetuximab treatment. Pembrolizumab was partly combined with other therapeutic approaches including intralesional administration of talimogene laherparepvec (T-Vec; oncolytic virus) into metastatic tumors, radiotherapy and anti-EGFR monoclonal antibody (panitumumab). Wound healing was not impaired during the late stage of the disease. The patient died due to tumor progression 18 months after starting pembrolizumab treatment [100]. Most recently, Khaddour et al. report on an RDEB patient with metastatic SCC who was treated with cemiplimab (monoclonal anti-PD1) every 3 weeks in combination with radiotherapy. During the 14-month follow-up the patient showed a durable response with no signs of immune-related adverse events [113]. These observations support conducting controlled clinical trials to assess the efficacy of PD-1 blockade in this patient group. Noteworthy, another anti-PD1 antibody (nivolumab) is currently under evaluation in a multi-centre phase II clinical trial for the palliative treatment of EB patients with advanced or metastatic squamous cell

#### *Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

expected to minimize such adverse effects.

*2.3.2 JAK1/2 inhibition*

*2.3.3 Polo-like kinase 1 inhibition*

mutations are resistant to treatment [97]. EB-associated SCCs frequently express EGFR, but with noticeable differences in expression level which could impact responsiveness to treatment [98]. To date, only a handful of cases have reported the use of cetuximab in EB patients with advanced cutaneous SCC, with limited beneficial effects on survival [88, 94, 98–100]. Progression-free survival in seven patients reported in the literature ranged from three weeks to nine months. Better outcomes have been observed when cetuximab was given as a first line treatment suggesting that treatment might be more efficacious when administered early. Of note, wound healing deficits and worsening of skin lesions, which negatively impact patient's QoL, were noted in three patients. For one patient, the negative effects on patient wound healing led to a dose reduction of cetuximab and subsequent tumor recurrence soon after [98]. This negative impact of cetuximab in RDEB is likely associated with the important roles of the aforementioned downstream signaling cascades in wound healing processes and exemplifies the challenge of treating tumors in the background of EB. Thus, more targeted strategies, aimed at inhibition of tumoressential pathways downstream of EGFR are already being investigated and are

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway mediates cellular responses to a variety of cytokines and growth factors, including IL-6 and EGF, downstream of these ligands binding their cognate receptors. These responses include proliferation, differentiation, migration, survival, and apoptosis, and are dependent on cell- and tissue-type, as well as the context of the signal [101]. In this respect JAK/STAT plays important roles in developmental and homeostatic processes, and is also aberrantly active in numerous cancers, including HN-SCC [102]. Increased levels of phosphorylated STAT3, a downstream effector of JAK, were observed in RDEB-SCC cells over normal keratinocytes, providing rationale for evaluating the effect of the JAK1/2 inhibitor ruxolitinib in a murine xenograft model of human RDEB-SCC [103]. In this preclinical study, ruxolitinib effectively reduced tumor mass, when administered either orally or topically onto tumors, because reduced STAT3 signaling led to decreased cell proliferation. These observations argue that ruxolitinib may be a promising anticancer drug for RDEB-SCC. When ruxolitinib was used with the aim to counteract the fibrotic processes in the skin of type VII collagen hypomorphic mice, a reduction of phosphorylated STAT3 in fibroblasts and SCC *in vitro*, but only limited therapeutic benefit against fibrosis-driven mitten deformities in type VII collagen hypomorphic mice was observed [104]. Additionally, the drug was not well tolerated by the mice and, even more importantly, treatment delayed wound healing, highlighting that caution and

rigorous evaluation is warranted before its clinical application in patients.

Due to the aggressive and metastasising nature of RDEB-SCC, which is atypical of UV-induced cutaneous SCCs that arise in the general population, gene expression assays were performed to identify differentially regulated genes that might account for this difference in tumor behavior. Among the handful of genes identified was polo-like kinase 1 (PLK1) [105], a serine/threonine protein kinase which was overexpressed in a number of different tumors [106]. Blocking PLK1 leads to mitotic arrest, inhibition of cell proliferation and apoptosis. Notably, cells were more sensitive to PLK1 inhibition when p53 was defective [107]. *TP53* is frequently mutated in RDEB-SCC [92] highlighting PLK1-inhibition as a potential strategy to selectively

**102**

target tumor cells over normal cells which exhibit normal p53 function. Several small molecules are under investigation for their ability to target PLK1 signaling in cancer, and results from these studies can be leveraged to facilitate the evaluation of these agents also for clinical use in RDEB patients. A study by Atansova et al., identified rigosertib (or ON-01910) among six different small-molecule inhibitors of PLK1 as a strong and selective inducer of apoptosis in RDEB-SCC cells. Its pre-clinical evaluation in a murine xenograft model demonstrated inhibition of tumor growth without obvious toxicity, laying the path for a multi-center phase II clinical trial in RDEB patients with late stage, metastatic, and/or unresectable SCC. [64] (NCT03786237).

#### *2.3.4 Immune checkpoint blocking*

Immune checkpoints are molecules that are either able to turn on (co-stimulatory molecules) or turn off (inhibitory molecules) immune signaling, generally referring to the activation of responses in T cells. Tumors have developed mechanisms to exploit these immune checkpoint molecules in order to evade immune surveillance and escape clearance by *e.g*. cytotoxic T cells [108]. Such immune checkpoint molecules include CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD-1 (programmed death-1). The latter is predominantly expressed on T cells, and by binding to its ligands PD-L1 and PD-L2 expressed on tumor cells, induces a negative signal that leads to effector T cell suppression [109]. Blocking these interactions using specific antibodies leads to reactivation of the immune system and improvement of anti-tumor immune responses. Remarkable antitumor effects were achieved with an antibody targeting CTLA-4 (ipilimumab), increasing median overall survival in metastatic melanoma patients [109]. Even better outcomes in survival rate have been observed with pembrolizumab, an anti-PD1 receptor antibody [110]. Furthermore, beneficial evidence in clinical trials using anti-PD1 treatment in advanced HN-SCC [111], and also locally advanced/ metastatic SCC [112] support PD-1 blocking as well in RDEB SCC. In this respect, 2 reports can be found in the literature describing the use of anti-PD1 blocking antibodies in RDEB-SCC patients. The first case described the use of pembrolizumab (PD-1 antibody) as second line therapy after cetuximab treatment. Pembrolizumab was partly combined with other therapeutic approaches including intralesional administration of talimogene laherparepvec (T-Vec; oncolytic virus) into metastatic tumors, radiotherapy and anti-EGFR monoclonal antibody (panitumumab). Wound healing was not impaired during the late stage of the disease. The patient died due to tumor progression 18 months after starting pembrolizumab treatment [100]. Most recently, Khaddour et al. report on an RDEB patient with metastatic SCC who was treated with cemiplimab (monoclonal anti-PD1) every 3 weeks in combination with radiotherapy. During the 14-month follow-up the patient showed a durable response with no signs of immune-related adverse events [113]. These observations support conducting controlled clinical trials to assess the efficacy of PD-1 blockade in this patient group. Noteworthy, another anti-PD1 antibody (nivolumab) is currently under evaluation in a multi-centre phase II clinical trial for the palliative treatment of EB patients with advanced or metastatic squamous cell carcinomas (EudraCT: 2016–002811-16) [6].

#### **3. Conclusions**

In the last decade, the number of clinical trials registered for the evaluation of therapies against various primary and secondary pathologies associated with the various forms of EB has risen dramatically (>70 clinical trials; clinicaltrials.

gov). They reflect the progress in the optimization of previous gene therapeutic approaches, discovery and advancement of novel gene editing technologies, and the increase in our understanding of the molecular and cellular mechanisms underpinning the nature of these EB-related complications. Drug repositioning has largely been prioritized, as leveraging the existing pharmacological and safety data represents the fastest and most economical route to clinical trials, and when successful, to marketing approval for EB. To further support the development of new therapy options for rare diseases like EB, several programs, like the orphan designation program by the European Medicines Agency (EMA) have been launched.

This is good news for patients, but also creates challenges for the recruitment of sufficient participants to the various trials, due to the rarity of the disease, strict inclusion criteria, and the disinclination of patients to participate. This heightens the risk of recruitment failure and the inability to meet statistical endpoints, resulting in extended trials that come at increased costs. Surmounting these challenges will require close collaboration within the entire EB community, to establish an international patient registry, incentivize patient participation, address logistical and regulatory aspects of multi-center trials, and allow for new outcome measures and the development of statistical methods for small cohorts. In parallel, applying current state-of-the-art methods that maximize acquisition of multi-modal data from patient samples, alongside the continuing advances in artificial intelligence, will further support the development of new therapies at various levels, starting from *in silico* drug discovery to establishing new means for measuring patientrelevant trial outcomes.

#### **Acknowledgements**

The authors would like to thank DEBRA Austria for their continued support and funding of our work. This work is generated within the ERN skin.

**105**

**Author details**

Iris Gratz1,2†

Josefina Piñón Hofbauer1†

and Ulrich Koller1†

\*Address all correspondence to: v.wally@salk.at

provided the original work is properly cited.

† All authors contributed equally.

, Verena Wally1

Paracelsus Medical University Salzburg, Salzburg, Austria

\* †

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Dermatology and Allergology, EB House Austria, Research Program for Molecular Therapy of Genodermatoses, University Hospital of the

2 Department of Biosciences, University of Salzburg, Salzburg, Austria

, Christina Guttmann-Gruber1†

,

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

#### **Conflict of interest**

The authors declare no conflict of interest.

*Therapy Development for Epidermolysis Bullosa DOI: http://dx.doi.org/10.5772/intechopen.97437*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

relevant trial outcomes.

**Acknowledgements**

**Conflict of interest**

gov). They reflect the progress in the optimization of previous gene therapeutic approaches, discovery and advancement of novel gene editing technologies, and the increase in our understanding of the molecular and cellular mechanisms underpinning the nature of these EB-related complications. Drug repositioning has largely been prioritized, as leveraging the existing pharmacological and safety data represents the fastest and most economical route to clinical trials, and when successful, to marketing approval for EB. To further support the development of new therapy options for rare diseases like EB, several programs, like the orphan designation program by the European Medicines Agency (EMA) have been launched.

This is good news for patients, but also creates challenges for the recruitment of sufficient participants to the various trials, due to the rarity of the disease, strict inclusion criteria, and the disinclination of patients to participate. This heightens the risk of recruitment failure and the inability to meet statistical endpoints, resulting in extended trials that come at increased costs. Surmounting these challenges will require close collaboration within the entire EB community, to establish an international patient registry, incentivize patient participation, address logistical and regulatory aspects of multi-center trials, and allow for new outcome measures and the development of statistical methods for small cohorts. In parallel, applying current state-of-the-art methods that maximize acquisition of multi-modal data from patient samples, alongside the continuing advances in artificial intelligence, will further support the development of new therapies at various levels, starting from *in silico* drug discovery to establishing new means for measuring patient-

The authors would like to thank DEBRA Austria for their continued support and

funding of our work. This work is generated within the ERN skin.

The authors declare no conflict of interest.

**104**

#### **Author details**

Josefina Piñón Hofbauer1† , Verena Wally1 \* † , Christina Guttmann-Gruber1† , Iris Gratz1,2† and Ulrich Koller1†

1 Department of Dermatology and Allergology, EB House Austria, Research Program for Molecular Therapy of Genodermatoses, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria

2 Department of Biosciences, University of Salzburg, Salzburg, Austria

\*Address all correspondence to: v.wally@salk.at

† All authors contributed equally.

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[103] Jackow J, Rami A, Hayashi R, Hansen C, Guo Z, DeLorenzo D, et al. Targeting the Jak/Signal Transducer and Activator Of Transcription 3 Pathway with Ruxolitinib in a Mouse Model of Recessive Dystrophic Epidermolysis Bullosa-Squamous Cell Carcinoma. J

[104] Mittapalli VR, Kuhl T, Kuzet SE, Gretzmeier C, Kiritsi D, Gaggioli C, et al. STAT3 targeting in dystrophic epidermolysis bullosa. Br J Dermatol.

[105] Watt SA, Pourreyron C, Purdie K, Hogan C, Cole CL, Foster N, et al. Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma. Oncogene.

[106] Takai N, Hamanaka R, Yoshimatsu J, Miyakawa I. Polo-like kinases (Plks) and cancer. Oncogene. 2005;24(2):287-291.

[107] Degenhardt Y, Lampkin T. Targeting Polo-like kinase in cancer therapy. Clin Cancer Res. 2010;16(2):384-389.

[108] He X, Xu C. Immune checkpoint signaling and cancer immunotherapy.

Cell Res. 2020;30(8):660-669.

DeGrave KA, Wheeler S, Freilino ML, et al. JAK kinase inhibition abrogates STAT3 activation and head and neck squamous cell carcinoma tumor growth.

Lacroix L, Gelly J, et al. Phase II study of cetuximab as first-line single-drug therapy in patients with unresectable squamous cell carcinoma of the skin. J Clin Oncol. 2011;29(25):3419-3426.

[95] Dai W, Li Y, Zhou Q, Xu Z, Sun C, Tan X, et al. Cetuximab inhibits oral squamous cell carcinoma invasion and metastasis via degradation of epidermal growth factor receptor. J Oral Pathol

[96] Uribe P, Gonzalez S. Epidermal growth factor receptor (EGFR) and squamous cell carcinoma of the skin: molecular bases for EGFR-targeted therapy. Pathol Res Pract. 2011;207(6):

[97] Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, et al. KRAS mutations as an independent prognostic

factor in patients with advanced

J Clin Oncol. 2008;26(3):374-379.

[98] Diociaiuti A, Steinke H,

cell carcinoma in dystrophic

Dis. 2019;14(1):278.

colorectal cancer treated with cetuximab.

Nystrom A, Schwieger-Briel A, Meiss F, Pfannenberg C, et al. EGFR inhibition for metastasized cutaneous squamous

epidermolysis bullosa. Orphanet J Rare

[99] Arnold AW, Bruckner-Tuderman L, Zuger C, Itin PH. Cetuximab therapy of metastasizing cutaneous squamous cell carcinoma in a patient with severe recessive dystrophic epidermolysis bullosa. Dermatology. 2009;219(1):80-83.

[100] Medek K, Koelblinger P, Koller J, Diem A, Ude-Schoder K, Bauer JW, et al. Wound healing deficits in severe

2015;51(14):1989-2007.

[94] Maubec E, Petrow P, Scheer-Senyarich I, Duvillard P,

Med. 2014;43(4):250-257.

337-342.

**112**

[110] Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med. 2015;372(26):2521-2532.

[111] Chow LQM, Haddad R, Gupta S, Mahipal A, Mehra R, Tahara M, et al. Antitumor Activity of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort. J Clin Oncol. 2016;34(32):3838-3845.

[112] Migden MR, Rischin D, Schmults CD, Guminski A, Hauschild A, Lewis KD, et al. PD-1 Blockade with Cemiplimab in Advanced Cutaneous Squamous-Cell Carcinoma. N Engl J Med. 2018;379(4):341-351.

[113] Khaddour K, Gorell ES, Dehdashti F, Tang JY, Ansstas G. Induced Remission of Metastatic Squamous Cell Carcinoma with an Immune Checkpoint Inhibitor in a Patient with Recessive Dystrophic Epidermolysis Bullosa. Case Rep Oncol. 2020;13(2):911-915.

**Chapter 7**

*Piotr Fiedor*

genetic diseases.

Rare Diseases

**115**

**Abstract**

Bullosa (EB)

Surgical Treatment of Wounds

*Magdalena Nita, Jacek Pliszczyński, Andrzej Eljaszewicz, Marcin Moniuszko,Tomasz Ołdak, Katarzyna Woźniak,*

*Sławomir Majewski, Cezary Kowalewski, Artur Kamiński,*

Epidermolysis bullosa (EB) is a group of hereditary skin diseases, or genodermatoses, characterized by the formation of severe, chronic blisters with painful and life-threatening complications. Despite the previous and ongoing progress in the field, there are still no effective causative treatments for EB. The treatment is limited to relieving symptoms, which—depending on disease severity —may involve skin (blisters, poorly healing wounds caused by the slightest mechanical stimuli, contractures, scarring, pseudosyndactyly) and internal organ abnormalities (esophageal, pyloric, or duodenal atresia; renal failure; and hematopoietic abnormalities). The last decade saw a series of important discoveries that paved the way for new treatment methods, including gene therapy, bone marrow transplantation, cell therapy (allogenic fibroblasts, mesenchymal stem cells [MSCs], and clinical use of induced pluripotent stem cells. Tissue engineering experts are attempting to develop skin-like structures that can facilitate the process of healing to promote skin reconstruction in injuries that are currently incurable. However, this is incredibly challenging, due to the complex structure and the many functions of the skin. Below, we characterize EB and present its potential treatment methods. Despite the cure for EB being still out of reach, recent data from animal models and initial clinical trials in humans have raised patients', clinicians', and researchers' expectations. Consequently, modifying the course of the disease and improving the quality of life have become possible. Moreover, the conclusions drawn based on EB treatment may considerably improve the treatment of other

**Keywords:** biological dressing, human skin allograft, allogenic human skin equivalent, Advanced Therapy Medicinal Product, Epidermolysis Bullosa,

*Dariusz Śladowski, Zbigniew Zimek, Maciej Kosieradzki and*

Using Stem Cells in Epidermolysis

**Chapter 7**

## Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)

*Magdalena Nita, Jacek Pliszczyński, Andrzej Eljaszewicz, Marcin Moniuszko,Tomasz Ołdak, Katarzyna Woźniak, Sławomir Majewski, Cezary Kowalewski, Artur Kamiński, Dariusz Śladowski, Zbigniew Zimek, Maciej Kosieradzki and Piotr Fiedor*

#### **Abstract**

Epidermolysis bullosa (EB) is a group of hereditary skin diseases, or genodermatoses, characterized by the formation of severe, chronic blisters with painful and life-threatening complications. Despite the previous and ongoing progress in the field, there are still no effective causative treatments for EB. The treatment is limited to relieving symptoms, which—depending on disease severity —may involve skin (blisters, poorly healing wounds caused by the slightest mechanical stimuli, contractures, scarring, pseudosyndactyly) and internal organ abnormalities (esophageal, pyloric, or duodenal atresia; renal failure; and hematopoietic abnormalities). The last decade saw a series of important discoveries that paved the way for new treatment methods, including gene therapy, bone marrow transplantation, cell therapy (allogenic fibroblasts, mesenchymal stem cells [MSCs], and clinical use of induced pluripotent stem cells. Tissue engineering experts are attempting to develop skin-like structures that can facilitate the process of healing to promote skin reconstruction in injuries that are currently incurable. However, this is incredibly challenging, due to the complex structure and the many functions of the skin. Below, we characterize EB and present its potential treatment methods. Despite the cure for EB being still out of reach, recent data from animal models and initial clinical trials in humans have raised patients', clinicians', and researchers' expectations. Consequently, modifying the course of the disease and improving the quality of life have become possible. Moreover, the conclusions drawn based on EB treatment may considerably improve the treatment of other genetic diseases.

**Keywords:** biological dressing, human skin allograft, allogenic human skin equivalent, Advanced Therapy Medicinal Product, Epidermolysis Bullosa, Rare Diseases

#### **1. Introduction**

Epidermolysis Bullosa (EB) is a group of heterogeneous genetic conditions (genodermatoses) characterized by skin fragility and blister formation. These blisters, or bullae, may form spontaneously or as a result of slight mechanical injuries. EB is estimated to occur in 1 person per 50,000 live births.

pathological gene variants in 20 different genes. The genes associated with EB encode intracellular, transmembrane, or extracellular proteins that constitute structural components of the cytoskeleton (keratin 5 and 14), extracellular matrix (integrin α6β4, collagen XVII, laminin 332, collagen VII, α3 integrin, kindlin-1),

The key clinical manifestation of EB is a tendency to develop skin lesions in response to mechanical stimuli, even those of a very low magnitude. The most common lesion types include blisters, milia, pigmented lesions, erosions, epidermal defects, and scars. Other characteristic features of the condition are nail plate changes, ranging from dystrophy to a complete loss. Another common symptom is hair loss and—in severe cases—alopecia. Blisters, erosions, and scars developing near joints may result in contractures and tissue adhesions due to scarring. The lesions that develop on hands and feet (which are most prone to mechanical injuries) may result in pseudosyndactyly. Contractures exacerbate hand and foot deformities, leading to disability ("cocoon hand", or "mitten hand" deformities).

*Ultrastructural sites of blister formation in major forms of epidermolysis bullosa EB. 1. In intact skin, the ultrastructural regions of the epidermal basement membrane zone consist of basal keratinocytes and the hemodesmosomal plaque, the lamina lucida, the lamina densa, the upper papillary dermis 2. in eb simplex (EBS), blisters arise within the lower portion of basal keratinocytes 3. In junctional EB (JEB) blisters form within the lamina lucida 4. In dystrophic EB (DEB), blisters develop below the lamina densa. Anchoring fibris are reduced in number in dominant DEB (DDEB) and absent or rudimentary in recessive DEB (RDEB). KRT5,KRT14 and keratin 5 and keratin14 respectively (s. https://plasticsurgerykey.com/epidermolysis-b*

or intercellular adhesions (desmoplakin, plakophilin, plakoglobin).

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

**3. Epidermolysis Bullosa**

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

**Figure 2.**

*ullosa/).*

**117**

EB constitutes a group of conditions with diverse clinical courses. Depending on the type of abnormalities in the specific genes, the course, severity, and location of lesions may vary. EB is a result of abnormal connection between the epidermis and dermis. The epidermis, which is the most superficial layer of the skin, constitutes an important barrier between the body and its external environment. The epidermis prevents the loss of water and protects the body against ultraviolet radiation and pathogens. The dermis contains blood vessels, nerve endings, and skin appendages. Under normal conditions, the epidermis and dermis are tightly connected via protein molecules [1–6].

#### **2. The epidermis – structure and functions**

The epidermis is the outermost part of the skin and serves as a barrier protecting the body against pathogens, ultraviolet radiation, and excessive loss of water. The epidermal layers, listed from the deepest to the most superficial, include the basal, spinous, granular, and cornified layers. The basal layer is composed of keratinocytes, which undergo intense cell divisions. The newly formed cells differentiate as they progress towards the epidermal surface, eventually becoming dead, anuclear cells (corneocytes) that have no mitochondria. Since they are surrounded by a lipid layer, corneocytes form an impermeable barrier. The epidermis is strongly and permanently connected to the dermis via a cytoskeleton and hemidesmosomes. (**Figure 1**) [7–9].

The course of EB may be severe if the condition is due to a lack of key adhesion proteins, for example as a result of loss-of-function mutations in laminin 332 or collagen VII genes. Conversely, isolated amino acid substitutions typically lead to a mild fragility of the skin. The genetic and allelic heterogeneity of EB is due to

**Figure 1.** *Skin structure. (from private sources MN).*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

pathological gene variants in 20 different genes. The genes associated with EB encode intracellular, transmembrane, or extracellular proteins that constitute structural components of the cytoskeleton (keratin 5 and 14), extracellular matrix (integrin α6β4, collagen XVII, laminin 332, collagen VII, α3 integrin, kindlin-1), or intercellular adhesions (desmoplakin, plakophilin, plakoglobin).

#### **3. Epidermolysis Bullosa**

**1. Introduction**

protein molecules [1–6].

(**Figure 1**) [7–9].

**Figure 1.**

**116**

*Skin structure. (from private sources MN).*

Epidermolysis Bullosa (EB) is a group of heterogeneous genetic conditions (genodermatoses) characterized by skin fragility and blister formation. These blisters, or bullae, may form spontaneously or as a result of slight mechanical injuries.

EB constitutes a group of conditions with diverse clinical courses. Depending on the type of abnormalities in the specific genes, the course, severity, and location of lesions may vary. EB is a result of abnormal connection between the epidermis and dermis. The epidermis, which is the most superficial layer of the skin, constitutes an important barrier between the body and its external environment. The epidermis prevents the loss of water and protects the body against ultraviolet radiation and pathogens. The dermis contains blood vessels, nerve endings, and skin appendages. Under normal conditions, the epidermis and dermis are tightly connected via

The epidermis is the outermost part of the skin and serves as a barrier protecting the body against pathogens, ultraviolet radiation, and excessive loss of water. The epidermal layers, listed from the deepest to the most superficial, include the basal,

keratinocytes, which undergo intense cell divisions. The newly formed cells differentiate as they progress towards the epidermal surface, eventually becoming dead, anuclear cells (corneocytes) that have no mitochondria. Since they are surrounded by a lipid layer, corneocytes form an impermeable barrier. The epidermis is strongly and permanently connected to the dermis via a cytoskeleton and hemidesmosomes.

The course of EB may be severe if the condition is due to a lack of key adhesion proteins, for example as a result of loss-of-function mutations in laminin 332 or collagen VII genes. Conversely, isolated amino acid substitutions typically lead to a mild fragility of the skin. The genetic and allelic heterogeneity of EB is due to

spinous, granular, and cornified layers. The basal layer is composed of

EB is estimated to occur in 1 person per 50,000 live births.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

**2. The epidermis – structure and functions**

The key clinical manifestation of EB is a tendency to develop skin lesions in response to mechanical stimuli, even those of a very low magnitude. The most common lesion types include blisters, milia, pigmented lesions, erosions, epidermal defects, and scars. Other characteristic features of the condition are nail plate changes, ranging from dystrophy to a complete loss. Another common symptom is hair loss and—in severe cases—alopecia. Blisters, erosions, and scars developing near joints may result in contractures and tissue adhesions due to scarring. The lesions that develop on hands and feet (which are most prone to mechanical injuries) may result in pseudosyndactyly. Contractures exacerbate hand and foot deformities, leading to disability ("cocoon hand", or "mitten hand" deformities).

#### **Figure 2.**

*Ultrastructural sites of blister formation in major forms of epidermolysis bullosa EB. 1. In intact skin, the ultrastructural regions of the epidermal basement membrane zone consist of basal keratinocytes and the hemodesmosomal plaque, the lamina lucida, the lamina densa, the upper papillary dermis 2. in eb simplex (EBS), blisters arise within the lower portion of basal keratinocytes 3. In junctional EB (JEB) blisters form within the lamina lucida 4. In dystrophic EB (DEB), blisters develop below the lamina densa. Anchoring fibris are reduced in number in dominant DEB (DDEB) and absent or rudimentary in recessive DEB (RDEB). KRT5,KRT14 and keratin 5 and keratin14 respectively (s. https://plasticsurgerykey.com/epidermolysis-b ullosa/).*

Severe forms of EB additionally involve internal anomalies in the oral cavity, esophagus, trachea, lungs, urinary catheter, or urinary bladder. Intestinal tract erosions, ulcerations, and scarring lead to strictures, which may result in difficulty swallowing (dysphagia) and necessitate a feeding jenunostomy to provide enteral nutrition. Oral manifestations of EB may include the tongue adhering to the floor of the mouth (ankyloglossia); a narrowed oral opening (microstomia); and difficulties in chewing and swallowing, which result in malnourishment, osteopenia, osteoporosis, growth retardation, and eating disorders, leading to cachexia. Oral lesions may cause oral hygiene problems, which leads to caries. Perianal erosions and ulcerations cause severe pain during defecation, which contributes to constipation. Possible ocular manifestations involve marginal blepharitis, eyelash loss, ectropion, adhesions between the palpebral and bulbar conjunctivae (symblepharon), and corneal blistering, which may lead to blindness. Other manifestations include treatment-refractory anemia, iron deficiency, and hypoalbuminemia. Due to chronic ulcerations and an impaired protective function of the epidermis, EB patients may develop skin cancer (squamous cell carcinoma [SCC]) in their thirties or forties (**Figure 2**) [10–15].

• dystrophic epidermolysis bullosa (DEB) involves anomalies of the dermis

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

**Subtype Phenotype Inheritance Gene affected**

AD *KRT5 or KRT14*

AD *KRT5 or KRT14*

AD *KRT5 or KRT14*

AD *Predominantly*

AD *KRT5*

AD *KLHL24*

AD or AR *PLEC*

AR *PLEC*

*KRTS; less frequently KRT14*

patient is diagnosed with DEB (**Table 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

EB simplex, localized Palmoplantar blistering from birth or

EB simplex, severe Early generalized blistering at or soon

dystrophy common

dystrophy may develop

nail dystrophy possible

early infancy, with subsequent keratoderma in affected areas

after birth; congenital areas of denuded skin may be present; can be life threatening in first year of life; classically, tense clustered 'herpetiform' blisters arise with minimal trauma or spontaneously; development of confluent palmoplantar keratoderma; nail

Generalized, although less severe blistering than EB simplex, severe

Blistering from birth of intermediate severity; additional mottled or reticulate macular pigmentation typically of the neck, upper trunk and acral skin; punctate keratoderma; nail

Vesicles from birth, on a background of inflammatory migratory circinate erythema that fades to leave postinflammatory hyperpigmentation;

Marked erosions in limbs at birth, healing with dyspigmentation and atrophic burn-like scars; keratoderma, nail-thickening and onychogryphosis possible; diffuse alopecia has occasionally been reported; dilated cardiomyopathy develops later in young adulthood

*Autosomal dominant disease is mild with mainly acral blistering; autosomal recessive has an intermediate*

Generalized blistering with variableonset myopathy including possible cardiomyopathy; focal plantar

*presentation*

**Overview of EB classification**

*EB simplex — Intraepidermal*

EB simplex, intermediate

EB simplex with mottled pigmentation

EB simplex, migratory

circinate

EB simplex, intermediate with cardiomyopathy

*EB simplex, intermediate with PLEC mutations*

EB simplex, intermediate with muscular dystrophy

**119**

The diagnosis is made based on a thorough microscopic examination of a skin sample. The examination helps determine the exact layer of the skin where tissue separation causes blister formation. There are several layers that can be identified under a microscope in a skin cross-section. If the blisters form within the epidermis, the patient is diagnosed with SEB; if they form within the lamina lucida, the patient is diagnosed with JEB, and if they form just underneath the lamina densa, the

#### **3.1 Classification**

EB is a result of mutations in approximately 20 genes that encode structural and enzymatic proteins responsible for forming and maintaining the connections between the epidermis and dermis. The most common mutations occur in one of three genes: *KRT5*, *KRT14*, or *TGM5*.


EB can be classified into three main types, which can be further divided into subtypes. This classification is based on anomalies in various protein molecules and each of the resulting EB types has a different clinical course.


*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

• dystrophic epidermolysis bullosa (DEB) involves anomalies of the dermis

The diagnosis is made based on a thorough microscopic examination of a skin sample. The examination helps determine the exact layer of the skin where tissue separation causes blister formation. There are several layers that can be identified under a microscope in a skin cross-section. If the blisters form within the epidermis, the patient is diagnosed with SEB; if they form within the lamina lucida, the patient is diagnosed with JEB, and if they form just underneath the lamina densa, the patient is diagnosed with DEB (**Table 1**).


Severe forms of EB additionally involve internal anomalies in the oral cavity, esophagus, trachea, lungs, urinary catheter, or urinary bladder. Intestinal tract erosions, ulcerations, and scarring lead to strictures, which may result in difficulty swallowing (dysphagia) and necessitate a feeding jenunostomy to provide enteral nutrition. Oral manifestations of EB may include the tongue adhering to the floor of the mouth (ankyloglossia); a narrowed oral opening (microstomia); and difficulties in chewing and swallowing, which result in malnourishment, osteopenia, osteoporosis, growth retardation, and eating disorders, leading to cachexia. Oral lesions may cause oral hygiene problems, which leads to caries. Perianal erosions and ulcerations cause severe pain during defecation, which contributes to constipation. Possible ocular manifestations involve marginal blepharitis, eyelash loss, ectropion, adhesions between the palpebral and bulbar conjunctivae (symblepharon), and corneal blistering, which may lead to blindness. Other manifestations include treatment-refractory anemia, iron deficiency, and hypoalbuminemia. Due to chronic ulcerations and an impaired protective function of the epidermis, EB patients may develop skin cancer (squamous cell carcinoma [SCC]) in their thirties

EB is a result of mutations in approximately 20 genes that encode structural and

enzymatic proteins responsible for forming and maintaining the connections between the epidermis and dermis. The most common mutations occur in one of

• KRT5: The protein encoded by this gene is a member of the keratin gene family. The type II cytokeratins consist of basic or neutral proteins which are

• KRT14: This gene product, a type I keratin. At least one pseudogene has been

encoded protein catalyzes formation of protein cross-links between glutamine and lysine residues, often resulting in stabilization of protein assemblies. This reaction is calcium dependent. Mutations in this gene have been

associated with acral peeling skin syndrome (RefSeq, Oct 2009). [https://www.

EB can be classified into three main types, which can be further divided into subtypes. This classification is based on anomalies in various protein molecules and

• simple epidermolysis bullosa (SEB) involves epidermal anomalies

• junctional epidermolysis bullosa (JEB) involves basement membrane

each of the resulting EB types has a different clinical course.

• TGM5: This gene encodes a member of the transglutaminase family. The

arranged in pairs of heterotypic keratin chains coexpressed during differentiation of simple and stratified epithelial tissues. This type II cytokeratin is specifically expressed in the basal layer of the epidermis with family member KRT14. Mutations in these genes have been associated with a complex of diseases termed epidermolysis bullosa simplex. The type II cytokeratins are clustered in a region of chromosome 12q12-q13. (RefSeq,

or forties (**Figure 2**) [10–15].

three genes: *KRT5*, *KRT14*, or *TGM5*.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

**3.1 Classification**

Jul 2008)

identified at 17p12-p11.

genecards.org/]

anomalies

**118**


**Overview of EB classification**

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

Junctional EB, late

Junctional EB with interstitial lung disease and nephrotic syndrome

DDEB, selfimproving1,2

**121**

*Dystrophic EB — sublamina densa*

onset

Junctional EB–laryngo-onychocutaneous (LOC) syndrome

Junctional EB, inversa Flexural blistering from birth; dental

and visual loss

Intermediate DDEB1 Generalized skin fragility, scarring

Localized DDEB1 Predominantly acral blistering,

disease)

DDEB, pruriginosa1 Profoundly pruritic linear cords of

than in severe RDEB

Variable degree of cutaneous involvement; fatality in early childhood is common; nail dystrophy possible; hair loss may occur

and milia presenting from birth or early infancy, with prominence over acral sites, elbows and knees; involvement of the mucous membranes may lead to microstomia, ankyloglossia and oesophageal stenosis, although less commonly

scarring and milia seen from birth or early infancy; occasional nails-only presentation, with progressive dystrophy and eventual nail loss; rarely, cutaneous features may predominate over pretibial skin alone (and can present as late-onset

papules associated with fragility, scarring and milia on the shins, and occasionally progressing to arms; may present in childhood or adulthood;

Blistering evident at or shortly after birth, usually on extremities where there may be aplasia cutis, whilst scarring and milia may occur; spontaneous resolution of cutaneous fragility within the first 2 years of life

nail dystrophy is usual

abnormalities and nail loss

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

Onset in childhood, with often acral fragility; skin fragility is progressive and loss of dermatoglyphs may be seen owing to scarring; variable dental enamel and nail defects

Skin fragility from birth with marked exuberant granulation tissue (greater than that in junctional EB, severe), particularly on face and neck; nail dystrophy and loss with granulation tissue of nail beds; laryngeal granulation can lead to respiratory compromise and death; conjunctival and eyelid granulation with consequent symblepharon, scarring

**Subtype Phenotype Inheritance Gene affected**

AR *LAMA3, LAMB3*

AR *COL17A1*

AR *LAMA3*

AR *IGTA3*

AD *COL7A1*

AD *COL7A1*

AD *COL7A1*

AD *COL7A1*

*and LAMC2*


**Overview of EB classification**

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

EB simplex, severe with pyloric atresia

EB simplex, autosomal recessive, KRT5 or

EB simplex, localized or intermediate with BP230 deficiency

EB simplex, localized or intermediate with exophilin 5 deficiency

EB simplex, localized with nephropathy (CD151 deficiency)

Junctional EB, intermediate

Junctional EB with pyloric atresia

Junctional EB, localized

**120**

KRT14

**Subtype Phenotype Inheritance Gene affected**

AR *PLEC*

AR *DST*

AR *EXPH5*

AR *CD151*

AR *LAMA3, LAMB3*

AR *LAMA3, LAMB3,*

AR *ITGA6 and ITG84*

AR *LAMA3, LAMB3, LAMC2, COL17A1, ITGB4 and ITGA3*

*and LAMC2*

*LAMC2 and COL17A*

AR *KRT5 or KRT14*

keratoderma and nail dystrophy; mucosal involvement is common; upper respiratory tract stenosis has

More severe, widespread generalized blistering or loss of skin at birth with pyloric atresia; early mortality within

Generalized blistering, intermediate or severe; keratin 5 abnormalities tend to have a more severe phenotype; absence of keratin 5 associated with widespread skin disease and early mortality; improvement of blistering with age is

Early-onset blistering, relatively mild, usually with acral

predominance; plantar keratoderma

Generalized intermittent blistering and skin fragility; mild mottled pigmentation may be evident

localized to periungual, buttock and elbow regions; overgranulation develops, particularly on orofacial and periungual regions, with development of bulbous nail folds; alopecia is common; dental enamel defects are usual; a hoarse cry is often a feature; usually fatal within the first

Less severe than above, with a reduced tendency to develop exuberant granulation tissue; elevated risk of SCC in adulthood

Extensive areas of skin loss seen at birth with severe cutaneous fragility; early-onset pyloric atresia, a frequent cause of early mortality, within days or weeks of birth; duodenal and anal atresia may also feature; milder non-

lethal variants often show genitourinary involvement

normal hair

Limited cutaneous fragility, often acral; variable nail and dental defects;

Early blistering, with pretibial predominance; poikiloderma may be seen; early alopecia; extracutaneous involvement manifests as oesophageal webbing and

been reported

not expected

nephropathy

2 years of life

Junctional EB, severe Blistering may be mild at birth and

a few months of birth


**3.2 Heredity**

**3.3 SEB subtypes**

course.

**123**

contractures.

SEB primarily shows an autosomal dominant pattern of inheritance, with the most common mutations in genes *KRT5* and *KRT14*. Autosomal recessive inheritance is less common and caused by mutations in genes *KRT14*, *ITGA6*, *ITGB4* (*this genes encodes a member of the integrin alpha chain family of proteins. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain that function in cell surface adhesion and signaling. The encoded preproprotein is proteolytically processed to generate light and heavy chains that comprise the alpha 6 subunit. This subunit may associate with a beta 1 or beta 4 subunit to form an integrin that interacts with extracellular matrix proteins including members of the laminin family. The alpha 6 beta 4 integrin may promote tumorigenesis, while the alpha 6 beta 1 integrin may negatively regulate erbB2/HER2 signaling. Alternative splicing results in*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

*DSP* (This gene encodes a protein that anchors intermediate filaments to desmosomal plaques and forms an obligate component of functional desmosomes), or *PKP1 (Plakophilin proteins contain numerous armadillo repeats, localize to cell desmosomes and nuclei, and participate in linking cadherins to intermediate filaments in the cytoskeleton. This protein may be involved in molecular recruitment and stabilization during desmosome formation).* SEB caused by a *PLEC1(Plakins, with their multidomain structure and enormous size, not only play crucial roles in maintaining cell and tissue integrity and orchestrating dynamic changes in cytoarchitecture and cell shape, but also serve as scaffolding platforms for the assembly, positioning, and regulation of signaling complexes (reviewed in PMID: 9701547, 11854008, and 17499243)* mutation may show an autosomal recessive or autosomal dominant pattern of inheritance.

JEB is primarily caused by mutations in genes *LAMB3, LAMC2, LAMA3 (this genes is a laminin that belongs to a family of basement membrane proteins), COL17A1* (This gen encode *collagen XVII is a structural component of hemidesmosomes,*

*multiprotein complexes at the dermal-epidermal basement membrane zone that mediate adhesion of keratinocytes to the underlying membrane), ITGA*6 or *ITGB4* and is characterized by autosomal recessive inheritance. A recent study (2009) showed a possible autosomal dominant inheritance pattern in the case of a mutated *COL17A1*. DEB is caused by mutations in only one gene, *COLA1*. The location and type of mutation determine the inheritance pattern (autosomal recessive or dominant).

• Koebner type: mutated genes for keratin 5 and 4 (*KRT5*, *KRT4*); lesions are often present at birth or in infancy; characteristic features are hyperkeratotic

• Dowling-Meara type: mutated *KRT14* and *KRT5* genes, which encode keratin 14 and 5, respectively; autosomal dominant inheritance; lesions are located primarily on the feet, less commonly in other locations; a relatively mild

• Weber-Cockayne type: associated with mutated *KRT5* (region 12q13.13) and *KRT14* (region 17q21.2) genes; characterized by a severe course and

• SEB with muscular dystrophy: a mutated plectin-encoding *PLEC1* gene.

herpetiform blisters. Poorly healing blisters and erosions lead to scarring and

lesions, hemorrhagic bullae, and erosions.

*multiple transcript variants. [provided by RefSeq, Oct 2015]).*

*AD, autosomal dominant; AR, autosomal recessive; DDEB, dominant dystrophic epidermolysis bullosa; DEB; dystrophic epidermolysis bullosa; EB, epidermolysis bullosa; EM, electron microscopy; ER, endoplasmic reticulum; RDEB, recessive dystrophic epidermolysis bullosa; SCC, squamous cell carcinoma. 1Major type is DDEB. 2Previously known as transient bullous dermolysis of the newborn baby. 3Major type is RDEB. 4Major type is DEB (dominant and recessive compound heterotygosity). Adapted from consensus guidelines3.*

#### **Table 1.**

*Overview of EB classification [16].*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

#### **3.2 Heredity**

**Overview of EB classification**

RDEB, selfimproving3

**Table 1.**

**122**

*Overview of EB classification [16].*

Intermediate RDEB3 Phenotype similar to that of

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

striate keratoderma

with extensive scarring and development of microstomia, ankyloglossia, oesophageal stenosis, flexion contractures of limbs and pseudosyndactyly; nails are often lost early in disease course; high risk of cutaneous SCC arising in EB wounds.

Severe RDEB3 Widespread blistering from birth,

RDEB, inversa3 Generalized blistering from birth, of

flexural sites

RDEB, localized3 Skin fragility and blistering typically

DEB, severe4 Clinically indistinguishable from

None Generalized blistering and variable

*and recessive compound heterotygosity). Adapted from consensus guidelines3.*

*Kindler EB — variable and mixed*

severe RDEB, with severe mucocutaneous fragility from birth

photosensitivity from birth or early childhood, with mucosal fragility; blistering gives way to progressive poikiloderma, initially most marked over dorsal hands and neck; confluent palmoplantar keratoderma and adermatoglyphia may occur; gingivitis and dental disease is a feature; oesophageal narrowing and colitis has been reported; mucocutaneous SCC has been reported, with poor prognosis

*AD, autosomal dominant; AR, autosomal recessive; DDEB, dominant dystrophic epidermolysis bullosa; DEB; dystrophic epidermolysis bullosa; EB, epidermolysis bullosa; EM, electron microscopy; ER, endoplasmic reticulum; RDEB, recessive dystrophic epidermolysis bullosa; SCC, squamous cell carcinoma. 1Major type is DDEB. 2Previously known as transient bullous dermolysis of the newborn baby. 3Major type is RDEB. 4Major type is DEB (dominant*

**Subtype Phenotype Inheritance Gene affected**

AR *COL7A1*

AR *COL7A1*

AR *COL7A1*

AR *COL7A1*

Dominant and recessive compound heterozygosity

*AR FERMT1*

*COL7A1*

intermediate DDEB, although greater severity with flexion contractures, limited digital fusion and occasional

intermediate severity; subsequently, fragility tends to be displayed on

at birth or neonatal period, limited to acral sites such as hands and feet, or occasionally only to pretibial skin, where it may manifest as late-onset disease during adulthood; nail dystrophy and loss usual

RDEB, pruriginosa3 As for DDEB, pruriginosa AR *COL7A1*

As for DDEB, self-improving AR *COL7A1*

SEB primarily shows an autosomal dominant pattern of inheritance, with the most common mutations in genes *KRT5* and *KRT14*. Autosomal recessive inheritance is less common and caused by mutations in genes *KRT14*, *ITGA6*, *ITGB4* (*this genes encodes a member of the integrin alpha chain family of proteins. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain that function in cell surface adhesion and signaling. The encoded preproprotein is proteolytically processed to generate light and heavy chains that comprise the alpha 6 subunit. This subunit may associate with a beta 1 or beta 4 subunit to form an integrin that interacts with extracellular matrix proteins including members of the laminin family. The alpha 6 beta 4 integrin may promote tumorigenesis, while the alpha 6 beta 1 integrin may negatively regulate erbB2/HER2 signaling. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Oct 2015]).*

*DSP* (This gene encodes a protein that anchors intermediate filaments to desmosomal plaques and forms an obligate component of functional desmosomes), or *PKP1 (Plakophilin proteins contain numerous armadillo repeats, localize to cell desmosomes and nuclei, and participate in linking cadherins to intermediate filaments in the cytoskeleton. This protein may be involved in molecular recruitment and stabilization during desmosome formation).* SEB caused by a *PLEC1(Plakins, with their multidomain structure and enormous size, not only play crucial roles in maintaining cell and tissue integrity and orchestrating dynamic changes in cytoarchitecture and cell shape, but also serve as scaffolding platforms for the assembly, positioning, and regulation of signaling complexes (reviewed in PMID: 9701547, 11854008, and 17499243)* mutation may show an autosomal recessive or autosomal dominant pattern of inheritance.

JEB is primarily caused by mutations in genes *LAMB3, LAMC2, LAMA3 (this genes is a laminin that belongs to a family of basement membrane proteins), COL17A1* (This gen encode *collagen XVII is a structural component of hemidesmosomes, multiprotein complexes at the dermal-epidermal basement membrane zone that mediate adhesion of keratinocytes to the underlying membrane), ITGA*6 or *ITGB4* and is characterized by autosomal recessive inheritance. A recent study (2009) showed a possible autosomal dominant inheritance pattern in the case of a mutated *COL17A1*.

DEB is caused by mutations in only one gene, *COLA1*. The location and type of mutation determine the inheritance pattern (autosomal recessive or dominant).

#### **3.3 SEB subtypes**


### **3.4 JEB subtypes**

• JEB with pyloric atresia: this rare type of EB results from mutated *ITGB4* and *ITGA6* genes that encode α6 β4 integrin. Skin lesions are accompanied by esophageal, pyloric, and/or duodenal atresia. Enamel hypoplasia is common.

EB management involves primarily local care of chronic wounds, ulcers, erosions, and blisters. Treatment challenges involve frequent bacterial infections, due to their chronic character, and factors that inhibit healing, such as malnutrition, anemia, itching, or repetitive wound irritation with regular dressing changes, all of which disturb epithelialization. Moreover, wounds may cause severe pain, exacerbated by regular, frequent dressing changes. Importantly, the condition requires life-long care, with the cost of monthly treatment often exceeding several hundred dollars. Therefore, the process of selecting the optimal dressing should include the following parameters: the price, availability, effectiveness, and safety. Other important complementary treatments include physiotherapy, genetic counselling, aggressive treatment of infections, nutritional supplementation, and skin cancer

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

Despite the enormous advances in our understanding of molecular genetics and EB physiopathology that have taken place over the last several decades, a definitive cure is yet to be discovered. There are many ongoing studies aiming to develop an effective treatment. These studies focus on several potential lines of treatment, including disease modifying treatments to diminish disease severity. Gene therapies, bone marrow transplants, and tissue engineering are receiving the most

**Advanced therapy medicinal products (ATMPs**) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury. ATMPs can be classified into three main types: gene therapy medicines, somatic-cell therapy medicines, tissueengineered medicines. In addition, some ATMPs may contain one or more medical devices as an integral part of the medicine, which are referred to as combined ATMPs. An example of this is cells embedded in a biodegradable matrix or scaffold. **Gene therapy** involves cultures of keratinocytes (obtained from patients with

recessive DEB [RDEB]) that have been transduced with a retroviral vector

are promising treatment modalities with a potential therapeutic effect in

warnings for the use of ASCT in EB treatment.

**Bone marrow transplant (BMT) and allogenic stem cell transplantation (ASCT)** are other very promising treatment strategies. In 2010, Wagner et al. performed ASCT in children with RDEB. Although the patients were not completely cured, their skin blisters were reduced, and skin regeneration was accelerated. BMT in RDEB patients has been reported to improve the clinical status, despite the lack of collagen VII growth in the skin. BMT is an experimental therapy, which is used as part of clinical studies, and currently is not an approved treatment. The risk of death and the uncertain degree and mechanism of the clinical response should be viewed in light of the results of the most recent translational research in RDEB, which reports ASCT to be currently the only therapeutic approach that shows systemic effects in what essentially is a systemic disease. There is a clear need for reports presenting data from extensive clinical studies to establish guidelines and

As pluripotent cells, MSCs have a potential to differentiate into many different types of skin cells, including keratinocytes, endothelial cells, and monocytes. Due to

containing full-length cDNA of the *COL7A1* gene (for collagen VII). These cultures are, subsequently, placed onto the patient's wounds in the form of epidermal grafts [32]. Treatment efficacy and collagen VII expression were demonstrated; however, the response lasted up to 12 months. Nonetheless this therapy is safe. One disadvantage of this method is the fact that it can be used in limited areas (at chronic wound sites). This method has been also used in a patient with JEB, in whom the placement of genetically corrected keratinocytes onto chronic wound sites led to successful wound healing. Based on the available reports, gene therapies

monitoring [17–31].

attention.

genodermatoses.

**125**


#### **3.5 DEB subtypes**


#### **3.6 Diagnostic investigations**

A primary diagnosis of EB is based on the clinical presentation. The definitive diagnosis is established after skin samples are examined via immunofluorescence antigen mapping and transmission microscopy.

Diagnosis is confirmed via genetic analysis that determines the type of mutation.

#### **3.7 Differential diagnoses**

The differential diagnoses should include congenital dermatoses, herpes simplex virus infections, epidermolytic hyperkeratosis with erosions and blisters, staphylococcal scalded skin syndrome, bullous pemphigoid, neonatal pemphigoid, and gestational pemphigoid.

#### **3.8 Treatment**

Management is primarily symptomatic. Surgical treatment mainly involves skin grafting. Importantly, the use of autologous skin grafts is ineffective due to poor healing and chronic wound formation at the donor sites. Plastic surgery procedures play an important role in repairing contractures and pseudosyndactyly of the hands and feet. In the case of esophageal, pyloric or duodenal atresia, various surgical procedures are used to overcome the effects of gastrointestinal strictures (e.g. feeding jejunostomy, endoscopic balloon dilatation).

#### *Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

EB management involves primarily local care of chronic wounds, ulcers, erosions, and blisters. Treatment challenges involve frequent bacterial infections, due to their chronic character, and factors that inhibit healing, such as malnutrition, anemia, itching, or repetitive wound irritation with regular dressing changes, all of which disturb epithelialization. Moreover, wounds may cause severe pain, exacerbated by regular, frequent dressing changes. Importantly, the condition requires life-long care, with the cost of monthly treatment often exceeding several hundred dollars. Therefore, the process of selecting the optimal dressing should include the following parameters: the price, availability, effectiveness, and safety. Other important complementary treatments include physiotherapy, genetic counselling, aggressive treatment of infections, nutritional supplementation, and skin cancer monitoring [17–31].

Despite the enormous advances in our understanding of molecular genetics and EB physiopathology that have taken place over the last several decades, a definitive cure is yet to be discovered. There are many ongoing studies aiming to develop an effective treatment. These studies focus on several potential lines of treatment, including disease modifying treatments to diminish disease severity. Gene therapies, bone marrow transplants, and tissue engineering are receiving the most attention.

**Advanced therapy medicinal products (ATMPs**) are medicines for human use that are based on genes, tissues or cells. They offer groundbreaking new opportunities for the treatment of disease and injury. ATMPs can be classified into three main types: gene therapy medicines, somatic-cell therapy medicines, tissueengineered medicines. In addition, some ATMPs may contain one or more medical devices as an integral part of the medicine, which are referred to as combined ATMPs. An example of this is cells embedded in a biodegradable matrix or scaffold.

**Gene therapy** involves cultures of keratinocytes (obtained from patients with recessive DEB [RDEB]) that have been transduced with a retroviral vector containing full-length cDNA of the *COL7A1* gene (for collagen VII). These cultures are, subsequently, placed onto the patient's wounds in the form of epidermal grafts [32]. Treatment efficacy and collagen VII expression were demonstrated; however, the response lasted up to 12 months. Nonetheless this therapy is safe. One disadvantage of this method is the fact that it can be used in limited areas (at chronic wound sites). This method has been also used in a patient with JEB, in whom the placement of genetically corrected keratinocytes onto chronic wound sites led to successful wound healing. Based on the available reports, gene therapies are promising treatment modalities with a potential therapeutic effect in genodermatoses.

**Bone marrow transplant (BMT) and allogenic stem cell transplantation (ASCT)** are other very promising treatment strategies. In 2010, Wagner et al. performed ASCT in children with RDEB. Although the patients were not completely cured, their skin blisters were reduced, and skin regeneration was accelerated. BMT in RDEB patients has been reported to improve the clinical status, despite the lack of collagen VII growth in the skin. BMT is an experimental therapy, which is used as part of clinical studies, and currently is not an approved treatment. The risk of death and the uncertain degree and mechanism of the clinical response should be viewed in light of the results of the most recent translational research in RDEB, which reports ASCT to be currently the only therapeutic approach that shows systemic effects in what essentially is a systemic disease. There is a clear need for reports presenting data from extensive clinical studies to establish guidelines and warnings for the use of ASCT in EB treatment.

As pluripotent cells, MSCs have a potential to differentiate into many different types of skin cells, including keratinocytes, endothelial cells, and monocytes. Due to

**3.4 JEB subtypes**

**3.5 DEB subtypes**

are common.

**3.6 Diagnostic investigations**

**3.7 Differential diagnoses**

gestational pemphigoid.

**3.8 Treatment**

**124**

• JEB with pyloric atresia: this rare type of EB results from mutated *ITGB4* and *ITGA6* genes that encode α6 β4 integrin. Skin lesions are accompanied by esophageal, pyloric, and/or duodenal atresia. Enamel hypoplasia is common.

• Herlitz type: mutations in the *LAMA3*, *LAMB3*, and *LAMC2* genes, which encode the polypeptide subunits of laminin 5 (α-3, β-3, and γ-2, respectively). Fatal type of EB, characterized by blisters and erosions over the entire body, which causes multiple infections that may lead to sepsis, loss of proteins (malnourishment), scarring, contractures, defects of large areas of skin.

• Non-Herlitz type: mutations in genes *COL17A1*, *LAMB3*, *LAMC2*, or *LAMA3*

• Hallopeau-Siemens type: a mutated *COL7A1* gene, which encodes collagen VII. This type of EB is characterized by scarring, erosions, pseudosyndactyly of the hands and feet; nail plate involvement, esophageal atresia, and corneal ulcers

• non-Hallopeau-Siemens type: mutated *COL7A1* gene, encoding collagen VII.

• Cockayne-Toureine type: autosomal dominant inheritance; mutated *COL7A1*

A primary diagnosis of EB is based on the clinical presentation. The definitive diagnosis is established after skin samples are examined via immunofluorescence

Diagnosis is confirmed via genetic analysis that determines the type of mutation.

The differential diagnoses should include congenital dermatoses, herpes simplex virus infections, epidermolytic hyperkeratosis with erosions and blisters, staphylococcal scalded skin syndrome, bullous pemphigoid, neonatal pemphigoid, and

Management is primarily symptomatic. Surgical treatment mainly involves skin grafting. Importantly, the use of autologous skin grafts is ineffective due to poor healing and chronic wound formation at the donor sites. Plastic surgery procedures play an important role in repairing contractures and pseudosyndactyly of the hands and feet. In the case of esophageal, pyloric or duodenal atresia, various surgical procedures are used to overcome the effects of gastrointestinal strictures (e.g.

• Pasini type: possible nail plate involvement; oral and mucosal lesions.

encoding laminin 5 and collagen XVII.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

(collagen VII); skin lesion on the limbs.

antigen mapping and transmission microscopy.

feeding jejunostomy, endoscopic balloon dilatation).

their immunomodulatory and anti-inflammatory effects, MSCs may play a significant role in wound healing and tissue regeneration. Moreover, MSCs do not trigger an immune response in the recipient, hence there is no need to match the donor's and recipient's human leukocyte antigen (HLA) types [33]. Due to their multidirectional differentiation potential, MSCs have been shown to regenerate collagen VII, which has a beneficial effect on the healing of wounds (including chronic wounds) and improves skin stability. These effects were observed with intradermal administration, which—apart from presenting fewer challenges—does not require as many MSCs as intravenous administration. Most studies have focused on bone marrow-derived MSCs (BM-MSCs). However, their harvesting from the bone marrow is a relatively invasive procedure. Moreover, the multipotent differentiation potential of BM-MSCs diminishes with age. Therefore, MSCs are currently obtained from alternative sources, such as the umbilical cord [34], which can provide up to a billion cells in 30 days, obtained non-invasively. The umbilical cord consists of umbilical vessels surrounded by a connective tissue, referred to as Wharton jelly (WJ). WJ-derived MSCs have a higher proliferative potential and are more homogeneous than those derived from the bone marrow. WJ-MSCs are similar to BM-MSCs in their fibroblast-like phenotype, non-hematopoietic surface markers [35], low immunogenicity [36], multipotent plasticity, and the expression of CD90, CD73, CD105 markers [37]. Moreover, WJ-MSCs seem to have more pronounced pro-angiogenic properties than BM-MSCs; they promote neovascularization and perfusion by releasing paracrine factors and by playing the role of perivascular precursor cells [38]. WJ-MSCs are a highly efficient source of young, noncarcinogenic, and non-immunomodulatory cells [39]. All these properties and the fact that WJ-MSCs are easily available make these cells a promising strategy for treating wounds in EB patients.

vitro-generated skin substitutes. They are available in various forms, which include epidermal, dermal, and dermo-epidermal analogs or complex skin analogs, and can

High-quality, safe skin analogs should be cost-effective, biocompatible, biodegradable, and noncarcinogenic, carry no risk of infectious disease transfer, and provoke no activation of the recipient's immune system. Despite a whole spectrum of bioengineered products currently available on the market, there are scarcely any that meet all the requirements of natural skin. Natural skin is composed of the epidermis, dermis, and subcutaneous tissue. It contains appendages, such as sweat glands, nails, and hair, as well as nerve endings and blood vessels. Additionally, natural skin protects the body against the external environment via its thermoregulatory function and its role in maintaining water–electrolyte balance. It also facilitates the perception of pain, heat, and touch; manufactures vitamin D; and shields the body against ultraviolet radiation by the means of melanin-producing melanocytes responsible for skin pigmentation [52, 53]. Due to the wide range of functions performed by human skin, creating its analog is a challenge for tissue

The first product that has transferred the potential of bioengineering into reallife EB applications is an autologous cultured epidermal substitute (CES). The pioneering study by Rheinwald and Green demonstrated that epidermal

keratinocytes from a single-cell suspension can be cultured in the form of sheets, and the resulting multi-layered sheets have proven to be very effective in the treatment of burns and wounds in EB patients. There are many commercially available skin substitutes composed of both epidermal and dermal components. Bell et al. developed a cultured skin substitute (CSS) (an equivalent of living skin) composed of keratinocytes and fibroblasts in a collagen gel. Boyce and Hansbrough developed a CSS composed of a collagen-glycosaminoglycan composite scaffold populated with keratinocytes and fibroblasts. Kuroyanagi et al. developed another CSS, composed of a spongy collagen matrix with keratinocytes and fibroblasts. Such two-layered CSSs are intended to permanently cover full-thickness skin defects. There have been studies on wound healing in EB with the use of OrCelTM, Biobrane, and Apligraf dressings. OrCelTM is a bilayer dressing composed of a bovine-collagen I matrix populated with neonatal foreskin keratinocytes and fibroblasts [54–56]. Despite the fact that OrCelTM exhibits beneficial wound healing properties in RDEB patients—via cytokines and growth factors, such as tumor growth factor alpha (TGFα), fibroblast growth factor 1 (FGF-1), and keratinocyte growth factor 1 (KGF-1)—its bovine collagen component increases the risk of graft rejection and transfer of diseases to the donor [57]. Another bilayer skin substitute is Biobrane, which is composed of a 3D nylon fiber scaffold and an ultrathin semipermeable epidermis-mimicking silicone layer that controls fluid loss [56–59].

The nylon fibers are surrounded by porcine collagen type 1. Jutkiewicz and

Noszczyk [60] were the first to report the use of Biobrane in the postoperative hand care in a group of RDEB patients. Apligraf is another bilayer skin substitute composed of dermal and epidermal analogs. The epidermal and dermal layers contain cultured keratinocytes and neonatal foreskin fibroblasts. The dermal layer additionally contains bovine collagen type 1, which facilitates cell growth and differentiation. Apligraf has a short life span, and its use is associated with high costs [57]. Nonetheless, this dressing was reported to be effective in treating EB wounds

Safe and Effective Therapy in the Light of Clinical Trials - New Approach to Treatment by Innovative Method (BIOOPA-ATMP) grant no. STRATERMED2/

be composed of cellular or acellular scaffolds [42–51].

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

engineers.

[61, 62].

**127**

269807/14/NCBR/2015.

Sebastiano et al. propose an innovating cell therapy for RDEB treatment, by developing a state of the art protocol of genetically repaired induced pluripotent stem cells (iPSCs) as to generate sheets of normal skin tissue to treat affected skin areas [40]. Moreover, as numerous stem cells are needed in order to cover the affected surface area, authors outline the necessity for creating personalized iPSCs banks as to provide a constant long-term iPSCs source. Generally, human iPSCs can be generated by reprogramming differentiated somatic cells into pluripotent embryonic stem cells (ESCs) capable of differentiating into ectoderm, mesoderm or endoderm cells. Reprogramming involves the introduction of a known set of genes into the somatic cells, using integrating viral and non-integrating non-viral methods. Following successful reprogramming, somatic cells will express genes and surface proteins similar to ESCs in vitro and will be able to differentiate into any of the three embryonic germ layers.

**Tissue engineering**: Not unlike patients with extensive burns, patients with EB do not qualify for autologous skin grafts. One solution available to these patients involves the use of allogeneic grafts, which serve to temporarily cover the wound (after 7 days the graft is rejected by the recipient; [41]). Therefore, tissue engineering seems to be a promising solution, as it helps create biopolymer scaffolds to cover the wounds. The idea is to create skin substitutes, which can then be seeded with keratinocytes, fibroblasts, or stem cells. Such polymer materials constitute a microenvironment and provide adequate scaffolds for cell colonization and epithelial cell migration during wound epithelialization. The multi-disciplinary nature of tissue engineering has helped develop many bioengineered skin substitutes, with potential applications as a suitable dressing for treating refractory wounds, such as those in EB patients. The field of tissue engineering has been rapidly transferring from the realm of basic research to commercial applications. There are many in

#### *Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

vitro-generated skin substitutes. They are available in various forms, which include epidermal, dermal, and dermo-epidermal analogs or complex skin analogs, and can be composed of cellular or acellular scaffolds [42–51].

High-quality, safe skin analogs should be cost-effective, biocompatible, biodegradable, and noncarcinogenic, carry no risk of infectious disease transfer, and provoke no activation of the recipient's immune system. Despite a whole spectrum of bioengineered products currently available on the market, there are scarcely any that meet all the requirements of natural skin. Natural skin is composed of the epidermis, dermis, and subcutaneous tissue. It contains appendages, such as sweat glands, nails, and hair, as well as nerve endings and blood vessels. Additionally, natural skin protects the body against the external environment via its thermoregulatory function and its role in maintaining water–electrolyte balance. It also facilitates the perception of pain, heat, and touch; manufactures vitamin D; and shields the body against ultraviolet radiation by the means of melanin-producing melanocytes responsible for skin pigmentation [52, 53]. Due to the wide range of functions performed by human skin, creating its analog is a challenge for tissue engineers.

The first product that has transferred the potential of bioengineering into reallife EB applications is an autologous cultured epidermal substitute (CES). The pioneering study by Rheinwald and Green demonstrated that epidermal keratinocytes from a single-cell suspension can be cultured in the form of sheets, and the resulting multi-layered sheets have proven to be very effective in the treatment of burns and wounds in EB patients. There are many commercially available skin substitutes composed of both epidermal and dermal components. Bell et al. developed a cultured skin substitute (CSS) (an equivalent of living skin) composed of keratinocytes and fibroblasts in a collagen gel. Boyce and Hansbrough developed a CSS composed of a collagen-glycosaminoglycan composite scaffold populated with keratinocytes and fibroblasts. Kuroyanagi et al. developed another CSS, composed of a spongy collagen matrix with keratinocytes and fibroblasts. Such two-layered CSSs are intended to permanently cover full-thickness skin defects. There have been studies on wound healing in EB with the use of OrCelTM, Biobrane, and Apligraf dressings. OrCelTM is a bilayer dressing composed of a bovine-collagen I matrix populated with neonatal foreskin keratinocytes and fibroblasts [54–56]. Despite the fact that OrCelTM exhibits beneficial wound healing properties in RDEB patients—via cytokines and growth factors, such as tumor growth factor alpha (TGFα), fibroblast growth factor 1 (FGF-1), and keratinocyte growth factor 1 (KGF-1)—its bovine collagen component increases the risk of graft rejection and transfer of diseases to the donor [57]. Another bilayer skin substitute is Biobrane, which is composed of a 3D nylon fiber scaffold and an ultrathin semipermeable epidermis-mimicking silicone layer that controls fluid loss [56–59]. The nylon fibers are surrounded by porcine collagen type 1. Jutkiewicz and Noszczyk [60] were the first to report the use of Biobrane in the postoperative hand care in a group of RDEB patients. Apligraf is another bilayer skin substitute composed of dermal and epidermal analogs. The epidermal and dermal layers contain cultured keratinocytes and neonatal foreskin fibroblasts. The dermal layer additionally contains bovine collagen type 1, which facilitates cell growth and differentiation. Apligraf has a short life span, and its use is associated with high costs [57]. Nonetheless, this dressing was reported to be effective in treating EB wounds [61, 62].

Safe and Effective Therapy in the Light of Clinical Trials - New Approach to Treatment by Innovative Method (BIOOPA-ATMP) grant no. STRATERMED2/ 269807/14/NCBR/2015.

their immunomodulatory and anti-inflammatory effects, MSCs may play a significant role in wound healing and tissue regeneration. Moreover, MSCs do not trigger an immune response in the recipient, hence there is no need to match the donor's and recipient's human leukocyte antigen (HLA) types [33]. Due to their multidirectional differentiation potential, MSCs have been shown to regenerate collagen VII, which has a beneficial effect on the healing of wounds (including chronic wounds) and improves skin stability. These effects were observed with intradermal administration, which—apart from presenting fewer challenges—does not require as many MSCs as intravenous administration. Most studies have focused on bone marrow-derived MSCs (BM-MSCs). However, their harvesting from the bone marrow is a relatively invasive procedure. Moreover, the multipotent differentiation potential of BM-MSCs diminishes with age. Therefore, MSCs are currently obtained from alternative sources, such as the umbilical cord [34], which can provide up to a billion cells in 30 days, obtained non-invasively. The umbilical cord consists of umbilical vessels surrounded by a connective tissue, referred to as Wharton jelly (WJ). WJ-derived MSCs have a higher proliferative potential and are more homogeneous than those derived from the bone marrow. WJ-MSCs are similar to BM-MSCs in their fibroblast-like phenotype, non-hematopoietic surface markers [35], low immunogenicity [36], multipotent plasticity, and the expression of CD90, CD73, CD105 markers [37]. Moreover, WJ-MSCs seem to have more pronounced pro-angiogenic properties than BM-MSCs; they promote neovascularization and perfusion by releasing paracrine factors and by playing the role of perivascular precursor cells [38]. WJ-MSCs are a highly efficient source of young, noncarcinogenic, and non-immunomodulatory cells [39]. All these properties and the fact that WJ-MSCs are easily available make these cells a promising strategy for

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Sebastiano et al. propose an innovating cell therapy for RDEB treatment, by developing a state of the art protocol of genetically repaired induced pluripotent stem cells (iPSCs) as to generate sheets of normal skin tissue to treat affected skin areas [40]. Moreover, as numerous stem cells are needed in order to cover the affected surface area, authors outline the necessity for creating personalized iPSCs banks as to provide a constant long-term iPSCs source. Generally, human iPSCs can be generated by reprogramming differentiated somatic cells into pluripotent embryonic stem cells (ESCs) capable of differentiating into ectoderm, mesoderm or endoderm cells. Reprogramming involves the introduction of a known set of genes

into the somatic cells, using integrating viral and non-integrating non-viral

methods. Following successful reprogramming, somatic cells will express genes and surface proteins similar to ESCs in vitro and will be able to differentiate into any of

**Tissue engineering**: Not unlike patients with extensive burns, patients with EB do not qualify for autologous skin grafts. One solution available to these patients involves the use of allogeneic grafts, which serve to temporarily cover the wound (after 7 days the graft is rejected by the recipient; [41]). Therefore, tissue engineering seems to be a promising solution, as it helps create biopolymer scaffolds to cover the wounds. The idea is to create skin substitutes, which can then be seeded with keratinocytes, fibroblasts, or stem cells. Such polymer materials constitute a microenvironment and provide adequate scaffolds for cell colonization and epithelial cell migration during wound epithelialization. The multi-disciplinary nature of tissue engineering has helped develop many bioengineered skin substitutes, with potential applications as a suitable dressing for treating refractory wounds, such as those in EB patients. The field of tissue engineering has been rapidly transferring from the realm of basic research to commercial applications. There are many in

treating wounds in EB patients.

the three embryonic germ layers.

**126**

Alternative promising product for the treatment of chronic wounds that occur in EB and other genodermatoses, as well as in burns, is an allogeneic, acellular human skin equivalent sterilized with radiation, and seeded with Wharton's jelly-derived mesenchymal stem cells- WJ-MSCs about the acronym in polish BIOOPA (biological dressing) is an advanced therapy medicinal product composed of a decellularized matrix of the superficial layers of cadaveric human skin (10 cm 10 cm). Acellular dermal matrix (ADM) is a Chemically/enzymatically processed allograft. This processing removes all epidermal and dermal cells while preserving the molecular and physiological structure of collagen fibers. The scaffold is sterilized via radiation and then seeded with 30 million WJ-MSCs. As a result of decellularization, this skin substitute does not induce an immune response in the recipient and poses a lower risk of transmitting any diseases. In order to assess the safety and efficacy of the BIOOPA dressing, the relevant study was conducted in two stages. During the first stage, in vitro experiments showed BIOOPA viability. All examination techniques demonstrated graft infiltration by host cells and neovascularization of the biological dressing. Moreover, BIOOPA is characterized by low immunogenicity, which was confirmed in histopathology examinations and in vitro T-cell proliferation tests. The second stage of the study was conducted in a group of qualified volunteers with EB and approved by an ethics committee. The 6 month follow-up indicates the safety and efficacy of the BIOOPA dressing, with no infections or necrosis at the graft implantation site observed over the follow-up period. The subjects reported decreased pain and improved quality of life **Figures 3**–**8** [63, 64].

**Figure 4.**

**Figure 5.**

**129**

*(chronic wound in the knee area).*

*EB (allogenic,acellular, human skin equivalent).*

*Day 0, procedure: chronic wound in the knee area covered with prepared graft in the 20-years old patient with*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

*Bioopa dressing: The scaffold is seeded with 30 million WJ-MSCs in 5 mL of a 5% human albumin solution covered with chlorhexidine-impregnated dressings and collagen gel. The same 20-years old patient with EB*

**Figure 3.** *BIOOPA- Advanced Therapy Medicinal Product (ATMP) acellular human skin equivalent sterilized with ultraviolet radiation.*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

#### **Figure 4.**

Alternative promising product for the treatment of chronic wounds that occur in EB and other genodermatoses, as well as in burns, is an allogeneic, acellular human skin equivalent sterilized with radiation, and seeded with Wharton's jelly-derived mesenchymal stem cells- WJ-MSCs about the acronym in polish BIOOPA (biologi-

(10 cm 10 cm). Acellular dermal matrix (ADM) is a Chemically/enzymatically processed allograft. This processing removes all epidermal and dermal cells while preserving the molecular and physiological structure of collagen fibers. The scaffold is sterilized via radiation and then seeded with 30 million WJ-MSCs. As a result of decellularization, this skin substitute does not induce an immune response in the recipient and poses a lower risk of transmitting any diseases. In order to assess the safety and efficacy of the BIOOPA dressing, the relevant study was conducted in two stages. During the first stage, in vitro experiments showed BIOOPA viability. All examination techniques demonstrated graft infiltration by host cells and neovascularization of the biological dressing. Moreover, BIOOPA is characterized by low immunogenicity, which was confirmed in histopathology examinations and in vitro T-cell proliferation tests. The second stage of the study was conducted in a group of qualified volunteers with EB and approved by an ethics committee. The 6 month follow-up indicates the safety and efficacy of the BIOOPA dressing, with no infections or necrosis at the graft implantation site observed over the follow-up period. The subjects reported decreased pain and improved quality of life

*BIOOPA- Advanced Therapy Medicinal Product (ATMP) acellular human skin equivalent sterilized with*

cal dressing) is an advanced therapy medicinal product composed of a decellularized matrix of the superficial layers of cadaveric human skin

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

**Figures 3**–**8** [63, 64].

**Figure 3.**

**128**

*ultraviolet radiation.*

*Day 0, procedure: chronic wound in the knee area covered with prepared graft in the 20-years old patient with EB (allogenic,acellular, human skin equivalent).*

#### **Figure 5.**

*Bioopa dressing: The scaffold is seeded with 30 million WJ-MSCs in 5 mL of a 5% human albumin solution covered with chlorhexidine-impregnated dressings and collagen gel. The same 20-years old patient with EB (chronic wound in the knee area).*

#### **Figure 6.**

*Results after 30-day follow-up in this patient with EB: All examination techniques revealed host-cell infiltration and neovascularization of the biological dressing. They are characterized by low immunogenicity, as confirmed by histopathology and in vitro T-cell proliferation assays.*

**4. Conclusion**

**Figure 8.**

*50 mm).*

[66–77].

**131**

To date, there is no causative treatment of EB, despite multiple ongoing studies

*Laser scanning confocal microscopic study using second-harmonic generation technique reveals the structure of collagen fibrils in acellular dermal matrix after decellularization and X-ray radiation 35 kG (Bar 1/4*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

The BiOOPA dressing is easily available, safe, and relatively inexpensive, all of which make it a promising therapy for EB-associated wounds. Preliminary results of the BIOOPA study indicate the dressing to be safe and effective to improve the quality of life in study subjects. Currently BIOOPA is evaluated as part of a phase

involving gene therapy and bone marrow transplantation. The standard of EB management still involves symptomatic conservative treatment. There is immense hope in therapies with the use of stem cells of various origins (bone marrow, umbilical cord, etc.). Advanced applications of various types of cells (embryonic, prenatal, and adult stem cells, endothelial cells, and melanocytes) and the rapid development of biomedical engineering, which contributes to refining biocompatible materials, such as collagen, hyaluronic acid, elastin, polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), and polyethylene glycol (PEG), bring hope of effective treatment for chronic wounds of various origin. The most recent developments allow for the manufacture of progressively better skin substitutes, which in the future may exhibit the fundamental characteristics of natural human skin (including sweat glands and hair follicles), more homogeneous pigmentation, and allow for the healing of scars [65]. Thus, further studies and efforts are crucial for creating skin substitutes truly mimicking natural skin. Despite the enormous progress in the treatment of EB, the current treatments are clearly not a definitive cure for this debilitating disease, and the risk associated with some of these procedures must be weighed against their potential benefits. Effective treatment of this, currently incurable, group of diseases requires advanced and innovative strategies with an improved safety profile, such as the ones that are currently being developed

#### **Figure 7.**

*Hematoxylin and eosin stain of scaffold populated with mesenchymal cells from Wharton's jelly. After 72 hours of culture mesenchymal stem cells create a multilayer structure on the scaffold resembling human epithelium.*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

#### **Figure 8.**

*Laser scanning confocal microscopic study using second-harmonic generation technique reveals the structure of collagen fibrils in acellular dermal matrix after decellularization and X-ray radiation 35 kG (Bar 1/4 50 mm).*

#### **4. Conclusion**

To date, there is no causative treatment of EB, despite multiple ongoing studies involving gene therapy and bone marrow transplantation. The standard of EB management still involves symptomatic conservative treatment. There is immense hope in therapies with the use of stem cells of various origins (bone marrow, umbilical cord, etc.). Advanced applications of various types of cells (embryonic, prenatal, and adult stem cells, endothelial cells, and melanocytes) and the rapid development of biomedical engineering, which contributes to refining biocompatible materials, such as collagen, hyaluronic acid, elastin, polylactic acid (PLA), poly lactic-co-glycolic acid (PLGA), and polyethylene glycol (PEG), bring hope of effective treatment for chronic wounds of various origin. The most recent developments allow for the manufacture of progressively better skin substitutes, which in the future may exhibit the fundamental characteristics of natural human skin (including sweat glands and hair follicles), more homogeneous pigmentation, and allow for the healing of scars [65]. Thus, further studies and efforts are crucial for creating skin substitutes truly mimicking natural skin. Despite the enormous progress in the treatment of EB, the current treatments are clearly not a definitive cure for this debilitating disease, and the risk associated with some of these procedures must be weighed against their potential benefits. Effective treatment of this, currently incurable, group of diseases requires advanced and innovative strategies with an improved safety profile, such as the ones that are currently being developed [66–77].

The BiOOPA dressing is easily available, safe, and relatively inexpensive, all of which make it a promising therapy for EB-associated wounds. Preliminary results of the BIOOPA study indicate the dressing to be safe and effective to improve the quality of life in study subjects. Currently BIOOPA is evaluated as part of a phase

**Figure 7.**

**130**

**Figure 6.**

*Hematoxylin and eosin stain of scaffold populated with mesenchymal cells from Wharton's jelly. After 72 hours of culture mesenchymal stem cells create a multilayer structure on the scaffold resembling human epithelium.*

*Results after 30-day follow-up in this patient with EB: All examination techniques revealed host-cell infiltration and neovascularization of the biological dressing. They are characterized by low immunogenicity, as*

*confirmed by histopathology and in vitro T-cell proliferation assays.*

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

I/II clinical study during the second year of observation. Our preliminary results of clinical trial strongly suggest, that our innovative dressing is a promising strategy and a tool for clinicians in the search for new opportunities of treatment for this rare condition.

### **Acknowledgements**

This is unpublished Original Article.The study has been approved by an ethics committee (KB/ 2019 14/01/2019; KB/177/2015). Supported by The National Center for Research and Development in terms of the project "Prevention Practices and Treatment of Civilizations Diseases-STRATEGMED" (grant no. STRATERMED2/ 269807/14/NCBR/2015).

**Author details**

Magdalena Nita<sup>1</sup>

Tomasz Ołdak<sup>4</sup>

Artur Kamiński<sup>7</sup>

\*

Białystok, Białystok, Poland

Warsaw, Warsaw, Poland

Warsaw, Warsaw, Poland

Warsaw, Poland

**133**

Piotr Fiedor<sup>1</sup>

, Jacek Pliszczyński<sup>1</sup>

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

, Katarzyna Woźniak<sup>5</sup>

, Dariusz Śladowski<sup>7</sup>

Warsaw, Infant Jesus Clinical Hospital, Warsaw, Poland

University of Białystok, Białystok, Poland

4 PBKM, Polish Stem Cells Bank, Warsaw, Poland

\*Address all correspondence to: piotrfiedor@wp.pl

provided the original work is properly cited.

, Andrzej Eljaszewicz<sup>2</sup>

, Sławomir Majewski<sup>6</sup>

, Zbigniew Zimek8

1 Department of General and Transplantation Surgery at Medical University of

2 Department of Regenerative Medicine and Immune Regulation at Medical

3 Department of Allergology and Internal Medicine at Medical University of

5 Department of Dermatology and Immunodermatology at Medical University of

6 Department of Dermatology and Venereology at Medical University of Warsaw,

7 Department of Transplantology and Central Tissue Bank at Medical University of

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

8 Institute of Nuclear Chemistry and Technology, Warsaw, Poland

, Marcin Moniuszko2,3,

, Cezary Kowalewski<sup>5</sup>

, Maciej Kosieradzki<sup>1</sup> and

,

EudraCT Number: 2018-003890-91. Protocol number BIOOPA DBL.474.317.2020.

#### **Author details**

I/II clinical study during the second year of observation. Our preliminary results of clinical trial strongly suggest, that our innovative dressing is a promising strategy and a tool for clinicians in the search for new opportunities of treatment for this rare

This is unpublished Original Article.The study has been approved by an ethics committee (KB/ 2019 14/01/2019; KB/177/2015). Supported by The National Center for Research and Development in terms of the project "Prevention Practices and Treatment of Civilizations Diseases-STRATEGMED" (grant no. STRATERMED2/

condition.

**132**

**Acknowledgements**

269807/14/NCBR/2015).

EudraCT Number: 2018-003890-91.

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Protocol number BIOOPA DBL.474.317.2020.

Magdalena Nita<sup>1</sup> , Jacek Pliszczyński<sup>1</sup> , Andrzej Eljaszewicz<sup>2</sup> , Marcin Moniuszko2,3, Tomasz Ołdak<sup>4</sup> , Katarzyna Woźniak<sup>5</sup> , Sławomir Majewski<sup>6</sup> , Cezary Kowalewski<sup>5</sup> , Artur Kamiński<sup>7</sup> , Dariusz Śladowski<sup>7</sup> , Zbigniew Zimek8 , Maciej Kosieradzki<sup>1</sup> and Piotr Fiedor<sup>1</sup> \*

1 Department of General and Transplantation Surgery at Medical University of Warsaw, Infant Jesus Clinical Hospital, Warsaw, Poland

2 Department of Regenerative Medicine and Immune Regulation at Medical University of Białystok, Białystok, Poland

3 Department of Allergology and Internal Medicine at Medical University of Białystok, Białystok, Poland

4 PBKM, Polish Stem Cells Bank, Warsaw, Poland

5 Department of Dermatology and Immunodermatology at Medical University of Warsaw, Warsaw, Poland

6 Department of Dermatology and Venereology at Medical University of Warsaw, Warsaw, Poland

7 Department of Transplantology and Central Tissue Bank at Medical University of Warsaw, Warsaw, Poland

8 Institute of Nuclear Chemistry and Technology, Warsaw, Poland

\*Address all correspondence to: piotrfiedor@wp.pl

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Epidermolysis bullosa – pęcherzowe oddzielanie się naskórka. Etiopatogeneza, dziedziczenie, diagnostyka, leczenie.Katarzyna Wertheim-Tysarowska, Cezary Kowalewski, Katarzyna Woźniak, Jerzy Bal, Wyd. Continuo;

[2] Genetyczne uwarunkowanie choroby skóry-przegląd wybranych genodermatoz, Medycyna Wieku Rozwojowego, 2012, XVI, 3, © IMiD, Wydawnictwo Aluna.

[3] Tantcheva-Poór I, Oji V, Has C (2016) A multistep approach to the diagnosis of rare genodermatoses. J Dtsch Dermatol Ges 14: 969-986 2. www.genodermatoses-network.org

[4] Schaffer JV (2016) Practice and Educational Gaps in Genodermatoses. Dermatol Clin 34: 303-310

[5] Smith FJD, McLean WHI (2011) Genodermatoses: Inherited Deisease of the Skin, W: Murphy MJ (red) Molecular Diagnostics in Dermatology and Dermatopathology, Estados Unidos : Humana Press, str 379- 410

[6] Jabłońska S, Majewski S (2006) Budowa i czynności skóry W: Jabłońska S, Majewski S (red) Choroby skóry i choroby przenoszone drogą płciową PZWL, str 15-28

[7] Tsuruta D, Hashimoto T, Hamill KJ, Jones JC (2011) Hemidesmosomes and focal contact proteins: functions and cross-talk in keratinocytes, bullous diseases and wound healing. J Dermatol Sci 62: 1-7

[8] Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127: 2499-2515

[9] Coulombe PA, Kerns ML, Fuchs E (2009) Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility. J Clin Invest 119: 1784-1793

[10] Fine JD, General cutaneous manifestations (2009), W: Fine JD, Hintner H (red) Life with Epidermolysis Bullosa, SpringerWienNewYork, str 99-106

Epidermolysis bullosa. Nature Reviews Disease Primers, 6(1). doi:10.1038/

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

healing and inflammation in moist, wet, and dry environ- ments. Adv Wound Care (New Rochelle). 2013;2:348–356.

[25] Rakel BA, Bermel MA, Abbott LI, et al. Split-thickness skin graft donor site care: a quantitative synthesis of the research. Appl Nurs Res. 1998;11:174–

[26] Wiechula R. The use of moist wound-healing dressings in the management of split-thickness skin graft donor sites: a systematic review. Int J Nurs Pract. 2003;9:S9–17.

[27] Moher D, Shamseer L, Clarke M, et al.; PRISMA-P Group. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4:1.

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[29] Rees TD, Swinyard A. Rehabilitative digital surgery in epidermolysis bullosa. Plast Reconstr Surg 1967;40(2):169–74.

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dermolysis bullosa. Br J Plast Surg 1993;

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[32] Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Khuu, P., Furukawa, L. K., … Marinkovich, M. P. (2016). Safety and Wound Outcomes Following Genetically Corrected

With Recessive Dystrophic

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[17] Blount AL, Foster S, Rapp DA, et al. The use of bioelectric dressings in skin graft harvest sites: a prospective case series. J Burn Care Res. 2012;33:354–357.

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preserved wound covering gel. Eur Surg

[19] Hasatsri S, Angspatt A, Aramwit P.

Randomized clinical trial of the innovative bilayered wound dressing made of silk and gela- tin: safety and efficacy tests using a split-thickness skin graft mod- el. Evid Based Complement Alternat Med. 2015;2015:206871.

[20] Metelmann HR, Brandner J, Schumann H, et al. Accelerating the aesthetic benefit of wound healing by triterpene. J Craniomaxillofac Surg.

[21] Potocká D, Kevická D, Koller J. Clinical trial of the temporary biosynthetic dermal skin substitute based on a collagen and hy- aluronic acid named Coladerm H/HM, first part.

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[12] Fine JD, Other internal complications (2009), W: Fine JD, Hintner H (red) Life with Epidermolysis Bullosa, SpringerWienNewYork; str 185-196

[13] Fine JD, Bruckner-Tuderman L, Eady RA, Bauer EA, Bauer JW, Has C, Heagerty A, Hintner H, Hovnanian A, Jonkman MF, Leigh I, Marinkovich MP, Martinez AE, McGrath JA, Mellerio JE, Moss C, Murrell DF, Shimizu H, Uitto J, Woodley D, Zambruno G (2014). Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification. J Am Acad Dermatol 70: 1103-1126 14.

[14] Has C (2018) Advances in understanding the molecular basis of skin fragility. F1000Res 7: 279

[15] Wertheim-Tysarowska K, Ołdak M, Giza A, Kutkowska-Kaźmierczak A, Sota J, Przybylska D, Woźniak K, Śniegórska D, Niepokój K, Sobczyńska-Tomaszewska A, Rygiel AM, Płoski R, Bal J, Kowalewski C (2016) Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype-phenotype correlation. J Appl Genet 57: 175-81

[16] Bardhan, A., Bruckner-Tuderman, L., Chapple, I. L. C., Fine, J.-D., Harper, N., Has, C., … Heagerty, A. H. (2020).

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*Rare Diseases - Diagnostic and Therapeutic Odyssey*

[10] Fine JD, General cutaneous manifestations (2009), W: Fine JD, Hintner H (red) Life with Epidermolysis Bullosa, SpringerWienNewYork, str

Dermatol 60: 203-211

[12] Fine JD, Other internal

complications (2009), W: Fine JD, Hintner H (red) Life with Epidermolysis Bullosa, SpringerWienNewYork; str

[13] Fine JD, Bruckner-Tuderman L, Eady RA, Bauer EA, Bauer JW, Has C, Heagerty A, Hintner H, Hovnanian A, Jonkman MF, Leigh I, Marinkovich MP, Martinez AE, McGrath JA, Mellerio JE, Moss C, Murrell DF, Shimizu H, Uitto J, Woodley D, Zambruno G (2014). Inherited epidermolysis bullosa:

updated recommendations on diagnosis and classification. J Am Acad Dermatol

[15] Wertheim-Tysarowska K, Ołdak M, Giza A, Kutkowska-Kaźmierczak A, Sota J, Przybylska D, Woźniak K, Śniegórska D, Niepokój K, Sobczyńska-Tomaszewska A, Rygiel AM, Płoski R, Bal J, Kowalewski C (2016) Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype-phenotype correlation. J Appl

[16] Bardhan, A., Bruckner-Tuderman, L., Chapple, I. L. C., Fine, J.-D., Harper, N., Has, C., … Heagerty, A. H. (2020).

[14] Has C (2018) Advances in understanding the molecular basis of skin fragility. F1000Res 7: 279

[11] Fine JD, Johnson LB, Weiner M, Li KP, Suchindran C (2009) Epidermolysis bullosa and the risk of life-threatening cancers: the National EB Registry experience, 1986-2006 J Am Acad

99-106

185-196

70: 1103-1126 14.

Genet 57: 175-81

Kowalewski, Katarzyna Woźniak, Jerzy

[2] Genetyczne uwarunkowanie choroby

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[4] Schaffer JV (2016) Practice and Educational Gaps in Genodermatoses.

[5] Smith FJD, McLean WHI (2011) Genodermatoses: Inherited Deisease of

Molecular Diagnostics in Dermatology and Dermatopathology, Estados Unidos

[6] Jabłońska S, Majewski S (2006) Budowa i czynności skóry W: Jabłońska S, Majewski S (red) Choroby skóry i choroby przenoszone drogą płciową

[7] Tsuruta D, Hashimoto T, Hamill KJ, Jones JC (2011) Hemidesmosomes and focal contact proteins: functions and cross-talk in keratinocytes, bullous diseases and wound healing. J Dermatol

[8] Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127: 2499-2515

[9] Coulombe PA, Kerns ML, Fuchs E (2009) Epidermolysis bullosa simplex: a

paradigm for disorders of tissue fragility. J Clin Invest 119: 1784-1793

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: Humana Press, str 379- 410

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oddzielanie się naskórka. Etiopatogeneza, dziedziczenie, diagnostyka, leczenie.Katarzyna Wertheim-Tysarowska, Cezary

Bal, Wyd. Continuo;

Wydawnictwo Aluna.

skóry-przegląd wybranych genodermatoz, Medycyna Wieku Rozwojowego, 2012, XVI, 3, © IMiD, [17] Blount AL, Foster S, Rapp DA, et al. The use of bioelectric dressings in skin graft harvest sites: a prospective case series. J Burn Care Res. 2012;33:354–357.

[18] Goertz O, Abels C, Knie U, et al. Clinical safety and efficacy of a novel thermoreversible polyhexanidepreserved wound covering gel. Eur Surg Res. 2010;44:96–101.

[19] Hasatsri S, Angspatt A, Aramwit P. Randomized clinical trial of the innovative bilayered wound dressing made of silk and gela- tin: safety and efficacy tests using a split-thickness skin graft mod- el. Evid Based Complement Alternat Med. 2015;2015:206871.

[20] Metelmann HR, Brandner J, Schumann H, et al. Accelerating the aesthetic benefit of wound healing by triterpene. J Craniomaxillofac Surg. 2012;40:e150–e154.

[21] Potocká D, Kevická D, Koller J. Clinical trial of the temporary biosynthetic dermal skin substitute based on a collagen and hy- aluronic acid named Coladerm H/HM, first part. Acta Chir Plast. 2012;54:31–38.

[22] Baser NT, Yuksel A, Unzile B, et al. High-valve vapor-permeable film dressing versus fine mesh gauze dressing on skin graft donor areas in diabetic patients: a prospective randomized controlled trial. European J Plast Surg. 2008;31:219–228.

[23] Siritientong T, Angspatt A, Ratanavaraporn J, et al. Clinical potential of a silk sericin-releasing bioactive wound dressing for the treatment of split-thickness skin graft donor sites. Pharm Res. 2014;31: 104–116.

[24] Junker JP, Kamel RA, Caterson EJ, et al. Clinical impact upon wound

healing and inflammation in moist, wet, and dry environ- ments. Adv Wound Care (New Rochelle). 2013;2:348–356.

[25] Rakel BA, Bermel MA, Abbott LI, et al. Split-thickness skin graft donor site care: a quantitative synthesis of the research. Appl Nurs Res. 1998;11:174– 182.

[26] Wiechula R. The use of moist wound-healing dressings in the management of split-thickness skin graft donor sites: a systematic review. Int J Nurs Pract. 2003;9:S9–17.

[27] Moher D, Shamseer L, Clarke M, et al.; PRISMA-P Group. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015;4:1.

[28] Fine JD. Musculoskeletal deformities. In: Fine JD, Hintner H: Life with epidermolysis bullosa (EB): Etiology, diagnosis, multidisciplinary patient care, and therapy. Wien, NewYork: Springer, 2009: 178–84.

[29] Rees TD, Swinyard A. Rehabilitative digital surgery in epidermolysis bullosa. Plast Reconstr Surg 1967;40(2):169–74.

[30] Vozdivzhensky SI, Albanova VI. Surgical treatment of contracture and syndactyly of children with epidermolysis bullosa. Br J Plast Surg 1993; 46:314–6.

[31] Ciccarelli AO, Rothaus KO, Carter DM, et al. Plastic and reconstructive surgery in epidermolysis bullosa: clinical experience with 110 procedures in 25 patients. Ann Plast Surg 1995;35:254–61.

[32] Siprashvili, Z., Nguyen, N. T., Gorell, E. S., Loutit, K., Khuu, P., Furukawa, L. K., … Marinkovich, M. P. (2016). Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic

Epidermolysis Bullosa. JAMA, 316(17), 1808. doi:10.1001/jama.2016.15588

[33] Kühl, T., Mezger, M., Hausser, I., Guey, L. T., Handgretinger, R., Bruckner-Tuderman, L., & Nyström, A. (2016). Collagen VII Half-Life at the Dermal-Epidermal Junction Zone: Implications for Mechanisms and Therapy of Genodermatoses. Journal of Investigative Dermatology, 136(6), 1116–1123. doi:10.1016/j.jid.2016.02.002

[34] Nekanti, U., Rao, V. B., Bahirvani, A. G., Jan, M., Totey, S., & Ta, M. (2010). Long-Term Expansion and Pluripotent Marker Array Analysis of Wharton's Jelly-Derived Mesenchymal Stem Cells. Stem Cells and Development, 19(1), 117–130. doi: 10.1089/scd.2009.0177

[35] Karahuseyinoglu, S., Cinar, O., Kilic, E., Kara, F., Akay, G. G., Demiralp, D. Ö., … Can, A. (2007). Biology of Stem Cells in Human Umbilical Cord Stroma: In Situ and In Vitro Surveys. Stem Cells, 25(2), 319– 331. doi:10.1634/stemcells.2006-0286

[36] Tipnis, S., Viswanathan, C., & Majumdar, A. S. (2010). Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells: role of B7-H1 and IDO. Immunology and Cell Biology, 88(8), 795–806. doi:10.1038/icb.2010.47

[37] Shen, Y., Zhu, Y.-M., Fan, X., Shi, J.-Y., Wang, Q.-R., Yan, X.-J., … Chen, S.-J. (2011). Gene mutation patterns and their prognostic impact in a cohort of 1185 patients with acute myeloid leukemia. Blood, 118(20), 5593–5603. doi:10.1182/blood-2011-03-343988

[38] Choi, S.-E., & Kemper, J. K. (2013). Regulation of SIRT1 by MicroRNAs. Molecules and Cells, 36(5), 385–392. doi: 10.1007/s10059-013-0297-1.

[39] Nekanti, U., Mohanty, L., Venugopal, P., Balasubramanian, S., Totey, S., & Ta, M. (2010). Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications. Stem Cell Research, 5(3), 244–254. doi:10.1016/j. scr.2010.08.005

[48] MacNeil S (2007) Progress and opportunities for tissue- engineered

*DOI: http://dx.doi.org/10.5772/intechopen.97036*

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB)*

Expert Review of Dermatology, 6(3), 255–262. doi:10.1586/edm.11.27

[59] Varkey, M., Ding, J., & Tredget, E. (2015). Advances in Skin Substitutes— Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing. Journal of Functional Biomaterials, 6(3), 547–563. doi:10.3390/jfb6030547

[60] Jutkiewicz, J., Noszczyk, B. H., & Wrobel, M. (2010). The use of Biobrane for hand surgery in Epidermolysis

Reconstructive & Aesthetic Surgery, 63

[61] Fivenson DP, Scherschun L, Cohen LV. Apligraf in the treatment of severe mitten deformity associated with recessive dystrophic epidermolysis bullosa. Plast Reconstr Surg 2003;112

[62] Falabella, A. F., Valencia, I. C., Eaglstein, W. H., & Schachner, L. A. (2000). Tissue-Engineered Skin (Apligraf) in the Healing of Patients With Epidermolysis Bullosa Wounds. Archives of Dermatology, 136(10). doi:

10.1001/archderm.136.10.1225

[63] Nita, M., Pliszczyński, J., Kowalewski, C., Woźniak, K.,

[64] Pliszczyński, J., Nita, M., Kowalewski, C., Woźniak, K.,

Eljaszewicz, A., Moniuszko, M., Fiedor, P. (2020). New Treatment of Wound Healing With Allogenic Acellular Human Skin Graft: Preclinical Assessment and In Vitro Study. Transplantation Proceedings. doi: 10.1016/j.transproceed.2020.02.115

Eljaszewicz, A., Moniuszko, M., Fiedor, P. (2020). Transplantation of a New

bullosa. Journal of Plastic,

bjps.2009.06.038

(2):584–8.

(8), 1305–1311. doi:10.1016/j.

[58] Varkey M, Ding J, Tredget EE (2015a) Advances in skin substitutespotential of tissue engineered skin for facilitating antifibrotic healing. J Funct

Biomater 6 (3):547–563

[49] Böttcher-Haberzeth S, Klar AS, Biedermann T, Schiestl C, Meuli-Simmen C, Reichmann E, Meuli M (2013) "Trooping the color": restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs. Pediatr Surg Int29(3):239–247

skin. Nature 445(7130):874

[50] Biedermann T, Boettcher-Haberzeth S, Reichmann E (2013) Tissue engineering of skin for wound coverage. Eur J PediatrSurg 23(5):375–

[51] Lovett M, Lee K, Edwards A, Kaplan DL (2009) Vascularization strategies for tissue engineering. Tissue Eng B Rev 15

[52] Catalano E, Cochis A, Varoni E, Rimondini L, Azzimonti B (2013) Tissue-engineered skin substitutes: an over- view. J ArtifOrgans 16(4):397–403

[53] Damanhuri M, Boyle J, Enoch S (2011) Advances in tissue-engineered skin substitutes. Wounds Int 2 (1):27–34

[54] Supp, D. M. & Boyce, S. T. 2005 Engineered skin substitutes: practices and potentials. Clin. Dermatol. 23, 403–

[55] Halim AS, Khoo TL, Mohd Yussof SJ (2010a) Biologic and synthetic skin substitutes: An overview. Indian J Plast

[56] Halim AS, Khoo TL, Yussof SJM (2010b) Biologic and synthetic skin substitutes: an overview. Indian J Plastic

[57] Lepow, B. D., Downey, M.,

Yurgelon, J., Klassen, L., & Armstrong, D. G. (2011). Bioengineered tissues in wound healing: a progress report.

382

(3):353–370

98;51:608–13

412. (doi:10.1016/j.

clindermatol.2004.07.023)

Surg 43(Suppl):S23–S28

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[41] Chen, H., Wang, W., Xie, H., Xu, X., Wu, J., Jiang, Z., … Zheng, S. (2009). A pathogenic role of IL- 17 at the early stage of corneal allograft rejection. Transplant Immunology, 21 (3), 155–161. doi:10.1016/j. trim.2009.03.006

[42] Gough MJ, Page RE. Surgical correction of the hand in epidermolysis bullosa dystrophica. Hand 1979; 11(1): 55–8.

[43] Eastwood DS. Autografting in the treatment of squa- mous cell carcinoma in epidermolysis bullosa dys- trophica. Plast Reconstr Surg 1972;49(1):93–5.

[44] Eisenberg M, Llewelyn D. Surgical management of hands in children with recessive dystrophic epider- molysis bullosa: use of allogeneic composite cultured skin grafts. Br J Plast Surg 19 Damanhuri M, Boyle J, Enoch S (2011) Advances in tissue-engineered skin substitutes. Wounds Int 2 (1):27–34

[45] Nicholas MN, Yeung J (2017a) Current status and future of skin substitutes for chronic wound healing. J Cutan Med Surg 21(1):23–30

[46] Nicholas MN, Jeschke MG, Amini-Nik S (2016a) Cellularized bilayer pullulan-gelatin hydrogel for skin regeneration. Tissue Eng A 22(9–10): 754–764

[47] Sheikholeslam M, Wright ME, Jeschke MG, Amini-Nik S (2017) Biomaterials for Skin Substitutes. AdvHealthcare Mater

*Surgical Treatment of Wounds Using Stem Cells in Epidermolysis Bullosa (EB) DOI: http://dx.doi.org/10.5772/intechopen.97036*

[48] MacNeil S (2007) Progress and opportunities for tissue- engineered skin. Nature 445(7130):874

Epidermolysis Bullosa. JAMA, 316(17), 1808. doi:10.1001/jama.2016.15588

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

Totey, S., & Ta, M. (2010).

scr.2010.08.005

Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications. Stem Cell Research, 5(3), 244–254. doi:10.1016/j.

[40] Koster MI (2009) Making an epidermis. Ann N Y Acad Sci 1170: 7-10

(3), 155–161. doi:10.1016/j.

[42] Gough MJ, Page RE. Surgical correction of the hand in epidermolysis bullosa dystrophica. Hand 1979; 11(1):

[43] Eastwood DS. Autografting in the treatment of squa- mous cell carcinoma in epidermolysis bullosa dys- trophica. Plast Reconstr Surg 1972;49(1):93–5.

[44] Eisenberg M, Llewelyn D. Surgical management of hands in children with recessive dystrophic epider- molysis bullosa: use of allogeneic composite cultured skin grafts. Br J Plast Surg 19 Damanhuri M, Boyle J, Enoch S (2011) Advances in tissue-engineered skin substitutes. Wounds Int 2 (1):27–34

[45] Nicholas MN, Yeung J (2017a) Current status and future of skin substitutes for chronic wound healing. J

[46] Nicholas MN, Jeschke MG, Amini-Nik S (2016a) Cellularized bilayer pullulan-gelatin hydrogel for skin regeneration. Tissue Eng A 22(9–10):

[47] Sheikholeslam M, Wright ME, Jeschke MG, Amini-Nik S (2017) Biomaterials for Skin Substitutes.

AdvHealthcare Mater

Cutan Med Surg 21(1):23–30

754–764

trim.2009.03.006

55–8.

[41] Chen, H., Wang, W., Xie, H., Xu, X., Wu, J., Jiang, Z., … Zheng, S. (2009). A pathogenic role of IL- 17 at the early stage of corneal allograft rejection. Transplant Immunology, 21

[33] Kühl, T., Mezger, M., Hausser, I., Guey, L. T., Handgretinger, R.,

Bruckner-Tuderman, L., & Nyström, A. (2016). Collagen VII Half-Life at the Dermal-Epidermal Junction Zone: Implications for Mechanisms and Therapy of Genodermatoses. Journal of Investigative Dermatology, 136(6), 1116–1123. doi:10.1016/j.jid.2016.02.002

[34] Nekanti, U., Rao, V. B., Bahirvani, A. G., Jan, M., Totey, S., & Ta, M. (2010). Long-Term Expansion and Pluripotent Marker Array Analysis of Wharton's Jelly-Derived Mesenchymal

Stem Cells. Stem Cells and

10.1089/scd.2009.0177

Development, 19(1), 117–130. doi:

[35] Karahuseyinoglu, S., Cinar, O., Kilic, E., Kara, F., Akay, G. G., Demiralp, D. Ö., … Can, A. (2007). Biology of Stem Cells in Human Umbilical Cord Stroma: In Situ and In Vitro Surveys. Stem Cells, 25(2), 319– 331. doi:10.1634/stemcells.2006-0286

[36] Tipnis, S., Viswanathan, C., &

Immunosuppressive properties of human umbilical cord-derived

mesenchymal stem cells: role of B7-H1 and IDO. Immunology and Cell Biology, 88(8), 795–806. doi:10.1038/icb.2010.47

[37] Shen, Y., Zhu, Y.-M., Fan, X., Shi, J.-Y., Wang, Q.-R., Yan, X.-J., … Chen, S.-J. (2011). Gene mutation patterns and their prognostic impact in a cohort of 1185 patients with acute myeloid leukemia. Blood, 118(20), 5593–5603. doi:10.1182/blood-2011-03-343988

[38] Choi, S.-E., & Kemper, J. K. (2013). Regulation of SIRT1 by MicroRNAs. Molecules and Cells, 36(5), 385–392. doi:

10.1007/s10059-013-0297-1.

[39] Nekanti, U., Mohanty, L., Venugopal, P., Balasubramanian, S.,

**136**

Majumdar, A. S. (2010).

[49] Böttcher-Haberzeth S, Klar AS, Biedermann T, Schiestl C, Meuli-Simmen C, Reichmann E, Meuli M (2013) "Trooping the color": restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs. Pediatr Surg Int29(3):239–247

[50] Biedermann T, Boettcher-Haberzeth S, Reichmann E (2013) Tissue engineering of skin for wound coverage. Eur J PediatrSurg 23(5):375– 382

[51] Lovett M, Lee K, Edwards A, Kaplan DL (2009) Vascularization strategies for tissue engineering. Tissue Eng B Rev 15 (3):353–370

[52] Catalano E, Cochis A, Varoni E, Rimondini L, Azzimonti B (2013) Tissue-engineered skin substitutes: an over- view. J ArtifOrgans 16(4):397–403 98;51:608–13

[53] Damanhuri M, Boyle J, Enoch S (2011) Advances in tissue-engineered skin substitutes. Wounds Int 2 (1):27–34

[54] Supp, D. M. & Boyce, S. T. 2005 Engineered skin substitutes: practices and potentials. Clin. Dermatol. 23, 403– 412. (doi:10.1016/j. clindermatol.2004.07.023)

[55] Halim AS, Khoo TL, Mohd Yussof SJ (2010a) Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg 43(Suppl):S23–S28

[56] Halim AS, Khoo TL, Yussof SJM (2010b) Biologic and synthetic skin substitutes: an overview. Indian J Plastic Surg 43(Suppl):S23

[57] Lepow, B. D., Downey, M., Yurgelon, J., Klassen, L., & Armstrong, D. G. (2011). Bioengineered tissues in wound healing: a progress report.

Expert Review of Dermatology, 6(3), 255–262. doi:10.1586/edm.11.27

[58] Varkey M, Ding J, Tredget EE (2015a) Advances in skin substitutespotential of tissue engineered skin for facilitating antifibrotic healing. J Funct Biomater 6 (3):547–563

[59] Varkey, M., Ding, J., & Tredget, E. (2015). Advances in Skin Substitutes— Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing. Journal of Functional Biomaterials, 6(3), 547–563. doi:10.3390/jfb6030547

[60] Jutkiewicz, J., Noszczyk, B. H., & Wrobel, M. (2010). The use of Biobrane for hand surgery in Epidermolysis bullosa. Journal of Plastic, Reconstructive & Aesthetic Surgery, 63 (8), 1305–1311. doi:10.1016/j. bjps.2009.06.038

[61] Fivenson DP, Scherschun L, Cohen LV. Apligraf in the treatment of severe mitten deformity associated with recessive dystrophic epidermolysis bullosa. Plast Reconstr Surg 2003;112 (2):584–8.

[62] Falabella, A. F., Valencia, I. C., Eaglstein, W. H., & Schachner, L. A. (2000). Tissue-Engineered Skin (Apligraf) in the Healing of Patients With Epidermolysis Bullosa Wounds. Archives of Dermatology, 136(10). doi: 10.1001/archderm.136.10.1225

[63] Nita, M., Pliszczyński, J., Kowalewski, C., Woźniak, K., Eljaszewicz, A., Moniuszko, M., Fiedor, P. (2020). New Treatment of Wound Healing With Allogenic Acellular Human Skin Graft: Preclinical Assessment and In Vitro Study. Transplantation Proceedings. doi: 10.1016/j.transproceed.2020.02.115

[64] Pliszczyński, J., Nita, M., Kowalewski, C., Woźniak, K., Eljaszewicz, A., Moniuszko, M., Fiedor, P. (2020). Transplantation of a New

Biological Product in Rare Diseases, Such as Epidermolysis Bullosa: Response and Clinical Outcome. Transplantation Proceedings. doi:10.1016/j. transproceed.2020.02.119

[65] MacNeil S (2008) Biomaterials for tissue engineering of skin. Mater Today 11(5):26–35

[66] Conget P, Rodriguez F, Kramer S, et al. Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa. Cytotherapy 2010;12:429–31.

[67] Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001;166: 7556–62.

[68] Ito M, Liu Y, Yang Z, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med 2005;11: 1351–4.

[69] Leoni G, Neumann PA, Sumagin R, Denning TL, Nusrat A. Wound repair: role of immune-epithelial interactions. Mucosal Immunol 2015;8: 959–68.

[70] Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing. J Cell Physiol 2003;196:245–50.

[71] Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 2008;180:2581–7.

[72] Inokuma D, Abe R, Fujita Y, et al. CTACK/CCL27 accelerates skin regeneration via accumulation of bone marrow-derivedkeratinocytes. Stem Cells 2006;24:2810–6.

[73] Sebastiano V, Zhen HH, Haddad B, Bashkirova E, Melo SP, Wang P, Leung TL, Siprashvili Z, Tichy A, Li J, Ameen M, Hawkins J, Lee S, Li L, Schwertschkow A, Bauer G, Lisowski L, Kay MA, Kim SK, Lane AT, Wernig M, Oro AE; Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Sci Transl Med. 2014 Nov 26; 6(264):264ra163.

[74] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S, Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell. 2007 Nov 30; 131(5):861-72

[75] S Spiliopoulos, N Davans,Induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Ann Transl Med. 2015 Dec;3 (22):349. doi: 10.3978/j.issn.2305- 5839.2015.09.42.

[76] Horwitz EM, Prockop DJ, Gordon PL, Koo WW, Fitzpatrick LA, Neel MD, McCarville ME, Orchard PJ, Pyeritz RE, Brenner MK. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood. 2001 Mar 1;97(5):1227-31.

[77] Dominici M, Marino R, Rasini V, Spano C, Paolucci P, Conte P, Hofmann TJ,Horwitz EM. Donor cell-derived osteopoiesis originates from a selfrenewing stem cell with a limited regenerative contribution after transplantation. Blood. 2008 Apr 15;111 (8):4386-91. doi: 10.1182/blood-2007- 10-115725. Epub 2008 Jan 8.

Biological Product in Rare Diseases, Such as Epidermolysis Bullosa: Response and Clinical Outcome. Transplantation

*Rare Diseases - Diagnostic and Therapeutic Odyssey*

marrow-derivedkeratinocytes. Stem

[73] Sebastiano V, Zhen HH, Haddad B, Bashkirova E, Melo SP, Wang P, Leung TL, Siprashvili Z, Tichy A, Li J, Ameen

Schwertschkow A, Bauer G, Lisowski L, Kay MA, Kim SK, Lane AT, Wernig M, Oro AE; Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Sci Transl Med. 2014 Nov 26; 6(264):264ra163.

[74] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S, Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell. 2007 Nov 30;

[75] S Spiliopoulos, N Davans,Induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Ann Transl Med. 2015 Dec;3 (22):349. doi: 10.3978/j.issn.2305-

[76] Horwitz EM, Prockop DJ, Gordon PL, Koo WW, Fitzpatrick LA, Neel MD, McCarville ME, Orchard PJ, Pyeritz RE, Brenner MK. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood.

[77] Dominici M, Marino R, Rasini V, Spano C, Paolucci P, Conte P, Hofmann TJ,Horwitz EM. Donor cell-derived osteopoiesis originates from a selfrenewing stem cell with a limited regenerative contribution after

transplantation. Blood. 2008 Apr 15;111 (8):4386-91. doi: 10.1182/blood-2007-

Cells 2006;24:2810–6.

M, Hawkins J, Lee S, Li L,

131(5):861-72

5839.2015.09.42.

2001 Mar 1;97(5):1227-31.

10-115725. Epub 2008 Jan 8.

[65] MacNeil S (2008) Biomaterials for tissue engineering of skin. Mater Today

[66] Conget P, Rodriguez F, Kramer S, et al. Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa. Cytotherapy

[67] Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001;166:

[68] Ito M, Liu Y, Yang Z, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med 2005;11:

[69] Leoni G, Neumann PA, Sumagin R, Denning TL, Nusrat A. Wound repair: role of immune-epithelial interactions. Mucosal Immunol 2015;8: 959–68.

[70] Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation

cutaneous wound healing. J Cell Physiol

[71] Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 2008;180:2581–7.

[72] Inokuma D, Abe R, Fujita Y, et al. CTACK/CCL27 accelerates skin regeneration via accumulation of bone

of bone marrow derived cells in

2003;196:245–50.

**138**

Proceedings. doi:10.1016/j. transproceed.2020.02.119

11(5):26–35

2010;12:429–31.

7556–62.

1351–4.

### *Edited by Mani T. Valarmathi*

A rare disease is any disease or condition that affects a small percentage of the population. Many rare conditions are life-threatening or chronically debilitating, and unfortunately do not have appropriate treatments, rendering them incurable. In recent years, there has been substantial development in the area of rare disease research and its clinical applications, for instance, rare disease biology and genomics, epidemiology and preventions, early detection and screening, and diagnosis and treatment. In this context, this book consolidates the recent advances in rare disease biology and therapeutics, covering a wide spectrum of interrelated topics, and disseminates this essential knowledge in a comprehensible way to a greater scientific and clinical audience as well as patients, caregivers, and drug and device manufacturers, especially to support rare disease product development. Chapters cover such diseases as Felty's syndrome, Löfgren's syndrome, mesothelioma, epidermolysis bullosa, and more. This book is a valuable resource not only for medical and allied health students but also for researchers, clinical and nurse geneticists, genetic counselors, and physician assistants.

Published in London, UK © 2021 IntechOpen © CIPhotos / iStock

Rare Diseases - Diagnostic and Therapeutic Odyssey

Rare Diseases

Diagnostic and Therapeutic Odyssey

*Edited by Mani T. Valarmathi*