Histopathological Characteristics

#### **Chapter 3**

## Histological Hallmarks of Malignant Melanoma

*Gerardo Cazzato, Anna Colagrande, Lucia Lospalluti, Giuseppe Ingravallo, Eliano Cascardi, Miriam Dellino, Saverio Capodiferro, Eugenio Maiorano, Caterina Foti and Leonardo Resta*

#### **Abstract**

The histopathological diagnosis of malignant melanoma remains the gold standard to allow the patient to access the entire process of the diagnostic-therapeutic-assistance path. Despite the continuous search for markers that can assist in the diagnostic process, there are cases that remain complex to diagnose, and the presence of different criteria among dermatopathologists further complicates the issue. This section will focus on the state of the art of dermatopathological diagnostics of melanoma, starting from the morphological bases up to the latest acquisitions of immunohistochemistry for diagnostic purposes, and molecular biology for therapeutic purposes. Furthermore, we will focus on particularly "challenging" MM histotypes and on what are the current guidelines for a correct diagnosis.

**Keywords:** malignant melanoma, histopathology, skin, immunohistochemistry, diagnosis

#### **1. Introduction**

Malignant melanoma (MM) is known in the medical literature as the "Great Mime" of pathological anatomy, as it can simulate, in different ways, other neoplasms, both of an epithelial nature and of a mesenchymal nature [1]. From the point of view of the location, MM may originate de novo on healthy skin or represent the malignant transformation of a preexisting melanocytic nevus [2, 3]. In the vast majority of cases, MM represents a sporadic event, while in less than 10% it is linked to alterations of tumor suppressor genes (encoded by the chromosomal region 9p21) and shows a hereditary character, for this reason, it is defined as "familial" [4]. Histopathologically, the MM of the skin and/or mucous membranes consists of neoplastic melanocytic elements, fused or epithelioid, with the presence of atypia and, often, mitotic figures. An accurate histological diagnosis is the basis for a clinical management of the patient affected by MM since all the histopathological parameters reported in the report have important implications not only from a diagnostic but also from a prognostic point of view.

#### **2. Histological report of malignant melanoma**

In the first instance, it is very important to determine whether the lesion we are analyzing constitutes an MM and is properly differentiated from atypical pigmented lesions that can closely simulate MM [5]. Morphologically the cutaneous MM can be classified into four histological subtypes such as superficial spreading type, lentigo maligna, acral lentiginous, and nodular type. If at first we tended to think that this was a mere histological description, in fact, more recent studies have correlated (sometimes very exhaustively) histological subtypes with particular molecular signatures of MM. For example, you can easily appreciate from the last WHO blue book "Classification of Skin Tumour"2018, IARC, that melanomas on skin chronically exposed to the sun have different chromosomal aberration patterns than melanomas that arise on skin with intermittent exposure to ultraviolet (UV) rays or in areas of acral skin or mucous membranes. Therefore, a correct histological recognition of the MM subtype is of great importance and must always be reported in the pathological report of an MM [6].

**Figure 1** shows an example of MM with features consistent with "Spitzoid" MM. Note that the lesion is composed of nests of melanocytes with some mitotic figures; top left is possible to appreciate pagetoid spreading of single melanocytes (another useful clue to diagnosis of MM).

Thickness according to Breslow is the strongest and most reliable prognostic factor in MM and is defined as the measurement of the vertical thickness of the neoplasm [7]. The American Joint Committee on Cancer (AJCC) 8th Edition criteria for staging accurately predict sentinel lymph node positivity in clinically melanoma-negative lymph node patients. In fact, when grouped by AJCC cutting points, there was an increased incidence of positive sentinel lymph nodes with increasing tumor thickness: 4% in melanomas below 1.00 mm, 12% in melanomas 1.01–2.00 mm, 28% in melanomas 2.01–4.00 mm and 44% in melanomas over 4.00 mm [8]. But it is important to consider that there are, however, cases in which the thickness of Breslow does not impact perfectly on the prognosis: it is the case of thin melanomas that are able not only to metastasize but also to bring the subject to death with some ease [9]. By

#### **Figure 1.**

*Histological photomicrograph showing an example of MM (Haematoxylin-Eosin, Original Magnification 10×).*

#### *Histological Hallmarks of Malignant Melanoma DOI: http://dx.doi.org/10.5772/intechopen.106638*

convention, the thickness of Breslow is measured from the granulous layer to the last cell of MM, except in the case of ulcerated lesions, where the base of the ulceration is taken as a reference to the last point where the neoplasm is evident [10].

A parameter related to the thickness of Breslow is the Clark level, which, unlike the first, is a topographical criterion, which is based simply on the definition of which area of skin is affected by MM: superficial (or papillary) dermis, reticular, and subcutaneous. In recent years, the low reproducibility and repeatability of this parameter, due to its rather "subjective" nature of measurement, has in fact caused its disuse, until it is no longer considered mandatory to be included in a histopathological report of melanoma. However, we still consider it correct to include this finding in the MM report as an additional indicator parameter, never to be substituted for Breslow [11].

Another important element that can never be missing in the description of a melanoma is the number of mitoses/mmq. In fact, various studies have shown how the number of mitoses can be a rather reliable indicator of the biological behavior of a given MM (staging and prognosis). Although there is no universally accepted method for counting mitotic figures in melanoma, the AJCC 8′Edition recommends making this measurement in the "hot spot" areas of the lesion, so as to ensure a faithful count of the maximum number of mitoses present in Ref [12].

It is very important to consider one parameter on which there is still no precise agreement in the literature, which is the regression of melanoma. In fact, among the various theories, the most accredited considers the regression as a result of aggression of melanoma cells by the immune system with the substitution of the same with fibrosis, melanophages, lymphocytic infiltrate, and angiectasis. Regression phenomena can be from focal to extended until the complete disappearance of the primary lesion (so-called "burn-out melanoma"). The difficulty of reproducibility of this parameter consists in the variability of what is considered as regression: some studies have considered regression as the total absence of melanoma cells, whereas other studies have also considered more or less partial regression areas in the measurement of regression proper [13]. Regression is now considered to be an independent prognostic factor for worsening prognosis, and according to the College of American Pathologists (CAP) guidelines, regression is measured by a cutoff at 75% [14].

An integral part of a histological report of MM is the evaluation of ulceration, considered an independent prognostic factor for survival associated with melanoma. Ulceration is defined as a continuous "true" solution at full thickness, of the epidermis, with the presence of fibrin deposition or neutrophil granulocytes, and/ or reactive hyperplasia of the surrounding epidermis in the absence of trauma or surgical manipulation prior to or in progress. In recent years, more and more evidence supports the need to express the radial extent of ulceration (in mm), as it would seem that more extensive ulcerations correspond to reduced survival values. It should also be remembered that it is necessary to be sure of the presence or absence of this parameter, as over-staging of the MM in question can affect the entire diagnostictherapeutic-care path of the patient [15].

Lymph vascular invasion (LVI) is a very useful parameter in predicting possible disease diffusion in a metastatic setting. For example, now included in the AJCC 8′Edition, this parameter has been shown to significantly increase the risk of recurrence, lymph node involvement, distant metastases, and death, just like the ulceration parameter [16]. From a histopathological point of view, consideration should be given to the possibility that tissue artifacts can simulate and/or obscure the morphology of lymphatic and/or blood vessels, and, therefore, studies have been carried out [17], which have shown that the use of immunomarkers such as D2–40 (Podoplanin)

can help to evaluate true lymph-vascular permeations that could otherwise give false-negatives.

Perineural invasion (PNI) is defined as the infiltration of nerve fibers by melanoma cells, resulting in an extension of the tumor along the surrounding nerves. This parameter is of some importance and should be included in the histological report of the MM, as it (from the literature examined) [18] is a pejorative prognostic factor. In addition, it is good to remember that there are some types of melanoma that are more easily neurotropic, such as desmoplastic melanoma or fused cell melanoma, and, therefore, this aspect is to be considered when diagnosing one of these types of lesions. Indeed, the high local recurrence rate of desmoplastic or fused-cell melanoma requires more aggressive surgery to reduce this risk [19].

Microsatellites are defined in the current CAP recommendations as microscopic and discontinuous cutaneous and/or subcutaneous metastases having a diameter larger or equal to 0.05 mm in the largest dimension, adjacent to a primary melanoma [14]. The presence of microsatellites increases from 4.6% in tumors less than 1.5 mm to 65% in those greater than 4 mm [20]. Furthermore, microsatellites are also associated with an increased frequency of regional lymph node metastasis in tumors greater than 1.5 mm [5, 20].

Lymphocytes infiltrating the tumor (TILs) are a type of lymphocytes capable of attacking neoplastic cells and, depending on the mode and distribution, are divided in ascending order into: absent, non-brisk, or brisk [14]. Several studies tend to point out that an increase in TLLs would be more correlated to an improvement in survival, but also in this area (similar to what we have seen for regression) inter-observer variability does not help to obtain a clearer concordance of studies.

#### **3. Subtypes of malignant melanoma**

Melanoma can be considered in the radial growth phase (when melanomatous cells grow in the horizontal plane without giving rise to a vertical growth phase) and in the vertical growth phase, in turn, divisible into tumorigenic (nests/cases deeper than the surface) or non-tumorigenic (melanocytes that do not form a net lesion). It is also very important to consider that melanoma in situ (by definition limited to the basal membrane, therefore, pTis according to AJCC 8′Edition) is different from melanoma in a radial growth phase that, if not called "in situ," suggests the possibility of the presence of some melanocyte minutes present below the basal membrane (c.d. microinvasive).

#### **3.1 Superficial spreading type melanoma (SSM)**

Superficial spreading melanoma is the most frequent histotype in the Caucasian population (70–80% of all melanomas). Usually, it is localized to the back in men and to the lower limbs in women. It is the most common type of melanoma in patients with dysplastic nevus syndrome, with familial melanoma and multiple melanomas. Histologically it is characterized, in the intraepidermal component of the horizontal growth phase, by the presence of pagetoid cells (**Figure 2**) and by a proliferation, in the dermal area, of neoplastic cells frequently associated with epitheliod type often to other cytotypes. In many cases, it can observe a horizontal intraepithelial proliferation of the dysplastic nevus type. Atypical cells in single or in small nests can

*Histological Hallmarks of Malignant Melanoma DOI: http://dx.doi.org/10.5772/intechopen.106638*

**Figure 2.**

*Histological photomicrograph that shows the features of pagetoid spreading, a major criteria for diagnosing MM (Haematoxylin-Eosin, Original Magnification 10×).*

be observed throughout the thickness of the epidermis arranged in an irregular way up to the most superficial layers. In the beginning, the phase of horizontal growth is characterized by invasion of the papillary dermis by single cells or small cell nests. Parallel to the initial invasion, the appearance of a marked inflammatory lymphocytic reaction can be noted. There may be scattered macrophages mixed with inflammatory infiltrate, fibroplasia, and vascular neogenesis. The atypical cells present in the most superficial portion of the papillary dermis, in the horizontal growth phase, have the same cytological characteristics as the cells present in the epidermis, and also the nests of cells are similar in size to those present in the epidermis. In the horizontal growth phase, no cell nest is present in the dermal area, it presents prevailing characteristics compared to the other nests adjacent. The cells of the vertical component, on the other hand, have different cytological characteristics and are predominantly epithelioid-type cells aggregated in nests with cohesive characters. Often a prevailing and expansive type of growth with a tendency for cell proliferation is necessary to develop in a centrifugal direction. The nests of atypical cells, which form the vertical growth, are in size greater than intraepithelial ones. The nuclei are bulky, hyperchromatic, and polymorphic with prominent nucleoli and irregular nuclear membranes. An always evident feature is the lack of cellular maturation with cells with identical cytological features both in the most superficial portion and in the deepest portion of the vertical phase. Mitoses can be in number variable, sometimes even numerous, but above all mitosis is also present in the plus portion of deep melanoma. Isolated cells or groups of necrosing cells are often seen in the dermal component [21, 22].

#### **3.2 Acral lentiginous melanoma (ALM)**

ALM is defined as the presence of a horizontal proliferative component characterized by a proliferation of atypical melanocytes, often with dendritic aspects, mainly localized to the basal layer of the epidermis and extended to the skin appendages associated with a lentiginous appearance with a marked elongation of the spurs epithelial (**Figure 3**). The vertical proliferative component is characterized by a proliferation of atypical melanocytes often type epithelioid, spindle, or polymorphic; sometimes the cells can be arranged in bundles such as to simulate a Schwannian differentiation. More frequently it affects the plantar skin and the palmar skin, especially that of the thumb [23].

#### **Figure 3.**

*Photomicrograph showing acral lentiginous MM with the characteristic disposition of melanocytes at the dermoepidermal junction, with some aspects of pagetoid spreading (Haematoxylin-Eosin, Original Magnificaton 10×).*

#### **3.3 Nodular melanoma (NM)**

The term nodular melanoma defines a tumor characterized by the appearance of a nodule that develops rapidly without a preexisting phase of horizontal growth (**Figure 4**). Nodular melanoma appears on apparently normal skin. Although no lesion is observed existing or a horizontal phase, however, there may be evidence of an association with a preexisting nevus. Nodular melanoma accounts for about 10–12% of all types of melanoma in Caucasian subjects. This neoplasm is, by definition, already growing vertically, is usually diagnosed at a fairly advanced stage, and has a worse prognosis than other melanomas [24].

#### **3.4 Lentigo maligna melanoma (LMM)**

LMM occurs most frequently on the face and sun-exposed upper extremities of elderly people. Macroscopically, there is great variation in color, with tan-brown, black, and even pink areas present. LMM can become an invasive malignancy (vertical growth phase) and it is characterized by thickening of the lesion with the development of elevated plaques or discrete nodules [25]. Histologically, LMM is characterized by an epidermal component of atypical melanocytes, singly and in nests, usually confined to the basal layer and with a little pagetoid invasion of the epidermis (**Figure 5**) [26].

#### **Figure 4.** *Example of nodular melanoma (Haematoxylin-Eosin, Original Magnification 10×).*

#### **Figure 5.**

*LMM characterized by some atypical melanocytes that conglomerate in a few atypical nests (Haematoxylin-Eosin, Original Magnification 10×).*

#### **4. Other histotypes of MM**

#### **4.1 Desmoplastic melanoma (DM)**

DM is properly recognized as a variant of MM, with its distinct histological, immunophenotypic, and molecular characterization. The injury is usually considered "scar-like" because, often, the first impression you have at low (panoramic) magnification of this injury is that of a scar. At higher magnification, it is possible to recognize fused cells, immersed in a desmoplastic stroma and focal with myxoid aspects, endowed with mild cytological characteristics, even if at times presenting a "random" pleomorphism (**Figure 6**). Rather characteristically DM cells are quite rich in mast cells and have a tendency to infiltrate the surrounding nerves. This variant of MM prefers the head/neck of older subjects, thus disproving it as a high cumulative solar damage (H-CSD)-related melanoma. Molecular studies have also confirmed a much higher rate of mutations for this lesion histotype than common MM variants [27].

#### **4.2 Nevoid melanoma (NM)**

Nevoid melanoma is classically referred to as the "tomb" of the dermatopathologist for its intrinsic ability, even in the eyes of a dermatopathologist with years of

#### **Figure 6.**

*Histological photomicrograph showing a bland-appereance proliferation that reveals a DM (Haematoxylin-Eosin, Original Magnification 10×).*

experience, to mimic a benign melanocytic nevus and, therefore, to pass as unknown. It is a rare type of MM, representing no more than 1% of all melanomas, which is commonly more frequent between the fourth and fifth decades of life, without a gender predominance. Clinically, NM presents as a slowly growing lesion, most commonly on the proximal extremities, trunk, neck, and head [28].

From a histological point of view, NM has an architectural pattern that looks very similar to that of a compound or intradermal nevus, with almost complete symmetry, good circumscription, and minimal or no pagetoid spreading. The most important morphological characteristic distinguishing an NM from a standard nevus is the presence of a monomorphic population of small nevus-like melanic cells, present both in the superficial and deep portions of the dermis. It is of absolute and indispensable importance to conduct a very careful research of mitotic figures, able to make us suspect that we are in front of an NM [29].

#### **5. Immunohistochemical features of malignant melanoma**

Melan-A/MART-1 is one of the most important markers of melanocytic differentiation, used in the dermatopathological field. This protein is expressed by melanoma cells and recognized by cytotoxic T cells. Its application is mainly intended for the differential diagnosis of melanocytic tumors, but also for particular other types of tumors, including metastatic since it is more sensitive than another marker, Human Melanoma Black-45 (HMB-45) [30].

HMB-45 is a monoclonal antibody that reacts with an antigen present in melanocytic tumors such as melanomas, hence it is full name of Human Melanoma Black. It is known in pathological anatomy as the best diagnostic marker for melanomas. HMB-45 was discovered in 1986 by doctors Allen M. Gown and Arthur M. Vogel and from then on, its diffusion has become widespread. The specific antigen recognized by HMB-45 is Pmel 17 and reacting positively to melanocytic tumors but not to others is configured for its high specificity and sensitivity.

Despite its high sensitivity HMB-45 has some negative aspects. HMB-45 can be detected only in 50–70% of melanomas. It does not react well to intradermal nevi, normal melanocytes of adult life, fused cell melanoma, and desmoplastic melanoma.

HMB-45 does not react to most malignant tumors that are not melanomas, except for tumors that show melanogenesis (example: pigmented schwannoma, clear-cell sarcoma, or tumors associated with the tuberous sclerosis complex (angiomyolipoma and lymphangiomyoma) [31].

S100 proteins are a family of low molecular weight proteins found in vertebrates, characterized by two binding sites for calcium with helix-loop-helix (EF-hand) structure. There are at least 21 types of S100 proteins and their name "S100" derives from the 100% solubility of these in ammonium sulfate at neutral pH. Most S100 have a homodimeric structure, that is, two identical polypeptides bound together by non-covalent bonds. Although S100 proteins are structurally similar to calmodulins, they differ from these in that they are cell-specific, expressed in particular cells at different levels depending on environmental factors. The calmodulins, on the other hand, are ubiquitous and universal Ca2+ receptors, present in many cells. S100 proteins are normally present in neural crest-derived cells (Schwann cells, melanocytes, and glia cells), chondrocytes, adipocytes, myoepithelial cells, macrophages, Langerhans cells, and dendritic cells.

S100 proteins are implicated in various intracellular and extracellular functions. They are also involved in the regulation of protein phosphorylation, transcription

#### *Histological Hallmarks of Malignant Melanoma DOI: http://dx.doi.org/10.5772/intechopen.106638*

factors, Ca++ homeostasis, the dynamics of cytoskeleton constituents, enzyme activities, cell growth and differentiation, and the inflammatory response. Some members of the S100 family are useful as markers for certain tumors and for the differentiation of cells in the epidermal sense. They can be found in melanomas, malignant peripheral nerve sheath tumors, schwannomas, paraganglioma stromal cells, and clear cell sarcomas [32].

p16 belongs to the family of CDKI, proteins that have the function of inhibiting the action of cyclin-dependent kinase (CDK) and then block the cell cycle. The function of p16 as an inhibitor of cyclin-dependent kinases configures it as a tumor suppressor gene since in its absence it is minus negative control over cell proliferation. It is currently considered another reliable but indicative parameter in differential diagnostics of ambiguous melanocytic lesions [33].

Finally, PRAME (preferentially expressed antigen in melanoma) is a tumor-associated antigen that was first identified through analysis of the specificity of tumorreactive T-cell clones derived from a patient with metastatic cutaneous melanoma. It was subsequently found that PRAME is not only expressed in cutaneous melanoma but also in ocular melanoma and various nonmelanocytic malignant neoplasms, including non-small cell lung cancer, breast carcinoma, renal cell carcinoma, ovarian carcinoma, leukemia, synovial sarcoma, and myxoid liposarcoma. Normal healthy tissues are not known to express PRAME except for testis, ovary, placenta, adrenals, and endometrium. Because of its expression profile, PRAME is a member of the family of cancer-testis antigens (CTA) and an attractive target for immunotherapy. A number of clinical trials are underway trying to exploit CTAs, including PRAME, for cancer treatment [34]. Although at the beginning there were high expectations of this marker in the dermatopathological diagnosis of malignant melanoma, in reality, with the development of the first studies and the first more numerous cases, the possibility that PRAME can be another indicator within other markers and the always essential morphology has been increasingly outlined, but that its positivity or negativity does not confirm and/or exclude a given diagnosis.

In recent years, there has been an increasing interest in the study of the microenvironment of MM [35]. Research has, in fact, led to the acquisition of new information about cells in the immune system that can control and potentially destroy cancer cells [36]. In this way, it was possible to study how T cells (labeled by the antibody anti-CD4 and/or CD8) were more or less capable of performing their own immunosurveillance functions. It has been possible, therefore, to find that the immune systems are able (as demonstrated earlier in other forms of cancer) to express a receptor, programmed cell death protein 1 or PD1, that binds its ligand called programmed cell death protein 1 ligand or PDL1, going to inhibit the functions of T lymphocyte cells against neoplastic cells [37]. These findings made it possible to formulate pharmacological principles that are known as Immunotherapy. Currently, in 2022, several adjuvant therapies in MM in clinical stage III-IV are considered unresectable. For example, it is important to mention the drug nivolumab, anti-PD1, which by blocking this receptor on T cells, prevents its inhibition mediated by PDL-1 binding and, therefore, enhances the antitumor response of the immune system [38].

#### **6. Next generation sequencing in diagnosis of malignant melanoma**

In recent years, the advent of next-generation sequencing (NGS) has made important contributions in the field of pathology, and of neoplasms in general, but also, therefore, in the field of MM [39]. In the case of MM, NGS allowed to highlight molecular alterations that had not previously been discovered, or confirmed, and which had the merit of allowing a greater ability to develop drugs capable of targeting them (for example BRAF) [40]. Although they are mainly used for cancer therapy purposes, NGS can, in some selected cases, also contribute during the diagnostic workout of MM. It is not uncommon, as mentioned above, that the dermatopathological diagnosis of MM is challenging, complex, and not always adequately reachable. For this reason, molecular confirmation of a possible mutation for BRAF can further support a given diagnostic hypothesis, while recognizing that there are cases in which NGS are not of certain diagnostic aid [41].

#### **7. Mucosal melanoma**

The mucosal melanoma occurs in the mucosa (mouth, vagina, penis, and conjunctiva) and presents with a quite large, irregularly pigmented macula, representing the growth phase horizontal and with a corresponding nodule or plaque to the vertical growth phase [42]. From the macroscopic point of view, the macular component usually has irregular edges and fairly regular color, but sometimes with a set of brown and brown and bluish-black shades. Sometimes due to the presence of regression, areas of gray-whitish color are noted. The invasive component of vertical proliferation is characterized by a bluish-black nodule but if it is amelanotic it may appear red or whitish-pinkish [43]. From a histological point of view, in addition to the proliferation of uniformly atypical melanocytes placed close to each other along the junction dermoepidermal, sometimes with elements with evident dendritic extensions, we can observe the presence of invasion by single cells or small nests. Single cells are often fusiform and may be erroneously interpreted as fibroblasts, especially in oral and vaginal seats. Melanoma presents an infiltrate inflammatory scattered, this aspect can be confused with an inflammatory lesion especially if there are macrophages without evidence of pigment. As soon as the proliferation begins vertical, begin to appear more voluminous nests of cells with cytological characters different from those of the proliferative component cells horizontal. Epithelioid cells may be noted as rather large or fused cells and sometimes the vertical component can assume characters desmoplastic. Neurotropism may be present with or without evidence of desmoplasia. These melanomas are almost never associated with or superimposed on a preexisting nevus [44].

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

In memory of Antonietta Cimmino (A.C.).

*Histological Hallmarks of Malignant Melanoma DOI: http://dx.doi.org/10.5772/intechopen.106638*

#### **Author details**

Gerardo Cazzato1 \*, Anna Colagrande1 , Lucia Lospalluti<sup>2</sup> , Giuseppe Ingravallo1 , Eliano Cascardi1,3, Miriam Dellino4 , Saverio Capodiferro5 , Eugenio Maiorano1 , Caterina Foti<sup>2</sup> and Leonardo Resta1

1 Section of Pathology, Department of Emergency and Organ Transplantation (DETO), University of Bari "Aldo Moro", Bari, Italy

2 Section of Dermatology and Venereology, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari "Aldo Moro", Bari, Italy

3 Unit of Pathology, Candiolo Cancer Institute, Candiolo, Italy

4 Section of Gynecology and Obstetrics, Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari "Aldo Moro", Bari, Italy

5 Department of Interdisciplinary Medicine, University of Bari "Aldo Moro", Bari, Italy

\*Address all correspondence to: gerardo.cazzato@uniba.it

© 2022 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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#### **Chapter 4**

## The Value of Histopathological Characteristics and BRAF and NRAS Mutations for the Diagnosis, Risk Stratification, and Prognosis of Malignant Invasive Melanoma

*Tatjana Zablocka and Sergejs Isajevs*

#### **Abstract**

In recent years, the direction of personalized medicine, which is based on a disease-specific targeting therapy, as well as the early diagnosis of tumors and the identification of high-risk individuals, is rapidly developing in the world. Invasive melanoma is a tumor with high impact for its rapidly growing incidence, high mortality, increased complexity, and high care costs in advanced stages. Recent studies demonstrated the significant value of both conventional histopathological characteristics and genetic alterations in melanoma. This review focuses on the value of conventional histopathological characteristics including histological tumor subtype, Clark level, Breslow thickness, solar elastosis, ulceration, regression, lymphovascular invasion, mitotic counts, peritumoral lymphocyte infiltration, clinical characteristics such as age, gender, length of follow-up after surgery, recurrence, or metastasis, and progression-free survival, and tumor BRAF and NRAS mutations.

**Keywords:** melanoma, histopathology, tumor infiltration lymphocytes, BRAF, NRAS

#### **1. Introduction**

More than 97% of all melanomas are diagnosed with a known primary site, most often on the skin [1–3]. Melanoma can also present within the eye or in the mucosae of internal organs [3]. In the rare cases in which it is diagnosed without an obvious primary site, it is referred to as melanoma of unknown primary (MUP) [3]. The predominant hypothesis to explain MUP involves the spontaneous regression of melanoma from a known primary site [3]. Metastatic melanoma could develop synchronously with a subclinical or otherwise unrecognized cutaneous, ocular, or mucosal melanoma.

Ultraviolet (UV) radiation is the most significant risk factor in the pathogenesis of melanoma, directly damaging DNA [1–3]. Multiple somatic and epigenetic alterations have also been implicated in the pathogenic process, along with the immune response and disturbances of immune tolerance [3].

There is a little evidence for early detection and risk stratification in malignant melanoma [4, 5]. The gold standard for melanoma diagnosis is still histopathological examination of tissues. Histopathological diagnosis involving the qualitative and quantitative assessment of biomarkers is susceptible to substantial interobserver variability, limiting its usefulness for individual patients. Specialized dermatopathologists are likely to be more consistent; however, their expertise is not widely available. Therefore, the standardization of the assessment is important [3].

Deep learning, an automated approach using labeled images to train a network with no other assumptions, has proven useful in many similar areas of digital pathology. In recent years, significant progress has been made in proteomics, metabolomics, and genomics. However, histopathological examination remains the gold standard for the diagnosis and prognosis of melanoma [3, 5–7].

The current World Health Organization (WHO) classification of skin tumors subdivides melanoma on the basis of solar elastosis assessed by dermal elastic fibers to measure cumulative sun damage (CSD) [3]. According to this WHO classification, there are currently three classes of melanomas: those associated with high CSD, those associated with low CSD, and those associated with nodular melanomas [3]. Solar elastosis is usually apparent in superficially spreading melanoma and lentigo maligna melanoma, the so-called high CSD melanoma. Desmoplastic melanoma is associated with increased solar elastosis. The most common subtype of high CSD melanoma is superficially spreading melanoma, which usually begins with early radial growth, followed by vertical growth and invasion of the dermis.

Acral, mucosal, uveal, and spitzoid melanomas are not associated with CSD or are characterized by low CSD. Nodular melanoma usually characterized as a low CSD type with early progression to vertical growth [3].

The development of melanoma is closely related to somatic and epigenetic changes. Different mutations have been implicated in its pathogenesis and evolution. Recent genomic classification subdivides melanoma into four subtypes based on the pattern of the most prevalent significantly mutated genes: BRAF, RAS and NF1 mutants, and triple-WT (wild type) [3, 5].

BRAF, CDKN2, and NRAS mutations are the most important and clinically relevant. The advent of novel personalized treatment for melanoma based on BRAF inhibitors and immunotherapy has reduced the mortality rate over the last decade, but advanced and metastatic melanomas remain difficult to treat [8–10]. Immune tolerance mechanisms are also important in the progression of melanoma.

Germline mutations in the cyclin-dependent kinase inhibitor 2A gene (CDKN2A) are frequently identified in familial melanoma; in 20–50% of such cases, three or more family members are diagnosed with melanoma [11]. Germline mutations in CDKN2A have also been associated with familial atypical multiple mole melanoma (FAMMM) syndrome, an autosomally dominant condition exemplified by a family history of melanoma and large numbers of atypical nevi [3, 11],

Immune responses are important in the pathogenesis of melanoma. Programmed cell death protein 1 ligand 1 (PDL1) and PDL2 are usually expressed by melanoma cells, T cells, B cells, and natural killer cells. This observation led to the development of specific antibodies against programmed cell death protein 1 (PD1) for the personalized treatment of melanoma (for example, nivolumab and pembrolizumab). Combinations of different targeting treatments that influence immune response mechanisms had beneficial effects on melanoma treatment, including PDL1 and CTLA4 targeting and immunotherapy with oncolytic viruses [8–12].

#### *The Value of Histopathological Characteristics and BRAF and NRAS Mutations for the… DOI: http://dx.doi.org/10.5772/intechopen.105722*

Clinicopathological characteristics, such as tumor size, tumor type, tumor invasiveness (Breslow thickness, Clark level, lymphovascular invasion, and neurotropisms), ulceration, and tumor mitotic activity, are significant prognostic factors for the development and progression of melanoma [3, 11]. In addition, it has been demonstrated that tumor-infiltrating lymphocytes can stratify melanoma into lowand high-risk progression types [13–15].

Diagnostic and therapeutic molecular markers have been increasingly used to assist in the histopathological assessment of melanoma [16]. These markers are helpful not only for diagnosing the condition, but also for distinguishing certain subtypes that could otherwise be difficult to identify [17–24]. BRAF-mutated melanoma is mostly associated with superficial spreading melanoma, younger patients, and non-CSD skin, whereas NRAS mutational melanoma is a nodular subtype associated with CSD skin [20, 25].

Generally, NRAS mutations are independent of BRAF mutations, but dual expression has been reported [25]. The association of NRAS mutations with the degree of solar elastosis suggests that NRAS is closely related to the mutations induced by UV irradiation. Previous studies showed that NRAS mutation is also associated with decreased immune responses in peritumoral melanoma tissue and a more advanced tumor stage [26]. However, the prognostic value of NRAS mutation is still controversial, especially in early-stage melanoma.

#### **1.1 Histopathological assessment of melanoma**

At present, the histopathological examination of melanoma is based on the current WHO classification and the College of American Pathologists (CAP) guidelines [3]. Such criteria as tumor type, ulceration, peritumoral lymphocytes, Clark invasion level, Breslow invasion level, lymphovascular invasion, neurotropism, regression, and mitotic activity are routinely assessed. In addition, the excision lines and distance from the tumor are recorded. The pathological tumor node metastasis (pTNM) staging is determined on the basis of histopathological assessment. **Table 1** summarizes the histopathological characteristics for assessing invasive melanoma.

Since Breslow thickness is of particular importance for TNM staging, digital slide analysis could provide better evidence for the measurement of invasions, especially in borderline cases. During recent years, digital pathology has been extensively used not only in research but also in clinical practice. Slide digitalization, scanning, and analysis by artificial intelligence have been suggested as a comprehensive tool to help pathologists construct a final report [27].

**Figure 1** shows superficial spreading melanoma. The slide was stained with hematoxylin and eosin, magnification ×100. The tumor cells are located in the epidermis and papillary dermis, with moderate cellular pleomorphism, epidermotropism, and asymmetry. There is prominent peritumoral lymphocyte infiltration.

Melanomas with an amelanotic appearance are more difficult to diagnose. Immunohistochemical staining positive for S100, SOX-10, HMB-45, Melan-A, Mart-1, and tyrosinase supports a diagnosis of melanoma [3].

Some melanomas, especially if regressed and metastatic, can cease to express HMB-45, Melan-A, and tyrosinase. In such cases, the immunohistochemical assessment of melanoma is straightforward; usually, only S-100 and vimentin expression is characteristic.

**Figure 2A** demonstrates S-100 expression in melanoma immunohistochemically. The arrow indicates positively stained cells. Note cytoplasmic biomarker expression.


#### **Table 1.**

*The protocol for routine clinical examination of melanoma.*

**Figure 2B** demonstrates SOX-10 expression in melanoma tissue immunohistochemically. The arrow indicates positively stained cells. Note the positive nuclear staining of melanoma cells.

Recently, it has been shown that p16 expression in melanoma is significantly lower than nevus [28]. PRAME has also been demonstrated as an immunohistochemical marker to aid the diagnosis of malignant melanoma [29].

*The Value of Histopathological Characteristics and BRAF and NRAS Mutations for the… DOI: http://dx.doi.org/10.5772/intechopen.105722*

#### **Figure 1.**

*Representative photomicrograph demonstrated superficial spreading melanoma. Hematoxylin-eosin staining method, magnification: ×100, and scale bar: 20 μm.*

#### **Figure 2.**

*Representative photomicrograph of biomarker expression melanoma. A. S-100, B. SOX-10. Immunohistochemical staining method, magnification: ×200, and scale bar: 50 μm.*

#### **1.2 Artificial intelligence in the histopathological assessment of melanoma**

Artificial intelligence (AI) and its subdisciplines of machine learning (ML) and deep learning (DL) are emerging as key technologies in healthcare with the potential to change lives and improve patient outcomes in many areas of medicine. While there is considerable promise for AI technologies in health, there are challenges ahead. These include recognition that it will be extremely difficult for AI to achieve full automation in the diagnostic/clinical pathway. Most efforts to date have focused on the development of neural network architectures to enhance the performance of different computational pathology tasks. U-Net has been used in several applications.

Recently, a deep learning network called MVPNet—multiviewing path deep learning neural networks for magnification invariant diagnosis in breast cancer—has been proposed for the digital analysis of breast cancer. MVPNet has significantly fewer parameters than standard deep learning models and combines local and global features.

During the past decade, advances in precision oncology have resulted in an increased demand for predictive assays that enable patients to be selected and stratified for treatment.

In the global market, there is a high demand for digital pathology and artificial intelligence software for consultations and automated data analysis. Recently, the Food and Drug Administration (FDA) approved the first digital pathology software for automated prostate cancer assessment.

The possibility of digitizing whole-slide images of tissue has led to the advent of artificial intelligence and machine learning tools in digital pathology, which enable subvisual morphometric phenotypes to be mined and could ultimately improve patient management [30].

#### **1.3 Tumor-infiltrating lymphocytes for stratifying the risk of melanoma progression**

Tumor-infiltrating lymphocytes (TILs) are considered a manifestation of the host immune response to the tumor [13–15].

Cell membrane-bound antigens different from those of normal cells are characteristic of tumor cells. These antigens are recognized as nonself by antigen-presenting cells, with subsequent activation of cellular and humoral immune responses. The key cells for cytotoxic immune responses are CD4, CD8, and NK cells; for humoral responses, they are B lymphocytes and plasma cells. However, a tumor can escape immune surveillance by unmasking its antigens and inducing apoptosis in the immune cells. The key characteristic of tumor immunity is the presence of peritumoral and intratumoral inflammatory cells. Tumor-infiltrating lymphocytes (TILs) arise from different inflammatory cells, mainly CD4 and CD8 T cells, plus CD20 B lymphocytes and NK cells. These cells have been extensively described in antitumor immunity. T-regulatory lymphocytes, which form the key cell population of peritumoral and intratumoral lymphocytes, have immunoregulatory features. They suppress the immune response and commonly express FOXP3, CD4, and CD25 [13–15].

It has been shown that peritumoral lymphocyte infiltration (TIL) is valuable for melanoma prognosis. It is also closely associated with tumor metastasis to lymph nodes. Patients with increased TIL infiltrate have a better prognosis [13]. Furthermore, increased TIL infiltration is a sign of longer progression-free survival and overall survival, and a lower mortality rate [31].

However, American Joint Committee for Cancer (AJCC) manuals have not included the assessment of TIL for tumor staging and prognosis, and some pathology guidelines do not require peritumoral lymphocyte infiltration to be assessed [3]. The College of American Pathologists (CAP) and the Royal College of Pathologists of Australasia (RCPA) protocols suggest that peritumoral lymphocyte infiltration be assessed as brisk and nonbrisk infiltration. The association of TIL with an improved prognosis for melanoma remains controversial [32–34]. Previous studies have shown that an increased TIL infiltrate is associated with more favorable survival outcomes [13, 30, 31].

A recent study showed that melanoma patients with high TIL grade had significantly better progression-free survival than patients with low TIL grade [15]. The authors recommend incorporating the assessment of TIL into a scoring system, for example from 0 to 3, by estimating the percentage cellular infiltration of the tissue.

#### *The Value of Histopathological Characteristics and BRAF and NRAS Mutations for the… DOI: http://dx.doi.org/10.5772/intechopen.105722*

The scoring system was defined as follows: 0 = absence of lymphocytes from the tissue, 1 = lymphocytes occupying <25% of the tissue, 2 = lymphocytes occupying 25–50% of the tissue, and 3 = lymphocytes occupying >50% of tissue. Low TIL infiltration was defined as scores of 0 and 1. High TIL infiltration was defined as scores of 2 and 3 [15]. This scoring system correlated significantly with progression-free survival and showed perfect concordance among pathologists; therefore, it could be recommended for routine clinical practice.

#### **1.4 Assessment of BRAF gene mutation for stratifying the risk of melanoma progression**

The BRAF gene is located on the seventh chromosome and encodes BRAF protein, one of the signaling kinases in the MAPK pathway. BRAF mutations are the most common genetic alterations in cutaneous melanoma. The prevalence of BRAF mutations among the different melanoma subtypes and populations ranges from 40% to 60% of cases [16–19, 25]. BRAF mutations lead to the constitutive activation of the MAPK pathway. The most common BRAF mutation (80% of all alterations in the gene) is V600E [20]. V600K and V600R mutations are other examples [21].

Previous studies have shown that the BRAF V600E mutation is associated with the superficial spreading melanoma subtype, solar elastosis, younger patients, and melanoma localization on the extremities and back. In contrast, BRAF V600K mutations are correlated with skin sites with high CSD, such as the head and neck, and with older patients [14–19, 35–40].

Recently, whole-genome sequencing of benign melanocytic nevi revealed BRAF mutations in addition to NRAS mutations, the mutational load being positively correlated with UV exposure. The mutational loads in congenital nevi were lower [23].

A recent study revealed associations between BRAF V600 mutational status and younger patient age, Clark invasion level, Breslow thickness, lymphovascular invasion, female gender, and TIL [15].

#### **1.5 Assessment of NRAS gene mutation for stratifying the risk of melanoma progression**

The importance of NRAS mutations for the progression of melanoma is controversial. Some studies have shown associations between NRAS mutation and melanoma prognosis, while others found that NRAS mutations have no value for assessing the prognosis [3, 11, 41, 42].

The RAS gene family includes genes that encode the G proteins responsible for cell growth and cell cycle regulation. Three major members of the RAS gene family are NRAS, KRAS, and HRAS. NRAS-mutant melanomas often have dysregulated cell cycles, characterized by the upregulation of cyclin D1 and loss of the tumor suppressor p16INK4A [43].

The NRAS gene is most frequently mutated at hotspots in exon 2 (codons 12 and 13) and exon 3 (codon 61) [42, 44–47]. Mutations of NRAS have previously been associated with the nodular subtype of the primary tumor and localization in sundamaged skin [45].

Some studies have shown that NRAS mutation is associated with a favorable prognosis [46]. In contrast, others have demonstrated that this mutation is associated with a worse prognosis [48, 49], and some found no significant association at all between NRAS mutation and a prognosis of melanoma [45, 50, 51].

Recent evidence showed that in up to 20–30% of cases, NRAS mutations coexisted with BRAF mutations. Patients with both BRAF and NRAS mutations had worse prognoses than those with BRAF mutant melanoma alone [25, 26]. Since the prognosis for co-mutations is worse, routine NRAS assessment of all the primary melanoma cases would seem to be beneficial.

The assessment of NRAS mutation in melanoma, especially in BRAF-wild-type melanoma, is beneficial since targeted treatment is considered for NRAS mutant melanoma [52]. Immune checkpoint inhibitors (anti-CTLA4 and/or anti-PD1) are the standard treatment in these cases. However, a recent clinical trial also showed promising results from targeted treatments of PI3K-AKT-mTOR, MEK, and CDK4/6.

#### **2. Conclusion**

In recent years, the direction of personalized medicine, which is based on diseasespecific targeting therapy, along with the early diagnosis of tumors and identification of high-risk individuals, has developed rapidly around the world.

The gold standard for melanoma diagnosis is histopathological investigation and routine evaluation of, e.g., tumor type and tumor invasiveness. Histopathological slide digitalization seems to be beneficial for standardizing the assessment of histopathological characteristics. In addition, the assessment of peritumoral lymphocyte infiltration and BRAF and NRAS mutation status in early-stage melanoma has proved to be of significant value for the risk stratification of disease progression and for personalized treatment.

The assessment of BRAF and NRAS mutations in melanomas is important not only for personalized targeting treatment, but also for prognosis and surveillance strategy. BRAF and NRAS mutations correlate with primary tumor type and disease stage. NRAS mutant melanoma has a significantly worse prognosis than BRAF mutant melanoma, and an active surveillance strategy should be applied to patients with this condition.

#### **Acknowledgements**

The study was supported by project "Strengthening of the capacity of doctoral studies at the University of Latvia within the framework of the new doctoral model" identification No. 8.2.2.0/20/I/006. The assistance of BioMedES UK (www.biomedes. biz) in the final drafting of this chapter is acknowledged.

#### **Conflict of interest**

The authors declare no conflict of interest.

*The Value of Histopathological Characteristics and BRAF and NRAS Mutations for the… DOI: http://dx.doi.org/10.5772/intechopen.105722*

#### **Author details**

Tatjana Zablocka1,2,3,4\* and Sergejs Isajevs1,2,4

1 University of Latvia, Faculty of Medicine, Riga, Latvia

2 Riga East University Hospital, Riga, Latvia

3 Pauls Stradins Clinical University Hospital, Riga, Latvia

4 Hospital of Traumatology and Ortopaedics, Riga, Latvia

\*Address all correspondence to: tatjana.zablocka@gmail.com

© 2022 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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