Section 2 Interferon in Therapy

## **Chapter 2**

## Interferons Horizon Therapeutics

*Ayesha Aiman, Seemi Farhat Basir and Asimul Islam*

#### **Abstract**

Interferons (IFNs) are a family of multi-functional proteins, called cytokines, that are produced by immune cells such as leukocytes, natural killer (NK) cells, macrophages, fibroblasts, and epithelial cells. The minute amount of these α-helical glycoproteins, produced by mammalian cells, are firm components of the innate arm of the immune system providing rapid and broad protection against numerous types of invading pathogens. Interferons, from their discovery in the 19th century, have always held out a promise of important clinical utility first as an antiviral agent and more recently holding anti-inflammatory and regenerative effects for treating various neurological diseases such as multiple sclerosis, encephalopathies, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), etc. IFNs elicit anti-viral and anti-inflammatory properties by inducing transcription of multiple IFN stimulated genes (ISG), a response that is partly mediated by Interferon regulatory factors (IRFs). This chapter provides a brief introduction of the interferon system as well as an in-depth assessment of the interferon signature and the various assay procedures for synthesizing non-natural interferon analogs for structural analysis, which may be helpful in designing improved products and act as a diagnostic tool for neurodegenerative disorders.

**Keywords:** cytokines, cancer, inflammation, interferons, interferon regulatory factors (IRFs), neurodegenerative disorders

### **1. Introduction**

The name of the interferons comes from their capability to intrude with the product of new contagion patches. When the vulnerable system is attacked, they get actuated due to viral infection or other unknown substances and the white blood cells in the body produces interferons, which are a group of proteins called cytokines. Interferons (IFNs) are a group of soluble α-helical glycoproteins [1] that are produced and released by the innate arm of immune cells such as leukocytes, natural killer (NK) cells, fibroblasts, and epithelial cells in response to virus infection (or any other stimuli). They bind to specific IFN receptors on cells to trigger multiple signaling pathways that result in the expression of IFN-stimulated genes (ISGs). The ISG products then render the cell resistant to subsequent virus infection. Interferons do not directly kill the virus or cancerous cells rather they boost the vulnerable system response and reduce the growth of cancer cells by regulating the exertion of several genes that control the stashing of multitudinous cellular proteins that affect their growth. In extreme cases, an

indecorous prosecution of these pathways or their inordinate activation can result in cell death. IFNs can play beneficial roles in the nervous system because of their tremendous capacity for upregulating immune responses. The three major types of interferons, that is, IFN-α, β and γ act as implicit curatives for a number of diseases. IFN-α has been used for the treatment of hepatitis B and C, and in several types of cancers, including hairy cell leukemia, chronic myeloid leukemia, Kaposi's sarcoma, and Erdheim–Chester disease (or polyostotic sclerotic histiocytosis), a rare complaint of bone marrow inflammation that can also affect the cerebellum [2]. IFN-β, an immunosuppressive cytokine, is the first medicine shown to promote clinical improvement in multiple sclerosis by inhibiting IL-12 production and inducing IL-10 [3]. Genetic research reveals the role of IFN-β in regulating mitochondrial dynamics to prevent neurodegeneration. IFN-β rescues mitochondrial abnormalities and neuronal survivance in Parkinson's disease *in vivo* [4]. Finally, IFN-γ has been used in the treatment of chronic granulomatous disease, a rare hereditable complaint in which the phagocytic cells have disabled capacity to kill ingested microbes, resulting in recurring bacterial and fungal infections [5]. IFNs, presumably in confluence with other cytokines, hold a prominent role in various therapies against diseases due to their incredible wideranging and pronounced immunological properties.

## **2. Origin and classification of interferons**

Among the major discoveries in science, the discovery of interferon was a fortuitous one. The 60-year history of exploration on IFN abounds with big and small breakthroughs and are been recorded in the literature. However, information on the succession in lines of thought that led from one discovery to the next is dispersed, and many of those linkages may only be recorded in the memory of 'veteran' interferon workers. New generations of interferon workers tend to rely on handbooks or laboratory manuals, whereas background about sophisticated pathways of discovery is usually omitted. Therefore, this historical section related to molecular structure, production, and action of IFN will be considered from the viewpoint of how our insights have grown within the environment of evolving tools and general knowledge in cellular and molecular biology.

The basic phenomenon of interference was first described in the year 1935 with the capability of one contagion to interfere with the replication of another (challenge) contagion [6]. Thus, the hunt was underway for the mediator of viral interference for about 20 years until Alick Isaacs and Jean Lindenmann coined the term interferon (IFN) to it, in 1957 [7]. When heat- or UV-inactivated influenza virus was injected into the 10-day-old fragmented chorioallantoic membrane of chick embryos, a substance was released that inhibited viral multiplication. Hemagglutination, or the virus's ability to interact with and agglutinate red blood cells, was used to quantify influenza viral production (or inhibition). The interfering chemical was given the name "interferon." The titration ended when a well (on a plate of tiny wells) was identified with partial agglutination; the reciprocal of the influenza dilution thus measured was used as the interferon titer (concentration). Interferon molecules produced by infected cells function via autocrine and paracrine signaling to transform host cells into antiviral cells [8]. They have profound immunomodulatory as well as antiviral properties. They were initially classified as leukocyte, fibroblast, or immune IFNs, based on their cellular origin. Type I IFN (leukocyte and fibroblast IFN) and

type II IFN (immune IFN) are two types of IFNs that are now known to be made up of over 20 distinct proteins [9].

## **3. IFNs family background**

Even though IFNs were initially classified as antiviral agents, Isaac and Lindenmann could not have anticipated the enormous impact their discovery would have, and the extent to which they would be pertinent far beyond the discipline of Virology. From the discovery in the 1960s that IFNs also played a role in the control of cell growth and animal tumors up to the recent findings that they are pivotal regulators of both innate and adaptive immune responses, the result is that vertebrate life would be permanently threatened without IFNs.

Multiple criteria, including sequence identity, genetic loci, cell of origin, receptor distribution, and downstream reactions, have been used to classify IFNs. Although IFNs are expressed at low levels in the body at rest, they are activated to varying degrees depending on the stimuli, as a result, they play a dynamic and pathogen-specific function in the immune response. IFNs modulate the immune system's ability by promoting transcription of interferon signaling genes (ISGs) after they are generated and released by immune cells.

IFNs were classified as type-I (pH stable) or type-II (pH unstable) based on their pH sensitivity. The designation of IFN-α/β and IFN-γ as type-I and type-II IFNs, respectively, was further verified by analysis of their unique amino acid sequences and crystal structures [10–13]. The type-I family has been expanded to 16 members which include 12 IFN-αs that are encoded by 13 genes (IFN-α1/13 encode the same protein) [14–18], IFN-β (the well-known IFNs and the first to be cloned, purified, and sequenced) [17, 19, 20], IFN-ϵ [21], IFN-κ [22], and IFN-ω [23]. Type-II family includes only one member, that is, interferon- γ, produced by NK cells and T-cells (in response to cytokines IL-12 and IL-18). Both types of IFN promote an "antiviral state" by snooping with cell proliferation and viral replication mechanisms. Moreover, IFNs render infected cells to become more susceptible to apoptosis (procaspase activity) and recognition by CD8+ cytotoxic T-cells by upregulating the expression of class I-major histocompatibility complex (MHC-I) on infected cells [24]. In 2003, the genome analysis discovered a novel type-III family of IFNs (IFN-λ), which were shown to be comparable to the IL-10 family of cytokines [16, 25–27], particularly IL-22 [28] based on sequence and subsequent structural studies. In humans, there are four different subtypes of Type III IFN, namely, IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4 [29]. These IFN present similar biological effects to type-I IFNs, playing an important role in host defense against viral infections.

After a brief introduction of some of the cardinal features of the three types of interferons, we will now discuss the type of receptors involved in signal transduction pathways and biological activities elicited by them and then focus on the regulation of these IFN responses using transcription regulatory factors.

### **4. IFN induction and signaling mechanism**

IFNs are incredibly effective at limiting virus replication and transmission, but because they are not normally expressed, IFN synthesis must be triggered promptly

**Figure 1.** *The Mechanism of production of IFNs [30–32].*

and strongly upon host contact with the virus. Because all viruses proliferate inside host cells, identifying bacterial or viral nucleic acids (e.g., RNA or DNA genome) upon microbial challenge, is an efficient technique for eliciting innate immune responses. These foreign substances are firstly identified by a specialized group of proteins known as Toll-like receptors (TLRs) which are, further, a type of patternrecognition receptors (PRRs), that are expressed on sentinel cells. These receptors are either cytosolic or endosomal membrane proteins [30]. The binding of dsRNA/ dsDNA to the helicase domain of RIG-I (Retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated gene-5), respectively, induces caspase activation following activation of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-associated NF-κB activator, TANK binding kinase 1 (TBK1) and inhibitor of NF-κB kinase IKKε **Figure 1** [31–33]. IRF-3 and IRF-7 are expressed ubiquitously as inactive monomers in the cytosol but when cells are stimulated with poly (I:C) or virus infection, they get phosphorylated by the serine/threonine kinases, homodimerized, and are then translocated from cytosol to nucleus and binds to responsive elements for IFN-β gene transcription. After this, the secreted IFN binds to their specific cognate cell surface receptors, the heterodimeric IFNAR1/IFNAR2 complex for type I IFNs, dimers of the heterodimeric IFNGR1/IFNGR2 complex for type II IFN and the heterodimeric IFNLR1/IL10R2 complex for type III IFNs as represented in **Table 1** [32], present on the infected cell's surface, causing an autocrine signaling cascade that mobilizes other interferon response components and changes the gene expression patterns, resulting in an interferon response. IFNs can also bind to the interferon receptor produced by nearby non-virus infected cells, operating in a paracrine manner to enhance interferon response and aid these cells in combating viral infection [33].

The IFN signal transduction pathway has been appropriately described in multiple comprehensive reviews **Figure 2** [17, 29, 38, 40–42, 44–57]. The type I IFNs bind to their related heterodimeric cell surface receptors, IFNAR1 and IFNAR2, which signals through the activation of Janus activated kinases (JAKs), specifically TYK2 (Tyrosine kinase 2) and JAK1, respectively, causing tyrosine phosphorylation of the


#### **Table 1.**

*Classification of interferons in humans.*

#### **Figure 2.**

*Signal transduction mechanism by Type I, Type II and Type III IFN receptors and production of ISGs [17, 29, 33, 38–40, 42, 43, 45–55].*

receptors' intracellular domains and recruitment of signal transducers and activator of transcription (STAT), STAT1 and STAT2 proteins, which in turn forms a trimeric complex, called ISGF3 (IFN stimulated gene factor 3) that consists of STAT1, STAT2, and IRF9 [17, 38]. The ISGF3 then translocate to the nucleus and binds to the IFN stimulated response element (ISRE) in the promoter region of IFN-stimulated genes (ISGs) and initiates transcription of antiviral genes. IRF2 acts as a transcriptional attenuator of ISGF3-mediated transcriptional activation within the nucleus, hence, the absence of IRF2 would result in increased Type I IFN signaling [46]. IFNAR activation also activates STAT1, STAT3, STAT4, STAT5, and STAT6 homodimers, as

well as STAT1–STAT2, STAT1–STAT3, STAT1–STAT4, STAT1–STAT5, STAT2–STAT3 and STAT5–STAT6 heterodimers which bind and activates GAS (IFN-γ activated sequence) motifs, found in the promoter region of ISGs resulting in their gene expression [47–51]. Type I IFN signaling may also activate other signaling pathways that do not rely on the so-called JAK/STAT pathway. They are the non-canonical modifiers of Type I signaling called the mitogen-activated protein kinase (MAPK)/c-Jun amino-terminal kinase (JNK) pathways and the phosphoinositide 3-kinase (PI3K) pathway, which leads to diverse effects on the cell [52]. Furthermore, there is sufficient evidence that the function of distinct STATs may be modulated to account for individual responses. For example, a recent study found that STAT1 inhibits the IFN-α dependent induction of IFN-γ expression, whereas surprisingly, IFN-α or IFN-β mediated activation of STAT4 is essential for the IFN-γ synthesis during viral infection [53]. As a result, the functional diversity of type I IFN-regulated pathways allows for the transcriptional activation of a plethora of genes that facilitate the induction of physiologic responses.

Type II IFNs or IFN-γ is biologically active in its noncovalently coupled homodimer form. The extracellular domain of the two IFNGR1/CD119 subunit attaches to this homodimer, which then interacts with IFNGR2 to form a functional IFN- γ receptor complex. The receptor complex's IFNGR1 subunits are linked to JAK1, while the IFNGR2 subunits are linked to JAK2 [40]. When JAK1 and JAK2 are activated, the receptor is phosphorylated, and STAT1 is recruited and phosphorylated. The phosphorylation of STAT1 causes it to homodimerize and translocate to the nucleus. STAT1 homodimers attach to Gamma activated sequence (GAS) sites in the promoters of target genes once they reach the nucleus, regulating their transcription [41, 42]. IFN-γ signaling is dependent on weak type I IFN signaling, which is mediated by low type I IFN constitutive production [54]. Many of the IFN-gamma/STAT1 signaling-induced target genes are transcription factors that cause the expression of secondary response genes. IFN-gamma signaling can also activate the MAPK, PI3K/ AKT/mTOR, and NF-kappa B signaling pathways, which control the expression of a variety of additional genes [55]. IFN-gamma signaling plays an important role in host defense by promoting macrophage activation, upregulating the expression of antigen processing and presentation molecules, driving the development and activation of Th1 cells, enhancing natural killer cell activity, regulating B cell functions, and inducing the production of chemokines that promote effector cell trafficking to sites of inflammation.

Type III interferons (IFN-λs) communicate with the body via a unique heterodimeric receptor complex, comprising of the IFN-λR1 subunit and interleukin-10R (IL-10R), shared by a number of cytokines in the IL-10 superfamily [29]. Despite the fact that IFN-λ and type I IFNs are structurally disparate and are engaged in different types of receptors, they both share the same JAK/STAT signal transduction pathway to trigger interferon-stimulated genes (ISGs), which have antiviral and immunoregulatory functions. So, these findings initially surmised that the Type I and Type III IFNs were functionally redundant. They were further distinguished by the kinetics of Type III interferons, which had a lower amplitude than Type I interferons while having long-lasting ISG expression [44, 45].

However, dysregulation of the IFN production and function would lead to immunological pathogenesis, such as inflammatory diseases, autoimmune and neurodegenerative disorders, via inappropriately stimulating inflammatory responses or dampening microbial controls. Thus, IFN response must be tightly regulated in order to develop protective immunity against microbial infections, curing autoimmune

disorders and neurodegenerative diseases while avoiding detrimental toxicity induced by improper or prolonged gene expression.

## **5. IRF-mediated regulation of IFNs**

Interferon regulatory factors (IRFs) are a group of transcription factors that are involved in a range of aspects of the innate and adaptive immune responses, including immune cell proliferation and differentiation, as well as modulating pathogenic responses [58]. Were first discovered and identified in the promoter region of the human interferon-β gene (IFN β1) during 1988, when a mouse cDNA clone encodes a protein that has specificity towards the IFN-β gene containing virus-inducible enhancer element, was identified [59]. During that period, there was no other homology present in accordance with this gene or other proteins. So, it was recognized and named as the IFN-regulatory factor 1 (IRF1). Further, cDNA clone that was identified later, subsequent cross-hybridization with IRF1 cDNA was named as IRF2. This signified the formal acknowledgment and birth of the massive IRF family [60]. IRFs specifically recognize the ISRE (Interferon-Stimulated Response Element), a conserved DNA consensus sequence, and become functionally active in the form of homodimers or heterodimers. The IRF family of transcription factors comprises of several members, namely, IRF1, IRF2, IRF3, IRF4 (also known as PIP, LSIRF or ICSAT), IRF5, IRF6, IRF7, IRF8 (also known as ICSBP), and IRF9 (also known as ISGF3γ/p48) were identified in Mus musculus and Homosapiens [61, 62]; IRF10 is observed in birds [63] and fishes [64], IRF11 is found in lower vertebrates, such as teleost fishes and zebrafish [65]. IRF1 and IRF2 have been extensively studied at the molecular level due to their unique properties of regulating gene expression despite having structural similarities. Although the former functions as a transcriptional activator and the latter repress IRF1 function by competing for the same cis-elements within type-I IFN (IFN-α/β) and IFN-inducible genes, they possess a high degree of structural similarity [66]. IRF-3, IRF-5, and IRF-7 are the three members of the IRF family which are induced by Type I IFNs, downstream of PRRs that detect viral DNA/RNA, resulting in a feedforward loop that maximally drives IFN expression [67]. Other members such as IRF-4, IRF-5, and IRF-8 are some of the key regulators of myeloid cell proliferation and phenotypic differentiation, which aids in modulating the inflammatory responses [68]. The fourth member of the family, IRF-9, controls the expression of a wide range of IFN stimulating genes. Researchers have discovered that, like the IFN-β gene, the IFN-λ1 gene is controlled by both IRF3 and IRF7, but IRF7 is the primary regulator of the IFN-λ2/3 genes [69, 70]. Understanding how their levels and activity are controlled is crucial since changes in either can lead to dysregulated immune responses and the development of autoimmune and neurodegenerative diseases.

## **6. Interferon system dysfunction and related disorders**

Interferons are used to treat a number of diseases such as those that are caused by viruses (such as hepatitis B and C virus) or due to inflammation (like multiple sclerosis and systemic sclerosis) as depicted in **Table 1** [34, 71, 72]. They also act as antineoplastic agents to treat malignancies (such as breast carcinoma, nodular lymphoma, chronic myelogenous leukemia (CML), Kaposi's sarcoma, and renal adenocarcinoma) [73]. The expression levels of IFNs, as well as their actions, are superbly controlled in

order to protect host cells from potential toxicity resulting from excessive responses. However, persistent and dysregulated IFN expression causes many diseases such as Type I interferonopathy, a type of inherited CNS disease. They have also been associated with the development or worsening of autoimmune diseases such as psoriasis, systemic lupus erythematosus (SLE), and, in rare cases, rheumatoid arthritis (RA) [74]. This was observed in their mRNA expression patterns that contain the interferon signature. Moreover, a lot of murine Alzheimer's disease (AD) models, as well as wild-type mouse brains challenged with generic nucleic acid-containing amyloid fibrils, showed an increased IFN-stimulated gene (ISG) signature [75]. These findings all point to IFNs that have a negative impact on the brain. Interferon overactivation is also associated with low levels of apoptotic particle clearance, resulting in an accumulation of apoptotic products (such as DNA-CpG motifs and U-RNAs). Similar abnormalities are seen in patients with primary Sjogren's syndrome, systemic scleroderma, and polymyositis, as well as a few cases of rheumatoid arthritis. Immunomodulation treatments aiming at lowering interferon overactivity are being tried in people with such diseases [76].

Shreds of evidence have shown that the development of autoimmune illnesses in certain people who were given IFN-α suggests that this cytokine plays a key role in breaking tolerance and triggering autoimmune responses in such patients [77]. Similarly, IFN-γ may also contribute to autoimmune disorders in addition to its host defense actions. Although IFN-γ production has been reported to be disease-limiting in experimental allergic encephalomyelitis (EAE), it may have a role in autoimmune nephritis [78]. Moreover, increased vulnerability to infection with certain viruses and intracellular bacteria appears to be linked to the loss of functioning IFNGR1 that is involved in Type II IFN signaling [79].

Recent research suggests that even mild-to-moderate acute COVID-19 infection results in a continuing, prolonged inflammatory response, which is not seen with common coronavirus infection [80]. After surviving acute coronavirus disease 2019 (COVID-19) infection, some individuals develop post-acute COVID syndrome (long COVID (LC)) that lasts longer than 12 weeks. The mechanisms behind this activation are still being investigated, but they might include antigen persistence, autoimmunity triggered by antigenic cross-reactivity, or a reflection of damage repair. These findings show that individuals with COVID-19 have an aberrant immunological profile at long intervals after infection, indicating the presence of an LC syndrome [80]. In this aspect, learning more about the immunological components of diverse pathologies has yielded common themes. Because of these unifying concepts, immune-based therapeutics for viral respiratory diseases, autoimmune and neurodegenerative disorders must be identified.

#### **7. Closing remarks and outlook**

There is a worldwide interest in repurposing existing drugs and understanding mechanisms against viral, autoimmune, and neurodegenerative diseases. Structural determination, interaction with different co-solutes, and binding studies can facilitate the process of vaccine development, help in understanding the mechanism of anti-inflammation, and design a potent inhibitor for drug discovery. The interaction studies with different proteins will stabilize and/or destabilize, allowing deeper insight into various interactions (attractive and repulsive forces) to maintain a high functional protein population as this can probably be helpful for pre-clinical

### *IFN as Therapeutics: Now, Then, and Forever DOI: http://dx.doi.org/10.5772/intechopen.104718*

toxicological studies. Furthermore, beyond the therapeutic benefit to the individual patient, IFN therapy may aid public health measures aimed at delaying the spread of pandemic diseases and also minimizing the deterioration of symptoms in cases of autoimmune diseases and neurodegenerative disorders by reducing the time it takes for their symptoms to deteriorate. However, the most difficult element of creating therapy options for immune modulation against such illnesses is disentangling beneficial from harmful signals. So, for that purpose, targeted immune regulation can temper maladaptive factors enabling beneficial immune response against disorders which might help reduce its severity in the future.

## **Author details**

Ayesha Aiman1 , Seemi Farhat Basir1 \* and Asimul Islam<sup>2</sup> \*

1 Department of Biosciences, Jamia Millia Islamia, New Delhi, India

2 Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India

\*Address all correspondence to: sbasir@jmi.ac.in and aislam@jmi.ac.in

© 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 3**

## Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic Myeloproliferative Disorders

*Anna Prochwicz and Dorota Krochmalczyk*

## **Abstract**

It has been noted that interferon can exert an antiproliferative effect by stimulating cells of the immune system. Interferon has been shown to be effective in the treatment of chronic myeloproliferative neoplasms. Over the years, interferon alpha-2a and interferon alpha-2b have been introduced into the treatment of chronic myeloproliferation, followed by their pegylated forms. Studies have been showing the effectiveness of interferon alpha in reducing the number of platelets in essential thrombocythemia, reducing the need for phlebotomies in patients with polycythemia vera and also in reducing the number of leukocytes. Additionally, it turned out to be effective in reducing the size of the spleen. Interferon has also been shown to be effective in inducing long-term molecular remissions. The introduction of new forms of interferon such as ropeginterferon and the combination of interferon alpha with newly introduced drugs from other groups causes that interferon remains an important drug in the field of chronic myeloproliferative disorders. The chapter presents the results of clinical trials and the experiences of various centers in its usage for mieloproliferative neoplasms.

**Keywords:** chronic myeloproliferative disorders, polycythemia vera, essential thrombocythemia, myelofibrosis, ropeginterferon, pegylated interferon alpha

### **1. Introduction**

Chronic myeloproliferative disorders are a group of clonal diseases of the stem cell. It is a group of several diseases with some common features. They derive from a multipotential hematopoietic stem cell. A clone of neoplastic cells in all these neoplams is characterized by a lower proliferative activity than that of acute myeloproliferative diseases. In each of these diseases, leukocytosis, thrombocythemia, and polyglobulia may appear at some stage, depending on the diagnosis [1, 2].

The research on interferon has been going on since the 1950s [3]. Then, the attention was paid to its influence on the immune system. It has been noted that it can exert an antiproliferative effect by stimulating cells of the immune system [4]. In 1987, a publication by Ludwig et al. was published, which reported the effectiveness of interferon alpha in the treatment of chronic myeloproliferative disorders [5].

More and more new studies have been showing the effectiveness of interferon alpha in reducing the number of platelets, reducing the need for phlebotomies in patients with polycythemia vera and also in reducing the number of leukocytes. Moreover, interferon reduced the symptoms of myeloproliferative disorders such as redness and itching of the skin. Additionally, it turned out to be effective in reducing the size of the spleen.

Further studies on the assessment of remission using molecular-level response assessments indicate that the interferon action in chronic myeloproliferation diseases targets cells from the mutant clone with no effect on normal bone marrow cells [6].

Over the years, interferon alpha-2a and interferon alpha-2b have been introduced into the treatment of chronic myeloproliferation, followed by their pegylated forms. The introduction of pegylated forms allowed for a reduction in the number of side effects and less frequent administration of the drug to patients. In recent years, monopegylated interferon alpha-2b has been used to further increase the interval between drug administrations while maintaining its antiproliferative efficacy.

The exact mechanism of action of interferon alpha in the treatment of chronic myeloproliferative disease is still not fully understood, but it has an impact on JAK2 (Janus Kinase) signal transducers and activates the STAT signal pathway (Janus Kinase/SignalTransducer and Activator of Transcription).

Interferon alpha binds to IFNAR1 and IFNAR2c, which are type I interferon receptors. Interferon alpha has an impact on JAK2(Janus Kinase) signal transducers and activates the STAT signal pathway. The disturbances in this signaling pathway are observed in chronic myeloproliferative disorders [7].

Interferon inhibits the JAK-STAT signaling pathway by directly inhibiting the action of thrombopoietin in this pathway [8].

So far, three driver mutations have been described in the course of chronic myeloproliferative diseases that affect the functioning of the JAK-STAT pathway.

JAK2 kinase and JAK1, JAK3, and TYK2 kinases belong to the family of non-receptor tyrosine kinases. They are involved in the intracellular signal transduction of the JAK-STAT pathway. It is a system of intracellular proteins used by growth factors and cytokines to express genes that regulate cell activation, proliferation, and differentiation. The mechanism of JAK activation is based on the autophosphorylation of tyrosine residues that occurs after ligand binds to the receptor. JAK2 kinase transmits signals from the hematopoietic cytokine receptors of the myeloid lineage (erythropoietin, granulocyte-colony stimulating factor thrombopoietin, and lymphoid lineage [9].

A somatic G/T point mutation in exon 14 of the JAK2 kinase gene converts valine to phenylalanine at position 617 (V617F) in the JAK2 pseudokinase domain, which allows constitutive, ligand-independent activation of the receptor to trigger a proliferative signal [10].

Mutation of the MPL gene, which encodes the receptor for thrombopoietin, increases the sensitivity of magekaryocytes to the action of thrombopoietin, which stimulates their proliferation [11].

Malfunction of calreticulin as a result of mutation of the CARL gene leads to the activation of the MPL-JAK/STAT signaling pathway, which is independent of the ligand, as calreticulin is responsible, for the proper formation of the MPL receptor. Consequently, there is a clonal proliferation of hematopoietic stem cells [12].

*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

Below, we provide an overview of some clinical studies on the efficacy of interferon in chronic myeloproliferative disorders.

### **2. Chronic myeloproliferative disorders**

#### **2.1 Polycythemia vera**

Polycythemia vera (PV) is characterized by an increase in the number of erythrocytes in the peripheral blood.

Polycythemia vera is caused by a clonal mutation in the multipotential hematopoietic stem cell of the bone marrow. The mutation leads to an uncontrolled proliferation of the mutated cell clone, independent of erythropoietin and other regulatory factors. As the mutation takes place at an early stage of hematopoiesis, an increase of the number of erythrocytes as well as of leukocytes and platelets is observed in the peripheral blood. The cause of proliferation in PV independent from external factors is a mutation in the Janus 2 (JAK2) tyrosine kinase gene. The V617F point mutation in the JAK2 gene is responsible for about 96% mutation, and in the remaining cases the mutation arises in exon 12. Both mutations lead to constitutive activation of the JAK-STAT signaling pathway [13].

As a result of the uncontrolled proliferation, blood viscosity increases, which generates symptoms such as headaches and dizziness, visual disturbances, or erythromelalgia. As the number of all hematopoietic cells, including the granulocytes ones, increases, the difficult to control symptoms of their hyperdegranulation may appear, among which gastric ulcer or skin itching is often observed. During the disease progression, the spleen and liver become enlarged.

The most common complication of the disease is episodes of thrombosis, especially arterial one. During the course of the disease, it can also evolve into myelofibrosis or acute myeloid leukemia.

The treatment of PV is aimed at preventing thromboembolic complications, relieving the general symptoms, the appearance of hepatosplenomegaly as well as preventing its progression.

Each patient should receive an antiplatelet drug chronically, and usually acetylsalicylic acid is the choice. Most often, the treatment is started with phlebotomy in order to rapidly lower the hematocrit level. If cytoreductive therapy is necessary, the drugs of first choice are hydroxycarbamide and interferon [2].

However, the research on the mechanism of the action of interferons is still ongoing. In vitro studies with CD34+ cells from peripheral blood of patients diagnosed with polycythemia vera showed that interferon inhibits clonal changed cells selectively. It was found that interferon alpha-2b and pegylated interferon alpha-2a reduce the percentage of cells with JAK2 V617F mutation by about 40%. Pegylated interferon alpha-2a works by activating mitogen-activated protein kinase P38. It affects CD34+ cells of patients with polycythemia vera by increasing the rate of their apoptosis [6].

A case of a patient with PV with a confirmed chromosomal translocation t(6;8) treated with interferon alpha-2b, which resulted in a reduction of the clone with translocation by 50% from the baseline value, was also described [14].

In 2019, the results of a phase II multicenter study were published, which aimed at assessing the effectiveness of recombinant pegylated interferon alpha-2a in cases of refractory to previously hydroxycarbamide therapy. The study included 65 patients

with essential thrombocythemia (ET) and 50 patients with polycythemia vera. All patients had previously been treated with hydroxycarbamide and showed resistance to this drug or its intolerance.

The assessment of the response was performed after 12 months of treatment. Overall response rate to interferon was higher in patients diagnosed with ET than in patients with polycythemia vera. In essential thrombocythemia, the percentage of achieved complete remissions was 43 and 26% of partial remissions. The remission rate in ET patients was higher if calreticulin CALR gene mutation was present. Patients with polycythemia vera achieved complete remission in 22% of cases and partial remission in 38% of cases.

Treatment-related side effects that follow to discontinuation of treatment were reported in almost 14% of patients [15].

The duration of response to treatment with pegylated interferon alpha-2a and the assessment of its safety in long-term use in patients with chronic myeloproliferative disorders was the goal of a phase II of the single-center study. Forty-three adult patients with polycythemia vera and 40 patients with essential thrombocythemia were enrolled in the study. The complete hematological response was defined as a decrease in hemoglobin concentration below 15.0 g/l, without phlebotomies, a resolution of splenomegaly, and no thrombotic episodes in the case of PV, and for essential thrombocythemia—a decrease platelet count below 440,000/μl and two other conditions as above. The assessment of the hematological response was performed every 3–6 months. The median follow-up was 83 months.

The hematological response was obtained in 80% of cases for the entire group. In patients with polycythemia vera, 77% of patients achieved a complete response (CR) while 7% a partial response (PR). The duration of response averaged 65 months for CR and 35 months for PR. In the group of patients diagnosed with essential thrombocythemia, CR was achieved in 73% and PR in 3%. The durance of CR was 58 months and PR was 25 months.

The molecular response for the entire group was achieved in 63% of cases.

The overall analysis showed that the duration of hematological remission and its achievement with pegylated interferon alpha-2a treatment is not affected neither by baseline disease characteristics nor JAK2 allele burden and disease molecular status. There was also no effect on age, sex, or the presence of splenomegaly.

During the course of the study, 22% of patients discontinued the treatment, because of toxicity. Toxicity was the greatest at the beginning of treatment. The starting dose was 450 μg per week and was gradually tapered off.

Thus, on the basis of the above observations, the researchers established that pegylated interferon alpha-2a may give long-term hematological and molecular remissions [16].

The assessment of pegylated interferon alpha-2a in group of patients diagnosed with polycythemia vera only was performed. The evaluation was carried out on a group of 27 patients. Interferon decreased the JAK2 V617F allele burden in 89% of cases. In three patients who were JAK2 homozygous at baseline, after the interferon alpha-2a treatment wild-type of JAK2 reappeared. The reduction of the JAK2 allele burden was estimated from 49% to an average 27%, and additional in one patient the mutant JAK2 allele was not detectable after treatment. It can therefore be postulated that the action of pegylated interferon alpha-2a is directed to cells of the polycythemia vera clone [17].

In 2005, the results of treatment by pegylated interferon alpha-2b of 21 patients diagnosed with polycythemia vera and 21 patients diagnosed with essential thrombocythemia were published. In the case of polycythemia vera in 14 patients, PRV-1 gene

#### *Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

mutation was initially detected. In 36% of cases, PRV-1 expression normalized after treatment with pegylated interferon alpha-2b. For the entire group of 42 patients, the remission assessment showed that complete remission was achieved in 69% cases after 6 months of treatment. However, only in 19 patients remission was still maintained 2 years after the start of the study. Pegylated interferon alpha-2b was equally effective in patients with PV and ET. The use and the type of prior therapy did not affect the achievement of remission [18].

Another study with enrolled only PV patients included 136 patients. They were divided into two arms. One group received interferon alpha-2b and the other group received hydroxycarbamide. Interferon dosage was administered in 3 million units three times a week for 2 years and then 5 million units two times a week. Hydroxycarbamide was administered at a dose between 15 and 20 mg/kg/day.

In the group of patients treated with interferon, a significantly lower percentage of patients developed erythromelalgia (9.4%) and distal parasthesia (14%) compared with the group treated with hydroxycarbamide, for whom these percentages were respectively: 29 and 37.5%. Interferon alpha-2b was found to be more effective in inducing a molecular response, which was achieved in 54.7% of cases, in comparison with hydroxycarbamide—19.4% of cases, despite the fact that the percentage of achieved general hematological responses did not differ between the groups and amounted about 70%. The 5-year progression free period in the interferon group was achieved in a higher percentage (66%) than in the hydroxycarbamide group (46.7%) [19].

#### *2.1.1 Ropeginterferon (monopegylated interferon alfa-2b)*

The most recent form of interferon approved by the *European Medicines Agency* (EMA) for the treatment of patients is ropeginterferon. It is human recombinant interferon alpha-2b. Ropeginterferon is a monopegylated form of interferon alpha-2b. Ropeginterferon is conjugated with a two-arm methoxypolyethylene glycol (mPEG).

Thanks to these changes to the structure of the molecule, it was possible to achieve a significant increase in its half-life. Ropeginterferon can be administered subcutaneously to patients every 14 days. The clinical trials conducted so far have assessed the ropeginterferon dose from 50 micrograms to a maximum dose of 500 microgams administered as standard every 2 weeks. The possible dose change in case of side effects includes not only the reduction of the drug dose itself, but also the extension of the interval between doses. The extension of the dosing interval up to 4 weeks was assessed.

Ropeginterforn was approved in 2019 by the EMA for the use in patients diagnosed with polycythemia vera without splenomegaly, as monotherapy.

Ropeginterferon, like the previous forms of interferons used in treatment, is contraindicated in patients with severe mental disorders, such as severe depression. It is also a contraindication in patients with noncompensatory standard treatment of disorders of the thyroid gland as well as severe forms of autoimmune diseases. The safety profile of ropeginterferon is similar to that of other forms of alpha interferons. The most common side effects are flu-like symptoms [20].

Ropeginterferon has been shown to exhibit in vitro activity against JAK2-mutant cells. The activity of ropeginterferon against JAK2-positive cells is similar to that of other forms of interferons used actually for standard therapy. Ropeginterferon has an inhibitory effect on erythroid progenitor cells with a mutant JAK2 gene. At the same time, it has almost no effect on progenitor cells without the mutated allele (JAK2-wile-type) and normal CD34+ cells. A gradual decrease of JAK2-positive cells was observed in patients

with PV during ropeginterferon treatment. The examination was performed after 6 and 12 months of treatment. In comparison, the reduction in the percentage of JAK2 positive cells in patients treated with hydroxycarbamide was significantly lower.

These results may suggest that ropeginterferon may cause elimination of the mutant clone, but further prospective clinical trials are needed to confirm this theory. The evaluation was performed on a group of patients enrolled in the PROUD-PV study who were treated in France [21].

In 2017, a multicenter study was opened in Italy. The study was of the second phase. In total, 127 patients with polycythemia vera were included in the study. All patients enrolled on the study had low-risk PV. The clinical trial consisted of two arms. Patients received phlebotomies and low-dose aspirin in one arm and ropeginterferon in the other arm. The aim of the study was to achieve a hematocrit of 45% or lower without any evidence of disease progression. Ropeginterferon was administered every 2 weeks at a constant dose of 100 μg.

The response to the treatment was assessed after 12 months. The reduction of hematocrit to the assumed level was achieved in significantly higher percentage of patients in the ropeginterferon group than of patients who received only phlebotomies and aspirin. In addition, none of the patients treated with ropeginterferon experienced disease progression during the course of the study, while among those treated with phlebotomies, 8% of patients progressed.

Grade 4 or 5 adverse events were not observed in patients treated with ropeginterferon, and the incidence of remaining adverse event (AE) was small and comparable in both arms. The most common side effects in the ropeginterferon group were flulike symptoms and neutropenia; however, the third-grade neutropenia was the most common (8% of cases) [22].

One of the most important clinical studies on the use of ropeginterferon was the PROUD-PV study and its continuation: the CONTINUATION-PV study. These were three-phase, multicenter studies. The aim of the study was to compare the effectiveness of ropeginterferon in relation to hydroxycarbamide. The study included adult patients diagnosed with polycythemia vera treated with hydroxycarbamide for less than 3 years and no cytoreductive treatment at all. In total, 257 patients received this treatment. The patients were divided into two groups: those receiving ropeginterferon or the other being given hydroxycarbamide.

During the PROUD-study, drug doses were increased until the hematocrit was achieved below 45% without the use of phlebotomies, and the normalization of the number of leukocytes and platelets was reached.

The PROUD-PV study lasted 12 months. After this time, the patients continued the treatment under the CONTINUATION-PV study for further 36 months. After the final analysis performed in the 12th month at the end of PROUD study, it was found that the hematological response rates did not differ between the ropeginterferon and hydroxycarbamide treatment groups. These were consecutively 43% in the ropeginterferon arm and 46% in the control arm.

However, after analyzing the CONTINUATION- PV study, it turned out that after 36 months of treatment, the rates of hematological responses begin to prevail in the group of patients receiving ropeginterferon, 53% versus 38% in the control group. Thus, from the above data, it can be seen that the response rate to ropeginterferon increases with the duration of treatment [23].

Another analysis of patients participating in the PROUD and CONTINUATION studies was based on the assessment of treatment results after 24 months, dividing patients into two groups according to age (under and over 60 years).

*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

The initial comparison of both groups of patients showed that older patients had a more aggressive course of the disease. Patients over 60 years of age had a higher percentage of cells with a mutant JAK2 allele. They experienced both general symptoms and some complications, such as thrombosis, more frequently. Both patients under 60 years of age and over 60 years of age in the ropeginterferon arm had a higher rate of molecular response, namely 77.1 and 58.7% compared with the HU remission: 33.3 and 36.1%, respectively. Significantly higher reductions in the JAK2 allele were observed in both groups of patients after ropeginterferon treatment: it was 54.8% for younger patients and 35.1% for elderly patients. For comparison, this difference in the group of patients treated with HU was 4.5 and 18.4%, respectively.

What is more, the age did not affect the frequency of ropeginterferon side effects. In addition, the incidence of adverse ropeginterferon disorders was similar to that observed in the hydroxycarbamide group [24].

#### **2.2 Essential thrombocythemia**

Essential thrombocythemia is a clonal growth of multipotential stem cells in the bone marrow. The consequence of this is increased proliferation of megakaryocytes in the bone marrow and an increase in the number of platelets in the peripheral blood. The level of platelets above 450,000/μl is considered a diagnostic criterion.

Essential thrombocythemia may progress over time to a more aggressive form of myeloproliferation, i.e., myelofibrosis. The disease can also evolve into acute myeloid leukemia or myelodysplastic syndrome, both with very poor prognosis. Thromboembolic complications are serious, and they concern over 20% of patients. Thrombosis occurs in the artery and venous area. Moreover, in patients with a very high platelet count, above 1,000,000/μl, bleeding may occur as a result of secondary von Willebrand syndrome [1, 2].

The treatment of ET is primarily aimed to prevent thrombotic complications.

In low-risk patients, only acetylsalicylic acid is used. In cases of high-risk patients, hydroxycarbamide is the first-line drug for most patients. Anagrelide and interferon are commonly used as second-line drugs.

Due to the possible effects of hydroxycarbamide of cytogenetic changes in the bone marrow cells after long-lasting usage, some experts recommend the use of interferon in younger patients in the first line. Interferon is also used as the drug of choice in patients planning a pregnancy [25].

The efficacy of pegylated interferon alpha-2a was assessed on the basis of the group of 39 patients with essential thrombocythemia and 40 patients with polycythemia vera.

Of the overall group, 81% of patients were previously treated prior to the study entry. The patients received pegylated interferon alpha-2a in a dose of 90 μg once a week. The dose of 450 μg was associated with a high percentage of intolerance.

In patients with essential thrombocythemia, the complete remission was achieved in 76%, while the overall hematological response rate brought 81%. Moreover, the molecular remission was achieved in 38%, in 14% of cases, JAK2 transcript became not detectable.

Patients diagnosed with polycythemia vera achieved 70% complete hematological remission and 80% general hematological response to treatment. JAK2 transcript was undetectable in 6% of patients. Molecular remission was achieved in 54% of cases.

Pegylated interferon alpha-2a at the dose of 90 μg per week was very well tolerated. In total, 20% of patients experienced a grade of 3 or 4 of adverse reaction, which was neutropenia. In addition, an increase in liver function tests was observed. Grade 4 of AE was not observed among patients who started the treatment with 90 μg/week while grade 3 neutropenia was an adverse event in only 7% of cases [26].

The effect of interferon alpha-2b treatment in patients with ET and PV was investigated. The study was prospective. Some of the results concerning the group of patients with polycythemia vera are presented in the subsection on polycythemia vera. In total, 123 patients with diagnosed essential thrombocythemia participated in the study. All of them received interferon alpha-2b. The patients were divided into two groups depending on the presence of the JAK2 V617F mutation. The enrolled patients were between 18 and 65 years of age. The treatment they received was, sequentially, interferon alpha-2b in the dose of 3 million units three times a week for the first 2 years, after which time the dose was changed into a maintenance dose, which amounted to 5 million units two times a week.

The analysis showed that the patients with the JAK2 V617F mutation present in a higher percentage achieved an overall hematological response as well as a complete hematological response. The overall hematological response was achieved in 83% of patients with JAK2 mutation, and the complete hematological remission was achieved in 23 cases. In the group of ET patients without the JAK2 V617F mutation, overall hematological response was achieved in 61.4%, while the complete hematological remission was achieved in 12 patients. The 5-year progression-free survival was obtained in 75.9% in the JAKV617F group and only in 47.6% without the mutation.

A significant proportion of patients experienced mild side effects. Grade 3 and 4 of adverse events were severe, most of them being a fever. The isolated cases of elevated liver tests and nausea have also been reported [19].

Pegylated interferon alpha-2b in patients with essential thrombocythemia who were previously treated with hydroxycarbamide, anagrelide, and other forms of interferon alpha, however, due to the lack of efficacy or toxicity, the patients required a change of treatment, was assessed. Pegylated interferon alpha-2b turned out to be effective in these cases. It led to the complete hematological remission in 91% of patients after 2 months of therapy, and in 100% of patients after 4 months. However, merely 11 patients participated in the study. Also only two patients required treatment discontinuation due to the side effects such as depression and general fatigue grade 3 [27].

#### *2.2.1 Pregnancy*

In case of pregnant patients, interferon is currently considered the only safe cytoreductive drug. Over the years, several analyses of the results of interferon treatment during pregnancy have been carried out.

The assessment of 34 pregnancies in 23 women diagnosed with ET was performed retrospectively. All the pregnancies included in the analysis were of high risk. This high risk was associated with a high platelet count above 1,500,000/μl, a history of thrombotic episode, severe microcirculation disorders, or a history of major hemorrhage.

It turned out that the use of interferon allowed the birth of an alive child in 73.5% of cases. There was no difference in efficacy between the basic and pegylated forms of interferon alpha. In pregnancies without interferon treatment, the percentage of live births was only 60%. Moreover, it was not found if the presence of the JAK2 V617F mutation had any influence on the course of pregnancy [28].

An analysis of the course of pregnancy in patients with ET was assessed in Italy. Data from 17 centers were taken into account. Data from 122 pregnancies were

*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

collected from 92 women. In patients diagnosed with essential thrombocythemia, the risk of the spontaneous loss of pregnancy is about 2.5 times higher than among the general population. In the contrary to the study quoted above, it was found that the presence of the JAK2 mutation increases the risk of pregnancy loss. The proportion of live births in patients exposed to interferon during pregnancy was 95%, compared with 71.6% in the group of patients not treated with interferon.

The multivariate analysis also showed that the use of acetylsalicylic acid during pregnancy had no effect on the live birth rate of patients with ET [29].

Whatever its form, interferon is the drug of first choice in pregnancy. Hydroxycarbamide and anagrelide should be withdrawn for about 6 months, and at least for 3 months, before the planned conception. Experts recommend the use of interferon in high-risk pregnancies [30]. A Japanese analysis of 10 consecutive pregnancies in ET patients showed 100% live births in patients who received interferon [31].

#### **2.3 Myelofibrosis**

In myelofibrosis (MF), monoclonal megakaryocytes produce cytokines that stimulate the proliferation of normal, non-neoplastic fibroblasts and stimulate angiogenesis. The consequence of this is the gradual fibrosis of the bone marrow, impaired hematopoiesis in the bone marrow, and the formation of extramedullary location mainly in the sites of fetal hematopoiesis, i.e., in the spleen and the liver.

The production of various cytokines by neoplastic megakaryocytes leads to the proliferation of normal, noncancerous fibroblasts as well as to increased angiogenesis.

Progressive bone marrow fibrosis leads to worsening anemia and thrombocytopenia. On the other hand, the production of proinflammatory cytokines by megakaryoblasts leads to the general symptoms such as weight loss, fever, joint pain, night sweats, and consequently, progressive worsening of general condition.

The prognosis for myelofibrosis is poor. In about 20% of patients, myelofibrosis evolves into acute myeloid leukemia with poor prognosis.

Currently, the only effective method of treatment that gives a chance to prolong the life is allogeneic bone marrow transplantation. However, this method is only available to younger patients.

The goal of treatment of patients who have not been qualified for allotranspalntation is to reduce the symptoms and to improve the patient's quality of life. In case of leukocytosis cytoreducing drugs, such as hydroxycarbamide, melphalan, or cladribine can be used. They cause a reduction in the number of leukocytes and may, to some extent, inhibit splenomegaly. Interferon alpha has been used successfully for the treatment of myelofibrosis for many years. The results of its effectiveness will be presented below [2].

Currently, the JAK2 inhibitor ruxolitinib is approved for the treatment of myelofibrosis with enlarged spleen in intermediate and high-risk patients. Ruxolitinib reduces the size of the spleen, reduces general symptoms, and improves the quality of life; however, it does not prolong the overall survival of patients [32].

In 2015, the results of a retrospective study were published to compare the histological parameters of the bone marrow before and after interferon treatment. Twelve patients diagnosed with primary myelofibrosis as well as post-PV MF and post-ET MF were enrolled in the study. Patients were treated with pegylated recombinant interferon alpha-2a or recombinant interferon alpha-2b in standard doses. The time of treatment was from 1 to 10 years. Some patients had previously been treated with hydroxycarbamide or anagrelide. In all cases, karyotype was normal. The prognostic

factor of Dynamic International Prognostic Scoring System (DIPSS) was assessed at the beginning as well as during the treatment.

Bone marrow cellularity decreased in cases with increased bone marrow cellularity before the treatment. After the interferon treatment, a reduction in the degree of bone marrow fibrosis was found. The parameters, such as the density of naked nuclei and the density of megakaryocytes in the bone marrow, also improved.

It proves that if the JAK2 V617F mutation had been present, DIPSS was decreased after interferon treatment. This relationship was not observed in patients without the JAK2 V617F mutation. The improvement in peripheral blood morphological parameters and the overall clinical improvement correlated with the improvement in the assessed histological parameters of the bone marrow.

Before the initiation of interferon, seven patients had splenomegaly. During the treatment with interferon, the complete resolution of splenomegaly was achieved in 17% of patients (two cases), and its size decreased in 25% (three cases). A good clinical response was achieved in 83% during interferon therapy. There was no significant difference in response between the two types of interferon used [33].

A prospective study was also conducted in patients with low and intermediate-1 risk group myelofibrosis. Seventeen patients were enrolled. Patients received interferon alpha-2b (0.5–3 milion units/three times a week) or pegylated interferon alpha-2a (45–90 μg/week). The duration of therapy was on average 3.3 years.

Most of the patients responded to the treatment. Partial remission was found in seven patients and complete remission in two patients. Moreover, in four cases, the disease was stabilized and in one case the clinical improvement was achieved. Three patients did not respond to treatment at all and progressed to myelofibrosis. Additionally, the assessment in reducing spleen size was performed. At baseline, 15 patients have splenomegaly, nine of them achieved the compete regression of spleen size [34].

However, the efficacy of interferon in the treatment of myelofibrosis appears to be limited only to a less advanced form, when the bone marrow still has an adequate percentage of normal hemopoiesis and the marrow stroma is not significantly fibrotic. In more advanced stages, interferon was not shown to have any significant effect on the regression of the fibrosis process [35].

In 2020, the results of the COMBI study were published. That was a two-phase, multicenter, single-arm study that investigated the efficacy and safety of the combination of ruxolitinib and pegylated interferon alpha. Thirty-two patients with PV and 18 patients with primary and secondary myelofibrosis participated in the study. The patients were at age 18 and older. Remission was achieved in 44% of myelofibrosis cases, including 28% (5 patients) of complete remission. In patients with PV, the results were slightly worse: 31% of remissions, including 9% of complete remissions. Patients received pegylated interferon alpha-2a (45 μg/week) or pegylated interferon alpha-2b (35 μg/week) in low doses and ruxolitinib in doses of 5–20 mg twice a day.

For the entire group of patients (with PV and MF), the initial JAK2 allele burden was 47% at baseline, and after 2 years of treatment with interferon and ruxolitinib, it decreased to 12%.

The treatment toxicity was low. The highest incidence of side effects occurred at initiation of therapy. It was mostly anemia and thrombocytopenia.

The observations from the COMBI study show that, for the combination of interferon in lower doses with ruxolitinib, it may be effective and well tolerated even in the group of patients who had intolerance to interferon used as the only drug in higher doses. The combined treatment improved the bone marrow in terms of fibrosis and its cellularity. It also allowed to improve the value of peripheral blood counts [36].

#### *Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

It is currently known that some of the additional mutations are associated with a worse prognosis in patients with myelorpoliferation, including patients with myelofibrosis. Some of these mutations have been identified as high-risk molecular mutations. These are ASXL1, EZH2, IDH1/2, or SRSF2. Earlier studies have shown their association with a more aggressive course of the disease, worse prognosis, and shorter survival of patients, as well as a poorer response to treatment. Due to their importance, they have been included in the diagnostic criteria of myelofibrosis [37].

It is also known that the presence of driver mutations, i.e., JAK2, CALR, and MPL or triple negativity, may affect the course of myeloproliferation, including the incidence of thromboembolic complications.

The assessment of the influence of driver mutations and a panel of selected additional mutations on the effectiveness of interferon treatment in patients with myelofibrosis was performed on a group of 30 patients. Only the patients with low- and intermediate-1-risk were enrolled in the study. The treatment with pegylated interferon alpha-2a or interferon alpha-2b resulted in a complete remission in two patients and partial remission in nine patients. The disease progressed in three cases. One patient relapsed and four died. The remaining patients achieved a clinical improvement or disease stabilization. In the studied group, it was not found if the effectiveness of interferon treatment was influenced by the lack of driver mutations. Among the group of four patients with additional mutations, two died and one had disease progression. It was a mutation of ASXL1 and SRSF2. The treatment with interferon in patients without additional molecular mutations in the early stages of the disease may prevent further progression of the disease [38].

The side effects of interferon in the group of patients with myelofibrosis are similar to those occurring after the treatment of other chronic myeloproliferative diseases. The most frequently described are hematological toxicity- anemia and thrombocytopenia, less often is the appearance of leukopenia. Hematological toxicity usually resolves with dose reduction or extension of the dose interval. The most frequently nonhematological toxicity was fatigue, muscle pain, weakness, and depression symptoms. All symptoms are usually mild and do not exceed grade 2 [38].

However, the use of interferon in the treatment of myelofibrosis has not been recommended as a standard therapy. Interferon is still being evaluated in clinical trials, or it is used in selected patients as a nonstandard therapy in this diagnosis.

#### **2.4 Mastocytosis**

Mastocytosis is characterized by an excessive proliferation of abnormal mast cells and their accumulation in various organs.

The basis for the development of mastocytosis is ligand-independent activation of the KIT receptor, resulting from mutations in the KIT proto-oncogene. The KIT receptor is a trans membrane receptor with tyrosine kinase's activity. Its activation stimulates the proliferation of mast cells. That excessive numbers of mast cells infiltrate tissues and organs and release mediators such as histamine, interleukine-6, tryptase, heparin, and others, which are responsible for the appearance of symptoms typical of mastocytosis. In addition, the infiltration of tissues for mast cells itself causes damage to the affected organs.

The prognosis of mastocytosis depends on the type of the disease. In the case of cutaneous mastocytosis (CM), in the majority of cases prognosis is good and the disease does not shorten the patient's life, but in aggressive systemic mastocytosis

(ASM), the average follow-up is about 40 months. Mast cell leukemia has a poor prognosis with a median follow-up of approximately 1 year.

Systemic mastocytosis usually requires the implementation of cytoreductive therapy. The first line of therapy is interferon alone or its combination with corticosteroids. In aggressive systemic mastocytosis, the first line in addition to interferon 2-CdA can be used. An effective drug turned out to be midostaurin in the case of the present KIT mutation. In patients without the KIT D816V mutation, treatment with imatinib may be effective. In the case of mast cell leukemia, multidrug chemotherapy is most often required, as in acute leukemias, followed by bone marrow transplantation [39].

Systemic mastocytosis requiring treatment is a rare disease, this is why the studies available in the literature evaluating various therapies concern mostly small groups of patients.

In 2002, the French authors presented their experiences on the use of interferon in patients with systemic mastocytosis. They included 20 patients. The patients received interferon alpha-2b in gradually increased doses.

The patients were assessed after 6 months. In cases in which bone marrow was infiltrated for mast cells at baseline, it still remained infiltrated after 6 months of treatment.

However, the responses were obtained in terms of symptoms related to mast cell degranulation. Partial remission was achieved in 35% of patients and minor remission in 30%. It concerns mainly skin lesions and vascular congestion. Moreover, the assessment of the histamine level in the plasma revealed a decrease of it in patients who previously presented symptoms related to the degranulation of mast cells, such as gastrointestinal disorders and flushing.

A high percentage of side effects were found during treatment. They concerned 35% of patients. Depression and cytopenia were most frequent ones [40].

Another analysis was a report of five patients with systemic mastocytosis treated with interferon and prednisolone. All patients received interferon alpha-2b in a dose of 3 million units three times a week and four patients additionally received prednisolone. Four patients responded to interferon treatment at varying degrees. One patient, who at baseline had bone marrow involvement by mast cells in above 10%, progressed to mast cell leukemia. In two patients, the symptoms C resolved completely and in one of them they partially disappeared. In one case, stabilizing disease was achieved [41].

In 2009, a retrospective analysis of patients treated with cytoreductive therapy due to mastocytosis was published. The authors collected data from 108 patients treated at the Mayo Clinic. This analysis allowed for the comparison of the efficacy of four drugs used in systemic mastocytosis. There were interferon alpha alone or in the combination with prednisone—among 40 patients, hydroxycarbamide—among 26 ones, imatinib—among 22 persons, and 2-chlorodeoxyadenosine (2-CdA)—among 22 patients.

After dividing the patients into three additional groups on the basis of the type of mastocytosis—indolent systemic mastocytosis, aggressive systemic mastocytosis, and systemic mastocytosis associated with another clonal hematological nonmast cell lineage disease (SM-AHNMD)—the effectiveness of each of type of therapy was assessed.

The highest response rates in indolent and aggressive mastocytosis were achieved with interferon treatment. They were 60% of the responses in both groups, and in the SM-AHNMD group of patients, the percentage was also one of the highest and amounted to 45%. The second most effective drug was 2-CdA. The response rates were 56% for indolent MS, 50% for aggressive MS, and 55% for SM-AHNMD. The

#### *Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

patients treated with imatinib achieved response in 14, 50, and 9% by following groups, respectively. In contrast, patients with indolent and aggressive systemic mastocytosis did not respond to hydroxycarbamide treatment at all. The response rate in both groups was 0%. However, patients with MS associated with another clonal hematological nonmast cell lineage disease achieved 21% response to hydroxycarbamide. Additionally, it was found that only interferon relieved symptoms caused by the release of inflammatory mediators by mast cells.

The additional analysis showed no influence of the TET 2 mutation on the response to treatment [42].

In the literature, there are also single cases of mastocytosis presenting trials of nonstandard treatment. That is description of a patient with systemic mastocytosis with mast cell bone marrow involvement. Mutation of c-kit Asp816Val was present. Patient progressed despite treatment with dasatinib and 2-chlorodeoxyadenosine. The patient developed symptoms related to the degranulation of mast cells and increased ascites.

The patient was treated with pranlukast, which is an anti-leukotriene receptor antagonist due to an asthma episode. The rate of ascites growth decreased significantly after one administration. The patient required paracentesis every 10 days and not every 3 days, as before starting to take the drug. After 15 days of treatment with pranlukast, the patient received interferon alpha, which resulted in complete regression of ascites, resolution of pancytopenia, and complete disappearance of the c-kit mutation clone. The infiltration of mast cells in the bone marrow significantly decreased [43].

Interferon alpha was also effective in a patient with systemic mastocytosis associated with myelodysplastic syndrome with the c-kit D816V mutation, which was refractory to imatinib treatment [44].

Interferon alpha also proved to be effective in the treatment of osteoporotic lesions appearing in the course of mastocytosis.

The series of 10 cases with resolved mastocytosis and osteoporosis-related fractures was presented in 2011. The patients received interferon alpha in a dose of 1.5 million units three times a week as well as pamindronic acid. The patients were treated for an average of 60 months. For the first 2 years, pamindronate was given at a dose of 1 mg/kg every month, and then every 3 months.

During the course of the study, no patient had a new-bone fracture. The level of alkaline phosphatase decreased by 25% in relation to the value before treatment and tryptase by 34%. Bone density increased during treated with interferon and pamindronate. The increase was on average 12% in the spine bones and 1.9% in the hip bones. At the same time, there was no increase in the density of the hip bone and a minimal increase in the density of the spine in patients treated with pamindronate alone.

The results of this observation suggest that it is beneficial to add low doses of interferon alpha to pamindronate treatment in terms of bone density increase [45].

That experiences show that interferon used in systemic mastocytosis significantly improves the quality of life of patients by inhibiting the symptoms caused by degranulation of mast cells. They prevent bone fractures and, in some patients, they cause remission of bone marrow infiltration by mast cells.

#### **2.5 Chronic neutrophilic leukemia**

Chronic neutrophilic leukemia (CNL) is a very rare disease. It is characterized by the clonal proliferation of mature neutrophils.

The diagnostic criteria proposed by the World Health Organization (WHO) comprise leukocyte counts above 25,000/μl (including more than 80% of rod and segmented *neutrophils* in the bone marrow blast cells count below 5%), normal neutrophils maturation, and an increase of neutrophilopoiesis. Also the presence of the CSF3R gene mutation is required.

Physical examination often shows enlargement of the liver and spleen, moreover, patients complain on weight loss and weakness [1].

The prognosis varies. The average survival time for patients with CNL is less than 2 years.

Only few descriptions of chronic neutrophilic leukemia are available in the literature, and these are mostly single case reports.

Because it is an extremely rare disease, there are no established and generally accepted treatment standards. In most cases, patients are given hydroxycarbamide or interferon. Patients who are eligible for a bone marrow transplant may benefit from this treatment. Bone marrow allotransplantation remains the only method that gives a chance for a significant extension of life.

The German authors presented a series of 14 cases of chronic neutrophilic leukemia. The group of patients consisted of eight women and six men. The average age was 64.7 years. From the entire group of patients, longer survival was achieved only in three cases. One of these patients was treated with interferon alpha and achieved hematological remission, the other underwent bone marrow allotransplantation from a family donor, and the third one was treated with hydroxycarbamide and transfusions as needed. The follow-up period of the patient after allogeneic matched related donor transplantation (allo-MRD) was 73 months, and for the patient after interferon treatment it was 41 months.

The remaining patients died within 2 years of diagnosis. Six patients, the largest group, died due to intracranial bleeding, three patients died because of leukemia cell tissue infiltration, one patient because of the disease transformation into leukemia, and one patient because of pneumonia [46].

It can be seen from these experiences that treatment with interferon alpha can significantly extend the survival time of patients.

The case of a 40-year-old woman diagnosed with chronic neutrophilic leukemia is presented by Yassin and coauthors. Initially, the patient had almost 41,000 leukocytes in the peripheral blood. In a physical examination, splenomegaly and hepatomegaly were not present. Patient received pegylated interferon alpha-2a. The initially dose was 50 μg once a week for the first 2 weeks, then the dose was increased to 135 μg weekly for 6 weeks, and then the dose interval was extended to another 2 weeks. As a result of the treatment, the general condition of the patient improved and the parameters of peripheral blood counts were normalized [47].

Another case report presented in the literature describes a 41-year-old woman diagnosed with CNL accompanied by focal segmental glomerulosclerosis (FSGS). The patient had increasing leukocytosis for several months. On the admission to the hospital, leukocytosis was 94,000/μl. Moreover, the number of platelets in the morphology exceeded 1,000,000/μl. More than a year earlier, the patient had splenectomy due to splenomegaly and spleen infraction.

Additionally, JAK2 V617F mutation was found. Some authors suggest that the presence of JAK2 mutation may be associated with longer survival in CNL.

The patient received hydroxycarbamide for 3 months and reduction in the number of leukocytes was achieved. After this time, interferon alpha-2b was added to hydroxycarbamide. As a result, focal segmental glomerulosclerosis disappeared and the renal tests improved [48].

*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

Another case of chronic neutrophilic leukemia with a JAK2 gene mutation concerns a 53-year-old man. The patient's baseline leukocytosis was 33,500/μl, including the neutrophil count of 29,700/μl. The patient also had splenomegaly.

The treatment with interferon alpha-2b at a dose of 3 million units every other day was started. After a month of treatment, the number of leukocytes was reduced to less than 10,000/μl. Then the patient was treated chronically with interferon alpha-2b in doses of 3 million units every 2 weeks. As a result of the therapy, the number of leukocytes remains between 8 and 10,000/μl. The patient remains in general good condition [49].

A series of two CNL cases are also shown. The first patient was a 70-year-old woman with stable leukocytosis of about 35,000/μl and the remaining morphology parameters in normal range. The patient was only observed for 5 years until hepasplenomegaly progressed rapidly. Then, interferon alpha-2b was included. Due to the treatment, the rapid regression of hepatosplenomegaly was achieved.

The second case is a 68-year-old woman with baseline leukocytosis of almost 14,000/μl. In this case, the treatment with hydroxycarbamide was started immediately. However, no improvement was achieved. After 6 weeks of HU treatment, interferon alpha-2b 3 million units 3 times a week was implemented and leukocytosis decreased. Due to the interferon treatment, the disease stabilized for a long time. Because the patient experienced an adverse reaction, a severe flu-like syndrome, interferon was discontinued. After interferon withdrawal, the disease progressed gradually and the treatment attempts by busulfan and 6-mercaptopurine were unsuccessful. Therefore, interferon was readministered and the disease went into remission. Interferon treatment was continued at a reduced dose. The disease regression was achieved again.

Additionally, the patient showed an improvement in the function of granulocytes in terms of phagocytosis and an improvement in neutral killer (NK) cell function after treatment with interferon [50].

The above examples show that interferon alpha is effective in the treatment of chronic neutrophilic leukemia. The side effects are rare and can be managed with dose reductions. Moreover, in these cases, interferon is also effective in a reduced dose. Disease remission or regression can be achieved without typical of CNL complications, such as intracranial bleeding.

#### **2.6 Another**

Interferon has been used in the past to treat chronic myeloid leukemia. The treatment with tyrosine kinase inhibitors is now a standard practice. However, in a small number of patients, they are ineffective or exhibit unmanageable toxicity. Therefore, the attempts are underway to use interferon in combination with TKI in lower doses, which is to ensure the enhancement of the antiproliferative effect while reducing the toxicity.

There are ongoing attempts to use ropeginterferon in patients diagnosed with chronic myeloid leukemia, in whom treatment with imatinib alone has not led to deep molecular response (DMR). The first phase study was conducted in a small group of patients with chronic myeloid leukemia. The patients in first chronic phase treated with imatinib who did not achieve DMR, but in complete hematologic remission and complete cytogenetic remission, were included in the study. Patients have been treated with imatinib for at least 18 months. Twelve patients were enrolled in the study, and they completed the study according to the protocol. These patients


## *Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*


## **Table 1.**

*Comparison of the effectiveness of interferon in chronic myeloproliferative disorders.*

received additional ropeginterferon to imatinib and four achieved DMR. Low toxicity was observed during the treatment. Among the hematological toxicities, neutropenia was the most common. There was no nonhematological toxicity with a degree higher than 1/2 during the treatment. Moreover, it has been found that better effects and fewer side effects are obtained when ropeginterferon is administered for a longer time, but in lower doses. The comparison of the effectiveness of interferon in chronic myeloproliferative disorders based on selected articles is presented in **Table 1** [51].

## **3. Conclusions**

Interferon alpha appears to be an effective and safe drug in the most type of chronic myeloproliferative disorders. Nowadays, all forms of its using have similar effectiveness. Interferon alpha can be effective even in cases of resistance for first-line treatment. Trial research is currently underway to combine it with some new drugs, such as ruxolitinib, and to add it to the already well-established therapy, it is a promising option for patients with refractory disease.

From time to time, new forms of interferon, such as ropeginterferon, are introduced, which gives hope for better effectiveness, better safety profile, and greater comfort in its use for patients who have to be treated for many years. In the case of the use of interferons alpha in the treatment of chronic myeloproliferative diseases, there are still opportunities to extend its use and to study its combination with newly introduced drugs.

## **Author details**

Anna Prochwicz1 \* and Dorota Krochmalczyk<sup>2</sup>

1 Department of Hematology, Specialized Hospital, Nowy Sącz, Poland

2 Department of Hematology, Jagiellonian University Medical College, Cracow, Poland

\*Address all correspondence to: annaprochwicz@interia.pl

© 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.

*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

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*Perspective Chapter: Impact of Interferon Alpha/Beta in the Management of Chronic... DOI: http://dx.doi.org/10.5772/intechopen.104501*

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Section 3
