Description of role of autophagy along with disease implications in various ocular tissues.
\r\n\tThe WHO classification in 2007; was based on the histogenesis and cell origin of the tumor. In the latest classification made in 2016; to better characterize the tumor and obtain better data on its prognosis; The combination of molecular and genetic biomarkers and histopathological features of the tumor was used. Despite all current treatment approaches, the median survival time is around 12 months in most GBM patients. Compared with the situation of some types of successfully treated cancers; the survival time of GBM patients is not at an acceptable level today. In the treatment of CNS tumors; surgery, chemotherapy, and radiation treatments (x-rays, gamma rays, electron and proton beams) are used. The therapeutic potential of chemotherapy; New strategies are needed to increase drug concentration at the diseased site, as this largely depends on the ability of the chemotherapeutic agent to achieve effective concentrations at tumor localization. Based on our better understanding of the genetic and molecular characteristics of CNS tumors; Targeted therapies, including vaccines, and treatment protocols such as immunotherapy are promising developments.
\r\n\r\n\tThis book supposes to be written by many authors who have an internationally honored place in their field to share their ideas about the treatment of CNS tumors. Surgery, Radiotherapy, Chemotherapy and Antiangiogenic Therapy Protocols, Immunotherapy, Molecular Therapy, Specific target-agents therapy with Nanoparticles and Gene Therapy for CNS tumors among the book chapters.
\r\n\tIn these sections; there are many practical pieces of information that can help the students who graduated from the Medicine Faculty and specialist doctors who are interested in Neurosurgery.
As a housekeeping cellular degradative and recycling process, autophagy is indispensable for the maintenance of ocular physiology. Since the early 2000s, research in understanding the mechanism and role of autophagy in development and disease has received a tremendous boost [1]. A rapidly growing wealth of data, focused on the diverse role of autophagy in ocular development, physiology, or disease, has enabled researchers to begin understanding this complex process with the hope of manipulating it as a therapeutic tool in treating a myriad of disorders that often lead to loss of visual acuity or complete blindness [2, 3].
\nThe eye has a complex anatomy (Figure 1A), with a plethora of specialized cells working together to create the visual perception [4]. Almost all cells in the developed eye have some common characteristics; they have high metabolic rates; are highly differentiated and are either post-mitotic or slowly dividing [5–10]. In addition, owing to the high blood perfusion rates, the eye is an oxygen-rich organ that, along with stressors like UV radiation and visible light, provides a highly oxidative microenvironment leading to cellular damage [7, 11, 12]. In order to combat this onslaught of oxidative damage, cells require not only effective antioxidant defense mechanisms but also cellular repair both at the organellar and macromolecular levels. Autophagy (referring mainly to macroautophagy), along with proteasomal degradation and DNA repair mechanisms, provides this critical housekeeping service to almost every cell type in the eye from the cornea in the anterior part of the eye to the retina/choroid in the posterior [3].
\n(A) Basic anatomy of the eye: principal ocular tissue components are shown. (B) Diagrammatic cross section of the retina. (C) Fundus image of a healthy adult eye (kindly provided by Dr. Yang Sun, MD, PhD, Department of Ophthalmology, IU School of Medicine, Indianapolis) with no ocular disease history is shown. The ganglion cell axons exit the eye at the optic disk, which is a ‘blind spot’ due to the complete absence of photoreceptors. The macula is tightly packed with photoreceptors and is critical for central vision. The fovea is a small pit-like area within the macula highly enriched with cone photoreceptors.
Perhaps, the most convincing evidence of the importance of autophagic activity in the eye is the preferential expression of autophagic proteins in the ocular cells and diurnal variation in expression of autophagic protein expression in the retina [13, 14]. As early as 1977, Reme et al. showed increased autophagosome formation in the inner segments of the photoreceptor cells 3 hours after maximum photoreceptor disk shedding in the rat retina [15, 16]. Diurnal variation in autophagosome formation rates was shown to be strongly dependent on light and amplitudes were severely dampened in animals kept under constant darkness [17, 18]. Recent reports have shown autophagic activity in the retinal pigmented epithelium (RPE) to be strongly correlated to the phagocytosis of photoreceptor outer segments (POS) underlining the importance of autophagy not just in being a housekeeping process but as an essential component of RPE function [19, 20].
\nBecause of its dual role in cell survival and death, autophagy has often been referred to as a ‘double-edged sword’ [21, 22]. As a degradative and recycling pathway, autophagy is essential for sequestration and digestion of toxic waste that could otherwise lead to loss of cell function and eventually lead to cell death. Autophagy (specifically macroautophagy) remains the only known process by which damaged cellular organelles as large as the mitochondria can be digested and recycled [23]. Metabolites generated from autophagic digestion and recycling serve as essential components for new macromolecules and organelles. Because of the ability of this process to be upregulated when the cell is subjected to stress such as nutrient starvation, oxidative stress, hypoxia, and growth factor depletion, autophagy can be thought of as an adaptive process that can meet the energy demand under unfavorable conditions [24].
\nDue to the generally non-dividing nature of many of the constituent cells of the eye, most reports on autophagy in the eye have concluded it to be a necessary cytoprotective mechanism that prevents the accumulation of cellular damage and inflammation over the lifetime of an individual [25, 26]. However, reduced autophagic efficiency is implicated in a number of ocular pathologies such as age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), photoreceptor degeneration, and ocular infections. Because of its ‘destructive self-eating’ nature, when autophagic activity exceeds a certain threshold or duration, it may actually promote cellular demise. Moreover, autophagy can ‘cross talk’ with other cell death modalities like apoptosis to influence overall cell fate [26]. It is thus critical to understand the mechanism and functional role of autophagy in specific cells of the eye before autophagic modulation be incorporated in ocular therapeutic strategies.
\nIn this chapter, we summarize the overall understanding of the role of autophagy in development and normal aging of the eye. We then describe the aspects of autophagy with respect to ocular diseases.
\nThere is increasing evidence that autophagy plays a critical role in ocular development and homeostasis. Developmentally, the vertebrate eye derives from coordinated interactions between neuroepithelium, surface ectoderm, and extraocular mesenchyme (originating from neural crest and mesoderm) [27]. The major development of the eye occurs between the 3rd and 10th weeks of fetal development with the initial formation of the optic vesicles followed by the gradual formation of the lens and the optic nerve [27–30]. Development of the lens requires maturation of lens fibers by degrading the mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum to create the transparent organelle-free zone to allow passage of light into the ocular chamber (reviewed in [31]). During development, the hyaloid artery supplies the lens with much needed nutrition and eventually its distal end degrades in the inner vitreous of the eye bulb, while the proximal end becomes the central retinal artery [32]. While the pigmented layer and the retina form from the outer and inner layers of the posterior (4/5th) optic cup, respectively, iris and the ciliary body are formed from the anterior (1/5th) region. The sclera and choroid are formed from the mesenchyme on the outer side of the optic cup. The primary and secondary lens fibers form the lens. The vitreous humor is a gel-like substance formed from the mesenchymal cells of the neural crest (reviewed in [33]). In the normal human eye, the photoreceptors continue to mature after birth. Foveal cone photoreceptor cell size shrinks (from 7.5 to 2 μm diameter), while cell density increases (18–42/100 μm) until 3 years of age [34, 35].
\nProgrammed cell death plays a crucial role in neuroretinal development [36]. Autophagic proteins AMBRA1 and Beclin1 are strongly expressed in chicken embryonic (E5) neural retina [37]. Autophagy supplies ATP to energize the externalization of phosphatidyl serine on the dying cell surface, an essential step in the clearance of cell corpses from the developing retinal neuroepithelium. Pharmacological inhibition (3-Methyladenine) of autophagy increases TUNEL-positive apoptotic cells [38]. It will be interesting to investigate the role of autophagy in the development of mammalian neuroretinal cells. Autophagy has also been shown to reduce cell size in other cell types [39–41]. Antagonistic mTOR and autophagic pathways control activity of YAP/TAZ transcription factors, thereby influencing cell size and proliferation [42]. Differential cellular signaling to modulate autophagy and mTOR in both dividing and differentiated photoreceptors during pre- and postnatal cone photoreceptor enrichment in the macular fovea is an area hitherto unexplored.
\nAutophagic vacuoles engulfing mitochondria were reported in the lens in 1984 [43, 44]. Autophagy was the expected pathway of choice for the developing lens’ fiber cells to degrade cell organelles to create the organelle-free zone (reviewed in [45]). Autophagosomes were reported in both differentiating primary and secondary lens fiber cells [43, 46]. However, during the embryonic period, deletion of either Atg5 or Pik3c3 genes in mice did not affect lens organelle clearance [43, 46]. Costello et al. put forward an alternative hypothesis that since both Atg5- and Pik3c3-independent autophagy have been reported and that mutation in autophagy gene FYCO1 causes autosomal recessive congenital cataract, the role of autophagy (and mitophagy) cannot be ruled out in organelle clearance [47–50]. ATG5-independent non-canonical autophagy has also been implicated in the mitochondrial clearance required during metabolic reprogramming of induced pluripotent cells (iPSC) [51]. Perhaps a simpler approach, where overall lens autophagic flux is inhibited (possibly by inhibiting lysosomal fusion), needs to be adopted to confirm that organelle clearance in developing lens is not dependent on autophagy. Furthermore, it remains to be seen if the various proteolytic mechanisms active during lens fiber differentiation can compensate autophagic deficiency (for further reading, please refer [30]).
\nThe adult eye is enclosed in the outer fibrous tunic, composed of the sclera (posterior 5/6th of the eye bulb) and cornea (transparent anterior part) (Figure 1A). The middle layer is known as the vascular tunic (or uvea) comprising of the choroid, ciliary body, and iris. The ciliary body supports the lens and controls the shape of the lens with the ciliary muscle. The innermost layer is the retina with ten distinct layers. Moving in a direction from the inside of the eye, these layers are arranged as (1) the inner limiting membrane; (2) the nerve fiber layer; (3) the ganglion cell layer; (4) the inner plexiform layer; (5) the inner nuclear layer; (6) the outer plexiform layer; (7) the outer nuclear layer; (8) the outer limiting membrane; (9) the photoreceptor (rods and cones) layer; and (10) the retinal pigmented epithelium (RPE) (Figure 1B) [52]. The innermost layers of the neural retina comprise of different classes of neuronal cells such as the ganglion cells, the Müller, horizontal, bipolar, amacrine cells, and the photoreceptor cells (rods and cones). Together, these cells constitute a complex network of visual sensory synapses that communicate the visual signals to the brain via the central nervous system.
\nThe extraocular muscles (EOMs) control eye directional movement and eyelid movements and contain cells that are likely to accumulate mitochondrial damage during aging, resulting in slower eye muscle movements [53–55]. McMullen et al. reported that autophagy was severely impaired in 18- and 30-month-old compared with 6-month old Fisher 344-Brown Norway rat EOMs supported by their observation of decline in LC3, ATG5, and ATG7 [56].
\nThe cornea, being a non-keratinized epithelial surface, requires to be kept moist by tear secretions from the lacrimal, meibomian (or tarsal) glands, and the conjunctival goblet cells [57, 58]. Basal autophagy-lysosomal activity in the constituent fibroblasts (also known as keratocytes) of the corneal stroma is critical for the clearance of transforming growth factor β-induced protein (TGF-βIp) (discussed in detail in next section along with other corneal abnormalities) [59].
\nThe lacrimal glands produce the aqueous components of the tears (i.e., lacritin, lysozymes, lactoferrin, lipocalin, secretory immunoglobulin A (IgA) and complements), which protect the cornea against a large number of infectious agents (reviewed in [60]). The meibomian glands produce the lipid components of tears called meibum consisting of a variety of esters and fatty acids that prevent evaporation of the tears from the conjunctiva (reviewed in [58]). The mucous secretion from the conjunctival goblet cells allows for even distribution of the tears over the conjunctival surface (reviewed in [61]).
\nEarliest data on autophagic activity in the acinar cells of the lacrimal glands showed dramatic buildup of autophagosomes upon treatment with vinblastine (microtubule inhibitor that blocks autophagosome–lysosome fusion), strongly suggesting the existence of basal autophagy [62, 63]. Autophagy (along with apoptosis) is upregulated in response to inflammation induced in BALB/c mice lacrimal glands, resulting in acinar cell death. It remains to be investigated whether this phenomenon is critical for tissue repair and remodeling post-inflammation injury [64]. The tear component glycoprotein, lacritin, has been reported to protect
The earliest publication on autophagy in the conjunctiva described autophagic structures in guinea pigs [69].
\nThe trabecular meshwork (TM) is located in the iridocorneal junction of the eye and is responsible for draining the aqueous humor from the eye via the anterior chamber (Figure 1B) [70]. Porcine TM cells under hypoxic conditions show increased autophagy, perhaps as a response to increase reactive oxygen species (ROS) [71]. TM cells when chronically exposed to oxidative stress tend to develop lysosomal basification and membrane permeabilization owing to the increased lysosomal iron content. Although autophagic activity is elevated in oxidatively stressed TM cells, the levels of ATG5, ATG7, and ATG12 are significantly reduced [72, 73]. This paradoxical observation hints at the possibility of the existence of active and potentially novel non-canonical autophagic pathways that may use another enzymatic network to modify LC3.
The iris modulates the amount of light entering the retina by controlling the size and diameter of the pupil. The iris contains pigmented epithelial cells that have the same origin as RPE and contain melanosomes [75].
During normal aging, basal LC3 levels are elevated in the lens of human cadaver eyes [79]. Analysis of autophagic gene expression by a combined approach of microarray, qRT-PCR, and Western blotting revealed as many as 42 autophagy-related genes in microdissected human lens epithelium and fiber cells (age range: 47–69 years) providing convincing evidence of autophagy in the lens [82, 83]. Two independent reports suggest the critical role of autophagy in maintaining lens homeostasis in mutant models of αβ-crystallin (R120G) and αA-crystallin (R120A) (discussed in detail in the next section) [84, 85].
\nThe mean retinal thickness of the human eye reduces by about 0.53 μm annually with concurrent loss of macular thickness, implying that there is a significant cell loss in the retina even with no pathology [86, 87]. It remains a challenge to researchers to determine whether changes in cell biology of the retinal cells that trigger onset of disease are different from the normal aging process. The nerve fiber layer shows substantial thinning over time (nearly 150/mm2 during an average lifetime) due to the significant loss of retinal ganglion cells (RGCs) that make up the ganglion cell layer of the retina and convey visual signals from the photoreceptor layer to the optic nerve that constitutes of RGC axons and glial cells [88–91]. The optic nerve also suffers some detrimental changes during aging due to the loss of ganglial axons [92]. The neuroretinal rim area reduces at a rate of 0.28–0.39% annually, while the optic cup area and the vertical cup diameter start to increase especially after the third decade of life [93]. Apoptosis is considered the primary cell death mode for RGC loss [94–96]. Reports suggest that autophagy may promote RGC survival after optic nerve axotomy in mice [97]. Atg5, 7 and 12, LC3, and Beclin-1 expression is elevated in the mouse RGCs up to 7 days after optic nerve injury [98]. It has been suggested that autophagic flux impairment in the RGC axons may lead to age-associated changes in the optic nerve [99]. We will elaborate the role of autophagy in terms of RGC and disease in Section 3.
\nPhotoreceptor density decreases at a rate of 0.2–0.4% annually with a greater degree of rod cell loss than cones causing reduced dark adaptation in aged individuals [100, 101]. This loss is mostly in the peripheral and the parafoveal retina rather than at the fovea (Figure 1C) [102]. We have previously shown strong expression of Atg9 and LC3 in the ganglion cell layer, retinal vessels, a subpopulation of the inner nuclear layer, the outer nuclear layer of rods and cones, and the RPE [103]. Deletion of Beclin1 or Atg7 or mitophagy-specific Parkin gene in mice causes severe retinal degeneration along with accumulation of abnormal mitochondria [104].
\n\nThe RPE monolayer consists of perhaps the most multifunctional cells of the eye. The RPE has a plethora of functions such as phagocytosis of photoreceptor outer segments, renewal of chromophores in visual transduction cycle, supplying nutrients from the choroidal side to the photoreceptors, ion, and metabolite exchange and light absorption (reviewed in [7]). Like the entire retina, the RPE is also prone to age-associated decline in function and vitality and accumulate massive cell damage even during normal aging [104, 105]. Additionally, the RPE has to combat light and reactive oxygen species induced damage not just to its own cellular components but also to those of the photoreceptors. Aging changes in the RPE layer is not uniform across the retina [105, 106]. It appears that the peripheral cell area increases while that at the central retina declines. Cell density decreases with increasing distance from the fovea, but the foveal RPE cell density is relatively very stable. Surprisingly, the aged non-diseased macula shows a population of apoptotic RPE [107]. Both the RPE and photoreceptors are highly metabolic and a healthy pool of mitochondria is required to meet this energy demand. There is a significant reduction in the number of healthy mitochondria and extensive damages to mitochondrial cristae, and matrices are observed [108]. A number of publications together have shown RPE and photoreceptors expressing autophagy proteins (p62, LC3, ATG7, ATG9, and Beclin1) in both human cadaver and rodent model retina sections [13, 14, 19, 103, 109]. Furthermore, as mentioned earlier, diurnal oscillations of autophagic proteins and autophagosomes in the RPE/photoreceptor layers confirm a functional role of autophagy that is integrally linked to POS phagocytosis [19, 110, 111]. Autophagic digestion of rhodopsin light pigment in rod photoreceptors is also necessary for adaptation to changes in light intensity (3–200-lx) [110]. Lentiviral shRNA-mediated silencing of autophagic genes (Beclin1 and ATG7) or 3-Methyadenine (3-MA)-mediated autophagy inhibition in human RPE in cell culture increases susceptibility to oxidative stress with compromised mitochondria, increased lipofuscin, and reduced cell viability [13]. Deletion of RB1CC1 in rodent RPE caused severe retinal degeneration underlining the importance of basal autophagy in the RPE [109]. Levels of autophagic protein such as ATG7, ATG9, and LC3 increase with aging in the retinal layers including RPE and photoreceptors in both human cadaver donor and c57Bl/6 mouse retina sections [13].
\nNon-canonical LC3-associated phagocytosis (LAP), dependent on Atg5 and Beclin1 but independent of the autophagy pre-initiation complex consisting of Ulk1/Atg13/Fip200, was reported to be critical for degradation of POS and renewal of retinoids required for chromophore synthesis for optimal visual function (Figure 2) [111, 112]. Melanoregulin, a 28 KDa membrane-associated protein is critical for lysosomal hydrolase activity in the RPE as well as for RILP-p150Glued complex-mediated retrograde melanosome transport via actin filaments in melanocytes [113, 114]. Frost et al. demonstrated a diurnal variation in melanoregulin expression in the RPE and its distinct association with the ATG5-dependent LAP [111]. Loss of melanoregulin causes accumulation of phagosomes and lipofuscin in the RPE with elevated cathepsin-D secretion that could injure not only the RPE but also the adjacent ocular layers [111, 114]. Furthermore, ROS generated from NADPH oxidase activity resulting from the delayed clearance of all-trans retinal (activated visual chromophore of the visual transduction cycle) shows severe RPE cytotoxicity [104]. LC3 association with phagosomes is signaled by elevated NAPDH oxidase activity in other ‘professionally’ phagocytic cells like macrophages [115]. Park2 (mitophagy receptor protein) and LC3 activity are indispensable for RPE defense against all-trans retinal induced cytotoxicity [104]. It is now evident that while basal rate canonical autophagy is critical for quality control and stress adaptation, non-canonical forms of autophagy where some but not all components and mechanisms of the canonical form participate, supports the very core of retinal visual function.
\nThe ocular vasculature has several indispensable functions including supply of oxygen and nutrients to the ocular components; transportation of ions and metabolites; circulation of immune-surveillant cells; and exclusion of pro-inflammatory cytokines and molecular toxins [116]. The study of autophagic flux and its role in ocular vascular endothelial physiology is still at rudimentary stages. However, recently it has been reported that conditional deletion of endothelial Atg7 in ApoE−/− mice results in accumulation of oxidized LDL within the RPE and choroidal vascular endothelium of the eye underscoring the importance of autophagy in vascular lipid homeostasis [117]. Conflicting opinions exist regarding the role of autophagy in angiogenesis most likely due to the different tissue source of the endothelial cells under study. Lee et al. have recently reported that Beclin1 deficiency leads to increased hypoxia-induced angiogenesis in human pulmonary artery endothelial cells [118]. On the other hand, in bovine aortic endothelial cells, Du et al. suggest that autophagy promotes angiogenesis and elevated ROS levels [119]. Autocrine vascular endothelial growth factor (VEGF) suppresses autophagy in human umbilical vascular endothelial cells (HUVECs) to maintain cell viability. The role of αvβ3 and αvβ5 integrins has long been implicated in retinal neovascularization [120, 121]. αvβ5 integrins act downstream of VEGF activating focal adhesion kinases (FAKs) that are critical for cell migration [122]. Recent reports suggest a critical role of autophagy in restricting integrin activity and thus inhibiting cell migration [123]. Autophagy receptor NBR1 has been shown to be a specific cargo receptor for targeting focal adhesion components to the lysosome for degradation [124].
\nClassic and non-canonical LC3-associated phagocytosis (LAP) in the RPE: basal autophagy is essential in the RPE to maintain organelle and protein quality. Phagocytosis of ingested outer segments may be mediated by autophagy components LC3, ATG5-12-16 complex, and delivery of the phagosome to the lysosome is dependent on these proteins underlining the existence of a non-canonical autophagic pathway in the eye that supports RPE phagocytic function. Furthermore, melanoregulin (MREG) facilitates LC3 recruitment to the phagosomes. Phagocytosis is essential for renewal of all-trans retinol to 11-cis retinal visually active chromophore that is sent to the photoreceptors for enabling the visual cycle. Hence, while basal canonical autophagy is essential for basic housekeeping of the RPE, non-canonical autophagy supports at least in part the visual cycle and photoreceptor disk processing.
These mechanisms need to be reinvestigated in retinal endothelial cells in order to elucidate the role of autophagy in maintenance of retinal vasculature. Inhibiting autophagy in the RPE
One of the inevitable consequences of oxidative damage in the aging retina is the accompanying inflammatory response and elevated levels of damage-associated molecular patterns (DAMP) [126]. Although the eye was considered immune privileged for a long time, immunocompetent cells like the monocyte-derived cells, microglia, dendritic cells, and perivascular macrophages have been detected in the retina [127–130]. Ample evidence suggests that the inflammation observed during normal tissue aging is an adaptive response and the word coined for this inflammation is ‘para-inflammation’ [129, 131–133]. Para-inflammation is required for retinal tissue homeostasis plays a crucial role in tissue repair/remodeling, but when para-inflammation becomes chronic or progresses to destructive inflammation, retinal damage and pathology may ensue (reviewed in [134]). As mentioned earlier, the aging retina shows an increase in apoptotic cells. However, several reports have recently indicated that other cell death modalities like autophagy and necrosis may also exist in the eye that may become particularly active in retinal degenerative conditions [135, 136]. While apoptosis restricts the release of inflammatory danger signals, late-stage apoptosis and necrosis can initiate DAMP-mediated inflammation. Autophagy, at least in the early stages, has been considered a protective response that suppresses inflammatory signals [137]. Shi et al. showed the activation of autophagy by sterile inflammation (NLRP3- and AIM2-mediated inflammation) limited caspase-1-mediated maturation of IL-1β and IL-18 [138]. Impairing autophagy in RPE leads to not only inflammasome activation but also macrophage-mediated angiogenesis [139–141]. The age pigment, lipofuscin, is a common feature of many post-mitotic cells throughout the body and is largely derived from autophagic removal of damaged organelles [142]. Lipofuscin accumulation occurs in an age-dependent manner in both photoreceptor cells and the RPE. In both cell types, lipofuscin is derived at least in part via autophagy of damaged organelles (e.g., mitochondria) [142], but the situation is more complicated in the RPE where (a) lipofuscin is also an inevitable consequence of phagocytosis of spent photoreceptor outer segments [143] and (b) phagocytosis is linked to a non-canonical autophagy pathway [111, 112]. Lipofuscin is both a cause and consequence of oxidative stress and oxidative stress-mediated accumulation of lipofuscin increases dramatically in the RPE when autophagy is pharmacologically inhibited [13].
\nConsiderable cross talk exists between apoptosis and autophagy. p53 and Bc1-2 family proteins and calpain have been classically considered as apoptotic proteins but can also modulate autophagy [144–146]. For example, Beclin1 is cleaved by caspase upon depletion of IL-3 in Ba/F3 cells leading to inactivation of autophagy and release of proapoptotic cytochrome c from the mitochondria [147]. Direct cleavage of ATG3 (a ubiquitin-like-conjugating enzyme involved in autophagosome biogenesis) by activated caspase-8 can lead to inhibition of autophagy and cell death [148]. Yet other reports show autophagy prevents necrosis by reducing metabolic stress [149]. Although cross talk between cell death pathways needs to be confirmed in ocular cells, it is safe to assume that autophagy in the aging eye plays a critical role in maintaining balance between the cell death modalities to avoid a pathological scenario. Arrested autophagic flux by lysosomal disruption enhances buildup of ubiquitinated protein aggregates and cell death under oxidative stress that cannot be prevented by apoptotic caspase inhibitor (zVAD-FMK) [150].
\nSeveral congenital deformities of the eye occur such as coloboma, congenital glaucoma, congenital cataracts, congenital detached retina, partially persistent iridopupillary membrane, persistent hyaloid artery, microphthalmia and Peter’s anomaly, Leber’s Congenital Ameurosis [151–156]. While the cause of most of these diseases is rooted deep in mutation of genes such as PAX2, PAX6, CYP1B1, GLC3A, GLC3B, GLC3C, FOXC1, CEP290, CRB1, GUCY2D, RPE65, several reports suggest autophagy may be compromised in some of these diseases [157]. Persistent hyaloid artery and persistent hyperplastic primary vitreous (PHPV) could result from incomplete involution of the hyaloid vessel [32, 158]. Studies have shown that hypoxic conditions, as seen in the developing eye, increase autophagic activity in vascular endothelial cells. Hypoxia plays a major role in triggering the hyaloid vessel regression and activation of autophagy seems to enhance hyaloid regression in the developing eye [159]. Recessive mutations in EPG5 cause a rare inherited congenital multisystem disorder called Vici syndrome with defective systemic autophagy [160]. EPG5, the human homolog of the
Autophagy is an integral part of developmental cell biology that is coordinated by a vast network of genes [167]. Although still at rudimentary stages, research on the role and fate of autophagy in ocular development must be intensified in the search of more promising therapies in debilitating congenital eye disorders.
\nSeveral topical eye ointments contain benzalkonium chloride (BAC) as a preservative that has been shown to induce caspase-independent cell death in a conjunctival cell line, was reversible by autophagy induction [168].
\nThe consequences of corneal infection can be devastating with corneal scars that would require corneal transplant [20].
Granular Corneal Dystrophy 2 (GCD2), an autosomal dominant disorder caused by mutation R124H in the transforming growth factor β-induced gene (TGFBI) on chromosome 5q31, shows dramatic accumulation of mutant transforming growth factor β-induced protein (TGF-βIp) in autophagosomes and/or lysosomes of corneal fibroblasts [59]. Autophagy is activated, but the rate of autophagic degradation is not sufficient to inhibit the accumulation of the aberrant protein or polyubiquitinated proteins that are also digested in part by autophagy [59, 181].
\nDefects in fluid drainage by the TM can lead to elevated IOPs and eventually cause irreversible damage to the optic nerve leading to glaucoma. Glaucoma is manifested with loss of peripheral vision leading eventually to complete blindness [182–184]. Both elevated IOP and biaxial TM stretching have been independently shown to promote autophagosome formation [185, 186]. Additionally, aging TM is subjected to both hypoxic and highly oxidative conditions that cause increased ROS production and accumulation of non-degradable material along with lipofuscin in lamina cribrosa as well as TM cells [187]. In both cell types, autophagic flux is severely impaired contributing to glaucoma pathogenesis [73, 188]. Autophagy seems to be protective from apoptotic caspase signals in TM cells [189].
\nOptic nerve damage is a commonality in all glaucoma subtypes [190]. Other retinal neuropathies such as optic neuritis, hereditary optic atrophy, and traumatic injury may also lead to degeneration of retinal ganglion cell axons in the optic nerve [191]. Optineurin overexpression in retinal ganglion cells (RGC-5)
As mentioned in the earlier section, there still seems to be considerable debate over the role of autophagy in digestion of organelles of differentiating lens fiber cells to create the organelle-free zone. However, even the reports that argue against the role of autophagy in organelle clearance suggest that it is indispensable for lens quality control. Morishita et al. showed that Pik3kc3/VPS34 deletion in mouse caused congenital lens defects including cataract and that in ATG5 deletion mice lens, the lens developed age-related cataracts although not congenital cataracts [46]. Mutations in FYCO1 (facilitates microtubule-dependent directional transport of autophagosome vesicles) show severe autosomal-recessive congenital cataracts in patients [48, 193]. αβ-Crystallin mutation (R120G) in hereditary cataract mouse model causes concurrent increase in autophagosome fractional volumes and p62-positive aggregates in lens suggesting impaired autophagic flux that leads to increased lens opacity [194]. Similar results were also observed in a hereditary mutant double knock-in (R49C+/+) mouse model where autophagic flux also seemed impaired [85]. Recently, an ESCRT-III subunit CHMP4B has been proposed to be involved in autophagosomal clearance of extranuclear DNA and chromatin [195]. CHMP4B mutation is associated with autosomal dominant posterior polar cataract formation [196].
\nMany studies have investigated the implications of autophagy in retinal degenerative diseases [12–14, 103, 109, 125, 150, 194, 197, 198]. Age-related macular degeneration (AMD) is an aging-associated neuropathy that affects primarily the photoreceptors and RPE in the macula resulting in loss of peripheral vision and eventual legal blindness [199]. Early in AMD pathology, sub-RPE deposits known as drusen are observed on Bruch’s membrane (BM) by fundoscopy. There are two types of AMD: ‘dry AMD’ characterized by geographic atrophy (GA) and ‘wet AMD’ characterized by neovascularization. Although a heterogenous disease, the key reason behind the pathology is the increased susceptibility of the RPE to oxidative stress [200, 201]. Diseased retina shows a significantly greater extent of damaged organelles (mitochondria, peroxisome, melanosomes, etc.) and protein aggregates compared to age-matched healthy retina [108]. The overall accumulation of damaged organelles and macromolecules suggests a collapse of overall antioxidant and proteolytic capacity of the RPE that sets up the stage for disease [202]. Not surprisingly, autophagy has been shown to be severely impaired in AMD retinas of both human cadaver eyes compared with age-matched donors as well as in AMD mouse models [13, 14, 198].
Diabetic retinopathy (DR) is a retinal complication characterized by pericyte loss, microvascular instability, blood retinal barrier (BRB) leakage, and abnormalities in the retinal vasculature [206, 207]. Since pericyte loss is a key feature of DR, the effect of autophagy was investigated in a combination mouse model of diabetes and hypercholesterolaemia. The authors showed that autophagy promoted pericyte survival under mild stress but under chronic stress conditions resulted in pericyte death [208]. This may be considered as a perfect example of the dual role of autophagy both as a protector and as a destructive pathway. Extravascular oxidized low-density lipoprotein (LDL) has been reported to be damaging to the BRB and to cause apoptotic pericyte loss [209]. Du et al. suggested that oxidized LDL may cause RPE injury by excessive oxidative stress, ER stress, autophagy, and apoptosis [210]. High glucose (30 mM) conditions in the RPE result in higher levels of p62 and LC3 accompanied by an increase in the number of autophagosomes [211]. This increase in autophagosomes is possibly to accommodate for the increased ROS damage sustained by the mitochondria, but it needs to be determined whether autophagic flux is reduced as lysosomal pH is reported to be elevated under high glucose conditions [212]. As described earlier, circadian rhythmicity and diurnal variations in expression amplitudes of autophagic proteins is a prominent feature of the retina. Disruption of the peripheral clock has been reported in DR pathology affecting cellular processes such as regulation of inflammation and lipid metabolism [213–216]. Our unpublished data show dramatic phase-shift and amplitude dampening of key autophagic proteins in the retina of rodent models of diabetes (manuscript under preparation). It remains to be elucidated how disruption of diurnal rhythm dysregulates the normal balance between retinal cell metabolism and autophagy, which contributes to DR pathology.
\nPhotoreceptor degeneration is widely observed both in AMD as well as in retinitis pigmentosa (RP). The latter is a highly hetergenous disease with hereditary mutations in multiple gene loci [217]. Both caspase-dependent and caspase-independent pathways are involved in photoreceptor cell death in RP [218–220]. rd/rd mouse, the rds/rds mouse, and the light-damage model in albino mice show several elements of the autophagic pathway to be upregulated. This induction seemed secondary to an increase in oxidative stress markers, suggesting that autophagy may be upregulated specifically to remove damaged photoreceptors [221].
\nInherited lysosomal storage disease Niemann–Pick type C (NPC) disease is caused by mutations in genes NPC 1 and 2 [222]. LC3 and autophagosmal numbers are elevated in the ganglion cell layer of Balb/cNctr-Npc1m1N/J mouse model possibly because of disruption of autophagic flux and reduced degradation of autophagosomes in the lysosome [223].
\nRetinal detachment has a number of causes and could be rhegmatogenous or may due to other causes such as traumatic brain injury, severe myopia, retinal tear, or vascular abnormalities frequently encountered in diabetic retina and hypertension [224–227]. In rodent models, retinal detachment induced by subretinal injection of 1% hyaluronic acid resulted in a rapid increase in autophagic activity 3 days after insult. However, 7 days post-injury the autophagic response declined with a simultaneous rise in calpain activity resulting in photoreceptor cell death. Calpain inhibition resulted in increased autophagy and prolonged the survival of photoreceptors [228]. Furthermore, activating autophagy in the same model in Fas-dependent manner inhibited apoptotic death of photoreceptors [229]. Unpublished results suggest hypoxia (increased Hif1α and Hif2α protein levels) induced by the retina-RPE separation is a key inducer of autophagy
The uvea (consisting of the choroid and the ciliary body) may be affected in some disease conditions such as uveitis and uveal melanoma. Autoantigen-induced experimental autoimmune uveitis (EAU) in Lewis rats shows an increased autophagic activity in infiltrating T lymphocytes that was required for disease recurrence [231].
\nUveal melanoma results from malignant tumors arising from melanocytes in the uvea and is the most common intraocular cancer [232]. Mutations in GNAQ and GNA11 genes contribute to a majority of uveal melanoma cases [233, 234]. Ambrosini et al. showed that mutant GNAQ promoted AKT activation via phosphorylation and deletion of mutant GNAQ upregulated AMP kinase-dependent autophagic cell death in primary choroidal uveal melanoma cell lines [235].
\nInflammation is an unavoidable phenomenon of aging. Elevated inflammation in the eye contributes to disease pathologies including uveitis, diabetic retinopathy, or maculopathies [236, 237]. As discussed earlier, chronic inflammation in diseased eye is destructive and detrimental to ocular health compared with para-inflammatory immune surveillance that responds to, and repairs, localized tissue injuries. In AMD, drusen deposits play a major role in eliciting inflammation via both the inflammasomes and the complement pathway [238]. While mild upregulation of NLRP3 inflammasome has been shown to be protective, accumulation of lipofuscin, drusen, and damaged mitochondrial DNA have all been implicated in pathological upregulation of inflammasome activity [239]. Furthermore, complement pathway element C5a has been shown to prime the RPE cell for upregulating NLRP3 inflammasome activity in response to light-induced damage [240].
\nOcular tissue | \nAutophagic role | \nDisease implications | \nReferences | \n
---|---|---|---|
Lens | \nCritical for proteolytic digestion and lens quality control. Role in lens organelle clearance during eye development controversial. | \nDisrupted autophagy due to Hereditary cataract mouse models show disruption of autophagic flux. | \nCostello et al. [47] Chen et al. [48] Wignes et al. [194] Shiels et al. [196] | \n
Cornea | \nCellular housekeeping and defense against infectious pathogens | \nInsufficient autophagic degradation leads to accumulation of TGF-βIp in autolysosomes. | \nChoi et al. [251, 252, 254] | \n
Optic nerve | \nPromotes cell survival in RGC post-optic nerve axotomy. Optineurin mediated RGC Alternative opinions exist of autophagy effecting cell death in chronic hypertensive model of glaucoma. | \nSome BPAN patients have optic nerve atrophy possibly due to defective autophagy. | \nPark et al, [186] Gregory et al. [166] Sternberg et al. [189] | \n
Trabecular meshwork | \nAutophagy upregulated under hypoxia, elevated IOP and biaxial TM stretching. Oxidative stress increases autophagy but ATG5, ATG7, and ATG12 reduced. Autophagic markers increase during aging in donor eyes. | \nDisruption of endoplasmic reticulum autophagy may be a key feature of myocilin accumulation seen in a majority of glaucoma cases. | \nPulliero et al. [74] Porter et al. [185] McElnea et al. [187] | \n
Ocular vasculature | \nCritical for proteolytic and lipid homeostasis. Potential regulator of vascular stability by prevention of angiogenesis. Autophagy essential for hyaloid regression and clearance during development. | \nDiabetic Retinopathy: Accumulation of extravascular oxidized LDLs due to disrupted autophagy leads to blood-retina barrier damage and causes pericyte loss. | \nTorisu et al. [117] Lee et al. [118] Kim et al. [159] Fu et al. [208] Wu et al. [209] | \n
Retina | \nDiurnal modulation of Autophagic proteins. Non-canonical LC3 associated Autophagy is essential for phagocytosis is critical for degradation of POS. Basal autophagy indispensable for maintenance of RPE and photoreceptor homeostasis. Restricts sterile inflammation in the retina | \nAMD: Autophagy initiation and flux are disrupted in human donors and mouse models for AMD. Diabetic Retinopathy: Disrupted autophagy may cause pericyte loss. | \nKim et al. [112] Yao et al. [19, 109]; Mitter et al. [13] Viiri et al. [14] Fu et al. [208] Liu et al. [139] | \n
Uvea | \nAutophagy promotes cellular survival | \nUveal Melanoma: Inhibition of autophagy may be effective in Uveal melanoma therapy | \nAmbrosini et al. [232] | \n
Description of role of autophagy along with disease implications in various ocular tissues.
Autophagy plays a critical role in controlling NLRP3 inflammasome activity in the retina. Several
Since autophagy is a pathway with a dual role in cell maintenance as well as cell death; multiple stages such as initiation, maturation, and lysosomal fusion; and cross talk with multiple cellular pathways, its manipulation is a challenging therapeutic option in diseases. Nevertheless, autophagy has received special attention in cancer, metabolic, neurodegenerative, and infectious diseases [242–245]. Since overall proteolytic capacity is attenuated in a majority of ocular diseases, autophagy modulation must be incorporated in current therapeutic regimens to achieve a better outcome.
\nTreatment strategies for immature cataracts have been sought after for more than a century [245, 246]. Topical solutions with antioxidants glutathione, cysteine ascorbate, l-taurine, riboflavin, and 2% N-acetyl carnosine showed some promise in reducing immature cataracts [247]. Including autophagy stimulation may improve this treatment strategy. Posterior capsule opacification, a common post-surgical complication of mature cataracts, results from the remnant lens fibers and epithelial cells that proliferate and damage the new lens implant [248]. Laser capsulotomy surgery although usually successful may in rare occasions give rise to retinal detachment and is also extremely challenging to execute when treating congenital cataracts in younger patients [249]. It is not known whether autophagy (as well as other mechanisms) supports cell survival of the remnant lens epithelial cells. Pharmacologically stimulating cell death may involve autophagy that either promotes or inhibits survival of these cells.
\nAs mentioned earlier, removal of TGF-βIp deposits is a focus in granular corneal dystrophy 2 (GCD2) research. Lithium, which has shown some success in removing these deposits from
Non-infectious uveitis treatment with subconjunctival injections of rapamycin as an immunosuppressive agent has shown promise in clinical studies with patients showing improved visual acuity and reduced vitreous haze with no noticeable adverse effects [255]. Mechanistic studies may reveal that at least a part of this immune suppressive ability of rapamycin may be credited to autophagy stimulation.
\nAntitumor activity is seen in combinatorial therapy involving mTOR inhibition and autophagy inhibition with hydroxychloroquine has been shown to restrict melanoma and these treatments are currently under phase-1 trial [256, 257]. Such treatment strategies may be adopted in treatment of uveal melanoma although the fact that chloroquine may induce cataract formation, demands that careful dose-response studies be conducted to ensure no adverse effects [258, 259].
\nAutophagic degradation is attenuated in AMD. Lipofuscin accumulation in the disease has been shown to perturb the lysosomes that have serious implications on RPE health [142, 260–262]. Lysosomal activity disruption affects both autophagic flux and phagocytosis [263]. Hence, putative therapies should first focus on restoring lysosomal activity to improve degradation of existing autophagosomes. Rapamycin administration to senescence-accelerated OXYS rats improved the RPE morphology in the retina [264]. Clinical trials using rapamycin to treat GA in advanced stages of dry AMD showed ‘no positive anatomic or functional effects’ [265]. The treatment failure may partly be attributed partly to the fact that the intervention may have been attempted at a time when the disease was well underway with well-developed AMD lesions. An earlier intervention in addition to stimulating lysosomal activity may produce better results.
\nTo include autophagy in ocular therapeutic strategy for better treatment outcomes, the following aspects must be considered. (1) Stimulating autophagy initiation: Several pharmacological activators of autophagy have been identified for possible therapeutic treatments. Rapamycin and its analogs (CCI-779, RAD001 and AP23573) act via inhibiting the mTOR pathway. Metformin mediates AMP kinase activity which stimulates autophagy initiation. Yet other drugs such as lithium and valproic acid have been identified that stimulate autophagy induction. Studies using rapamycin or resveratrol have shown promising results in treatment of cardiac hypertrophy [266, 267]. Clearance of α-synuclein and polyQ mutant Huntingtin aggregates has also resulted from using rapamycin in Parkison’s and Huntingtin disease, respectively [268–270]. Also, small molecule enhancers of rapamycin have also been reported that show positive results in neuroprotection. However, whether stimulation of autophagy would be at all beneficial in retinal diseases perspective depends significantly on the status of lysosomal machinery at the stage of the disease when intervention is attempted. Stimulating autophagosome biogenesis when lysosomes are destabilized will not alleviate the cytotoxic burden resulting from damaged protein aggregates. (2) Stimulating lysosomal activity: An effective strategy to clear aggregate proteins may be attempted by improving lysosomal activity and thereby increasing (or restoring) the autophagic flux. Transcription Factor EB (TFEB) is considered a ‘master regulator of autophagy’ and drives the expression of several autophagy and lysosomal genes including p62, Atg9b, LC3B, Wipi1, and Lamp1 [271]. Gene therapy with TFEB in mouse model of hepatic disease improved clearance of protein aggregation and rescued alpha-1-anti-trypsin deficiency [272]. High efficiency gene transfers have been achieved to specific retinal layers previously with different adeno-associated virus (AAV) serotypes. TFEB gene transfer may dramatically improve lysosomal biogenesis and overall autophagic flux in the RPE and may be of particular importance in AMD therapeutic strategies.
\nThe autophagic machinery consists of a fine-tuned complex network of genes whose mysteries are still being unraveled by researchers. Autophagy research in the eye so far has established it as an essential housekeeping pathway indispensable for ocular homeostasis. While therapeutic strategies to regulate autophagy in ocular diseases are still in rudimentary stages, promising results from initial trials have raised hope of autophagic modulation moving gradually from bench to clinic. The challenge lies in modulation of autophagy to the levels required in the particular disease scenario, that is do we want cell death in malignant conditions or just to restore autophagy to levels where it can not only clear cellular waste but also effectively reverse inflammation and contain cell death signals.
\nChronic 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].
Below, we provide an overview of some clinical studies on the efficacy of interferon in chronic myeloproliferative disorders.
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 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].
The most recent form of interferon approved by the
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 flu-like 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).
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].
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].
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 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].
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].
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.
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 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.
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
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].
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.
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 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].
Source | Type of trial | Interferon | Diagnosis | No. | Prior treatment status | Response rate |
---|---|---|---|---|---|---|
Yacoubet al. [15] | Phase II, multicenter | Pegylated IFN alfa-2a | PV | 50 | Resistance to HU or HU intolerance | CR:22% PR:38% |
ET | 65 | CR:43% PR:26% | ||||
Masarova et al. [16] | Phase II, single-center | Pegylated IFN alfa-2a | PV | 43 | Untreated or previously treated with cytoreductive therapy | CR:77% PR:7% |
ET | 40 | CR:73% PR:3% | ||||
Samuelsson et al. [18] | Phase II | Pegylated IFN alfa-2b | PV | 21 | Untreated or previously treated with cytoreductive therapy | CR: 69% for the entire group |
ET | 21 | |||||
Huang BT et al. [19] | Open label, multicenter | IFN alfa-2b | PV | 136 | Untreated or previously treated with cytoreductive therapy | OHR:70% Molecular response:54.7% |
ET | 123 | OHR (JAK2+ patients):83% CHR:23 cases OHR (JAK2-patients): 61.4% CHR:12 cases | ||||
Gisslinger et al. [23] | phase III, multicenter | Ropeginterferon | PV | 257 | Previously treated | OHR:53% |
Quintás-Cardama et al. [26] | phase II | Pegylated IFN alfa-2a | PV | 40 | Untreated or previously treated with cytoreductive therapy | OHR:80% CR:70% Molecular remission:54% |
ET | 39 | OHR:81% CR:76% Molecular remission:38% | ||||
Sørensen et al. [36] | Phase III, multicenter, COMBI | Pegylated IFN alfa-2a with ruxolitinib or Pegylated IFN alfa-2b with ruxolitinib | PV | 32 | Untreated or previously treated with cytoreductive therapy | OHR:44% CR:28% |
MF | 18 | OHR:31% CR:9% | ||||
Casassus et al. [40] | Open label, multicenter | IFN alpha-2b | Mastocytosis | 20 | Untreated and previously treated | PR:35% Minor remission: 30% |
Comparison of the effectiveness of interferon in chronic myeloproliferative disorders.
PV: polycythemia vera; ET: essential thrombocythemia; MF: myelofibrosis; HU: hydroxycarbamide/hydroxyurea; CR: complete remission; PR: partial remission; and OHR: overall hematological response.
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.
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