Merkel Cell Polyomavirus in Merkel Cell Carcinoma: Clinical and Therapeutic Perspectives Mahtab Samimi,a,b,c Charlotte Gardair,d Jérome T.J. Nicol,a,c Francoise Arnold,a,c Antoine Touzé,a,c and Pierre Coursageta Merkel cell carcinoma (MCC) is a rare and often aggressive cutaneous cancer with a poor prognosis. The incidence of this cancer increases with age, immunodeficiency and sun exposure. Merkel cell polyomavirus (MCPyV), a new human polyomavirus identified in 2008, is detected in the majority of the MCCs and there is a growing body of evidence that healthy human skin harbors resident or transient MCPyV. A causal link between MCPyV and MCC has been evidenced and this is the first polyomavirus to be clearly implicated as a causal agent underlying a human cancer, and MCPyV was recently classified as a 2A carcinogen. MCC is thus a rare tumor caused by a very common viral skin infection. The aim of this review is to provide a basic overview of the epidemiological, clinical, and pathological characteristics of MCC, to present the current knowledge on MCPyV polyomavirus and its causal association with MCC development, and to describe the therapeutic implications of this causal link. Semin Oncol 42:347-358 & 2015 Elsevier Inc. All rights reserved.

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erkel cell carcinoma (MCC), also known as primary cutaneous neuroendocrine carcinoma, was first described in 1972 by Toker as being composed of “solid trabeculae” and displaying an aggressive clinical course.1 Ultrastructural examination of these “trabecular carcinomas” subsequently revealed the presence of intracytoplasmic neurosecretory granules, similar to those observed in Merkel cells, which are mechanosensory cells that tune mammalian touch receptors located in the basal layer of the epidermis.2 It was therefore hypothesized that these “trabecular carcinomas” were derived from resident Merkel cells, leading to the current denomination “Merkel cell carcinoma”. Subsequent reports, especially of the absence of proliferative potential of human Merkel cells and the

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Université François Rabelais, Tours, France. CHRU de Tours–Hôpital Trousseau, Service de Dermatologie, Tours, France. c Unité Mixte de Recherche INRA-Univerity of Tours N11282, Tours, France. d CHRU de Tours–Hôpital Trousseau, Service d'Anatomie et Cytologie Pathologiques, Tours, France. Conflicts of interest: none. Address correspondence to Pierre Coursaget, Pharm D, Faculté de Pharmacie, 31 avenue Monge, 37200 Tours, France. E-mail: [email protected] 0093-7754/ - see front matter & 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminoncol.2014.12.021 b

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heterogeneity in MCC differentiation patterns, have argued against this hypothesis. It is more likely that a common progenitor, located in the epidermis or dermis, with the capacity for both neuroendocrine and epidermoid differentiation, is at the origin of MCCs, but this putative stem cell remains to be identified.3 MCC accounts for less than 1% of non-melanoma skin cancers.4 Its incidence rate was estimated to be 0.24 per 100,000 person-years in the United States5 and the European age-standardized incidence rate is 0.3 per 100,000 person-years.4 Epidemiological data from US6 and European7 databases indicate a predominance of such tumors in white people aged above 50 years, with a slightly greater predominance in males. The sun-exposed areas are the most affected sites. The primary tumor is located on the head and neck area in nearly 50% of cases, on the limbs in one third and on the trunk in 10% of cases.5,7 In addition, primary MCC can be observed on any part of the non-exposed skin surface, including the oral and genital mucosae.5,8 Clinically, MCC usually presents as a painless erythematous or violaceous, rarely ulcerated nodule (Figure 1). Median size on diagnosis was found to be 1.7 cm (range 0.1–7.0 cm) in a large database.6 This clinical presentation is nonspecific and MCC nodules may be misdiagnosed as a benign cutaneous tumor (cyst, fibroma) or another malignant neoplasm (basal cell and spindle cell carcinoma, achromic melanoma, B-cell lymphoma, cutaneous metastases, etc). 347

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Prognosis is related to the MCC staging (Figure 3) currently based on the 2010 American Joint Committee on Cancer (AJCC) classification.14 There is no consensus regarding the initial disease staging assessment in the European and American guidelines.15–17 A careful clinical examination, focusing on detection of cutaneous, lymph node, or distant metastases, should be combined with appropriate imaging based on clinical findings. Due to the high propensity of MCC to metastasize, regional lymph node ultrasonography and thoracic-abdominal-pelvic tomodensitometry (computed tomography [CT] scan) are usually recommended at baseline. Other forms of imaging should be discussed according to clinical findings, including brain magnetic resonance imaging (MRI), 18-flouro deoxyglucose (18-FDG) positron emission tomography (PET), or radiolabeled scan using a somatostatin analog. Figure 1. Clinical appearance of Merkel cell carcinoma lesion. A 1-cm, dome-shaped Merkel cell carcinoma on the left temple of a 87-year-old woman.

Dermatoscopic examination reveals polymorphous vascular patterns suggestive of a malignant tumor. However, this is not specific for MCC.9 A cutaneous biopsy is mandatory for diagnosis. On hematoxylin and eosin (H&E) staining, MCC appears as a hyperbasophilic round tumor located in the dermis with variable depth extension, separated from the epidermis by a “grenz” zone composed of dermal collagen. The tumor cells are small, round, and monomorphic, with a scant rim of cytoplasm and hyperchromatic nuclei. Immunohistochemistry assessing epithelial and neuroendocrine markers is mandatory for a positive diagnosis of MCC (Figure 2). MCC usually displays dot-like paranuclear positivity for CK20 and is negative for CK7, contrary to neuroendocrine carcinomas of other organs that are usually positive for both markers. Additional positivity for neuroendocrine markers (chromogranin and synaptophysin) confirms the diagnosis of MCC. Other immunostaining markers that may be helpful for differential diagnosis are thyroid transcription factor 1 (TTF-1), PS100, HMB45, and leukocyte common antigen CD45, in order to exclude a cutaneous metastasis of neuroendocrine carcinoma of the lung, a melanoma, or a lymphoma, respectively. MCC usually follows an aggressive course. At diagnosis, the majority of patients have local disease (66%) followed by nodal (27%) and distant metastatic disease (7%).10 MCC also can be diagnosed on node metastases from an unknown primary tumor, with frequency varying from 5%–55% of cases.7,11 In the majority of studies, relative 5-year survival ranges between 50%– 60%.5,10,12 Five-year relative survival rates for local disease, regional nodal disease, and metastatic disease are 64%, 39%, and 18%, respectively.13

MERKEL CELL POLYOMAVIRUS The Virus The first polyomavirus was identified in 1953, as a virus causing salivary gland tumors in mice.18 Simian virus 40 (SV40) was identified 7 years later19 in rhesus monkey cell lines, and then several polyomaviruses were identified in mammalian species and birds. SV40 does not cause tumors in the rhesus monkey, its natural host. However, SV40 immortalizes several cell types and induces tumors when inoculated in newborn rodents. Only a limited number of polyomaviruses induce tumors in their natural host, including murine polyomavirus (MPyV), hamster polyomavirus (HaPyV), raccoon polyomavirus (RaPyV), and Merkel cell polyomavirus (MCPyV). The first two human polyomaviruses identified in 1970, ie, the BK and JC viruses, are common infections of childhood and are pathogenic only when they are reactivated in immunosuppressed subjects. These two polyomaviruses were shown to induce tumors when injected into newborn rodents, but as for SV40, neither epidemiological nor molecular studies have provided clear evidence of their role in human cancers. A dramatic boost to polyomavirus research was achieved in 2008 by the identification by Chang and Moore20 of a polyomavirus (MCPyV) associated with MCC. This discovery was a major finding in human tumor virology and was followed by the identification of eight new human polyomaviruses within 6 years. As with other human polyomaviruses, MCPyV is a non-enveloped double-stranded DNA virus of 45–55 nm (Figure 4) belonging to the family Polyomaviridae, genera Orthopolyomavirus. The viral life cycle of polyomaviruses is divided into an early phase, taking place before the viral genome replication, and a late phase occurring after. The MCPyV genome of about

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Figure 2. Histological features of Merkel cell carcinoma. H&E staining: dermal proliferation of blue cells separated from the epidermis by a “grenz” zone. Positive immunostaining with CK20 antibodies with a paranuclear dot-like pattern. Positive nuclear immunostaining for MCPyV LT-Ag in a MCPyV-positive MCC.

5.4 kb is similar to those of other human polyomaviruses. It is divided into the early region that encodes three oncoproteins [large T (LT), small T (sT), and 57-kT] and the late region that encodes three capsid proteins (VP1, VP2, and VP3) (Figure 5). There is an orderly gene expression cascade for MCPyV during viral replication, Large T-antigen (LT-Ag) being expressed first followed by the expression of small T antigen (sT-Ag) and then VP1 proteins.21 LT is essential for viral replication

and regulates the expression of early and late genes. In addition, the genome of MCPyV encodes miRNA that downregulates expression of the LT antigen.22

Epidemiology of MCPyV Infection in the General Population Detection of current MCPyV infection is based on the detection of the viral DNA by polymerase chain reaction (PCR) techniques and the detection of TAg

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Primary tumor (T) Tx. Primary tumor cannot be assessed T0. No evidence of primary tumor Tis. In situ primary tumor T1. Less than or equal to 2 cm maximum tumor dimension T2. Greater than 2 cm but not more than 5 cm maximum tumor dimension T3. Over 5 cm maximum tumor dimension T4. Primary tumor invades bone, muscle, fascia, or cartilage Regional lymph nodes (N) Nx. Regional lymph nodes cannot be assessed N0. No regional lymph node metastasis CN0. Nodes negative on clinical examination (no pathology examination performed) PN0. Nodes negative on pathology examination N1.Metastasis in regional lymph node(s) N1a.Micrometastases

Stage 0 Stage IA Stage IB Stage IIA Stage IIB Stage IIC Stage IIIA Stage IIIB Stage IV

TisN0M0 T1pN0M0 T1cN0M0 T2/T3pN0M0 T2/T3cN0M0 T4N0M0 Any T, N1aM0 Any T, N1b/N2M0 Any T, Any N, M1

N1b.Macrometastases N2.In transit metastases Distant metastases M0. No distant metastases M1. Metastases beyond regional lymph nodes M1a. Metastases to skin, subcutaneous tissues or distant lymph nodes M1b. Metastases to lung M1c. Metastases to all other visceral sites

Figure 3. AJCC 2010 Merkel cell carcinoma staging.

by immunochemistry. End point PCR amplification and quantitative PCR are used to detect viral DNA, and qPCR allows quantification of the viral load per cell. Detection of the viral DNA on the skin of almost all adults has suggested that most MCPyV infections

5386 bp

Figure 4. MCPyV on the surface of Cos-7 cells. (Photography: Pierre-Yves Sizaret, Faculty of Medicine, Tours, France.)

Figure 5. Genome organization of Merkel cell polyomavirus. The genome encodes early proteins including LTAg (purple), sT-Ag (blue) and three late proteins (capsid proteins): VP1 (red), VP2 (orange), and VP3 (yellow). These two coding regions are separated by the promotor/replication origin region (Ori ¼ replication origin).

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occur by direct skin to skin contact or from the environment, since MCPyV DNA seems to be detected frequently on environmental surfaces,23 but the exact mode(s) of MCPyV transmission and site(s) of initial infection have not yet been characterized.24,25 Present or past exposure to MCPyV is assessed by the detection of specific antibodies against the major capsid protein (VP1). Anti-VP1 antibodies are detected using recombinant proteins, virus-like particles (VLPs), and MCPyV pseudovirions. VP1 is a highly immunogenic protein and serum anti-VP1 antibodies are frequently detected in healthy adults with stable antibody titers. Seroprevalence of 50%– 95% has been reported in adults.26–32 The skin is the primary site of chronic MCPyV infection in healthy adults and a positive correlation between MCPyV anti-VP1 antibody titers and viral load has been reported for all anatomical sites investigated, indicating that MCPyV antibody titers are correlated with the viral load on the skin surface.31,33 In addition, the age-specific seroprevalence of MCPyV indicates that exposure occurs early in life, with seroprevalence of 20%–40% in children aged 1–5 years.

Oncogenesis of MCPyV Polyomaviruses are known to induce cancer through direct mechanisms at the cell genome level. LT protein is a multifunctional protein necessary for viral DNA replication and viral gene expression, and is able to induce oncogenic transformation of cells. LT contains several functional domains such as a nuclear localization signal, a DNA binding domain, and a helicase domain. It also contains oncogenic functions associated with the DnaJ, pRB and p53 binding domains through interactions with HSc70, pRb and p53, respectively. These interactions lead either to functional inactivation or to degradation of key regulation proteins, thus affecting the cell cycle and gene expression. In addition, LT protein inhibits apoptosis, stimulates telomerase activity, affects signaling pathways and gene expression and induces angiogenesis. MCPyV was the first polyomavirus to be clearly identified as being associated with a human cancer, but the oncogenic mechanisms remain largely unknown. The first argument for a causal link between MCPyV and MCC was the fact that MCPyV DNA is detected in around 80% of MCC tumors investigated. The second was that the virus is clonally integrated in the cell DNA of both primary tumors and metastases,20,34,35 indicating that integration precedes metastasis. In addition, epidemiological data have suggested that viral infection takes place prior to the clonal expansion of the tumor cell. The third argument is that the integration pattern

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argues against the hypothesis that the virus is a passenger of the tumor cell. Integration of the MCPyV in MCC tumor cells was found to be systematically associated with occurrence of stop-mutations or deletions in the sequence encoding the LT-Ag. Thus, MCC tumor cells harbor truncated forms of the LT antigen, resulting in the loss of domains required for viral DNA replication (origin of replication, helicase) and P53 binding, but leaving the DnaJ and Rb binding domains intact.36 Viral integration and LT-Ag deletions and/or mutations therefore result into the elimination of T antigen replication capacity, the permanent overexpression of the LT-Ag and the loss of expression of the highly antigenic major capsid protein.26,36,37 These steps are required for MCC cell survival38 and thus probably constitute the basis for MCPyVinduced oncogenesis. Accumulated findings suggest that the MCPyV oncogenic process is unique compared to other polyomaviruses. Expression of SV40 LT alone leads to the in vitro transformation of cells. In contrast, MCPyV LT (wild type or truncated) is unable to initiate transformation. In addition, only the MCPyV sT-Ag is sufficient to transform rodent cells.39 Fulllength LT-Ag seems not to bind directly to p53 but to reduce p53-dependent transcription significantly,40 whereas truncated LT-Ag does not bind to p53 or reduce p53-dependent transcription. In addition, truncated LT is expressed at very high levels compared to full-length LT and exhibits higher binding affinity for Rb.40 As observed for other polyomaviruses, MCPyV sT interacts with PP2A cellular protein but, in contrast to SV40, mutations within the PP2A domain do not significantly inhibit its capacity to promote cell cycle progression and transformation. One specific property of MCPyV sT is its capacity to activate capdependent translation by maintaining hyperphosphorylation of the cellular transformation factor 4E-BP1,39 a downstream target of the Akt-mTOR signaling pathway, thus enhancing cell transformation. p53 protein is rarely detected in MCC tumors and is rarely mutated, with the exception of MCPyVnegative MCCs. In addition, in MCPyV-positive MCCs the expression of p53 is inversely correlated with viral load.41 These findings suggest that p53 does not play a major role in MCPyV tumorigenesis. In addition, because MCC regularly appears on sun-exposed areas, UV radiation is regarded as a major risk factor. UV radiation could cause multiple DNA damage, with as a consequence either cell cycle arrest and then DNA repair or apoptosis resulting in cell death. It is also evident that UV and MCPyV act in synergy to transform cells.42 Moreover, it is evident that immunosuppression is involved in the development MCC and clearly

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predisposes to reactivation of latent MCPyV. These viruses have the ability to exploit a number of immune evasion strategies, and innate and adaptative immune system pathways are impaired, thus helping to initiate or maintain cancer development. Such immune evasion enables them to persist within host cells, including during a latent phase, and then to be reactivated under immunosuppression or UV irradiation.

MCPyV IMPLICATIONS IN MANAGEMENT OF MCC PATIENTS Detection of MCPyV Markers in MCC Patients MCPyV DNA has been detected in 59%–100% of MCC tumors,20,25,35–37,43,44,45 a proportion similar to that observed in patients without MCC and in nonlesional tissue of MCC patients. However, when assessed by quantitative PCR, MCC tumor tissues usually harbor much higher viral loads. The mean viral load (1–5 copies per cell) is in agreement with integration of the viral genome into chromosomal DNA,20,34,35,44 with higher viral loads reflecting the presence of episomal viral genomes. Elimination of the virus’s replicative capacities results in the absence of expression of the highly immunogenic VP1 capsid protein in MCC tumor cells.26,37,46 In contrast, immunohistochemistry demonstrates the expression of LT-Ag (Figure 2) in the nuclei of 58%–96% of MCCs in nearly every tumor cell.36,46–48 Tag expression in tumor cells is specific of MCCs, and has rarely been reported in non-tumor or other tumor tissues. Detection of the MCPyV LT antigen has been reported as an aid in the positive diagnosis of MCC when the tumor displays an unusual differentiation pattern.49 However, its use as a diagnostic tool by pathologists in the diagnosis of MCC remains to be evaluated. Absence of detection of MCPyV DNA and MCPyV LT in around 20% of MCCs has raised the possibility of a subset of MCPyV-negative MCCs. However, a recent report on the detection of LT-Ag in almost 100% of MCC cases48 suggests that MCPyV is also the etiologic agent of such “MCPyV-negative“ cases. Serologic studies in MCC patients have yielded very different results when assessing the humoral response against the VP1 viral capsid protein or the oncogenic proteins (T antigens). Anti-VP1 antibodies have been detected in up to 100% of MCC cases,26,27,37 at higher frequences and higher antibody titers than age-matched controls.26,37 The correlation between high antibody levels and high viral load31,33 in MCC patients, and the absence of virion production by the tumor cells,26,37 suggest that individuals who develop MCC have persistent and active skin shedding of MCPyV virions prior to

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the development of MCC. In contrast to anti-VP1 antibodies, LT antibodies are rarely detected in the general population. Indeed, the T-Ag is expressed very transiently during the virus replication cycle. However, overexpression of LT-Ag in MCC tumors results in the production of specific antibodies in approximately 40% of MCC patients. Moreover, antiLT antigen antibody detection has been found to be related to the tumor burden, and antibodies are progressively cleared after tumor removal. As a consequence their reappearance or an increase in their levels after surgical treatment is associated with disease recurrence or progression.50 Therefore, detection of anti–T-antigen antibodies could be a useful marker for monitoring tumor evolution. Compared to anti-VP1 antibodies, anti-T-Ag antibodies are more specific of MCC and better correlated with viral load and expression of T-Ag.

Viral Status and MCC Prognosis Whether the MCPyV status influences MCC outcome is still a matter of debate, although a range of findings suggests a more favorable prognosis for MCPyV-positive MCCs. Detection of MCPyV DNA in MCC tumor samples was not found to be associated with a better MCC outcome in three studies,51–53 whereas high levels of viral DNA copies in MCC tumors seemed to be a favorable prognostic factor in three others.54–56 Similarly, there is lack of agreement regarding the prognostic value of detection of the LT-Ag. Detection of LT expression together with high MCPyV DNA levels was reported to be associated with better prognosis in two studies,41,57 whereas LT Ag expression did not influence MCC outcome in another.58 Serological investigations also have suggested that patients with higher anti-VP1 antibody titers have a better prognosis.37 In contrast, persistence or reappearance of anti-LT antibodies after successful treatment of patients has been reported to be associated with poor prognosis.50

Immune Responses Against MCPyV in MCC Patients As for other virus-induced cancers, host immunity interferes with tumor promotion and development. This is demonstrated by the fact that immunosuppressed patients are at higher risk of MCC and have worse outcomes,59,60 especially when the cellmediated immune responses are compromised (human immunodeficiency virus [HIV] infection, organ transplantation, chronic lymphocytic leukemia, immunosuppressive therapy). Spontaneous regression of MCC tumors also has been reported in patients with increased lymphocyte infiltration of the tumors,61 although it is not known whether this

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may be related to an efficient anti-tumor host response. MCC often develops despite the presence of humoral and cellular immune responses against MCPyV, which are detected in most patients. Strong humoral responses are evidenced against the MCPyV capsid protein VP1 and the oncoprotein LT. The humoral response against anti-VP1 is stable over time, and does not seem to be influenced by the clinical course of the MCC tumor once established. In contrast, antibodies directed against the LT-Ag are specifically produced in MCC patients at disease onset, reflect the tumor burden and are gradually cleared over time after remission.50 Although specific, this anti–T-antigen humoral response is not effective in disease control. Cellular immune responses against MCPyV antigens have been suggested to play a relevant role in the clinical course of MCC. MCC patients with prominent intratumoral CD3(þ), CD8(þ), FoxP3(þ) cell infiltration were reported to have better outcomes.62,63 However, it is unclear to what extent these tumor infiltrating lymphocytes control tumor growth in vivo. MCPyV-reactive CD8(þ) and CD4(þ) T cells have indeed been identified in the tumors and blood of MCC patients.64,65 Their presence is correlated with MCC disease burden and progression.66 However, these specific MCPyV- T cells display reduced activation and fail to induce cytotoxic responses,67 which is related to the expression of inhibitory markers such as PD-1 and Tim-3 leading to T-cell exhaustion.66 Other immune evasion mechanisms have been identified, such as downregulation of human leukocyte antigen (HLA) class I in MCC tumors,62 disruption of T-cell migration as evidenced by the downregulation of intratumoral vascular E-selectin, a factor critical for T-cell entry into the skin,68 and the downregulation of human Toll-like receptor 9 expression.69

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performed at the time of the surgery. However, there is a risk of false-negative SLNB in head and neck locations.17,70 Histological analysis of sentinel lymph nodes detects micrometastasis in about one third of patients, and is associated with poorer prognosis.13,71 When SLNB is positive, extension of the surgical procedure with radical regional lymph node dissection is recommended. When regional lymph node involvement is evident on initial evaluation (macrometastases), complete lymph node dissection also is recommended. MCC is a radiation-sensitive cancer, and postoperative radiotherapy has been reported to be associated with decreased risk of locoregional recurrence,8,72 though the benefit in terms of patient survival is a matter of debate. Radiation therapy at the tumor site (50 Gy) is performed systematically. Radiation of the regional lymph nodes (50 Gy) is recommended for micro- or macrometastases after complete lymph node dissection, and when SLNB cannot be performed. Management of patients with distant metastases (stage IV disease) is palliative, and should be decided with a multidisciplinary consultation. Surgery, radiation therapy, and/or various chemotherapy regimens can be considered. However, none has so far been demonstrated to have any benefit on overall survival. Standard chemotherapy regimens include cytotoxic drugs such as anthracyclines (doxorubicin), cyclophosphamide, etoposide, vincristine, and platinum derivatives, alone or in combination.15,17 Elderly patients and those with poor performance status may be treated with well-tolerated monotherapy regimens, including etoposide and liposomal doxorubicin, although abstention also is acceptable. Response to chemotherapy has been observed in up to 60% of stage IV patients, but the prognosis remains poor, with a median overall survival of 21.5 months.73

Therapeutic Perspectives MCC THERAPY Current Recommendations Although evidence-based information remains sparse because of the rarity of such tumors, guidelines for management of MCC have been established by American and European expert groups.15,17 Surgery remains the mainstay of treatment in the absence of distant metastases (stages I, II, and III). The primary tumor should be removed with wide margins (2–3 cm) to the investing fascia layer. Micrographic surgery techniques are an alternative in locations when such margins cannot be achieved (head and neck). In the absence of apparent regional lymph node involvement on initial evaluation, a sentinel lymph node biopsy (SLNB) should be

Despite optimization of the curative approach, the prognosis of MCC remains poor and there is a need for innovative therapies especially in metastatic patients. Identification of MCPyV has resulted in a regaining of interest in understanding the pathogenesis of MCC that combines disrupted oncogenetic cellular pathways and impaired anti-tumor immune responses. The resulting therapeutic possibilities include targeted molecular therapies and immunotherapy (Table 1).

Targeted Therapies There is no evidence that one key oncogenic pathway is involved in the pathogenesis of MCC. However, MCC cells express dysregulation of

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Table 1. Current MCC Treatments and New Treatments Under Investigation Type of Treatment

Specific Treatment

Current treatments Surgery Radiation

Wide local surgical excision 50 Gy delivered over a 5- to 6-week period to primary and lymph nodes in conjunction with surgery (alone in cases of inoperable tumors.) Chemotherapy In cases of metastatic disease and inoperable tumors Treatments under investigation Targeted therapies PI3K and mTOR inhibitors, apoptosis analogs (inhibitors of survivin expression, BCL2 antisense) Inhibitors of VEGFR and PDGFR Inhibitors of somatostatin Immunotherapy Cytokine therapy (interferon, interleukin-12) Adoptive immunotherapy (in vitro expression of tumor infiltrating lymphocytes or peripheral T cells) CD56 anti-cancer therapy Specific MCPyV Therapeutic vaccines (T-Ag recombinant protein) therapies Antisense therapy (T-Ag shRNA)

various cellular proteins with proliferative, proangiogenic, and anti-apoptotic properties. Thus, targeted therapies may be an alternative approach in patients in whom such dysregulation is identified. Survivin, a protein that exhibits anti-apoptotic and proliferative properties, has been found to be upregulated in MCCs.74 Survivin expression depends on the expression of MCPyV large T antigen, and is necessary for the survival MCPyV-positive MCC cells.74 YM-155, a survivin inhibitor, was reported to induce cell death in MCC cell lines and to improve survival in a mouse xenograft tumor model.75 MCC tumors also express high protein levels of prosurvival Bcl-2 members, and combined inhibition of multiple family members by using a small-molecule antagonist has been reported to result in apoptosis in MCC cell lines and growth suppression of MCC xenografts.76 However, a Bcl-2 antisense has not been found to be effective in MCC patients.77 There have been some conflicting results regarding the frequency of KIT overexpression in MCCs and the relevance of the use of KIT inhibitors. It has been evidenced that the detection of KIT expression in MCC tumors did not correlate with the presence of KIT-activating mutations, that remains a rare event in MCCs.78 Imatinib mesylate, a tyrosine receptor kinase inhibiting KIT and platelet-derived growth factor receptor-alpha (PDGFR-α), has yielded some encouraging results in case reports,79,80 but disappointing findings were reported in a phase II clinical trial.81 MCC cells also have been shown to express receptors to somatostatin, a natural hormone that exerts anti-tumoral effects in neuroendocrine tumors. Case reports suggest potential efficacy of somatostatin analogs in metastatic MCC patients,82–84

making it an interesting therapeutic option in somatostatin receptor-positive MCCs. MCC tumor cells also express various components of the vascular endothelial growth factor (VEGF)–VEGF-receptor (VEGF-R) family as well as PDGFR-α, both of which are involved in angio- and lymphangiogenesis.85,86 Potential efficacy of pazopanib, a tyrosine kinase receptor targeting VEGFR-1, -2, -3, PDGFR-α, -β, and c-kit, was reported in one MCC metastatic patient.87 In addition, activation of the PI3K/pAKT/mTOR pathway has been evidenced in the majority of MCPyV-positive MCCs.88,89 Moreover, MCC cell lines have been found to be sensitive to PI3K/mTOR inhibitors.88–90 However, these inhibitors could act in an opposite way to the MCV oncogene sT that has been found to act downstream in the mTOR signaling pathway to preserve 4E-BP1 hyperphosphorylation.39 Finally, MCC cells express high levels of the cellular heat shock protein 70 (HSP70), a protein that is thought to be necessary for MCPyV LT-Ag to achieve its oncogenic activity. In accordance with this, a HSP70 inhibitor was found to induce apoptosis in MCC cell lines and to exhibit anti-tumor activity in the MCC xenograft mouse model.91

Immunotherapy Immune evasion in many cancers is based on dysregulation of co-inhibitory and co-stimulatory receptors involved in host anti-tumor responses. Specific immune T cells infiltrating MCCs display reduced activation67 related to expression of inhibitory markers such as PD-1, PD-L1, and PD-L2. Agents that stimulate T-cell activity, block regulatory T-cell

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function, or inhibit PD-1 signaling thus may be effective in MCC treatment.67 Similarly, ipilimumab, an antibody directed against CTLA-4, has been shown to enhance T-cell–mediated anti-tumor responses in many cancers. Disruption of T-cell migration in MCC tumors has been evidenced by the downregulation of intratumoral vascular E-selectin. Existing therapeutic agents that modulate E-selectin expression may thus restore T-cell entry and could potentially synergize with other immune-stimulating therapies.68 Type I and II interferons (IFNs) also were found to reduce expression of MCPyV LT-Ag in association with apoptosis of MCC cell lines,92,93 although no clinical response was reported after systemic IFN treatment of two patients with advanced MCC.94 However, encouraging results were reported in one MCC metastatic patient using a combined approach including the local administration of IFNβ-1b followed by adoptive transfer of ex vivo–expanded polyclonal polyomavirus-specific T cells.95

MCPyV-Specific Therapies MCPyV oncoproteins are ideal targets for the development of MCC therapies since they are constitutively expressed in malignant tumors and their expression is mandatory for tumor development, and since sT antigen has been found to be necessary for survival of MCPyV-positive MCC cell lines.39,96 MCPyV-specific treatment based on T antigens could be used in the management of MCPyV-positive MCCs. Preliminary therapeutic approaches have investigated the efficacy of a MCPyV LT-Ag DNA vaccine in a mouse model. This vaccine was able to confer a LT-specific CD8(þ) T-cell–mediated therapeutic effect in mice bearing LT-expressing tumors and accordingly to prolong their survival.97 The recently reported identification of MCPyV T-cell epitopes65 also may be used to design peptidespecific vaccines, and to generate virus-specific T cells for adoptive immunotherapy. Antisense (siRNA) therapies could also be developed such as the design of shRNAs targeting MCPyV oncogenes in a similar way that shRNAs were used in an HPV oncogenic mice model.98

CONCLUSIONS MCC is a rare neuroendocrine skin cancer with increasing incidence due to greater age of the population, the increase in sun exposure and the increasing number of immunocompromised individuals. MCPyV is the etiological agent of MCC and is thus the first example of a human oncogenic polyomavirus. Seroepidemiology studies indicate that MCPyV infection is very common. The presence of

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MCPyV DNA on the skin surface of most adults suggests that MCPyV infection persists throughout life. However, the mode of transmission and the host cells of this virus remain to be elucidated. As for other human polyomaviruses, primary MCPyV exposure occurs early in life and the majority of adults have anti-MCPyV antibodies. Identifying the natural tropism of MCPyV would probably assist the understanding of the natural history and the cell origin of MCC. The etiologic role of MCPyV in MCC opens up opportunities to increase the understanding of this cancer and potentially to improve its treatment.

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Merkel cell polyomavirus in merkel cell carcinoma: clinical and therapeutic perspectives.

Merkel cell carcinoma (MCC) is a rare and often aggressive cutaneous cancer with a poor prognosis. The incidence of this cancer increases with age, im...
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