Review al carcinomului cu celule Merkel – ce se ştie şi ce nu se ştie?

Introduction

Merkel cell carcinoma (MCC) is a rare neu­ro­endocrine skin cancer – also known as trabecular carcinoma – and has the highest mortality rate among skin malignancies. These neuroendocrine cells, that were first discovered by Merkel in 1875, and their origin were difficult to establish, the cell having both epithelial and neuroendocrine elements and may have neural crest ori­gin. Regarding its role, it is believed that it acts to mo­du­late mechanoreception. Immunohistochemical stai­ning of the Merkel cell has demonstrated the pre­sence of NSE (neuron-specific enolase) and APUD (de­car­bo­xy­lation) cell marker and staining for cytokeratin 8, 18 and 19 and neuroendocrine markers, including sy­nap­to­physin and chromogranin^(1-4,7,8). The incidence is in­crea­sing over the past few decades (over 3000 new ca­ses diagnosed in the world). It frequently appears on areas exposed to sunlight: face (the most common site), neck and extremities (30-40%), other areas (mucosal sur­fa­ces like the nose or esophagus, trunk or genitalia), but the most Merkel cell carcinomas (MCCs) appear in the damaged areas of the head and neck, especially in elderly people. Other factors include advanced age and immunosuppression (organ transplant, HIV, he­ma­to­lo­gical malignancies, immunosuppressive drugs). In the carcinogenesis process, UV-induced mutations and clonal integration of the Merkel cell polyomavirus into the host genome, or both, can determine patterns of multiple DNA mutations (retinoblastoma [RB] protein se­que­stra­tion or in association with UV radiation-induced al­te­ra­tions involving the TP53 gene and mutations, he­te­ro­zygous deletion, and hypermethylation of the re­ti­no­­blas­toma or p53 inactivation). Over 60% of MCCs are as­so­ciated with the Merkel cell polyomavirus (MCPyV). In nonviral MCC, excessive sun exposure is the pre­do­mi­nant risk factor for the development of disease, and next-generation sequencing and whole exome sequencing have revealed a high tumour mutational burden (TMB), with recurrent mutations in TP53 and RB1. In MCPyV-positive patients, studies have shown low TNB. Immune escape is likely to be found in both groups, because immune checkpoint therapies involving PD-1/PD-L1 have shown to be effective in both viral and nonviral subtypes of Merkel cell carcinoma. MCC tumors can be classified into two groups, based on the frequency of tumor infiltrating lymphocytes (TIL) and PD-L1 expression: immune resistant (high TIL/high PD-L1 – higher mutation burden) and immune ignorant (low TIL/low PD-L1)^(1-4,6-8). In a larger study, that included 317 patients with MCC, comprehensive genomic profiling was performed to characterize the genomic landscape of MCC. The primary objective of the study was to evaluate both clinical and molecular correlations with immunotherapy response and patient survival. A retrospective evaluation demonstrated that both subsets respond similarly well to immunotherapy and the clinical response is associated with a long-term impact on survival. More importantly, the retrospective analyses suggested that the use of other therapies prior to immunotherapy negatively influences the response to immunotherapy and checkpoint inhibitors should be used as a first option when possible^(9-11,14).

The lesions usually appear as firm, indurated, red or purple lumps on the skin, are not usually painful and can grow quickly. The most common site of occurrence is the skin of the face (periorbital region, frontal area) and in 75 % of the cases they are located to the primary site. Approximately 25% of the patients can have regional lymph node metastasis and less than 2% of the patients can have an unknown primary site. Recurrence is frequent and can be local, through the lymphatic nodes or distant metastases. The most frequent sites for metastases are other areas of the skin, brain, liver, lungs and bones. The AEIOU acronym (Heath et al.) includes the clinical signs and risk factors for MCC: asymptomatic/lack of tenderness, expanding rapidly, immune suppression, older than 50 years of age, and UV-exposed site on a person with fair skin^(1-5). Three major histological subtypes can be identified: small cell (poor prognosis), trabecular (favorable prognosis) and intermediate cell. Staging is very important, and investigations include full-body CT scans, MRIs or PET-CT, with emphasis on clinical examination also, especially the lymph nodes areas. Octreotide scintigraphy can be useful in some cases. Sentinel node biopsy should be performed in patients with no clinical evidence of regional lymph node enlargement. Biological status and dermatological examination for other lesions or skin malignancies are mandatory. Guidelines regarding Merkel cell carcinoma were published in 2015 by the collaborative group of multidisciplinary experts from the European Dermatology Forum (EDF), the European Association of Dermato-Oncology (EADO), and the European Organization of Research and Treatment of Cancer (EORTC)^(1-5,10-13).

Patients with localized disease have the best survival rates (65-85% five-year survival), thus the extent of the disease is the most important prognostic factor. Several prognostic factors have been associated with poor prognosis: lymphovascular invasion, tumor growth pattern, number of lymph nodes, the presence of mitotic features and G2+ tumor nuclei, PDGF (platelet-derived growth factor), c-kit (CD117), and phosphoinositide 3-kinase (PI3K). Data are still insufficient to assign any biomarker to prognostic outcome, although research regarding PD-L1, p63, vascular endothelial growth factor receptor (VEGFR), Ki-67, CD34, Ep-CAM, nuclear fac­tor kappa B, sonic hedgehog pathway proteins and ma­trix metalloproteinase has suggested that these bio­mar­kers could predict disease outcomes in patients with MCC. The five-year survival rate for metastatic di­sease is approximately 25%, the mortality rate is be­t­ween 30% and 50% and, although there have been ma­jor advancements in understanding the MCC biology and the treatments have evolved, the survival rates have re­mained low. Unknown primary MCC has a higher tu­mor mutational burden and lower association with MCPyV than those with known primary MCC and fre­quen­tly presents as clinically positive nodal disease, being associated with an improved survival compared to those with concurrent primary tumor. Studies proved that nodal tumors with unknown primary MCC have UV-determined mutations, suggesting an im­mu­no­lo­gi­cal mechanism pattern between regression of primary tu­mor and better prognosis of unknown primary MCC^(1-5,14).

The therapeutic options for MCC depend on the cha­rac­­te­­ris­tics of the patients and the disease (stage, pri­ma­ry tumor site, lymph node involvement, co­mor­bi­dities of the patient and performance status) and it includes mul­­ti­­­mo­­dal treatments like surgery, chemotherapy or ra­dio­­the­­rapy (extracapsular extension of lymph nodes) and im­mu­notherapeutic treatments which are showing pro­­­mi­­sing early results. In order to improve the prognosis of pa­tients with (MCC), early diagnosis and further un­­­der­­­stan­ding of the roles of surgery, radiotherapy, che­mo­­the­rapy and immunotherapy are mandatory. The in­clu­­sion in clinical trials is recommended in dis­se­mi­na­ted Merkel cell carcinoma^{(1-3,5,9,13,15)}.

Surgical excision (Mohs microsurgery) is indicated in patients with affected areas that need tissue sparing (2-3 cm margins) and for the margin control (100% mar­gin assessment). Positive margins on histological re­port indicate a risk of recurrence and re-excision is re­com­men­ded. The use of Mohs microsurgery for MCC was ques­tioned because of the increase of in-transit me­ta­sta­ses. Patients unfit for excision can be treated with radio­the­ra­py as an option. If clinical regional lymph node in­vol­ve­ment is suspected, then radical lymphadenectomy is required. Sentinel node biopsy is recommended if there is no clinical evidence of regional lymph node en­lar­ge­ment. Large retrospective analyses (n>100) or meta-analyses of SLNB in patients with clinically node-negative localized MCC have reported rates of SLN positivity ranging from 30% to 38%. Results from re­tro­spec­tive studies evaluating the prognostic value of SLN status showed significant association between SLN ne­ga­ti­vity and lower risk of recurrence and improved MCC-specific survival or overall survival (OS). A SLN biopsy is very important as part of the comprehensive patho­­lo­gical staging/work up, and coordinating an ac­cu­rate SLN biopsy prior to Mohs surgery would be of utmost importance. The impact on overall survival is unknown. The standard treatment recommends wide lo­cal excision with adjuvant radiotherapy. Future stu­dies need to determine if radical lymphadenectomy provides an additional survival benefit compared to adjuvant RT in patients with a positive sentinel node biopsy^{(1,2,5,12,15-18)}.

Merkel cell carcinoma is considered a radiosensitive type of tumor and radiotherapy is indicated as adjuvant treatment after surgical excision. Adjuvant radiotherapy reduces the risk of local recurrence compared with surgery alone. Radiotherapy can be used like adjuvant therapy for better local control. The usual dose is 45-50 Gy given in 5 weeks and for high-risk tumors the recommended dose is 55-65 Gy given in 5 weeks (tumors with positive margins, primary tumor >1.5 cm, positive margins, margins <2 mm, evidence of lymphatic, vascular, or perineural invasion or regional lymph node involvement). In palliative setting, radiotherapy is used as treatment option for inoperable MCC (tumor control, pain, bleeding, ulceration and secondary infections)^(1,2,5,19). For stage I/II MCC receiving adjuvant RT, retrospective studies showed improved OS compared with those receiving only surgery; however, it was not demonstrated for stage III MCC. Studies have also shown that Merkel cell carcinoma of the head and neck adjuvant therapy (chemotherapy and radiotherapy) offers better improvement in the overall survival rate than does postoperative radiotherapy alone in patients with risk factors^(12,20-22).

Chemotherapy is indicated for advanced-stage or metastatic MCC due to high chemosensitivity, and pla­ti­num-based regimens have been frequently used, but with limited data efficacy. There is no standard regimen in­di­ca­ted, because randomized studies comparing dif­ferent chemotherapy regimens could not be performed due to the rarity of the disease. The role of adjuvant che­mo­therapy is not well established. It was associated with increased morbidity, resistance to chemotherapy on recurrence, decreased quality of life and immune sup­pres­sion, and it is not recommended by guidelines. Che­mo­the­ra­py is indicated as treatment for metastatic MCC (stage IV). The most used regimens include CAV (cyclophosphamide, doxorubicin, vincristine), CEV (cyclophosphamide, etoposide, vincristine), EP (eto­po­side, cisplatin) and topotecan, with no standard regimen es­ta­blished. Future large randomized studies need to de­ter­mine if patients with MCC with advanced disease be­ne­fit from chemotherapy treatments regarding pro­gre­ssion-free survival or overall survival. The enrollment in a clinical trial is usually recommended^{(1-3,5,9,11-13,23-25)}.

Due to the high immunogenicity of MCC, the efficacy of immune checkpoint inhibitors was demonstrated in several clinical trials. There are individual immune responses and molecular genetic signatures, with heterogeneity between different metastatic lesions. CTLA4, PD1, PD-L1 and CD28 are molecular receptors upregulated in the MCC tumor microenvironment, being associated with poor prognosis. Ongoing trials question the role of immunotherapeutic agents, targeted therapies and endocrine therapy with somatostatin analogues in adjuvant metastatic setting. Predictive biomarkers could help guide the multimodal treatment, such as intratumor levels of CD8+ T cells. Also, in the MCC patient blood and tumors, MCPyV oncoprotein-specific cells were identified and tumor burden was linked to higher rates. The CD8+ T cells (MCPyV-specific) express both PD-1 and Tim3, suggesting functional exhaustion, and MCPyV-negative MCC was linked to high levels of T-cell infiltrates. Higher expression rates of PD-L1 were observed in virus-positive tumors. These findings provided rationale for using immunotherapeutic agents targeting PD-1 or PD-L1 in advanced MCC and many studies followed this hypothesis^{(11,13,26-28,30,31)}.

Avelumab (Bavencio®) is a G1 immunoglobulin (IgG1) monoclonal antibody targeting programmed death 1 (PD-1) and was approved by the US Food and Drug Administration (FDA) for numerous types of can­cers, like lung cancer, urothelial cancer and MCC. The approval for metastatic MCC was in March 2017 (ac­ce­le­ra­ted approval), being the first drug approved by the FDA for metastatic MCC. The approval was based on the JAVELIN Merkel 200 study. This multicenter study en­rolled 88 patients diagnosed with metastatic MCC and di­sease progression on or after the administration of che­mo­therapy for distant metastatic disease. Data from the study indicated an objective response rate above 30%, with a complete response rate of 11%. At 6 months, pro­gres­sion-free survival was 40% and 86% of tumor res­pon­ses lasted at least 6 months (25 patients), while 45% las­ted at least 12 months (13 patients). It appeared to be ef­fec­tive in both MCPyV-positive and MCPyV-negative tumors (objective response rate 26% and 35%). Maturing PFS and OS data suggest a long-term benefit in some patients. In a phase 2 trial (NCT02155647 Part A) that included patients with stage IV MCC pretreated with chemotherapy, the quality of life and clinical outcome were monitored. The study revealed that no progression during avelumab treatment contributed to statistically and clinically meaningful improvements in health-rela­ted quality of life (HRQoL). In the ongoing phase II trial of avelumab (JAVELIN Merkel 200 part B), 39 patients with no prior systemic treatment for metastatic disease were enrolled until disease progression or unacceptable toxicity. The patients were not stratified for programmed cell death-ligand 1 expression or Merkel cell polyomavirus status. The primary endpoint was the duration of response (objective response persisting for ≥6 months) and the results of the study concluded that high rates of response to first-line avelumab therapy in patients with metastatic MCC are durable. The data sustain avelumab’s approval and use as a standard-of-care treatment for metastatic Merkel cell carcinoma^{(11,13,29,30,32-34)}. Data emerging in 2019 showed that the early objective response to avelumab treatment is associated with improved overall survival in patients with metastatic Merkel cell carcinoma. Twenty-nine patients had confirmed OR, but the median OS was not reached in patients with OR and was 8.8 months (95% CI 6.4-12.9) in patients without. OS seems to be influenced by early OR. Patients with OR by 7 or 13 weeks had significantly longer OS than patients without⁽³⁵⁾.

Studies investigated the potential associations of anti-PD-1 response and the presence of MCPyV, with the density and distribution of CD8+, PD-1+ and PD-L1+ in the tumor cell microenvironment (TME). The results presented in 2017 at the AACR Annual Meeting, although preliminary, suggested a relationship between PD-1 positive and response to anti-PD-1 therapy and highlighted that lymphocyte subsets other than CD8+ T-cells may contribute to the observed response. Pembrolizumab (anti-PD-1 antibody) demonstrated clinical efficacy in the treatment of naive patients with advanced or metastatic MCC. The results of the study that permitted the approval showed objective response rate (ORR) of 56% and 16% complete response (CR) rate, with a response duration ranging from at least 2.2 months to 9.7 months. The progression-free survival (PFS) was above 60% at six months. Pembrolizumab was effective regardless of Merkel cell polyomavirus status (ORR 62% and 44%, respectively, not significantly different). Pembrolizumab has been indicated as a treatment option for disseminated disease since 2017. In a phase II trial, 50 patients with MCC with no prior treatment were included and randomized to receive pembrolizumab (2 mg/kg every 3 weeks) for up to 2 years. The response was assessed with Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. The median follow-up period was 14.9 months. The results have shown higher OS compared with data from patients treated with chemotherapy. 64% of the patients had Merkel cell polyomavirus-positive tumors. The objective response rate for immune checkpoint was 56%, ORRs being 59% in virus-positive and 53% in virus-negative tumors. The 24-month PFS rate was 48.3%, and the median PFS period was 16.8 months. ORR, PFS and OS were not correlated with the presence of Merkel cell polyomavirus, and improved PFS and OS were noted in patients with PD-L1-positive tumors^{(13,36,37,38)}. A study on a small cohort (26 patients with advanced MCC re­cei­ving pembrolizumab) asserted the specific immune res­ponses in patients with Merkel cell carcinoma receiving anti-PD-1 therapy. In the study, MCPyV-oncoprotein anti­bodies were quantified and T cells were assessed for MCPyV-specificity. T-cell receptor clonality was determined by pretreatment tumor biopsies. The results did not identify any predictive biomarkers to determine the rate of response to pembrolizumab. MCPyV oncoprotein antibodies were detectable in 15 of 17 (88%) of virus-positive MCC (VP-MCC) patients. MCPyV-positive tumors had a lower intratumoral TCR than virus-negative tumors (p=0.0001). The titre of antibodies decreased in 91% of patients with response. Virus-specific T cells decreased over time in patients who had a complete response, and increased in patients who had progressive disease. The large number of mutations in virus-negative MCCs may result in the recruitment of a diverse T cell population and apparently lower clonality. The results of this study sustain the prognostic value of determining MCPyV oncoprotein antibodies in patients with advanced MCC who are at an increased risk for recurrent disease⁽⁴⁰⁾.

Also, the efficacy in MCC treatment of nivolumab was investigated. The trial of nivolumab (anti-PD-1 antibody), CheckMate358, included patients who had and those who had not received prior chemotherapy. The results showed that the progression-free survival at 3 months was 82% and 15 of 22 patients (68%) had objective responses. Nivolumab appeared to induce rapid and durable tumor regressions in the majority of treatment-naive and treatment-experienced patients with advanced or metastatic MCC, with a manageable safety profile. Ni­vo­lu­mab has been indicated as a treatment option for dis­se­minated MCC since 2018^(13,15,41).

The development of effective therapeutics agents is mandatory. Other therapies were studied regarding their efficacy in relapsed or metastatic MCC. These are interleukin 12 (IL-12) adoptive T-cell therapy, glu­co­py­ra­nosyl lipid adjuvant – stable emulsion (GLA-SE), ki­na­se inhibitors and somatostatin analogues. IL-12 is a pro­in­flammatory cytokine with an important role in cell-mediated immunity and by activating the na­tu­ral killer and T cells that produces interferon (IFN). Tar­gets like IL-12 gene using vaccine and electroporation tech­no­logy could provide additional information. Mo­le­cu­lar pathways can be overexpressed, such as PI3K/Akt, c-kit, VEGFA, VEGFC, VEGFR-2, PDGF-a and PDGF-b in MCC. Kinase inhibitors, like imatinib, cabozantinib or pazopanib, mTOR inhibitors or the PI3K inhibitor were investigated regarding their efficacy in MCC, with limited results within a few cases due to the low incidence of the disease. Ongoing studies for advanced or metastatic MCC need to validate if kinase inhibitors have a role in the treatment of MCC. In some MCC tumors, somatostatin receptor type 2 (sst2) has a high expression and ongoing studies will evaluate the activity of lanreotide in patients with locally advanced or metastatic MCC. There are no predictive biomarkers that could help select patients and additional research is essential, especially with the development of immunotherapeutic agents that have the potential to provide results for patients with advanced or recurrent MCC^(41-43).

Conclusions

No predictive biomarkers that could better select patients have been identified to be associated with the development of immunotherapeutic agents that have the potential to provide results for patients with metastatic MCC. However, due to the low incidence of MCC and the need to improve the prognosis of patients with MCC, the early diagnosis must be considered and the role of local surgical treatment, radiotherapy, chemotherapy and immunotherapy must be studied in large randomized trials, although this is very difficult due to the low in­ci­dence of this type of tumors. In the studies evaluating ave­lu­mab or pembrolizumab, responses were observed regard­less of Merkel cell polyomavirus status, proving that immunotherapy can provide benefit to all patients with advanced or metastic disease. Further studies of MCC-specific immune responses should provide some an­swers regarding the profile of the patients who are most likely to respond and which type of immunotherapy or combinations of checkpoint inhibitors and other treatments should be used to improve patients’ outcomes. Comprehensive genomic analyses need to be further assessed in MCC and the association between genomics and response to therapy should provide a more accurate indication on the profile of the patients who will have a maximum benefit from immunotherapy. The uniqueness of immunogenicity of this disease, MCPyV positive and  MCPyV negative subtypes, regarding re­spon­ses to tumour antigens, is not feasible for most can­cers. The results from a small cohort study sustained the prog­nostic value of determining MCPyV oncoprotein an­ti­bodies in patients with advanced MCC who are at an in­creased risk for recurrent disease, due to the enhanced titres of antibodies at disease progression according to the results of recent studies. Immunotherapeutic agents targeting PD-1 should be considered for first-line treat­ment options in patients with metastatic MCC due to higher response rates that were observed in patients with fewer lines of prior treatment. MCC patients which had a res­ponse on anti-PD-1 therapy showed higher densities of PD-1 positivity when compared with nonresponders. Fur­ther studies on large cohorts need to validate these results.