Association of Merkel cell polyomavirus infection with tumor p53, KIT, stem cell factor, PDGFR-alpha and survival in Merkel cell carcinoma

Most Merkel cell carcinomas (MCCs) contain Merkel cell polyomavirus (MCPyV) DNA, and the virus likely has a pivotal role in tumor pathogenesis. p53 and the KIT receptor tyrosine kinase have also been implicated in MCC pathogenesis, but little is known about their association with MCPyV infection. We identified 207 patients diagnosed with MCC in Finland in 1979-2004 and reviewed the histological diagnoses. Adequate clinical information, tumor tissue and DNA were available from 87 confirmed MCC cases. Presence of MCPyV DNA was assessed using quantitative PCR; p53, KIT, phospho-KIT, stem cell factor (SCF) and PDGFRα expression using immunohistochemistry and presence of mutations in KIT exons 9, 11, 13 and 17 and PDGFRA exons 10, 12, 14 and 18 using DNA sequencing. Most (77.0%) of the 87 tumors contained MCPyV DNA and 37 (42.5%) expressed KIT, whereas PDGFRα, p53, SCF and pKIT expression was less common (31.9, 22.8, 8.6 and 4.8%, respectively). No KIT or PFGFRA mutations were detected, but 10 (12.5%) of the 80 tumors studied harbored common PDGFRA exon 10 S478P substitution. Tumor p53 and KIT expression were associated with absence of MCPyV DNA (p = 0.01 and 0.009, respectively). Tumor p53 expression was associated with unfavorable MCC-specific survival (p = 0.021) and overall survival (p = 0.046), but tumor KIT expression only when stratified by presence of MCPyV DNA. The results suggest that p53 and KIT expression are associated with absence of MCPyV DNA in MCC, and that the molecular pathogenesis of MCC is multifactorial.     

Prokineticins and Merkel cell polyomavirus infection in Merkel cell carcinoma

Background:

Prokineticin-1 (PROK1) and prokineticin-2 (PROK2) are chemokine-like proteins that may influence cancer growth by regulating host defence and angiogenesis. Their significance in viral infection-associated cancer is incompletely understood. We studied prokineticins in Merkel cell carcinoma (MCC), a skin cancer linked with Merkel cell polyomavirus (MCPyV) infection.

Methods:

Carcinoma cell expression of PROK1 and PROK2 and their receptors (PROKR1 and PROKR2) was investigated with immunohistochemistry, and tumour PROK1 and PROK2 mRNA content with quantitative PCR from 98 MCCs. Subsets of tumour infiltrating leukocytes were identified using immunohistochemistry.

Results:

Merkel cell polyomavirus-positive MCCs had higher than the median PROK2 mRNA content, whereas MCPyV-negative MCCs contained frequently PROK1 mRNA. Cancers with high tumour PROK2 mRNA content had high counts of tumour infiltrating macrophages (CD68+ and CD163+ cells). Patients with higher than the median PROK2 mRNA content had 44.9% 5-year survival compared with 23.5% among those with a smaller content (hazard ratio (HR): 0.53; 95% confidence interval (CI): 0.34–0.84; P=0.005), whereas the presence of PROK1 mRNA in tumour was associated with unfavourable survival (P=0.052).

Conclusions:

The results suggest that prokineticins are associated with MCPyV infection and participate in regulation of the immune response in MCC, and may influence outcome of MCC patients.     

p63 expression as a new prognostic marker in Merkel cell carcinoma

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin tumor, mostly affecting elderly individuals. The histogenesis of this rare tumor as well as its prognostic criteria are a matter of dispute. METHODS: An immunohistochemical analysis of markers of proliferation (Ki-67/MIB-1), neuroendocrine differentiation (chromogranin A and synaptophysin), and basal cell differentiation (p63) was performed in a series of 47 cases of MCC. The significance of pathologic data and of immunoreactivity with different markers was evaluated using the chi-square test. Survival curves were calculated using the Kaplan-Meyer method. The survival difference was estimated using the Wilcoxon or Mantel-Cox test. RESULTS: Immunohistochemical evidence of neuroendocrine differentiation and immunopositivity for cytokeratin 20 were observed in all cases, whereas positivity for p63 was detected in approximately half of the cases (25 of 47 cases; 53.2%). Cases that were positive for p63 demonstrated a more aggressive clinical course than those that were negative (Z value of 2.93; P = .0003; hazards ratio of 22.22). CONCLUSIONS: Data from the current study indicate that p63 expression is associated with a worse prognosis in patients with MCC and represents a new independent marker of clinical evolution.     

Merkel cell carcinoma subgroups by Merkel cell polyomavirus DNA relative abundance and oncogene expression

Merkel cell polyomavirus (MCPyV) was recently discovered in Merkel cell carcinoma (MCC), a clinically and pathologically heterogeneous malignancy of dermal neuroendocrine cells. To investigate this heterogeneity, we developed a tissue microarray (TMA) to characterize immunohistochemical staining of candidate tumor cell proteins and a quantitative PCR assay to detect MCPyV and measure viral loads. MCPyV was detected in 19 of 23 (74%) primary MCC tumors, but 8 of these had less than 1 viral copy per 300 cells. Viral abundance of 0.06–1.2 viral copies/cell was directly related to presence of retinoblastoma gene product (pRb) and terminal deoxyribonucleotidyl transferase (TdT) by immunohistochemical staining (p ≤ 0.003). Higher viral abundance tumors tended to be associated with less p53 expression, younger age at diagnosis and longer survival (p ≤ 0.08). These data suggest that MCC may arise through different oncogenic pathways, including ones independent of pRb and MCPyV.     

Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma,lymphoid tissues and lymphoid tumors

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in ～80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8–14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV. © 2009 UICC     

The Merkel cell carcinoma challenge

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin that occurs primarily in elderly or immunocompromised patients. For this report, the authors reviewed the diagnostic challenges associated with MCC encountered on their fine‐needle aspiration (FNA) service and also conducted an in‐depth review of the literature on MCC. A computer search for patients who were diagnosed with MCC by FNA at the authors' institution from 2006 to 2010 was conducted, and 5 patients were selected for cytologic and immunochemical analyses based on their varied and diagnostically challenging clinical presentations. The 5 selected patients had clinical findings commonly associated with MCC, including advanced age (4 of the 5 patients were ages 75‐85 years) and a history of previous malignancies (3 of the 5 patients had a history of previous malignancy), and 1 patient was diagnosed with a concomitant low‐grade lymphoma. The patients and their disease illustrated the protean clinical presentation of MCC and the clinical and cytologic challenges associated with this neoplasm. The current findings indicate the need for cytopathologists to be aware of the deceptive presentation of this neoplasm and its cytologic and immunochemical features to correctly diagnose this insidious neoplasm. Cancer (Cancer Cytopathol) 2013;121:179–188. © 2012 American Cancer Society.     

Prospective study of Merkel cell polyomavirus and risk of Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare type of skin cancer that has a characteristically increased incidence among immunosuppressed subjects. The DNA of Merkel cell polyomavirus (MCV) is regularly found in most MCC tumors. We investigated whether Merkel cell polyomavirus (MCV) infection increases the risk for future MCC. Two large biobank cohorts (Southern Sweden Microbiology Biobank and the Janus Biobank), containing samples from 856,000 healthy donors, were linked to the Cancer Registries in Sweden and Norway to identify cases of MCC occurring up to 30 years after donation of a serum sample. For each of the 22 cases (nine males and 13 females), four matched controls were included. The serum samples were analyzed with an MCV neutralization assay and for IgG antibodies to MCV pseudovirions, using JC polyomavirus and cutaneous human papillomaviruses as control antigens. An increased risk for future MCC was associated both with high levels of MCV antibodies [OR 4.4, 95% CI 1.3–17.4] and with MCV neutralizing activity (OR 5.3, 95% CI 1.3–32.3). In males, MCV seropositivity was not associated to MCC risk, whereas the risk was strongly increased in females, both for high levels of MCV antibodies (OR 7.0, 95% CI 1.6–42.8) and for MCV neutralizing activity (OR 14.3, 95% CI 1.7–677). In conclusion, we found prospective evidence that MCV infection is associated with an increased risk for future MCC, in particular among females.     

Detection of Merkel cell virus and correlation with histologic presence of Merkel cell carcinoma in sentinel lymph nodes

Background:

Adjuvant treatment can dramatically improve the survival of patients with metastatic Merkel cell carcinoma (MCC), making early, accurate detection of nodal disease critical. The purpose of this study was to correlate Merkel cell virus (MCV) detection with histopathologic disease in sentinel lymph nodes (SLNs) of MCC.

Methods:

Merkel cell carcinoma cases with SLN (n=25) were compared with negative controls (n=27). Viral load was obtained by quantitative polymerase chain reaction (PCR) for regions VP1 and LT3 of MCV. Histopathologic disease and viral load were correlated.

Results:

Merkel cell virus was detected in 16 out of 17 (94%) of primary MCC (mean viral load (MVL)=1.44 copies per genome). Viral load in the negative controls was <0.01 copies per genome. Merkel cell carcinoma was present in 5 out of 25 (20%) SLN by histopathology, and MCV was detected in 11 out of 25 (44%) MCC SLN (MVL=1.68 copies per genome). In all, 15 out of 25 (60%) SLN showed correlation between histologic and MCV results. In all, 2 out of 25 (8%) samples were histopathologically positive and PCR negative. Of note, 8 out of 25 (32%) samples had detectable MCV without microscopic disease.

Conclusion:

Patients with positive SLN for MCV even if negative by histopathology were identified. The application of molecular techniques to detect subhistologic disease in SLN of MCC patients may identify a subset of patients who would benefit from adjuvant nodal treatment.     

An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC.     

皮肤Ｍｅｒｋｅｌ细胞癌的研究进展

Ｍｅｒｋｅｌ细胞癌（ＭＣＣ）是一种好发于皮肤的少见恶性神经内分泌瘤,预后较差,易局部复发和转移。ＭＣＣ的临床诊断可参考“ＡＥＩＯＵ”五元音法,但必须由组织病理学确诊。ＭＣＣ在高度紫外线暴露的地区较常见,且与免疫抑制密切相关。ＭＣＣ可能与Ｍｅｒｋｅｌ细胞多瘤病毒（ＭＣＰｙＶ）相关。目前ＭＣＣ尚无成熟的治疗方案,但首选手术治疗并常常辅助放疗或化疗。     

Solitary regional bony recurrence in Merkel cell carcinoma

This case report of recurrent Merkel cell carcinoma between radiotherapy fields for primary site and regional nodes supports the value of postoperative radiotherapy to the primary site, the intervening lymphatics and draining nodes.     

Predictors of immunotherapy benefit in Merkel cell carcinoma

Merkel cell carcinoma is a rare cancer for which immune checkpoint blockade is standard-of-care for recurrent/metastatic disease. However, not all patients benefit from immunotherapy. A greater understanding of molecular mechanisms and predictive biomarkers are unmet needs. We retrospectively analyzed electronic health records and next-generation sequencing data of 45 patients treated at our institution from 2013 to 2020 to understand clinical and genomic correlates of benefit from immunotherapy. Our cohort predominantly included individuals with stage III disease at primary disease diagnosis and individuals with stage IV disease at recurrent/metastatic disease diagnosis. Most received immunotherapy as first-line treatment. 43% experienced objective response (median duration of response 24.2 months, 95% confidence interval 8.8-not reached). Median overall survival was 15.5 months (95% confidence interval 9.0–28.7) (median follow-up 25.2 months). Less advanced stage at primary disease diagnosis and shorter disease-free interval between completion of initial treatment and recurrence were each associated with greater odds of response (odds ratio of 0.06, p = 0.04 for stage; odds ratio 0.75, p = 0.05 for disease-free interval). Single-nucleotide variants in ARID2 and NTRK1 were associated with response (p = 0.05, without Bonferroni correction), while none of Merkel cell polyomavirus status, total mutational burden, ultraviolet mutational signatures, and copy-number alterations predicted outcomes. Patients with shorter disease-free interval may be particularly suitable immunotherapy candidates. Our molecular findings point to ARID2 and NTRK1 as potential predictive markers and/or therapeutic targets (e.g., with Trk inhibitors), although this association needs to be confirmed in a larger sample.     

Merkel cell carcinoma: Clinical outcome and prognostic factors in 351 patients

Background

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine carcinoma of the skin.

Aim

To describe clinical outcome and prognostic factors of MCC patients in two expert‐centers.

Method

Patients with histologically confirmed MCC in 1990‐2014 were included. Data on patient, tumor characteristics and treatment were retrospectively collected.

Results

A total of 351 Patients were evaluated, 153 (44%) males, median age 74 years (range 28‐94). Median follow‐up time was 28 months (IQR 13‐58). Median primary tumor size was 17 mm (range 2‐135). At time of diagnosis 112 (32%) patients had lymph node metastases. The cohorts′ 5‐year overall survival (OS) was 58%. Using a competing risk analysis the 5‐year relapse and MCC related death was 42% and 22%. Adjuvant radiation therapy (XRT) was associated with reduced recurrence (SDH 0.54; CI 0.3‐0.9). Nodal involvement (SDH 2.7; CI 1.1‐6.6) and the male gender were associated with higher MCC related death (SDH 3.1; CI 1.2‐7.9)

Conclusion

In a large cohort a low MCC related death, in the presence of a low OS was seen. This indicates that a significant number of MCC patients die due to other causes than MCC. Adjuvant XRT was associated with relapse. Male gender and nodal metastasis were associated with MCC related death.     

TERT promoter mutations and gene amplification: Promoting TERT expression in Merkel cell carcinoma

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.     

Merkel cell carcinoma: histologic features and prognosis

BACKGROUND.

Currently, little is known regarding the potential prognostic value of histologic features in primary cutaneous neuroendocrine (Merkel cell) carcinomas (MCC).

METHODS.

In a retrospective review of the tumor histology and clinical outcome data (median follow‐up, 51 months; range, 3‐224 months) of 156 patients with a diagnosis of MCC, the following histologic features were evaluated: tumor thickness, tumor size (greatest dimension of the tumor), microanatomic compartment involved by tumor (dermis and/or subcutis and/or deeper), tumor growth pattern (nodular circumscribed vs infiltrative), lymphovascular invasion (LVI), tumor‐infiltrating lymphocytes, tumor necrosis, ulceration, and solar elastosis.

RESULTS.

The overall 5‐year survival rate was 67.5%. On univariate analysis, parameters that were associated significantly with survival were tumor thickness (P = .001), tumor size (P = .0002), deepest anatomic compartment involved by tumor (P = .0003), tumor growth pattern (P = .003), LVI (P < .00001), tumor‐infiltrating lymphocytes (P = .05), and solar elastosis (P = .04). On multivariate analysis, the presence of a nodular growth pattern, low tumor depth, and absence of LVI were associated with longer survival.

CONCLUSIONS.

In addition to the known prognostic value of tumor stage, 3 histologic features were identified to have prognostic significance: tumor thickness (depth of tumor invasion), the presence of LVI, and tumor growth pattern. Cancer 2008. © 2008 American Cancer Society.     

PD-L1 blockade with avelumab: A new paradigm for treating Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Until recently, no durable treatment options were available for patients with advanced disease. As an immunogenic cancer, MCC was hypothesized to be a candidate for PD-L1/PD-1 targeted therapy. On March 23, 2017 the US Food and Drug Administration granted accelerated approval for avelumab, an anti-PD-L1 monoclonal antibody, for the treatment of metastatic MCC on the basis of the JAVELIN Merkel 200 trial. Here we examine the results and implications of this pivotal study, published in Lancet Oncology by Kaufman et al., as well as current developments in the use of immune-checkpoint therapies for treating patients with MCC.