Merkel Cell Polyomavirus Status Research Articles

Patched 1 expression in Merkel cell carcinoma.

The relevance of Hedgehog signaling in Merkel cell carcinoma has only been addressed by a few studies with conflicting results. Thus, we aimed to establish the expression of Hedgehog signaling molecules in Merkel cell carcinoma to characterize causes of aberrant expression and to correlate these findings with the clinical course of the patients. Immunohistochemistry was performed for Sonic, Indian, Patched 1 (PTCH1) and Smoothened on patients' tumor tissue. Respective mRNA expression was analyzed in 10 Merkel cell carcinoma cell lines using quantitative real-time polymerase chain reaction. PTCH1 sequencing and DNA methylation microarray analyses were carried out on tumor tissues as well as cell lines. PTCH1 immunoreactivity in Merkel cell carcinoma was similar to that of basal cell carcinomas, which both significantly differed from PTCH1 immunoreactivity in healthy skin. Most PTCH1 mutations found were synonymous or without known functional impact. However, on average, the promoter regions of both PTCH1 were hypomethylated independently from PTCH1 gene expression or Merkel cell polyomavirus status. PTCH1 and GLI1/2/3 genes were differently expressed in different cell lines; notably, there was a significant correlation between GLI2 and PTCH1 mRNA expression. Similar to PTCH1 protein expression in patient tissues, PTCH1 gene expression in Merkel cell carcinoma cell lines is highly variable, but due to the similar methylation pattern across Merkel cell carcinoma cell lines, effects other than methylation seem to be the reason for the differential expression and PTCH1 appears to be upregulated by GLI as a classical Hedgehog target gene.

Clinical, FDG-PET and molecular markers of immune checkpoint inhibitor response in patients with metastatic Merkel cell carcinoma

BackgroundMetastatic Merkel cell carcinoma (mMCC) is an aggressive neuroendocrine malignancy of the skin with a poor prognosis. Immune checkpoint inhibitors (ICIs) have shown substantial efficacy and favorable safety in clinical...

Expression of Lefty predicts Merkel cell carcinoma-specific death.

Lefty and Nodal are transforming growth factor β-related proteins, which, beside their role in determination of laterality during embryogenesis, have also been linked with cancer progression. Prompted by the observed significant left-sided laterality of Merkel cell carcinoma (MCC), we addressed whether Lefty and Nodal are expressed in MCC and correlated expression patterns with clinical parameters such as MCC laterality and patient outcome. Expression of Lefty and Nodal in primary MCC was assessed in 29 patients by immunohistochemistry. The histology (H-)score was calculated and correlated with clinical parameters. The median (range) H-score of Lefty and Nodal was 17.6 (0-291) and 74.9 (0.7-272), respectively. There was a significant correlation between Lefty expression and Nodal expression (correlation coefficient of 0.60, P=0.0006). There was no significant correlation between Lefty expression and Nodal expression with either tumour laterality, gender, age, Merkel cell polyomavirus status, disease stage, anatomical localization of primary tumours or disease relapse. On univariate analysis, low Lefty expression and Nodal expression were significantly associated with MCC-specific death (P=0.010 and P=0.019, respectively). On univariate analysis, low Lefty expression was the only significant independent predictor for MCC-specific death (P=0.025) as indicated by an odds ratio of 14 (95% CI: 1.43-137.33). Lefty and Nodal are frequently expressed in MCC, but not correlated with tumour laterality. Importantly, our data suggest that a low level of Lefty expression in primary MCC is a strong predictor of MCC-specific death.

P63 expression in Merkel cell carcinoma: comparative immunohistochemistry invokes TAp63 as the dominant isoform involved

The literature suggests that p63 expression in Merkel cell carcinoma (MCC) is associated with a poor prognosis. p63 immunohistochemistry marks the 2 main isoforms of this transcriptional protein: TAp63 (tumor suppressor-like properties) and ∆Np63 (oncogenic properties). Little information about the isoform of relevance in MCC exists. p40 immunohistochemistry specifically marks ∆Np63, and using comparative, semiquantitative expression of p63 and p40, we sought to clarify the issue. Our cohort of 53 cases (28 men and 25 women, median age 79 years, interquartile range 71-88) was stratified by morphology and viral status. Immunohistochemistry (p63, p40, and cytokeratin 5/6) was performed, H-scores for nuclear expression of p63 and p40 were derived (2 observers; positivity ≥ 10), and interobserver agreement was evaluated. Clinical, pathological, and outcome data were documented. The results were analyzed statistically. Mortality amounted to 57% (median follow-up 686 days, interquartile range 292-1599). Positivity for Merkel cell polyomavirus was observed in 29 (55%) of cases. Expression of p63 and p40 was present in 36 (69%) and 4 (8%) of cases, respectively. Increased age (P = .0241), negative Merkel cell polyomavirus status (P = .0185), and p63 positivity (P = .0012) were significantly associated with mortality. The latter 2 variables were highly correlated (P = .004). The interclass correlation between the 2 sets of H-scores was 0.95. Our findings support an association between p63 expression and reduced overall survival in MCC and show consistency in scoring this prognostic parameter. TAp63 is the dominant isoform of the protein involved. The paradoxical tumor suppressor-like activity of this isoform in p63-positive MCCs with reduced overall survival requires further study.

A Systematic review and Meta-Analysis of the survival and clinicopathological features of p63 expression in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare skin tumour of neuroendocrine origin with aggressive behaviour. The aims of this study were to investigate the association of p63+MCC with clinicopathological features and to estimate survival through a systematic review and meta-analysis. A comprehensive search of PubMed, Embase, Scopus and Virtual Health Library following the PRISMA guidelines was conducted on September 2017. DerSimonian and Lard random-effects models were used to calculate survival-weighted means and their corresponding 95% confidence intervals (CI) among studies. Five studies met our inclusion criteria after screening 77 citations and 36 full-text articles. The included studies enrolled 413 patients with MCC. We observed that p63+MCC was significantly associated with mortality with OR 2.92 (95% CI [1.66-5.13]). The summary hazard ratio of multivariate analysis was 1.99 (95% CI [1.32-3.01]). The only clinicopathological feature associated with p63+MCC with statistical significance was the Merkel cell polyomavirus (MCPyV) status. The presence of MCPyV was associated as a protective factor for the expression of p63 (OR 0.25, 95% CI [0.08-0.73]). These results support that p63+MCC evaluated by immunohistochemistry has a poor outcome. Therefore, we suggest p63 to be performed when staging MCC.

341O - Clinical and FDG-PET markers of immune checkpoint inhibitor (ICI) response in patients with metastatic Merkel cell carcinoma (mMCC)

BackgroundmMCC is a rare, highly aggressive neuroendocrine cancer of the skin with a poor prognosis. ICIs have favourable efficacy and safety in clinical trials. We outline single centre experience utilising ICIs in mMCC. MethodsMedical records of patients (pts) with mMCC treated with ICIs from Aug 2015 to Dec 2018 at Peter MacCallum Cancer Centre in Australia were retrospectively analysed. RNA sequencing and immunohistochemistry for PD-L1, CD3 and Merkel cell polyomavirus (MCPyV) on tumor samples were performed. Baseline tumor volumes and responses were assessed with FDG-PET scans using the Hicks criteria. Results23 pts with mMCC were treated with ICIs. Pt characteristics are summarised in the table. A median of 8 cycles (range 1 to 47) were administered, with treatment ongoing in 6 pts. Objective responses (OR) were observed in 14 pts (61%); 10 (44%) complete metabolic responses (CMR) and 4 (17%) partial metabolic responses (PMR). Median time to response was 8 weeks (range 6 to 12) and 12-month progression-free survival (PFS) rate was 39%. Increased OR were seen in pts aged less than 75 (OR 8/10, 80% vs 46%), pts with normal baseline LDH (OR 5/7, 71% vs 50%), pts with an immune-related adverse event (irAE) (OR 6/6, 100% vs 47%) and in MCPyV negative pts (OR 11/16, 69% vs 43%). Pts with a CMR had lower mean-tumor volume on baseline FDG-PET scan (CMR: 35.7mL, no CMR: 187.8mL, p = 0.05). No correlation was seen between tumor PD-L1 positivity and response to ICI (p = 0.25) or MCPyV status (p = 0.76). Similarly, no association was seen between OR and CD3 staining within the tumor (p = 0.07). 10 pts received radiation (RT) during ICI: 4 pts started RT concurrently (OR 75%, CMR 50%), 3 pts had isolated ICI-resistant lesions successfully treated with RT and 3 pts with multisite progression continued to progress despite RT. 6 pts (26%) had a Grade 1-2 irAE.Table341O Baseline characteristicsTablePatient Characteristicsn = 23Age (years) Median (range)75 (64 to 91)Gender Male Female18 5ECOG 0 12 38 12 3ICI Avelumab Pembrolizumab Other (BGB-A317)15 5 3MCPyV status Positive Negative7 16Visceral disease17Prior chemotherapy10History of autoimmune disease2Previous immunosuppression2 ConclusionsICIs showed efficacy and safety consistent with trial data. Younger age, negative MCPyV status, normal LDH, lower baseline FDG-PET tumor volume and irAEs are potentially associated with enhanced response. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureR.J. Hicks: Shareholder / Stockholder / Stock options: Telix Pharmaceuticals; Travel / Accommodation / Expenses: Endocyte; Travel / Accommodation / Expenses: GE Medical Systems. R. Tothill: Honoraria (self), Travel / Accommodation / Expenses: Merck Serono. All other authors have declared no conflicts of interest.

Global PD-L1 Signals and Tumor-Infiltrating Lymphocytes: Markers of Immunogenicity in Different Subsets of Merkel Cell Carcinoma and Potential Therapeutic Implications.

We previously studied the genetic and immunohistochemical profiles of subsets of Merkel cell carcinoma (MCC) stratified by morphology and Merkel cell polyomavirus (MCPyV) status. Recent advances in the immunotherapy of this disease prompted us to examine markers of immunogenicity [PD-L1 expression and tumor-infiltrating lymphocytes (TILS) in these subsets]. The observed clinical responses to checkpoint inhibition of the PD-1/PD-L1 pathway have not correlated with PD-L1 expression by MCC cells, and recent evidence suggests that functions of this pathway within the immune tumor microenvironment may be relevant. We conducted a semiquantitative (high, moderate, and minimal) immunohistochemical evaluation of the global PD-L1 signal in 52 cases of MCC, segregated in 3 subsets [pure MCPyV-positive (n = 28), pure MCPyV-negative (n = 9), and combined MCPyV-negative (n = 15)]. TILS were categorized as brisk, nonbrisk, or absent. Intersubset comparisons revealed that high global PD-L1 signals were exclusively associated with pure MCPyV-positive MCCs contrasted with virus-negative cases (P = 0.0003). Moderate signals were seen across all 3 groups. Brisk TILS were significantly associated with MCPyV-positive MCCs compared with MCPyV-negative cases (P = 0.029). Neither parameter (PD-L1 or TILS) was significantly different between the MCPyV-negative groups. Of potential clinical relevance, MCPyV seems to convey greater immunogenicity to MCCs than the high mutational burden/greater neoantigen load of MCPyV-negative cases. Interesting too is the fact that subset-related profiles of these markers mirrored those noted at genetic and immunohistochemical levels, separating pure MCPyV-positive MCCs from the virus-negative subsets.

Decreased H3K27me3 Expression Is Associated With Merkel Cell Polyomavirus-negative Merkel Cell Carcinoma, Especially Combined With Cutaneous Squamous Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer, frequently infected with Merkel cell polyomavirus (MCPyV). H3K27me3 acts as a repressive histone modification that epigenetically controls gene transcription. The aim of this study was to examine H3K27me3 expression in MCC. H3K27me3 expression levels were immunohistochemically analyzed in 20 MCPyV-positive MCCs, 15 MCPyV-negative MCCs with squamous cell carcinoma (SCC) (combined MCCs), and six MCPyV-negative pure MCCs. Reduced H3K27me3 expression was variously observed in MCCs. H3K27me3 H-score was significantly lower in MCPyV-negative MCCs than in MCPyV-positive MCCs (p=0.002). H3K27me3 expression was significantly lower in MCPyV-negative combined MCC component than in MCPyV-positive MCCs (p<0.001), MCPyV-negative pure MCCs (p=0.036), or pure MCC histology (p<0.001). Kaplan-Meier analysis showed no association of H3K27me3 with outcome. Differential reduction in H3K27me3 expression was observed based on MCPyV status and morphological type. These results implicate H3K27me3-mediated epigenetic changes in tumorigenesis of MCC, especially in MCPyV-negative MCC combined with SCC.

Integrative molecular analysis of metastatic Merkel cell carcinoma to identify predictive biomarkers of response to avelumab.

9569 Background: Avelumab, an FDA-approved human anti–PD-L1 IgG1 monoclonal antibody for patients (pts) with metastatic Merkel cell carcinoma, showed an objective response rate (ORR; by RECIST v1.1) of 31.8% in a second-line phase 2 trial (NCT02155647). We assessed the association of tumor mutational burden (TMB; nonsynonymous somatic variants/megabase), PD-L1 expression, Merkel cell polyomavirus (MCPyV) status, and CD8+ tumor-infiltrating T-cell density with ORR and survival. Molecular profiles (RNAseq and WEX) also were analyzed. Methods: Baseline tumors (n = 36) were profiled using RNAseq and WEX sequencing. PD-L1 expression (≥1% cutoff), MCPyV status, and CD8+ T-cell density at the tumor invasive margin were evaluated by IHC. MHC locus expression was measured with OptiType and loss of heterozygosity (LOH) with LOHHLA. Results: Of 36 pts profiled, 12 had a response, 27 were PD-L1+, and 23 were MCPyV+. The TMB upper tertile and quartile values were 1.34 and 3.16, respectively. Consistent with literature, MCPyV− pts had a higher median TMB (2.72) than MCPyV+ pts (0.49). PD-L1+ tumors trended toward a higher TMB. An empirical cohort-specific TMB cutoff of ≥2 was chosen to include sufficient pts per subgroup. Pts with TMB ≥2 vs TMB &lt; 2 had higher ORR (5 of 11 [45.5%] vs 7 of 25 [28.0%]) and 6-mo PFS rates (60% vs 38%). Among pts with TMB ≥2, the highest ORRs were reported in MCPyV− (4 of 7 pts), PD-L1+ (5 of 9 pts), and CD8+ T-cell density higher than median (5 of 6 pts) subgroups. MHC expression trended with ORR and survival. Higher mean MHC expression was found in pts with CD8+ T-cell density higher than median (p &lt; 0.05). Mutations in antigen presentation genes were detected: LOH at the HLA locus in 9 of 30 pts (28%), including 4 with a response; an NK cell activation signature was also associated with response. These data may suggest that ADCC contributes to response. Factorial analysis of gene signature scores identified signatures (eg, IFNγ, TP53 pathway) associated with MCPyV status and response. Conclusions: Responses in this data set were not attributed to any specific biomarker alone. Future analysis is focused on validating these results and identifying rational drug combinations with avelumab. Clinical trial information: NCT02155647.

Prognostic differences and the effect of radiotherapy on survival in Merkel cell carcinoma: A retrospective analysis of a Swedish cohort.

e21047 Background: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. The pathogenesis is linked to the immune system and Merkel cell polyoma virus (MCPyV) is present in about 80% of MCC. Reports have shown that patients with a virus-negative MCC have a worse prognosis. Adjuvant radiotherapy given after surgery has previously been shown to give a survival benefit in retrospective data. Our aim is to analyze clinical variables and their prognostic impact in a Swedish cohort. Methods: All patients with confirmed diagnosis of MCC referred to Karolinska University Hospital in Sweden from 1989-2018 were included and retrospective data was collected. Results: 113 patients were included. Median age at operation was 75.4 years (range 19-99); 64 (57%) were female. Treatment included surgery with (n = 46) or without adjuvant radiotherapy (RT). 43% of female patients and 37% of male patients received RT. Median overall survival (OS) was 2,6 years (range 0,1-25,8), for female patients 3,4 years (range 0,1-25,8) and for male patients 1,8 years (range 0,1-19,0). Median OS in surgery group was 1,8 years (range 0,4-25,8). With adjuvant RT median OS improved with 1,7 years (p = 0,0001) to 3,5 years (range 0,2-22,1), for female patients 4,0 years (range 0,2-22,1) and for male patients 2,0 years (range 0,3-8,8). MCPyV status was available in 53 patients. 74% were MCPyV positive, 26% negative. MCPyV gave no difference in OS, however when gender was added in to multivariate analysis, female patients with virus negative disease had better outcome than virus negative males (p = 0.03). In virus positive MCC there was no difference in OS between female and male patients. Conclusions: Our data confirms the positive impact of adjuvant radiotherapy on survival and illustrates the difference in outcome between female and male patients, with women having a better outcome. In the MCPyV group the tendency is that men with virus negative MCC has the worst outcome. Our findings indicate that MCPyV positive and negative MCC act as two different diseases and it also raises questions if there is a difference in the disease itself or the immune response towards MCC between male and female patients.

Clinical and FDG-PET markers of immune checkpoint inhibitor (ICI) response in patients with metastatic Merkel cell carcinoma (mMCC).

9540 Background: mMCC is a rare, highly aggressive neuroendocrine cancer with a poor prognosis. ICIs have favourable efficacy and safety in clinical trials. We outline single centre experience utilising ICIs in mMCC. Methods: Medical records of patients (pts) with mMCC treated with ICIs from Aug 2015 to Dec 2018 at Peter MacCallum Cancer Centre in Australia were retrospectively analysed. RNA sequencing and immunohistochemistry for PD-L1, CD3 and Merkel cell polyomavirus (MCPyV) on tumor samples were performed. Baseline tumor volumes and responses were assessed with FDG-PET scans using the Hicks criteria. Results: 23 pts with mMCC were treated with ICIs. Pt characteristics are summarised in Table. A median of 8 cycles (range 1 to 47) were administered, with treatment ongoing in 7 pts. Objective responses (OR) were observed in 14 pts (61%); 10 (44%) complete metabolic responses (CMR) and 4 (17%) partial metabolic responses (PMR). Median time to response was 9 weeks (range 4 to 11) and 12-month progression-free survival (PFS) rate was 32%. Increased OR were seen in pts aged less than 75 (OR 8/10, 80% vs 46%), no prior history of chemotherapy (OR 10/14, 71% vs 44%), pts with an immune-related adverse event (irAE) (OR 6/6, 100% vs 47%) and in MCPyV negative pts (OR 9/11, 82% vs 50%). Pts with a CMR had lower mean-tumor volume on baseline FDG-PET scan (CMR: 35.7mL, no CMR: 187.8mL, p value 0.05). 10 pts received radiation (RT) during ICI: 4 pts started RT concurrently (OR 75%, CMR 50%), 3 pts had isolated ICI-resistant lesions successfully treated with RT and 3 pts with multisite progression continued to progress despite RT. 6 pts (26%) had a Grade 1-2 irAE. Conclusions: ICIs showed efficacy and safety consistent with trial data. Younger age, negative MCPyV status, no prior chemotherapy, lower baseline FDG-PET tumor volume and irAEs are potentially associated with better responses. [Table: see text]

ALK and EGFR expression by immunohistochemistry are associated with Merkel cell polyomavirus status in Merkel cell carcinoma.

Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.

Characterizing the Therapeutic Potential of a Potent BET Degrader in Merkel Cell Carcinoma

Studies on the efficacy of small molecule inhibitors in Merkel cell carcinoma (MCC) have been limited and largely inconclusive. In this study, we investigated the therapeutic potential of a potent BET degrader, BETd-246, in the treatment of MCC. We found that MCC cell lines were significantly more sensitive to BETd-246 than to BET inhibitor treatment. Therapeutic targeting of BET proteins resulted in a loss of “MCC signature” genes but not MYC expression as previously described irrespective of Merkel cell polyomavirus (MCPyV) status. In MCPyV+ MCC cells, BETd-246 alone suppressed downstream targets in the MCPyV-LT Ag axis. We also found enrichment of HOX and cell cycle genes in MCPyV− MCC cell lines that were intrinsically resistant to BETd-246. Our findings uncover a requirement for BET proteins in maintaining MCC lineage identity and point to the potential utility of BET degraders for treating MCC.

Merkel Cell Polyoma Viral Load and Intratumoral CD8+ Lymphocyte Infiltration Predict Overall Survival in Patients With Merkel Cell Carcinoma.

Introduction: Merkel cell carcinoma (MCC) is linked to the presence of clonally integrated Merkel cell polyomavirus (MCPyV) in up to 80% of the cases. The aim of the study was to determine the prognostic value of baseline MCPyV viral load and lymphocytic infiltration.Methods: MCPyV DNA prevalence, integration status and viral load were determined by specific quantitative real-time PCR in surgical specimens obtained from 49 patients with MCC treated with (n = 22, 45%) or without postoperative radiotherapy (RT). CD8+ tumor infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) status were assessed using immunohistochemistry. MCPyV characteristics and immune marker expression were correlated with clinicopathological factors and overall survival (OS).Results: Median age at diagnosis was 74 (range, 42–100); 51% of the patients were female. One-, three, and five-year OS rates were 83.8, 58.6, and 47.1%, respectively. A positive MCPyV status was associated with female gender (p = 0.042). Tumor localization (head/arms vs. trunk) positively correlated with PD-L1 status (p = 0.011) and combined CD8/PD-L1 expression (p = 0.038). Overall CD8+ infiltration was inversely associated with N-stage (p = 0.048). Stromal TILs correlated significantly with both PD-L1 expression (p = 0.010) and N-stage (p = 0.037). A high viral load (>median) was significantly associated with worse OS (p = 0.029) and high intratumoral CD8+ infiltration with improved OS for the entire cohort (p = 0.045).Conclusion: These data provide important insight on the role of MCPy DNA viral load and TILs in the context of PD-L1 in patients with Merkel cell carcinoma. Future clinical studies should aim to explore the effect of PD-1/PD-L1 immune-checkpoint inhibitors in combination with existing radiotherapy approaches.

Expression of Notch 3 and Jagged 1 Is Associated With Merkel Cell Polyomavirus Status and Prognosis in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine skin cancer and most MCCs are related to infection with Merkel cell polyomavirus (MCPyV). Notch signaling modulates cell fate in various tissues including the skin during development and homeostasis, and its aberrant activity relates to onset and progression of various malignancies. Therefore, association of NOTCH1/ NOTCH2/NOTCH3/jagged 1 (JAG1) expression with MCPyV status and prognosis in MCC was investigated. A total of 19 MCPyV-positive and 19 MCPyV-negative MCC samples from patients were stained immunohistochemically with antibodies against NOTCH1, NOTCH2, NOTCH3, and JAG1 and analyzed. Expression of NOTCH1 and NOTCH2 was not associated with MCPyV status or prognosis. However, higher JAG1 expression was found in MCPyV-negative than in MCPyV-positive MCC (p<0.001), and NOTCH3 expression was higher in MCPyV-positive MCC (p=0.062). Kaplan-Meier and multivariate analyses showed that patients with MCC with higher NOTCH3 expression had better overall survival than otherwise (p=0.001 and p=0.033, respectively). Expression of NOTCH3, as a tumor suppressor, is an independent predictor of MCC outcome.

Usefulness of ulceration and hyperkeratosis as clinical predictors of Merkel cell polyomavirus-negative and combined Merkel cell carcinoma: A retrospective study.

Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin that is associated with Merkel cell polyomavirus (MCPyV). The clinical appearance and demographic characteristics of this tumor have been described using the mnemonic AEIOU: asymptomatic, expanding rapidly, immune suppression, older than 50 years, and ultraviolet-exposed fair skin. In addition, MCC can be categorized based on morphology as pure MCC or combined MCC that exhibits neuroendocrine and other phenotypic elements. There is limited information regarding the clinical characteristics and prognosis of combined MCC. This retrospective study aimed to identify factors, such as ulceration or hyperkeratosis, that could predict MCPyV status and morphological variants. Twenty patients with MCC were divided into groups based on MCPyV status and morphology: MCPyV-positive or MCPyV-negative MCC and pure or combined MCC. The patients' MCPyV status was immunohistochemically determined using the CM2B4 antibody to the MCPyV large T-antigen. The patients' clinicopathological characteristics were evaluated to identify predictors of MCPyV-negative MCC and combined MCC. The presence of ulceration/hyperkeratosis predicted the presence of MCPyV-negative MCC (80% of cases) and combined MCC (50% of cases). None of the 10 patients with MCPyV-positive MCC had ulceration/hyperkeratosis. The clinical presence of ulceration/hyperkeratosis may help guide the diagnosis of MCPyV-negative MCC and combined MCC.

Expression of the IDO1/TDO2-AhR pathway in tumor cells or the tumor microenvironment is associated with Merkel cell polyomavirus status and prognosis in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer, with approximately 80% of cases related to Merkel cell polyomavirus (MCPyV). Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) are the key rate-limiting enzymes of the tryptophan-to-kynurenine metabolic pathway. With aryl hydrocarbon receptor (AhR), an intracellular transcription factor, they play a role in escaping the immunosurveillance process in several cancers. IDO1/TDO2/AhR expression associated with the MCPyV status and prognosis in MCC was investigated. Samples included 24 MCPyV-positive MCCs, 12 MCPyV-negative MCCs with squamous cell carcinoma, and 7 MCPyV-negative pure MCCs. They were stained immunohistochemically with IDO1, TDO2, and AhR antibodies and analyzed. Higher IDO1 expression in MCC tumor cells was found in MCPyV-negative than in MCPyV-positive MCC (P < .001). The tumor microenvironment (TME) in MCPyV-negative MCC expressed higher TDO2 than in MCPyV-positive MCC (P < .001). Kaplan-Meier and log-rank tests showed that MCC with lower IDO1 expression in tumor cells and with lower TDO2 and AhR expressions in TME had better overall survival than otherwise (P = .043, .008, and .035, respectively); lower TDO2 expression in TME was also associated with longer disease-specific survival (P = .016). This suggests that IDO1, TDO2, and AhR express differentially in tumor cells or TME and play different roles in tumorigenesis between MCPyV-positive and MCPyV-negative MCC that may affect the MCC biology. Evaluating IDO1/TDO2/AhR expression is important for selecting the most likely patients with MCC for immunotherapies targeting the IDO1/TDO2-AhR pathway.

469 Usefulness of ulceration and hyperkeratosis as clinical predictors of merkel cell polyomavirus-negative and combined merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma, typically associated with Merkel cell polyomavirus (MCPyV). Combined MCC is occurred with non-neuroendocrine components, particularly squamous cell carcinoma, and thought to represent a distinct disease process from pure MCC, with absent MCPyV. Combined MCC may be occasionally difficult to distinguish histologically from pure MCC, and the diagnosis of combined MCC is not highly-reproducible based only on histologic assessment. This study analyzed the predictive clinical factors for MCPyV status and its morphological variants. We aimed to evaluate the significance of clinical parameters of ulceration/hyperkeratosis as potential novel predictors for MCPyV-negative MCC patients. In our study, 20 MCC specimens from 20 patients were examined. Patients were divided into two groups based on MCPyV status and morphological subsets among those diagnosed with MCC: MCPyV- positive/negative MCCs and pure/combined MCCs. The clinicopathological variants that predict the presence of MCPyV-negative MCC and combined MCC were evaluated. Of the 20 patients studied, 10 cases (50%) were not immunohistochemically identified as showing MCPyV using the MCPyV large T-antigen antibody (CM2B4). The presence of ulceration/hyperkeratosis was a predictive factor for detection of MCPyV-negative and combined MCC, being observed in 80%/50% of cases in which MCPyV was not identified. Among 10 MCPyV-positive MCCs, absence of ulceration and hyperkeratosis was found in 10 cases demonstrating smooth surface. Clinically, presence of ulceration/hyperkeratosis is a predictive factor for diagnosing MCPyV-negative MCC and combined MCC. Cases presenting with clinical evidence of ulceration and hyperkeratosis possibly suggest the presence of MCPyV-negative and combined MCC.

Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay.

Purpose: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCCs. Detection of MCPyV in MCCs has diagnostic utility and prognostic potential. We investigated whether RNAscope, an RNA in situ hybridization (ISH) assay for detection of RNA transcripts in tissues, is useful for MCPyV detection.Experimental Design: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMA) and whole-tissue sections encompassing 87 MCCs from 75 patients, 14 carcinomas of other types, and benign tissues. For comparison, qPCR was performed on 57 cases of MCC from 52 patients.Results: RNA-ISH demonstrated the presence of MCPyV in 37 of 75 cases (49.3%). Notably, tumors from younger patients (<73 years) had a significantly higher virus positivity than those from elderly patients (≥73 years; 64.9% vs. 34.2%, P = 0.011). Female patients had a higher positive rate of MCPyV than male patients (66.7% vs. 39.6%, P = 0.032). Data from both RNA-ISH and qPCR were available for 57 samples. Considering MCPyV qPCR as the gold standard for determining MCPyV status, RNAscope had 100% sensitivity and 100% specificity. There was a strong correlation between qPCR copy number and RNA-ISH product score (Spearman correlation coefficient R2 = 0.932, P < 0.0001).Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV and allows for correlation with tissue morphology. This study also reveals a significant association between age, gender, and MCPyV positivity. Clin Cancer Res; 23(18); 5622-30. ©2017 AACR.

Exploratory biomarker analysis in avelumab-treated patients with metastatic Merkel cell carcinoma progressed after chemotherapy.

9557 Background: Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer. Tumor oncogenesis is linked to Merkel cell polyomavirus (MCPyV) integration and UV exposure. PD-L1 is often expressed in MCC tumors, suggesting that patients with MCC could benefit from anti-PD-L1 therapy. Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody that has demonstrated clinical efficacy in patients (pts) with metastatic MCC (mMCC) in a Phase 2 trial with an objective response rate (ORR) of 31.8% in the primary analysis. Assessment of candidate predictive biomarkers may help to identify patients with a greater probability of response to avelumab and to improve understanding of MCC biology. Methods: Patients in a Phase 2 trial (NCT02155647) with mMCC and tumor progression on prior chemotherapy received avelumab at 10 mg/kg Q2W. PD-L1 expression, MCPyV status and CD8+ T-cell infiltration in pretreatment tumor samples were evaluated by immunohistochemistry (IHC). MCPyV status was also evaluated by real-time PCR. Results: Tumor PD-L1 expression was evaluable in 74 of 88 (84.1%) pts with mMCC treated with avelumab, of which 58 (65.9%) and 19 (21.6%) pts were positive at 1% and 5% cut-offs. ORR was 34.5% and 18.8% for PD-L1 positive and negative pts at 1% cutoff, and 52.6% and 23.6% for PD-L1 positive and negative pts at 5% cutoff. MCPyV status was positive in 60% (46/77) pts evaluable by IHC and 63% (45/71) pts evaluable by PCR; of 66 pts tested by both IHC and PCR, concordance was 90.9%. MCPyV+ and MCPyV– pts had similar frequencies of PD-L1+ tumors (80% and 73%) with an ORR of 26.1% and 35.5% respectively. Baseline CD8+ T-cell infiltration was assessed at tumor invasive margin and tumor center in 53 pts, ORR was 44.4% vs 19.2% and 32.1% vs 28% for pts with high or low CD8+ T-cell density at respective locations. Conclusions: In an international cohort of pts with mMCC, avelumab had clinical activity among biomarker subgroups analyzed, including PD-L1 expression, MCPyV status and density of CD8+ tumor-infiltrating T-cells. The current biomarkers were not predictive of response but further research into understanding how avelumab mediates anti-tumor activity in MCC may identify novel biomarkers. Clinical trial information: NCT02155647.