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Background Immunotherapy has revolutionised the treatment of advanced cutaneous squamous cell carcinoma (cSCC). It is important to understand both safety and efficacy in a real-world and trial-ineligible cSCC population. We aimed to evaluate safety, efficacy and molecular insights among a broader cSCC population, including immunosuppressed patients, treated with immune checkpoint inhibitors (CPI). Methods We present a cohort of advanced cSCC patients (n = 61) treated from 2015 to 2020 evaluating the best overall response (BOR) (RECISTv1.1) to CPI therapy, immune-related adverse events (irAEs) and tumour mutational burden (TMB) to correlate with outcomes. A validated geriatric scoring index (CIRS-G) was utilised to assess comorbidities among patients ≥75. These data were compared with published clinical trial results among the broader cSCC population. Results BOR to CPI was lower among the entire cohort when compared with trial data (31.5 vs. 48%, P P P = 0.02), while pre-treatment lymphocyte count and TMB predicted response (P = 0.02). Conclusions We demonstrate comparatively lower response rates to CPI among real-world cSCC patients not explained by older age or immunosuppression history alone. Immune-related toxicity, absolute lymphocyte count and TMB predicted CPI response.

Limited data exists regarding the efficacy of curative hypofractionated radiotherapy (hypo-RT) regimens compared to conventionally-fractionated radiotherapy (conv-RT) for Merkel cell carcinoma (MCC).A retrospective analysis of 241 patients diagnosed with non-metastatic MCC from 2005-2021 and who received RT at Dana-Farber/BrighamWomen's Cancer Center. The primary outcome was cumulative incidence of in-field locoregional relapse using Gray's test with competing risks of death and isolated out-of-field recurrence. Secondary outcomes included overall survival (OS) and MCC-specific survival using log-rank tests, and risk factors of recurrence using Cox-proportional hazards regression.There were 50 (20.6 %) and 193 (79.4 %) courses of hypo-RT and conv-RT, respectively. The hypo-RT cohort was older (≥73 years at diagnosis: 78.0 % vs 41.5 %, p 0.01), and received a lower equivalent total RT dose in 2 Gy per fraction (50 Gy: 58.0 % vs 5.2 %, p 0.01). Median follow-up was 65.1 months (range: 1.2-194.5) for conv-RT and 25.0 months (range: 1.6-131.3) for hypo-RT cohorts. Two-year cumulative incidence of in-field locoregional relapse was low in both groups (1.1 % conv-RT vs 4.1 % hypo-RT, p = 0.114). While two-year OS was lower for the hypo-RT group (62.6 % vs 84.4 %, p = 0.0008), two-year MCC-specific survival was similar (84.7 % vs 86.6 %, p = 0.743). On multivariable analysis, immunosuppression, clinical stage III disease, and lymphovascular invasion were associated with any-recurrence when controlling for sex, age, and hypo-RT.There was no difference in cumulative incidence of in-field locoregional relapse or MCC-specific survival between hypo-RT and conv-RT. Prospective studies are needed to confirm hypo-RT as an efficacious treatment option for MCC.

Purpose/objective(s) Serum antibodies to the Merkel oncoprotein (AMERK) are expressed in approximately 50% of patients (pts) with MCC and can be used to monitor for recurrence. This study aimed to describe changes in AMERK levels after RT for MCC and identify patterns that predict disease recurrence. Materials/methods Pts with MCC were identified retrospectively if they had curative-intent postoperative or definitive RT at a single institution from 2010-2020. Pts treated with RT at other institutions were excluded due to inability to assess RT quality, previously shown to impact MCC outcomes. All patients had a pre-RT AMERK and at least one post-RT AMERK (median no. Tests 5, range 1-10). Changes in AMERK were described and compared using Wilcoxon rank sum test for two-sample non-parametric comparisons and the Chi-square test for recurrence association. Results Of 202 MCC pts treated at our institution, 61 curatively treated pts had a pre-RT AMERK test performed. AMERK was detectable in 27 of the 61 patients (44%) with a median value of 2950 (range 127 to 115,000 [upper limit of assay]). Detectable pre-RT AMERK was associated with decreased MCC recurrence compared to pts with undetectable pre-RT AMERK (OR 0.35, 95% CI 0.12,1.04). The 27 pts with positive pre-RT AMERK had Stages I (n = 5), II (n = 1), and III (n = 21) from sites of the extremity (n = 14), head/neck (n = 7), unknown primary (n = 4), and trunk (n = 2). Pts with gross disease at RT (n = 11) had higher median pre-RT AMERK than those without gross disease at RT (n = 16) (median 8550 vs. 1840, P = 0.06). With a median follow-up of 20 months (range 2.3-61.3), there were 7 recurrences, all were distant. All AMERK-positive pts were alive at last follow-up except for the single immunosuppressed pt who recurred and died of MCC. A ≥50% AMERK reduction by 6-months after RT had a sensitivity of 95% (95% CI 74-100) for detecting disease-free status and a negative predictive value of 83% (95% CI 41-97%), reflecting the probability of recurrence if 115,00 value, had initiated high-dose corticosteroids for a comorbid condition. Out of 8 recurrence-free patients with AMERK levels available more than 2-years post-RT, 50% (4 of 8) had detectable AMERK (range 239-1960, > 2 years post-RT). Conclusion Reduction in AMERK post-RT can be used to predict MCC recurrence, which rarely occurs with a sustained ≥50% reduction by 6-months post-RT. AMERK may be persistently detectable in recurrence-free pts beyond 2 years. Further research is needed to determine the impact of corticosteroid initiation on AMERK levels.

Background Merkel cell carcinoma is a rare cancer for which the standard-of-care is immune checkpoint blockade in the recurrent/metastatic setting. However, immunotherapy is not effective in all patients. A greater understanding of molecular mechanisms and potential predictive biomarkers are unmet needs for clinicians and researchers. Methods We undertook a retrospective analysis of 45 patients treated at our institution from 2013 to 2020 to understand the clinical and genomic correlates of clinical benefit from immunotherapy. We gathered data from the electronic health record, including provider notes and results from our institutional next-generation sequencing panel of actionable genomic alterations. Results Our cohort predominantly included individuals with stage III disease at diagnosis and stage IV disease at the time of diagnosis of recurrent/metastatic disease. Most patients received immunotherapy in the first line. 43% of patients experienced an objective response to immunotherapy (median duration of response 24.2 months, 95% confidence interval 8.8-not reached) and median overall survival was 15.5 months (95% confidence interval 9.0–28.7) (median follow-up 25.2 months). Lower stage at diagnosis of primary disease 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). The most common single-nucleotide variants among the sequenced cohort were those in TP53 (59%) and RB1 (51%). Single-nucleotide variants in the ARID2 and NTRK1 genes were associated with response without Bonferroni correction (p=0.05), while none of Merkel cell polyomavirus status, total mutational burden, ultraviolet mutational signatures, and copy-number alterations predicted outcomes (figure 1). Conclusions Patients with shorter disease-free interval after definitive treatment may be particularly suitable candidates for immunotherapy. 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. Acknowledgements AJK receives research funding from the American Society of Hematology and from the Pritzker School of Medicine. Ethics Approval The study was approved by the Dana-Farber institutional review board, protocol numbers 11–104 and 17–000. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

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.

Background: Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer. Prior to the advent of immunotherapy, treatment options were limited. In our study, we evaluate the impact of tumor cell PD-L1 expression and tumor immune microenvironment on survival in MCC patients who were not treated with immune checkpoint inhibitors. Methods: Clinical data and tissue samples were collected from 78 patients with confirmed MCC treated at Dana-Farber Cancer Institute. Specimens were analyzed for the distribution of PD-L1 by immunohistochemistry staining (IHC) and standardized analysis. Results were correlated with survival data. Results: In this study, membrane and cytoplasmic MCC tumor cell staining for PD-L1 was detected in 22.4% (15 of 67) of cases and PD-L1 staining of intratumoral microvessels and PD-L1 positive immune cells at the infiltrative margins of the tumor in 92.5% (62 of 67) of cases. In patients untreated with immune checkpoint inhibitors, median overall survival was not different for patients based on PD-L1 expression (PD-L1+ 64 months vs. PD-L1- not reached; HR = 1.26, 95% CI: 0.46–3.45; p = 0.60). Conclusion: PD-L1 expression is frequently detected in MCC tumor cells and tumor microenvironment. PD-L1 expression did not affect prognosis in this cohort that had not received PD-1/L1 blockade.

Background Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC. Methods In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure. Results Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival. Conclusions These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.

BackgroundThis study sought to determine the efficacy and safety profile of cabozantinib in patients with advanced Merkel cell carcinoma (MCC).Experimental DesignThis prospective, phase II, single-institution trial enrolled patients with platinum-failure, recurrent/metastatic MCC to receive cabozantinib 60 mg orally daily until disease progression, withdrawal from study, or severe toxicity. The primary endpoint was disease control rate. Secondary endpoints included overall survival (OS), progression-free survival (PFS), and toxicity. Immunohistochemistry for VEGFR-2, MET, and HGF expression and next-generation sequencing of tumor tissue were performed and correlated with outcome.ResultsEight patients were accrued from January 24, 2014, to June 8, 2016. The study was closed prematurely because of toxicity and lack of responses. The most frequent adverse events were grades 1 and 2 and included anorexia, fatigue, nausea, hypothyroidism, and dysgeusia. Two patients developed nonhealing, painful ulcers and tumor-skin fistula. One patient had stable disease for 8 months. One patient withdrew from the study after 2 weeks of therapy because of adverse events. Three patients required dose reduction because of toxicity. Median PFS and OS were 2.1 and 11.2 months, respectively. No expression of MET, HGF, or VEGFR-2 was identified in tumor cells by immunohistochemistry of patients’ tissue samples.ConclusionCabozantinib was poorly tolerated and did not demonstrate activity in patients with recurrent/metastatic, platinum-failure MCC. It is unclear whether preselection of patients with the specific upregulation or genetic alteration in the targets for cabozantinib would have changed the results of this study. (Clinical trial identification number: NCT02036476)Implications for PracticeThis phase II study demonstrated poor tolerability and lack of activity of cabozantinib in an unselected group of patients with advanced Merkel cell carcinoma. Although it is unclear whether preselection of patients with the specific upregulation and genetic alterations in targets for cabozantinib would have changed the results of this study, this would have likely led to an extremely rare patient population that would take many years to accrue.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKϵ inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo. Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196–215. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Deng et al., p. 216. This article is highlighted in the In This Issue feature, p. 127

BACKGROUND: Merkel cell carcinoma (MCC) is a rare cutaneous malignancy of neuroendocrine origin, with about 30 cases of brain metastasis (BM) reported in the literature. Historically, the treatment of neuro-metastatic MCC has largely included chemotherapy (CT) and radiotherapy (RT). The aim was to investigate predictors of overall survival (OS) in neuro-metastatic MCC. METHODS: In this retrospective study, we surveyed institutional databases and additionally conducted a systematic review of the literature to identify cases reporting on management of distant MCC BM. A pooled analysis was performed on the institutional and literature cases to assess predictors of OS. Survival analysis was done on R (ver 3.4.0) using a Log Rank statistic and cox proportional hazard ratio. RESULTS: Forty cases were included for analysis, describing operative (14) and non-operative (26) management. Median time from initial MCC diagnosis to CNS involvement was 17.0-mos (IQR 10.5- 26.5), and most patients had a single BM (62.5%). Management of intracranial disease included RT (84.2%), systemic therapy (59.5%) and surgical resection (35%). Operative management was associated with a lower intracranial burden of disease (BoD, single BM: op 92.9% vs. non-op 46.2%, p=0.004), but similar systemic BoD. Median OS was longer in patients treated with neurosurgery (73-mos, 95%CI:31–115 vs. 25-mos, 95%CI:17–44, p ‹ 0.001). Both neurosurgery (HR 0.18, 95%CI:0.06–0.54, p=0.002) and having a single BM (multiple BM or leptomeningeal disease: HR 2.51, 95%CI:1.12–5.6, p=0.03) conferred an OS benefit on risk-unadjusted analysis. On multivariable analysis, only neurosurgical resection was an independent predictor of OS (HR 0.16, 95%CI:0.04–0.59, p=0.006), controlling for age, BoD and RT. Systemic therapy and RT were not associated with OS. CONCLUSIONS: Resection of MCC BM may confer a survival benefit relative to non-operative management given appropriate patient selection. Prospective investigation of multimodal management of neurometastatic MCC is warranted, especially given the promise of new immunotherapy agents in treating MCC.

Background Merkel cell carcinoma (MCC) is a rare cutaneous malignancy of neuroendocrine origin, with about 30 cases of brain metastasis (BM) reported in the literature. Historically, the treatment of neurometastatic MCC has largely included chemotherapy and radiotherapy. The aim was to investigate predictors of overall survival (OS) in neurometastatic MCC. Methods In this retrospective study, we surveyed institutional databases and conducted a systematic review of the literature to identify cases reporting on management of distant MCC BM. A pooled survival analysis was performed on the institutional and literature cases to assess predictors of OS. Results Forty cases were included for analysis, describing operative [14] and non-operative [26] management. Median time to central nervous system involvement was 17.0-mos (interquartile range 10.5–26.5), and most patients had a single BM (62.5%). Management of intracranial disease included radiotherapy (82.5%), systemic therapy (59.5%) and surgical resection (35%). Operative management was associated with a lower intracranial burden of disease (BoD), but similar systemic BoD. Both neurosurgery (hazard ratio [HR] 0.18, 95% confidence interval [CI]: 0.06–0.54, p = 0.002), having RT (HR 0.37, 95% CI: 0.14:0.93, p = 0.04) and having a single BM (extensive intracranial BoD: HR 2.51, 95% CI: 1.12–5.6, p = 0.03) conferred an OS benefit on risk-unadjusted analysis. Only, neurosurgical resection was an independent predictor of OS (HR 0.12, 95% CI: 0.03–0.49, p = 0.003), controlling for age, BoD and radiotherapy. Conclusions Resection of MCC BM may confer a survival benefit given appropriate patient selection. Prospective investigation of multimodal management of neurometastatic MCC is warranted, especially given the promise of new immunotherapy agents in treating MCC.

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations., IMPORTANCE A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors.

Merkel cell carcinomas (MCCs) are rare but highly malignant skin cancers associated with a recently described polyomavirus. MCC tumors were infiltrated by T cells, including effector, central memory, and regulatory T cells. Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25. Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T-cell activation, proliferation, enhanced cytokine production, and loss of viable tumor cells from cultures. Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137. MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting that tumor-specific T cells capable of controlling tumor growth were present in MCC. Both CD4(+) and CD8(+) FOXP3(+) regulatory T cells were frequent in MCC. Fifty percent of nonactivated T cells in MCC-expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages. In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors. Agents that stimulate T-cell activity, block regulatory T cell function, or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with profound but poorly understood resistance to chemotherapy, which poses a significant barrier to clinical MCC treatment. Here we show that ATP-binding cassette member B5 (ABCB5) confers resistance to standard-of-care MCC chemotherapeutic agents and provide proof-of-principle that ABCB5 blockade can inhibit human MCC tumor growth through sensitization to drug-induced cell cytotoxicity. ABCB5 expression was detected in both established MCC lines and clinical MCC specimens at levels significantly higher than those in normal skin. Carboplatin- and etoposide-resistant MCC cell lines exhibited increased expression of ABCB5, along with enhanced ABCB1 and ABCC3 transcript expression. ABCB5-expressing MCC cells in heterogeneous cancers preferentially survived treatment with carboplatin and etoposide in vitro and in human MCC xenograft-bearing mice in vivo. Moreover, patients with MCC also exhibited enhanced ABCB5 positivity after carboplatin- and etoposide-based chemotherapy, pointing to clinical significance of this chemoresistance mechanism. Importantly, ABCB5 blockade reversed MCC drug resistance and impaired tumor growth in xenotransplantation models in vivo. Our results establish ABCB5 as a chemoresistance mechanism in MCC and suggest utility of this molecular target for improved MCC therapy.