Merkel cell carcinoma: updates in tumor biology, emerging therapies, and preclinical models.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma thought to arise via either viral (Merkel cell polyomavirus) or ultraviolet-associated pathways. Surgery and radiotherapy have historically been mainstays of management, and immunotherapy has improved outcomes for advanced disease. However, there remains a lack of effective therapy for those patients who fail to respond to these established approaches, underscoring a critical need to better understand MCC biology for more effective prognosis and treatment. Here, we review the fundamental aspects of MCC biology and the recent advances which have had profound impact on management. The first genetically-engineered mouse models for MCC tumorigenesis provide opportunities to understand the potential MCC cell of origin and may prove useful for preclinical investigation of novel therapeutics. The MCC cell of origin debate has also been advanced by recent observations of MCC arising in association with a clonally related hair follicle tumor or squamous cell carcinoma in situ. These studies also suggested a role for epigenetics in the origin of MCC, highlighting a potential utility for this therapeutic avenue in MCC. These and other therapeutic targets form the basis for a wealth of ongoing clinical trials to improve MCC management. Here, we review these recent advances in the context of the existing literature and implications for future investigations.

Durable radiologic and molecular complete response following nivolumab in an HNSCC patient with UV signature and HIV.

Cancer patients living with HIV (CPLWH) may experience increased mortality risk. Furthermore, they have been historically excluded from clinical trials due to safety concerns. Our patient with squamous cell carcinoma of the lower lip received radiotherapy and platinum-based chemotherapy but declined by multiple centers due to his accidental HIV status. Genomic profiling revealed CDKN2A/B, PBRM1, TP53, and TERT alterations corresponding to UV signature, and high tumor mutational burden with positive PD-L1 staining. Accordingly, we report a durable radiologic and molecular complete response upon nivolumab plus IVC and antiretroviral therapy (ART). We demonstrated the safety and efficacy of ICIs, and feasibility of managing adverse events caused by antitumor, antiviral, and integrative therapies.

Utility of UV Signature Mutations in the Diagnostic Assessment of Metastatic Head and Neck Carcinomas of Unknown Primary.

BACKGROUND: Metastatic carcinoma of unknown primary origin to the head and neck lymph nodes (HNCUP) engenders unique diagnostic considerations. In many cases, the detection of a high-risk human papillomavirus (HR-HPV) unearths an occult oropharyngeal squamous cell carcinoma (SCC). In metastatic HR-HPV-independent carcinomas, other primary sites should be considered, including cutaneous malignancies that can mimic HR-HPV-associated SCC. In this context, ultraviolet (UV) signature mutations, defined as ≥ 60% C→T substitutions with ≥ 5% CC→TT substitutions at dipyrimidine sites, identified in tumors arising on sun exposed areas, are an attractive and underused tool in the setting of metastatic HNCUP. METHODS: A retrospective review of institutional records focused on cases of HR-HPV negative HNCUP was conducted. All cases were subjected to next generation sequencing analysis to assess UV signature mutations. RESULTS: We identified 14 HR-HPV negative metastatic HNCUP to either the cervical or parotid gland lymph nodes, of which, 11 (11/14, 79%) had UV signature mutations, including 4 (4/10, 40%) p16 positive cases. All UV signature mutation positive cases had at least one significant TP53 mutation and greater than 20 unique gene mutations. CONCLUSION: The management of metastatic cutaneous carcinomas significantly differs from other HNCUP especially metastatic HR-HPV-associated SCC; therefore, the observation of a high percentage of C→T with CC →TT substitutions should be routinely incorporated in next generation sequencing reports of HNCUP. UV mutational signatures testing is a robust diagnostic tool that can be utilized in daily clinical practice.

Differential gradients of immunotherapy vs targeted therapy efficacy according to the sun-exposure pattern of the site of occurrence of primary melanoma: a multicenter prospective cohort study (MelBase).

Background: The tumor mutational burden (TMB) is high in melanomas owing to UV-induced oncogenesis. While a high TMB is a predictive biomarker of response to PD-1 inhibitors, it may be associated with the rise of resistant clones to targeted therapy over time. We hypothesized that survivals may depend on both the sun-exposure profile of the site of primary melanoma and the type of systemic treatment. Patients and methods: Patients were screened from MelBase, a multicenter biobank dedicated to the prospective follow-up of stage III/IV melanoma. All patients with a known cutaneous primary melanoma who received a 1st-line systemic treatment by immunotherapy or targeted therapy were included (2013-2019). Outcomes were progression-free survival (PFS) and overall survival (OS). Results: 973 patients received either anti PD-1(n=466), anti CTLA-4(n=143), a combination of both (n=118), or targeted therapies (n=246). Patients' characteristics at treatment initiation were: male (62%), median age of 62, AJCC stage IV (84%). Median follow-up was 15.5 months. The primary melanoma was located on chronically sun-exposed skin in 202 patients (G1: head neck), on intermittently sun-exposed skin in 699 patients (G2: trunk, arms, legs), and on sun-protected areas in 72 patients (G3: palms, soles). Median PFS was significantly higher in G1 under anti PD-1 treatment (8.7 months vs 3.3 and 3.4 months for G2 and G3, respectively) (p=0.011). PFS did not significantly differ in other groups. Similarly, median OS was significantly higher in G1 receiving 1st line anti PD-1 treatment (45.6 months vs 31.6 and 21.4 months for G2 and G3) (p=0.04), as opposed to 1st line targeted therapy (19.5 months vs 16.3 and 21.1 months for G1, G2 and G3 respectively). Conclusion: Our study confirms that immunotherapy with anti PD-1 is particularly recommended for melanomas originating from chronically sun-exposed areas, but this finding needs to be confirmed by further research.

Genetic Aspects of Conjunctival Melanoma: A Review.

Conjunctival melanoma (CM) is a rare but aggressive cancer. Over the past decade, molecular studies using rapidly advancing technologies have increasingly improved our understanding of CM genetics. CMs are mainly characterized by dysregulated MAPK and PI3K/AKT/mTOR pathways, driven by commonly mutated (BRAF, NRAS, NF1) or less commonly mutated (KIT, PTEN) genes. Another group of genes frequently mutated in CMs include TERT and ATRX, with known roles in telomere maintenance and chromatin remodeling/epigenetic regulation. Uveal melanoma-related genes (BAP1, SF3B1, GNAQ/11) can also be mutated in CMs, albeit infrequently. Additional CM-related mutated genes have increasingly been identified using more comprehensive genetic analyses, awaiting further confirmation in additional/larger studies. As a tumor arising in a partly sun-exposed mucosal tissue, CM exhibits a distinct genomic profile, including the frequent presence of an ultraviolet (UV) signature (and high mutational load) and also the common occurrence of large structural variations (distributed across the genome) in addition to specific gene mutations. The knowledge gained from CM genetic studies to date has led to new therapeutic avenues, including the use of targeted and/or immuno-therapies with promising outcomes in several cases. Accordingly, the implementation of tumor genetic testing into the routine clinical care of CM patients holds promise to further improve and personalize their treatments. Likewise, a growing knowledge of poor prognosis-associated genetic changes in CMs (NRAS, TERT, and uveal melanoma signature mutations and chromosome 10q deletions) may ultimately guide future strategies for prognostic testing to further improve clinical outcomes (by tailoring surveillance and considering prophylactic treatments in patients with high-risk primary tumors).

Clock-like Mutation Signature May Be Prognostic for Worse Survival Than Signatures of UV Damage in Cutaneous Melanoma.

Novel treatment modalities comprising immune checkpoint inhibitors and targeted therapies have revolutionized treatment of metastatic melanoma. Still, some patients suffer from rapid progression and decease within months after a diagnosis of stage IV melanoma. We aimed to assess whether genomic alterations may predict survival after the development of stage IV disease, irrespective of received therapy. We analyzed tumor samples of 79 patients with stage IV melanoma using a custom next-generation gene-sequencing panel, MelArray, designed to detect alterations in 190 melanoma-relevant genes. We classified the patients: first, as short survivors (survival ≤6 months after stage IV disease, n = 22) and long survivors (survival >6 months, n = 57); second, by using a cut-off of one year; and third, by comparing the longest surviving 20 patients to the shortest surviving 20. Among analyzed genes, no individual gene alterations, or combinations of alterations, could be dichotomously associated with survival. However, the cohort's mutational profiles closely matched three known mutational signatures curated by the Catalog of Somatic Mutations in Cancer (COSMIC): UV signature COSMIC_7 (cosine-similarity 0.932), clock-like signature COSMIC_5 (cosine-similarity 0.829), and COSMIC_30 (cosine-similarity 0.726). Patients with UV signature had longer survival compared to patients with clock-like and COSMIC 30 (p < 0.0001). Subgroup dichotomization at 6 months showed that 75% of patients with UV signature survived longer than 6 months, and about 75% of patients with clock-like signature survived less than 6 months after development of stage IV disease. In our cohort, clock-like COSMIC_5 mutational signature predicted poor survival while a UV signature COSMIC_7 predicted longer survival. The prognostic value of mutational signatures should be evaluated in prospective studies.

Clinical Next-Generation Sequencing Panels Reveal Molecular Differences Between Merkel Cell Polyomavirus-Negative Merkel Cell Carcinomas and Neuroendocrine Carcinomas.

OBJECTIVES: We aim to determine molecular differences between Merkel cell polyomavirus (MCPyV)-negative Merkel cell carcinomas (MCCs) and neuroendocrine carcinomas (NECs). METHODS: Our study included 56 MCCs (28 MCPyV negative, 28 MCPyV positive) and 106 NECs (66 small cell NECs, 21 large cell NECs, and 19 poorly differentiated NECs) submitted for clinical molecular testing. RESULTS: APC, MAP3K1, NF1, PIK3CA, RB1, ROS1, and TSC1 mutations, in addition to high tumor mutational burden and UV signature, were frequently noted in MCPyV-negative MCC in comparison to small cell NEC and all NECs analyzed, while KRAS mutations were more frequently noted in large cell NEC and all NECs analyzed. Although not sensitive, the presence of either NF1 or PIK3CA is specific for MCPyV-negative MCC. The frequencies of KEAP1, STK11, and KRAS alterations were significantly higher in large cell NEC. Fusions were detected in 6.25% (6/96) of NECs yet in none of 45 analyzed MCCs. CONCLUSIONS: High tumor mutational burden and UV signature, as well as the presence of NF1 and PIK3CA mutations, are supportive of MCPyV-negative MCC, whereas KEAP1, STK11, and KRAS mutations are supportive of NEC in the appropriate clinical context. Although rare, the presence of a gene fusion is supportive of NEC.

Immunohistochemical and mutational status of telomerase reverse transcriptase in conjunctival squamous cell carcinoma.

PURPOSE: Mutations in human telomerase reverse transcriptase (TERT) are associated with increased telomerase activity in cutaneous melanomas. Conjunctival squamous cell carcinoma, also referred to as ocular surface squamous cell carcinoma, is cancer on the surface of the eye. Recent studies have identified UV signat`ure mutations in TERT promoters in ocular melanoma and ocular surface squamous neoplasia. However, its immunohistochemical status has not been reported in ocular surface squamous cell carcinoma. This study aimed to explore the immunohistochemical and mutational status of TERT in ocular surface SCC. METHODS: The immunohistochemical expression of TERT and mutational status of TERT promoter was evaluated in 19 ocular surface squamous cell carcinoma cases. Conjunctival melanoma tissue was used as a positive control. RESULTS: The cytoplasmic overexpression of TERT was detected in 11/19 (57%), and TERT promoter mutations were identified in 6/19 (31%) of ocular surface squamous cell carcinoma. Out of these, 66% had a C228T mutation, and 33% had a C250T mutation. The TERT expression was found to be associated with a high (≥T3) AJCC category (P = 0.023), and TERT immunoexpression was significantly correlated with reduced disease-free survival (P = 0.024, log-rank analysis) in ocular surface squamous cell carcinoma patients. CONCLUSION: The present study demonstrates that TERT promoter mutations with UV signatures are frequent in ocular surface squamous cell carcinoma. The increased expression of TERT could be of biological significance in aggressive ocular surface squamous cell carcinoma.

Merkel Cell Carcinoma of Unknown Primary: Immunohistochemical and Molecular Analyses Reveal Distinct UV-Signature/MCPyV-Negative and High Immunogenicity/MCPyV-Positive Profiles.

BACKGROUND: Merkel cell carcinomas of unknown primary (MCC-UPs) are defined as deep-seated tumors without an associated cutaneous tumor. Although the distinction has important clinical implications, it remains unclear whether these tumors represent primary tumors of lymph nodes or metastatic cutaneous primaries. METHODS: We compared the immunohistochemical profiles of four groups of MCCs (Merkel cell polyomavirus (MCPyV)-positive UP, MCPyV-negative UP, MCPyV-positive known primary (KP), and MCPyV-negative KP) using B-cell and pre-B-cell markers, cell cycle regulating proteins, follicular stem cell markers, and immune markers, and performed next generation and Sanger sequencing. RESULTS: Virus-positive and virus-negative MCC-UPs exhibited an immunoprofile similar to virus-positive and virus-negative primary cutaneous MCCs, respectively. MCC-UP tumors (both virus-positive and -negative) were immunogenic with similar or even higher tumoral PD-L1 expression and intratumoral CD8 and FoxP3 infiltrates in comparison to MCPyV-positive cutaneous tumors. In addition, similar to primary cutaneous MCCs, MCPyV-negative MCC-UPs exhibited UV signatures and frequent high tumor mutational burdens, whereas few molecular alterations were noted in MCPyV-positive MCC-UPs. CONCLUSIONS: Our results showed distinct UV-signatures in MCPyV-negative tumors and high immunogenicity in MCPyV-positive tumors. Although additional studies are warranted for the MCPyV-positive cases, our findings are supportive of a cutaneous metastatic origin for MCPyV-negative MCC-UP tumors.

Error-Prone Replication through UV Lesions by DNA Polymerase θ Protects against Skin Cancers.

Cancers from sun-exposed skin accumulate "driver" mutations, causally implicated in oncogenesis. Because errors incorporated during translesion synthesis (TLS) opposite UV lesions would generate these mutations, TLS mechanisms are presumed to underlie cancer development. To address the role of TLS in skin cancer formation, we determined which DNA polymerase is responsible for generating UV mutations, analyzed the relative contributions of error-free TLS by Polη and error-prone TLS by Polθ to the replication of UV-damaged DNA and to genome stability, and examined the incidence of UV-induced skin cancers in Polθ-/-, Polη-/-, and Polθ-/- Polη-/- mice. Our findings that the incidence of skin cancers rises in Polθ-/- mice and is further exacerbated in Polθ-/- Polη-/- mice compared with Polη-/- mice support the conclusion that error-prone TLS by Polθ provides a safeguard against tumorigenesis and suggest that cancer formation can ensue in the absence of somatic point mutations.

Molecular Pathways in Melanomagenesis: What We Learned from Next-Generation Sequencing Approaches.

PURPOSE OF REVIEW: Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles. Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous. The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways. The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies. RECENT FINDINGS: Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis. After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAFmut, RASmut, and non-BRAFmut/non-RASmut.

Targeted molecular profiling reveals genetic heterogeneity of poromas and porocarcinomas.

The genetic landscape of rare benign tumours and their malignant counterparts is still largely unexplored. While recent work showed that mutant HRAS is present in subsets of poromas and porocarcinomas, a more comprehensive genetic view on these rare adnexal neoplasms is lacking. Using high-coverage next generation sequencing, we investigated the mutational profile of 50 cancer-related genes in 12 cases (six poromas and six porocarcinomas). Non-synonymous mutations were found in two-thirds of both poromas and porocarcinomas. Hotspot HRAS mutations were identified in two poromas (p.G13R and p.Q61R) and one porocarcinoma (p.G13C). While in poromas only few cases showed single mutated genes, porocarcinomas showed greater genetic heterogeneity with up to six mutated genes per case. Recurrent TP53 mutations were found in all porocarcinomas that harboured mutated genes. Non-recurrent mutations in porocarcinomas were found in several additional tumour suppressors (RB1, APC, CDKN2A, and PTEN), and genes implicated in PI3K-AKT and MAPK signalling pathways (ABL1, PDGFRA, PIK3CA, HRAS, and RET). UV-associated mutations were found in TP53, APC, CDKN2A, PTEN, and RET. In conclusion, our study confirms and extends the spectrum of genetic lesions in poromas and porocarcinomas. While poromas exhibited only few mutations, which did not involve TP53, the majority of porocarcinomas harboured UV-mediated mutations in TP53 with some of these cases showing considerable genetic heterogeneity that may be clinically exploitable.

The Prognostic Significance of Low-Frequency Somatic Mutations in Metastatic Cutaneous Melanoma.

Background: Little is known about the prognostic significance of somatically mutated genes in metastatic melanoma (MM). We have employed a combined clinical and bioinformatics approach on tumor samples from cutaneous melanoma (SKCM) as part of The Cancer Genome Atlas project (TCGA) to identify mutated genes with potential clinical relevance. Methods: After limiting our DNA sequencing analysis to MM samples (n = 356) and to the CANCER CENSUS gene list, we filtered out mutations with low functional significance (snpEFF). We performed Cox analysis on 53 genes that were mutated in ≥3% of samples, and had ≥50% difference in incidence of mutations in deceased subjects versus alive subjects. Results: Four genes were potentially prognostic [RAC1, FGFR1, CARD11, CIITA; false discovery rate (FDR) < 0.2]. We identified 18 additional genes (e.g., SPEN, PDGFRB, GNAS, MAP2K1, EGFR, TSC2) that were less likely to have prognostic value (FDR < 0.4). Most somatic mutations in these 22 genes were infrequent (< 10%), associated with high somatic mutation burden, and were evenly distributed across all exons, except for RAC1 and MAP2K1. Mutations in only 9 of these 22 genes were also identified by RNA sequencing in >75% of the samples that exhibited corresponding DNA mutations. The low frequency, UV signature type and RNA expression of the 22 genes in MM samples were confirmed in a separate multi-institution validation cohort (n = 413). An underpowered analysis within a subset of this validation cohort with available patient follow-up (n = 224) showed that somatic mutations in SPEN and RAC1 reached borderline prognostic significance [log-rank favorable (p = 0.09) and adverse (p = 0.07), respectively]. Somatic mutations in SPEN, and to a lesser extent RAC1, were not associated with definite gene copy number or RNA expression alterations. High (>2+) nuclear plus cytoplasmic expression intensity for SPEN was associated with longer melanoma-specific overall survival (OS) compared to lower (≤ 2+) nuclear intensity (p = 0.048). We conclude that expressed somatic mutations in infrequently mutated genes beyond the well-characterized ones (e.g., BRAF, RAS, CDKN2A, PTEN, TP53), such as RAC1 and SPEN, may have prognostic significance in MM.