B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. OBJECTIVES: To identify overexpressed oncogenes as potential drug targets for MCC. METHODS: NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. RESULTS: Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. CONCLUSIONS: Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I-III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

BRAF and MEK inhibition in melanoma patients enables reprogramming of tumor infiltrating lymphocytes.

BACKGROUND: Combined inhibition of BRAF/MEK is an established therapy for melanoma. In addition to its canonical mode of action, effects of BRAF/MEK inhibitors on antitumor immune responses are emerging. Thus, we investigated the effect of these on adaptive immune responses. PATIENTS, METHODS AND RESULTS: Sequential tumor biopsies obtained before and during BRAF/MEK inhibitor treatment of four (n = 4) melanoma patients were analyzed. Multiplexed immunofluorescence staining of tumor tissue revealed an increased infiltration of CD4+ and CD8+ T cells upon therapy. Determination of the T-cell receptor repertoire usage demonstrated a therapy induced increase in T-cell clonotype richness and diversity. Application of the Grouping of Lymphocyte Interactions by Paratope Hotspots algorithm revealed a pre-existing immune response against melanoma differentiation and cancer testis antigens that expanded preferentially upon therapy. Indeed, most of the T-cell clonotypes found under BRAF/MEK inhibition were already present in lower numbers before therapy. This expansion appears to be facilitated by induction of T-bet and TCF7 in T cells, two transcription factors required for self-renewal and persistence of CD8+ memory T cells. CONCLUSIONS: Our results suggest that BRAF/MEK inhibition in melanoma patients allows an increased expansion of pre-existing melanoma-specific T cells by induction of T-bet and TCF7 in these.

MHC class-I downregulation in PD-1/PD-L1 inhibitor refractory Merkel cell carcinoma and its potential reversal by histone deacetylase inhibition: a case series.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer in which PD-1/PD-L1 blockade has shown remarkable response rates. However, a significant proportion of patients shows primary or secondary resistance against PD-1/PD-L1 inhibition, with HLA class-I downregulation and insufficient influx of CD8+ T cells into the tumor as possible immune escape mechanisms. Histone deacetylase inhibitors (HDACi) have been demonstrated to reverse low HLA class-I expression caused by epigenetic downregulation of the antigen machinery (APM) in vitro and in pre-clinical models in vivo. CASE PRESENTATIONS: We report four cases of patients with metastatic MCC who did not respond to immunotherapy by PD-1/PD-L1 blockade. Two of the patients received, subsequently, the HDACi panobinostat in combination with PD-1/PD-L1 blockade. Tumor biopsies of the patients were analyzed for cellular and molecular markers of antigen processing and presentation as well as the degree of T-cell infiltration. RESULTS AND CONCLUSION: Low expression of APM-related genes associated with low HLA class-I surface expression was observed in all MCC patients, progressing on PD-1/PD-L1 blockade. In one evaluable patient, of the two treated with the combination therapy of the HDACi, panobinostat and PD-1/PD-L1 blockade, reintroduction of HLA class-I-related genes, enhanced HLA class-I surface expression, and elevated CD8+ T-cell infiltration into the MCC tumor tissue were observed; however, these changes did not translate into a clinical benefit. Our findings suggest that HDACi may be useful to overcome HLA class-I downregulation as a resistance mechanism against anti-PD-1/PD-L1 antibodies in MCC patients. Prospective clinical trials are needed to evaluate this notion.

Atypical fibroxanthoma and pleomorphic dermal sarcoma harbor frequent NOTCH1/2 and FAT1 mutations and similar DNA copy number alteration profiles.

Atypical fibroxanthomas and pleomorphic dermal sarcomas are tumors arising in sun-damaged skin of elderly patients. They have differing prognoses and are currently distinguished using histological criteria, such as invasion of deeper tissue structures, necrosis and lymphovascular or perineural invasion. To investigate the as-yet poorly understood genetics of these tumors, 41 atypical fibroxanthomas and 40 pleomorphic dermal sarcomas were subjected to targeted next-generation sequencing approaches as well as DNA copy number analysis by comparative genomic hybridization. In an analysis of the entire coding region of 341 oncogenes and tumor suppressor genes in 13 atypical fibroxanthomas using an established hybridization-based next-generation sequencing approach, we found that these tumors harbor a large number of mutations. Gene alterations were identified in more than half of the analyzed samples in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter. The presence of these alterations was verified in 26 atypical fibroxanthoma and 35 pleomorphic dermal sarcoma samples by targeted amplicon-based next-generation sequencing. Similar mutation profiles in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter were identified in both atypical fibroxanthoma and pleomorphic dermal sarcoma. Activating RAS mutations (G12 and G13) identified in 3 pleomorphic dermal sarcoma were not found in atypical fibroxanthoma. Comprehensive DNA copy number analysis demonstrated a wide array of different copy number gains and losses, with similar profiles in atypical fibroxanthoma and pleomorphic dermal sarcoma. In summary, atypical fibroxanthoma and pleomorphic dermal sarcoma are highly mutated tumors with recurrent mutations in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter, and a range of DNA copy number alterations. These findings suggest that atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically related, potentially representing two ends of a common tumor spectrum and distinguishing these entities is at present still best performed using histological criteria.

Liquid Profiling of Circulating Tumor DNA in Plasma of Melanoma Patients for Companion Diagnostics and Monitoring of BRAF Inhibitor Therapy.

BACKGROUND: The current standard for determining eligibility of patients with metastatic melanoma for BRAF-targeted therapy is tissue-based testing of BRAF mutations. As patients are rarely rebiopsied, detection in blood might be advantageous by enabling a comprehensive assessment of tumor mutational status in real time and thereby representing a noninvasive biomarker for monitoring BRAF therapy. METHODS: In all, 634 stage I to IV melanoma patients were enrolled at 2 centers, and 1406 plasma samples were prospectively collected. Patients were assigned to 3 separate study cohorts: study 1 for assessment of circulating tumor DNA (ctDNA) as part of companion diagnostics, study 2 for assessment of ctDNA for patients with low tumor burden and for follow-up, and study 3 for monitoring of resistance to BRAF inhibitor (BRAFi) or mitogen-activated protein kinase inhibitor therapy. RESULTS: Overall, a high degree of concordance between plasma and tissue testing results was observed at 90.9% (study 1) and 90.1% (study 2), respectively. Interestingly, discrepant results were in some cases associated with nonresponse to BRAFi (n = 3) or a secondary BRAF-mutant malignancy (n = 5). Importantly, ctDNA results correlated with the clinical course of disease in 95.7% and with response to treatment. Significantly, the detection of BRAF mutant ctDNA preceded relapse assessed by Response Evaluation Criteria in Solid Tumors, and was more specific than serum S100 and lactate dehydrogenase. CONCLUSIONS: Blood-based testing compares favorably with standard-of-care tissue-based BRAF mutation testing. Importantly, blood-based BRAF testing correlates with the clinical course, even for early-stage patients, and may be used to predict response to treatment, recurrence, and resistance before radioimaging under BRAFi therapy, thereby enabling considerable improvements in patient treatment.

Melanoma genome sequencing reveals frequent PREX2 mutations.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

Intraventricular melanocytoma diagnosis confirmed by gene mutation profile.

Primary leptomeningeal melanocytic tumors (PLMTs) are rare. They usually arise along the spinal cord and at the skull base. Here we report on a patient with a very rare intraventricular melanocytoma. Histologically, a melanocytic tumor was clearly diagnosed. However, to make the uncommon diagnosis of an intraventricular melanocytoma, metastatic melanoma needed to be excluded. Next generation sequencing covering gene mutations that may occur in PLMTs and cutaneous melanoma was performed. The unique gene mutation profile detected, consisting of an activating CYSLTR2 L129Q mutation and EIF1AX G9R mutation and a lack of mutations in genes known to occur in metastatic melanoma (i.e. BRAF or NRAS) confirmed the diagnosis of an intraventricular melanocytoma. This case report is the second intraventricular melanocytoma published to date and demonstrates the value of applying novel genetic assays to make this diagnosis.

IRF4 rs12203592 functional variant and melanoma survival.

Inherited genetic factors may modulate clinical outcome in melanoma. Some low-to-medium risk genes in melanoma susceptibility play a role in melanoma outcome. Our aim was to assess the role of the functional IRF4 SNP rs12203592 in melanoma prognosis in two independent sets (Barcelona, N = 493 and Essen, N = 438). Genotype association analyses showed that the IRF4 rs12203592 T allele increased the risk of dying from melanoma in both sets (Barcelona: odds ratio [OR] = 6.53, 95% CI 1.38-30.87, Adj p = 0.032; Essen: OR = 1.68, 95% CI 1.04-2.72, Adj p = 0.035). Survival analyses only showed significance for the Barcelona set (hazard ratio = 4.58, 95% CI 1.11-18.92, Adj p = 0.036). This SNP was also associated with tumour localization, increasing the risk of developing melanoma in head or neck (OR = 1.79, 95% CI 1.07-2.98, Adj p = 0.032) and protecting from developing melanoma in the trunk (OR = 0.59, 95% CI 0.41-0.85, Adj p = 0.004). These findings suggest for the first time that IRF4 rs12203592 plays a role in the modulation of melanoma outcome and confirms its contribution to the localization of the primary tumour.

Activating cysteinyl leukotriene receptor 2 (CYSLTR2) mutations in blue nevi.

Blue nevi are common melanocytic tumors arising in the dermal layer of the skin. Similar to uveal melanomas, blue nevi frequently harbor GNAQ and GNA11 mutations. Recently, recurrent CYSLTR2 and PLCB4 mutations were identified in uveal melanomas not harboring GNAQ or GNA11 mutations. All four genes (GNAQ, GNA11, CYSLTR2, and PLCB4) code for proteins involved in the same signaling pathway, which is activated by mutations in these genes. Given the related functional consequences of these mutations and the known genetic similarities between uveal melanoma and blue nevi, we analyzed a cohort of blue nevi to investigate whether CYSLTR2 and PLCB4 mutations occur in tumors lacking GNAQ or GNA11 mutations (as in uveal melanoma). A targeted next-generation sequencing assay covering known activating mutations in GNAQ, GNA11, CYSLTR2, PLCB4, KIT, NRAS, and BRAF was applied to 103 blue nevi. As previously reported, most blue nevi were found to harbor activating mutations in GNAQ (59%, n=61), followed by less frequent mutations in GNA11 (16%, n=17). Additionally, one BRAF (1%) and three NRAS (3%) mutations were detected. In three tumors (3%) harboring none of the aforementioned gene alterations, CYSLTR2 mutations were identified. All three CYSLTR2 mutations were the same c.386T>A, L129Q mutation previously identified in uveal melanoma that has been shown to lead to increased receptor activation and signaling. In summary, our study identifies CYSLTR2 L129Q alterations as a previously unrecognized activating mutation in blue nevi, occuring in a mutually exclusive fashion with known GNAQ and GNA11 mutations. Similar to GNAQ and GNA11 mutations, CYSLTR2 mutations, when present, are likely defining pathogenetic events in blue nevi.

SF3B1 and BAP1 mutations in blue nevus-like melanoma.

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

Phenotypic characterization and prognostic impact of circulating γδ and αß T-cells in metastatic malignant melanoma.

Human T cells carrying γδ T-cell receptors (TCRs) represent a minor population relative to those with αß TCRs. There has been much interest recently in the possibility of using these γδ T-cells in cancer therapy because they can kill tumor cells in vitro in an MHC-unrestricted manner, and possess potential regulatory capability and antigen-presenting capacity. The presence of γδ T-cells in late-stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T-cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T-cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αß T-cells. We found an increased Vδ1:Vδ2-ratio and a decreased CD4:CD8-ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per µL blood. Nonetheless, Kaplan-Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early-differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early-differentiated CD8+ αß T-cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T-cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival.

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

Targeted next generation sequencing reveals unique mutation profile of primary melanocytic tumors of the central nervous system.

Melanocytic tumors originating in the central nervous system (MT-CNS) are rare tumors that generally have a favorable prognosis, however malignant tumors do occur. Pathogenetically MT-CNS are not well characterized. Similar to uveal melanoma and blue nevi, they frequently harbor activating GNAQ or GNA11 mutations. Rare NRAS mutations have also been reported. Other mutations have not yet been described. We analyzed 19 MT-CNS, 7 uveal melanomas and 19 cutaneous melanomas using a targeted next generation sequencing approach analyzing 29 genes known to be frequently mutated in other melanocytic tumors (in particular uveal and cutaneous melanomas). In concordance with previous studies, cutaneous melanoma samples showed frequent NRAS or BRAF mutations, as well as mutations in other genes (e.g. NF1, RAC1, PIK3CA, ARID1A). Metastasized uveal melanomas exhibited mutations in GNAQ, GNA11 and BAP1. In contrast, MT-CNS almost exclusively demonstrated mutations in GNAQ (71 %) or GNA11 (12 %). Interestingly both GNA11 mutations identified were detected in MT-CNS diagnosed as intermediate grade melanocytomas which also recurred. One of these recurrent cases also harbored an inactivating BAP1 mutation and was found to have lost one copy of chromosome 3. Our findings show that while MT-CNS do have GNAQ or GNA11 mutations, they rarely harbor other recurrent mutations found in uveal or cutaneous melanomas. Considering chromosome 3 and BAP1 loss are robust markers of poor prognosis in uveal melanoma, it will prove interesting to determine whether these genomic alterations are also of prognostic significance in MT-CNS.

Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks.

BACKGROUND: Biobanks are becoming increasingly important for assessment of disease risk as well as identification and validation of new diagnostic biomarkers and druggable targets. The validity of data obtained from biobanks is critically limited by the biomaterial quality of the biological samples. External quality assessment (EQA) programs suitable to comprehensively measure the biomaterial quality in archived materials are currently lacking. We report on quantitative assay designs for the analysis of both structural and functional integrity of DNAs that were applied in a first pilot EQA within the priority program on tumor tissue biobanking funded by the German Cancer Aid. METHODS: Participating biobanks isolated DNAs from a standardized set of 10 samples comprising sections of four different formalin-fixed paraffin-embedded tissues using their standard operating procedures. Isolated DNAs and analytical results were returned and analyzed centrally for nucleic acids yield, purity, fragmentation and amplificability at a quantitative level using dedicated assay designs. RESULTS: The amount of extracted DNA varied in isolates ranging between 1.5 µg and 25.8 µg. Quantification of DNA fragmentation and amplificability allowed to highlight considerable discrepancies in DNA quality. Amplicons yielded from the isolates of these identical EQA samples ranged from 105 to 411 bp suggesting differences between residual inhibitors of downstream enzymatic reactions. CONCLUSIONS: The quality of extraction of bioanalytes from biomaterial archives is heterogeneous even for stable biomolecules like DNA isolated with highly standardized methods. EQAs are appropriate tools to uncover strengths and weaknesses in biobanks in a systematic fashion. Biomaterial integrity is insufficiently reflected by standard methods, but needs to be assessed to improve biobank interoperability. Finally, our results also point towards the problem of measuring the quality of more delicate biomolecules like proteins or metabolites.

Inherited variation in the PARP1 gene and survival from melanoma.

We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS). Results were combined using random effects meta-analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04-1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01-1.39, p = 0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS.

TERT promoter mutations are frequent in atypical fibroxanthomas and pleomorphic dermal sarcomas.

Activating mutations in the TERT promoter leading to increased telomerase expression were recently identified in cutaneous melanoma and subsequently in many other types of cancer. These mutations lead to increased telomerase expression, allowing cells to proliferate continuously without entering apoptosis or senescence. Atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically poorly understood tumors developing in the skin of older patients. Known genetic events in these tumors are mutations in TP53 (atypical fibroxanthoma and pleomorphic dermal sarcoma) and RAS (pleomorphic dermal sarcoma) genes, often having a UV signature. We analyzed a cohort of 27 atypical fibroxanthomas and 34 pleomorphic dermal sarcomas for the presence of TERT promoter mutations by conventional Sanger sequencing. TERT promoter mutations were identified in 25 (93%) atypical fibroxanthomas and in 26 (76%) pleomorphic dermal sarcomas. Mutations were found to have a UV signature (C>T or CC>TT) and were largely identical to those detected in cutaneous melanoma. Our data show that TERT promoter mutations are the most frequent mutations in atypical fibroxanthomas and pleomorphic dermal sarcomas reported to date. The identified mutations confirm the pathogenetic role of UV exposure in both atypical fibroxanthomas and pleomorphic dermal sarcomas and suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis of these tumors.

Genetic and clinico-pathologic analysis of metastatic uveal melanoma.

Uveal melanoma is the most common malignant tumor of the adult eye. Fifty percent of tumors will eventually metastasize, and there are no effective treatments for them. Recent studies of uveal melanoma have identified activating mutations in GNAQ and GNA11, loss-of-function mutations in the tumor suppressor gene BAP1, and recurrent mutations in codon 625 of SF3B1. Previous studies have reported the existence of a higher frequency of GNA11 than GNAQ mutations, frequent BAP1 loss, and rare SF3B1 mutations in metastatic uveal melanoma. We analyzed a cohort of 30 uveal melanoma metastases for the occurrence of GNAQ, GNA11, and SF3B1 mutations, as well as BAP1 loss, and correlated these parameters with clinical and histopathologic features. Most (92%) tumors were composed of cells with an epithelioid or mixed (<100% spindle cells) morphology. Tumor samples composed exclusively of spindle cells were rare (n=2, 8%). Most tumors showed a moderate to marked degree of nuclear pleomorphism (n=24, 96%), and contained hyperchromatic, vesicular nuclei with variably conspicuous nucleoli. GNA11 mutations were considerably more frequent than GNAQ mutations (GNA11, GNAQ, and wild-type in 18 (60%), 6 (20%), and 6 (20%) cases, respectively). SF3B1 mutation was found in 1 of 26 tumors (4%), whereas loss of BAP1 expression was present in 13 of 16 tumors (81%). Patients with GNA11-mutant tumors had poorer disease-specific survival (60.0 vs 121.4 months, P=0.03) and overall survival (50.6 vs 121.4 months, P=0.03) than those with tumors lacking GNA11 mutations. The survival data, combined with the predominance of GNA11 mutations in metastases, raises the possibility that GNA11-mutant tumors may be associated with a higher risk of metastasis and poorer prognosis than GNAQ-mutant tumors. Further studies of uveal melanoma are required to investigate the functional and prognostic relevance of oncogenic mutations in GNA11 and GNAQ.