Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors(35%) and losses of CDKN2A in9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas.

DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective.Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies.

Genomic locus at chromosome 9p21 that contains the CDKN2A and CDKN2B tumor suppressor genes is inactivated through mutations, deletions and promoter methylation in multiple human cancers. Additionally, the locus encodes an anti-sense RNA (ANRIL). Both hemizygous and homozygous deletions at the locus targeting multiple genes are fairly common in different cancers. We in this study investigated breakpoints in five melanoma cell lines, derived from metastasized tumors, with previously identified homozygous deletions using array comparative genomic hybridization (aCGH). For breakpoint mapping, we used primer approximation multiplex PCR (PAMP) and inverse PCR techniques. Our results showed that three cell lines carried complex rearrangements. In two other cell lines, with focal deletions of 141 kb and 181 kb, we identified fusion gene products, involving MTAP and ANRIL. We also confirmed the complex rearrangements and focal deletions in DNA from tumor tissues corresponding to three cell lines. The rapid amplification of 3'cDNA ends (3'RACE) carried out on transcripts resulted in identification of three isoforms of MTAP-ANRIL fusion gene. Screening of cDNA from 64 melanoma cell lines resulted in detection of fusion transcripts in 13 (20%) cell lines that involved exons 4-7 of the MTAP and exon 2 or 5 of the ANRIL genes. We also detected fusion transcripts involving MTAP and ANRIL in two of the seven primary melanoma tumors with focal deletion at the locus. The results from the study, besides identifying complex rearrangements involving CDKN2A locus, show frequent occurrence of fusion transcripts involving MTAP and ANRIL genes.

Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown.

Development and validation of robust molecular biomarkers has so far been limited in melanoma research. In this paper we used a large population-based cohort to replicate two published gene signatures for melanoma classification. We assessed the signatures prognostic value and explored their biological significance by correlating them with factors known to be associated with survival (vitamin D) or etiological routes (nevi, sun sensitivity and telomere length). Genomewide microarray gene expressions were profiled in 300 archived tumors (224 primaries, 76 secondaries). The two gene signatures classified up to 96% of our samples and showed strong correlation with melanoma specific survival (P=3 x 10(-4)), Breslow thickness (P=5 x 10(-10)), ulceration (P=9.x10-8) and mitotic rate (P=3 x 10(-7)), adding prognostic value over AJCC stage (adjusted hazard ratio 1.79, 95%CI 1.13-2.83), as previously reported. Furthermore, molecular subtypes were associated with season-adjusted serum vitamin D at diagnosis (P=0.04) and genetically predicted telomere length (P=0.03). Specifically, molecular high-grade tumors were more frequent in patients with lower vitamin D levels whereas high immune tumors came from patients with predicted shorter telomeres. Our data confirm the utility of molecular biomarkers in melanoma prognostic estimation using tiny archived specimens and shed light on biological mechanisms likely to impact on cancer initiation and progression.

Despite of highly effective new therapeutic strategies, chemotherapy still is an important treatment option in metastatic melanoma. Since predictors of chemotherapy response are rare, drugs and regimens are currently chosen arbitrarily. The present study was aimed at the identification of molecular markers predicting the outcome of chemotherapy in melanoma. Tumor biopsies from metastatic lesions were collected from 203 stage IV melanoma patients prior to chemotherapy onset and used for gene expression profiling (n = 6; marker identification set), quantitative real-time PCR (n = 127; validation set 1), and immunohistochemistry on tissue microarrays (n = 70; validation set 2). The results were correlated to the tumors' in-vitro chemosensitivity and to the patients' in-vivo chemotherapy outcome. SERPINB1 was found to correlate to the in-vitro sensitivity to cisplatin-containing chemotherapy regimens (p = 0.005). High SERPINB1 gene expression was associated with favorable tumor response (p = 0.012) and prolonged survival (p = 0.081) under cisplatin-based chemotherapy. High SERPINB1 protein expression in tumor tissue from cisplatin-treated patients was associated with a favorable survival (p = 0.011), and proved as an independent predictor of survival (p = 0.008) by multivariate analysis. We conclude, that SERPINB1 expression, although not functionally involved, is predictive for the outcome of cisplatin-based chemotherapy in melanoma, and thus may be useful to personalize melanoma chemotherapy.