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The evidence for T-cell–mediated regression of human cancers such as non–small-cell lung carcinoma, renal cell carcinoma, and—in particular—melanoma after immunotherapy is strong. Anti-CTLA4 (ipilimumab) treatment has been approved for treatment of meta-static melanoma,1 and antibody-mediated blockade of PD-1, a second inhibitory receptor on T cells, has shown highly encouraging results in early clinical trials.2,3 Although the clinical activity of these treatments is apparent, it is still unknown which T-cell reactivities are involved in immunotherapy-induced cancer regression.4 T-cell reactivity against nonmutated tumor-associated self-antigens has been analyzed in patients treated with ipilimumab or with autologous tumor-infiltrating T cells, but the magnitude of the T-cell responses observed has been relatively modest.5,6 In part on the basis of such data, recognition of patient-specific mutant epitopes (hereafter referred to as neoantigens) has been suggested to be a potentially important component.7 A potential involvement of mutated epitopes in T-cell control would also fit well with the observation that the mutation load in sun-exposed melanomas is particularly high.8-10 Intriguingly, on the basis of animal model data, it has recently been suggested that (therapy-induced) analysis of T-cell reactivity against patient-specific neoantigens may be feasible through exploitation of cancer genome data.11,12 However, human data have thus far been lacking. Here we report a case of a patient with stage IV melanoma who exhibited a clinical response to ipilimumab treatment. Cancer exome–guided analysis of T-cell reactivity in this patient revealed reactivity against two neoantigens, including a dominant T-cell response against a mutant epitope of the ATR (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exome–guided analysis to dissect the effects of melanoma immunotherapy.

Purpose Oncogenic activation of the BRAF gene has been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). In this study, we investigated the contribution of BRAF to melanoma susceptibility, also making a comparison with frequency of CDKN2A germline mutations in MM patients from different areas in Italy. Patients and Methods Using a combination of denaturing high-performance liquid chromatography analysis and automated sequencing on genomic DNA from peripheral blood or tumor tissue samples, 569 MM patients (211 from northern Italy and 358 from southern Italy) were screened for BRAF mutations. Results Three BRAF germline sequence variants (M116R, V599E, and G608H) were identified in four (0.7%) of 569 MM patients. The most common BRAF mutation, V599E, was detected in one germline DNA sample only; M116R and G608H were newly described mutations. A high frequency (59%) of BRAF mutations was instead observed in tumor samples from patients also undergoing germline DNA analysis; at the somatic level, substitution of valine 599 was found to account for the majority (88%) of BRAF mutations. We then estimated the germline mutation rates in BRAF and CDKN2A among 358 consecutively collected patient samples originating in southern Italy; a low (2.5%) or very low (0.29%) prevalence of CDKN2A and BRAF mutations, respectively, was detected. Conclusion Mutation analysis of either blood DNA from a large collection of MM patients or matched MM tissues from a subset of such patients revealed that BRAF is somatically mutated and does not play a major role in melanoma susceptibility. The present study further suggests that patient origin may account for different mutation rates in candidate genes.

10511 Background: Angiosarcoma is an aggressive tumour of presumed vascular endothelial origin that is commonly thought to be driven by aberrant angiogenesis. The somatic alterations underpinning a...

9085 Background: Evidence for T cell mediated regression of human cancer in particular melanoma following immunotherapy is strong. Anti-CTLA4 treatment has been approved for treatment of metastatic melanoma and blockade of PD-1 has shown encouraging results. However, it is unknown which T cell reactivities are involved in cancer regression. Reactivity against non-mutated tumor self-antigens has been analyzed in patients treated with Ipilimumab or with autologous TILs, but the size of these responses are modest. Therefore, T cell recognition of patient-specific mutant epitopes may be a potentially important component. Animal model data recently suggested that analysis of T cell reactivity against patient-specific neo-antigens may be feasible through exploitation of cancer genome data. However, human data have thus far been lacking. Methods: To address this we have used MHC class I peptide exchange technology allowing production of very large collections of pMHC complexes, together with a pMHC "combinatorial coding" strategy for parallel detection of dozens of different T cell populations within a single sample. Results: From a melanoma patient responding to ipilimumab treatment, we identified tumor specific mutations via exome sequencing of tumor material. The exome contained 1,075 non-synonymous mutations. Possible MHC epitopes covering these mutations were predicted based on; 1) predicted to bind the patient’s MHC; 2) predicted to be cleaved by the proteasome; 3) genes of which the mutated peptides arose had evidence of RNA expression. The analysis yielded 1,952 epitopes restricted to the HLA-A and HLA-B. To screen for T cell reactivity against these epitopes we used the pMHC combinatorial coding approach. We found T cell reactivity against 2 neo-antigens, including a dominant T cell response against a mutant epitope of the ATR gene product. Analysis of PBMC samples collected before and during Ipilimumab therapy showed that this particular response increased strongly after treatment from 0.06% to 0.28% of CD8 T cells after being stable in magnitude for 10 months. Conclusions: These data provide the first demonstration of cancer exome-guided analysis to dissect the effects of melanoma immunotherapy.

4571 Background: The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. Methods: A recent l...

10039 Background: Testicular germ cell tumors (TGCTs) account for 1% of all male cancers with increasing incidence rates documented. A family history of TGCT occurs in 2% of patients, conferring relative risks of 8–10 for brothers and 4–6 for fathers or sons of patients. These risks are high in comparison to other solid cancers and suggest that the risk of developing TGCT may be, at least in part, inherited rather than acquired. Methods: Pedigrees with >1 male family member with TGCT were identified from a tumor database and systematically collected from 1992. Patients donated samples and medical details with full informed consent and under ethical review board approval. Pedigree and medical data were obtained by interview, mail and telephone contact. Results: This series examines 78 pedigrees, containing 160 cases and 170 TGCTs. In the period 1994–2003 6% of all cases had a family history of TGCT. It includes 35 sib-pairs, 12 father-son pairs, 11 uncle-nephew pairs, 10 cousin pairs and 4 pedigrees with ≥ 3 cases of TGCT. The dataset contains 66 seminomas, 56 non-seminomas, 17 mixed histology, 1 CIS and 30 tumors of unconfirmed histology. The median age at diagnosis was 33 (range 17 to 87). The median difference in age at diagnosis between the cases in each pedigree was 6 years (0–58). Histology was concordant between family members in 22 of the 53 (41.5%) 2-case pedigrees. 11 pedigrees contained a case with bilateral TGCTs (6.9% of all cases, compared to 1.1% in the sporadic patient population, p No significant financial relationships to disclose.