Yuan J, Adamow M, Ginsberg BA, et al

Yuan J, Adamow M, Ginsberg BA, et al. also fit well with the observation that this 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 exomeCguided 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 (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exomeCguided analysis to dissect the effects of melanoma immunotherapy. Case Report A 56-year-old male was diagnosed in 2003 with a nodular melanoma with a Breslow thickness of 1 1.5 mm around the left upper arm. In April 2009, he developed lymph node metastases in both axillae and underwent dissection of involved nodes at the right side. Positron emission tomography showed [18F]fluorodeoxyglucose uptake in both axillae, in soft tissue at the right scapula, in the left liver lobe, and mesenterially cranial of the transverse colon. He was treated with dacarbazine but experienced clear disease progression after six courses. At that time (October 2009), as a result of pain, a palliative dissection of the left axillary nodes was performed. In June 2010, before enrollment in the ipilimumab Expanded Access Pro-gram, magnetic resonance imaging of the brain showed three lesions, of which one was resected and two others were treated with stereotac-tic radiotherapy. In August 2010, he started ipilimumab treatment (3 mg/kg) and received four infusions. All four courses of ipilimumab were tolerated well, except for grade 1 dermatitis. After completion, the patient displayed a marked regression of the tumor load (25%), as shown by computed tomography (Fig 1A) and close to normalization (upper limit of normal 0.10 g/L) of the S100b tumor marker after ipilimumab treatment (Fig 1B). Open in a separate window Physique 1 Patient characteristics To determine whether exome-guided analysis of antigen-specific T-cell responses against mutated antigens was feasible, we obtained both tumor cells and tumor-infiltrating lymphocytes (TILs) from the lesion resected in 2009 2009. Whole-exome sequencing of tumor cells Potassium oxonate and autologous healthy tissue was performed to identify tumor-specific mutations. This revealed a total of 1 1,657 somatic mutations, consisting of 1,075 nonsynonymous (1,036 single nucleotide and 39 nonsense variants) and 573 synonymous mutations with a false discovery rate of 0.07. In addition, the tumor harbored seven frame shifts and two codon deletions. Consistent with prior data, C T/G A mutations, reflective of UV-induced DNA damage, predominated (Fig 1C).8-10 To predict Potassium oxonate potential neoantigens from this set of PR55-BETA mutations, the data were first filtered for gene expression using RNAseq data. Subsequently, predictions for proteasomal processing and Potassium oxonate HLA class I bind-ing were performed on stretches of amino acid sequences that contained nonsynonymous mutations, using the NetChop Cterm3.0 and NetMHC3.2 algorithms.13-15 This analysis yielded a set of 448 potential CD8 T-cell epitopes (nine to 11 amino acids in length) with a predicted medium-to-high affinity binding for each HLA-A and -B allele (HLA-A*03:01, HLA-A*32:01, HLA-B*35:03, and HLA-B*40: 02). Predicted peptides were synthesized, and HLA multimers made up of these ligands were produced by micro-scale parallel UV-induced peptide exchange reactions.16,17 Subsequently, tumor-infiltrating lymphocytes from this patient were analyzed for reactivity against these predicted T-cell epitopes by a multiplexed major histo-compatibility complex (MHC) multimer staining strategy.16,18,19 An overview of the complete screening procedure can be found in Determine 2 (MHC, major histocompatibility Potassium oxonate complex). Open in a separate window Physique 2 Overview of screening procedure This analysis revealed a T-cell response against two patient-specific neoantigens (both confirmed in impartial analyses), a mi-nor T-cell response against a mutated.