Summary We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02–restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

Detection of lymphocytes that target tumor-specific mutant neoantigens--derived from products encoded by mutated genes in the tumor--is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

Immunotherapy using either checkpoint blockade or the adoptive transfer of antitumor lymphocytes has shown effectiveness in treating cancers with high levels of somatic mutations-such as melanoma, smoking-induced lung cancers and bladder cancer-with little effect in other common epithelial cancers that have lower mutation rates, such as those arising in the gastrointestinal tract, breast and ovary. Adoptive transfer of autologous lymphocytes that specifically target proteins encoded by somatically mutated genes has mediated substantial objective clinical regressions in patients with metastatic bile duct, colon and cervical cancers. We present a patient with chemorefractory hormone receptor (HR)-positive metastatic breast cancer who was treated with tumor-infiltrating lymphocytes (TILs) reactive against mutant versions of four proteins-SLC3A2, KIAA0368, CADPS2 and CTSB. Adoptive transfer of these mutant-protein-specific TILs in conjunction with interleukin (IL)-2 and checkpoint blockade mediated the complete durable regression of metastatic breast cancer, which is now ongoing for >22 months, and it represents a new immunotherapy approach for the treatment of these patients.

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors. Most importantly, we discovered that TRRAP harbored a recurrent mutation that clustered in one position (p. Ser722Phe) in 6 out of 67 affected individuals (~4%), as well as a previously unidentified gene, GRIN2A, which was mutated in 33% of melanoma samples. The nature, pattern and functional evaluation of the TRRAP recurrent mutation suggest that TRRAP functions as an oncogene. Our study provides, to our knowledge, the most comprehensive map of genetic alterations in melanoma to date and suggests that the glutamate signaling pathway is involved in this disease.

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. A mutational analysis of the Protein Tyrosine Kinase (PTK) gene family in cutaneous metastatic melanoma identified 30 somatic mutations in the kinase domain of 19 PTKs. The whole of the coding region of these 19 PTKs was further evaluated for somatic mutations in a total of 79 melanoma samples. This analysis revealed novel ERBB4 mutations in 19% of melanoma patients and that an additional two kinases (FLT1 and PTK2B) are mutated in 10% of melanomas. Seven missense mutations in the most commonly altered PTK (ERBB4) were examined and found to increase kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies might lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.Malignant melanoma is the most fatal skin cancer 1,2 . To develop personalized treatments for advanced disease, it is important to identify genetic alterations leading to melanoma. Protein tyrosine kinases (PTKs) are frequently mutated in cancer (http://www.sanger.ac.uk/genetics/CGP/Census/), and since they are amenable to pharmacologic inhibition 3,4 , further analysis of the PTK gene family may identify new therapeutic strategies. In this study, we used high-throughput gene sequencing to analyze the entire PTK gene family in melanoma, and have identified many novel somatic alterations.We initially sequenced the coding exons comprising the kinase domains of all 86 members of this gene superfamily in 29 melanomas (Supplementary Table 1). A total of 593 exons were extracted from genomic databases and amplified by polymerase chain reaction (PCR) * To whom correspondence should be addressed: National Human Genome Research Institute, 50 South Drive, MSC 8000, Building 50, Room 5140, Bethesda MD 20892-8000, Phone: 301-451-2628, Fax: 301-480-9864, samuelsy@mail.nih.gov. Author contributions T.DP. and Y.S. designed the study; J.R.W. and S.A.R. collected and analyzed the melanoma samples, N.S.A., J.C.C., K.E.Y., J.C.L., NISC., P.C and Y.S. analyzed the genetic data; T.D.P., X.W. and K.E.Y., performed and analyzed the functional data. All authors contributed to the final version of the paper. NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 July 6. Table 2) and directly sequenced with dye-terminator chemistry. We determined whether a mutation was somatic (i.e., tumor-specific) by examining the sequence of the gene in genomic DNA from normal tissue of the relevant patient. From the ~12 Mb of sequence information obtained, we identified 19 genes containing a total of 30 somatic mutations within their kinase domains. All coding exons of these 19 genes were then analyzed for mutations in a total of 79 melanoma samples using specific primers (Supplementary Table 3).We identified 99 non-synonymous, somatic mutations in ...

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Immunotherapy has clinical activity in certain virally-associated cancers. The tumor antigens targeted in successful treatments are not well defined and have important implications for the study and design of novel immunotherapies. We used a personalized immunogenomic approach to elucidate the global landscape of anti-tumor T-cell responses in complete regression of human papillomavirus-associated metastatic cervical cancer after tumor-infiltrating adoptive T-cell therapy. Remarkably, the immunodominant T-cell reactivity was directed against mutated neoantigens or a cancer-germline antigen, rather than the canonical viral antigens underlying the disease. T cells targeting viral tumor antigens did not display preferential in vivo expansion during cancer regression. T-cell clonotypes specific for both viral and non-viral tumor antigens resided predominantly in the programmed cell death 1 (PD-1) expressing T-cell compartment before treatment suggesting that PD-1 blockade may unleash diverse anti-tumor T-cell reactivities. These findings suggest a new paradigm of targeting non-viral antigens in immunotherapy of virally-associated cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors which may represent ideal targets for immunotherapy. Using high throughput immunologic screening of mutant gene products identified via whole exome sequencing, we identified neoantigen reactive tumor infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic non-synonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell based immunotherapies for patients bearing these cancers.