Cell surface marker-based capture of neoantigen-reactive CD8+ T-cell receptors from metastatic tumor digests.

BACKGROUND: Cellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority. METHODS: We analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8+ TILTP). RESULTS: TILTP TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TILTP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TILTP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TILTP clonotypes from four patients indicated that most in vitro expanded TILTP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition. CONCLUSIONS: While direct usage of in vitro-expanded CD8+ TILTP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TILTP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers.

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

Characteristics of Gastrointestinal Stromal Tumors incidentally discovered during abdominal surgery.

BACKGROUND: Gastrointestinal Stromal Tumors (GISTs) are rare sarcomas with 5000 new cases arising in the United States each year. Despite their low incidence, general surgeons should be familiar with GISTs since a quarter of these neoplasms are encountered incidentally. METHODS: A retrospective medical records review was conducted to create a database of all GISTs resected from January 2005 to May 2019. We isolated patients who had incidental discovery of GISTs intraoperatively or within final pathology. Characteristics of patient (Age, gender), index procedure (malignant vs. benign, elective vs. emergent) and tumor (location, size and mitotic rate) were analyzed. RESULTS: A total 48 patients were incidentally discovered to have a GIST excised during index operation. The mean age of these patients was 62 years, with 27 females and 21 males. The primary location of tumors in descending frequency was stomach (30), small bowel (15), colon/rectum (2) and esophagus (1). The average size of all tumors was 1.2 cm, with the average size of the stomach, small bowel, colon/rectum and esophagus at 0.9 cm, 1.7 cm, 0.9 cm and 0.3 cm respectively. Mitotic rate was less than 5 mitosis per 50 HPF in 96% of patients. Incidental tumors were identified during both bariatric (13) and non-bariatric stomach surgery (8), colorectal surgery (14), hernia repair (4), ampullary/pancreatic surgery (5), esophageal surgery (2) liver surgery (1) and uterine surgery (1). Most incidental-GISTs were identified during elective surgery (81%, 39). Finally, 15 of the tumors were identified during surgery for other malignancies. CONCLUSIONS: One quarter (25%) of the GISTs encountered at our academic community cancer center over a 15-year period were discovered incidentally. These tumors had less malignant characteristics overall and were likely cured with surgical resection.