TGF-ß Alters the Proportion of Infiltrating Immune Cells in a Pancreatic Ductal Adenocarcinoma.

PURPOSE: Immunotherapy, such as checkpoint inhibitors against anti-programmed death-ligand 1 (PD-L1), has not been successful in treating patients with pancreatic ductal adenocarcinoma (PDAC). Tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and the TGF-ß cytokine are critical in anti-cancer immunity. We hypothesized that TGF-ß enhances the immunosuppressive effects of TAM, MDSC, and DC presence in tumors. METHODS: Using a murine PDAC cell line derived from a genetically engineered mouse model, we orthotopically implanted treated cells plus drug embedded in Matrigel into immunocompetent mice. Treatments included saline control, TGF-ß1, or a TGF-ß receptor 1 small molecule inhibitor, galunisertib. We investigated TAM, MDSC, DC, and TAM PD-L1 expression with flow cytometry in tumors. Separately, we used the TIMER2.0 database to analyze TAM and PD-L1 gene expression in human PDAC tumors in TCGA database. RESULTS: TGF-ß did not alter MDSC or DC frequencies in the primary tumors. However, in PDAC metastases to the liver, TGF-ß decreased the proportion of MDSCs (P=0.022) and DCs (P=0.005). TGF-ß significantly increased the percent of high PD-L1 expressing TAMs (32 ± 6 % vs. 12 ± 5%, P=0.013) but not the proportion of TAMs in primary and metastatic tumors. TAM PD-L1 gene expression in TCGA PDAC database was significantly correlated with tgb1 and tgfbr1 gene expression (P<0.01). CONCLUSIONS: TGF-ß is important in PDAC anti-tumor immunity, demonstrating context-dependent impact on immune cells. TGF-ß has an overall immunosuppressive effect mediated by TAM PD-L1 expression and decreased presence of DCs. Future investigations will focus on enhancing anti-cancer immune effects of TGF-ß receptor inhibition.

Use of endoloop in video-assisted thoracoscopic enucleation of a very rare esophageal tumor.

A gastrointestinal stromal tumor is an infrequent tumor of the gastrointestinal tract with very rare involvement of the esophagus. We present a case of a patient with dysphagia and a 4 cm submucosal mass. The patient underwent thoracoscopic enucleation with complete resection of the mass. We present case details and operative video highlighting the important surgical steps of exposure and retraction. We believe that the Endoloop is a very useful tool in providing countertraction needed during minimally invasive resection of such lesions.

Routine Computer Tomography Imaging for the Detection of Recurrences in High-Risk Melanoma Patients.

BACKGROUND: The use of routine CT imaging for surveillance in asymptomatic patients with cutaneous melanoma is controversial. We report our experience using a surveillance strategy that included CT imaging for a cohort of patients with high-risk melanoma. METHODS: A total of 466 patients with high-risk cutaneous melanoma enrolled in adjuvant immunotherapy trials were followed for tumor progression by physical examination, labs, and CT imaging as defined by protocol. Evaluations were obtained at least every 6 months for year 1, every 6 months for year 2, and then annually for the remainder of the 5-year study. Time to tumor progression, sites of recurrence, and the method of relapse detection were identified. RESULTS: The patient cohort consisted of 115 stage II patients, 328 stage III patients, and 23 patients with resected stage IV melanoma. The medium time to progression for the 225 patients who developed tumor progression was 7 months. Tumor progression was detected by patients, physician examination or routine labs, or by CT imaging alone in 27, 14, and 59% of cases respectively. Melanoma recurrences were noted to be locoregional in 36% of cases and systemic in 64% of cases. Thirty percent of patients with locoregional relapse and 75% of patients with systemic relapse were detected solely by CT imaging. CONCLUSIONS: CT imaging alone detected the majority of sites of disease progression in our patients with high-risk cutaneous melanoma. This disease was not heralded by symptoms, physical examination, or blood work. Although the benefit of the early detection of advanced melanoma is unknown, this experience is relevant because of the rapid development and availability of potentially curative immunotherapies.

Durable Complete Response from Metastatic Melanoma after Transfer of Autologous T Cells Recognizing 10 Mutated Tumor Antigens.

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR.

Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.

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.

Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.