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Supplementary Figure 5

Supplementary Tables 1 and 2

Supplementary Table 3

Adoptive cell therapy (ACT) with T cells targeting neoantigens can mediate durable responses in patients with metastatic cancer. Cell therapies targeting common shared antigens for epithelial cancers are not yet broadly available. Here, we report the identification and characterization in one patient of T-cell receptors (TCRs) recognizing mutated p53 p.R175H, which is shared among a subset of patients with cancer. Tumor-infiltrating lymphocytes were screened for recognition of mutated neoantigens in a patient with metastatic colorectal cancer. HLA-A*0201–restricted recognition of mutated p53 p.R175H was identified, and the minimal peptide epitope was HMTEVVRHC. Reactive T cells were isolated by tetramer sorting, and three TCRs were identified. These TCRs mediated recognition of commercially available ovarian cancer, uterine carcinoma, and myeloma cell lines, as well as an NIH patient–derived esophageal adenocarcinoma line that endogenously expressed p53 p.R175H and HLA-A*0201. They also mediated recognition of p53 p.R175H+ colon, breast, and leukemia cell lines after transduction with a retrovirus encoding HLA-A*0201. This work demonstrates that common shared mutated epitopes such as those found in p53 can elicit immunogenic responses and that the application of ACT may be extended to patients with any cancer histology that expresses both HLA-A*0201 and the p53 p.R175H mutation.

Supplementary Figures 1, 2, 3, 4, 6 and 7

Phenotypic characterization of TIL.

HLA typing data.

Tumor mutation profile.

Mutation expression in metastatic lesions at autopsy.

Clonal architecture of 4062 tumor.

Recognition of mRNA-encoded RBPJ mutation.

Purpose: The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer.Experimental Design: A TNBC metastasis was resected for TIL generation and whole-exome sequencing. Tandem minigenes or long 25-mer peptides encoding selected mutations were electroporated or pulsed onto autologous antigen-presenting cells, and reactivity of TIL was screened by upregulation of CD137 and IFNγ ELISPOT. The nature of the T-cell response against a unique nonsynonymous mutation was characterized.Results: We identified 72 nonsynonymous mutations from the tumor of a patient with TNBC. CD4+ and HLA-DRB1*1501–restricted TILs isolated from this tumor recognized a single mutation in RBPJ (recombination signal binding protein for immunoglobulin kappa J region). Analysis of 16 metastatic sites revealed that the mutation was ubiquitously present in all samples.Conclusions: Breast cancers can express naturally processed and presented unique nonsynonymous mutations that are recognized by a patient's immune system. TILs recognizing these immunogenic mutations can be isolated from a patient's tumor, suggesting that adoptive cell transfer of mutation-reactive TILs could be a viable treatment option for patients with breast cancer. Clin Cancer Res; 23(15); 4347–53. ©2017 AACR.

4157 Background: The use of neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma (PDAC) has shown clear advantages in locally advanced and borderline resectable disease. The benefit in upfront resectable PDAC is debated. Moreover, in early clinical stages IA/IB, potential benefits including improved R0 resection rate, decreased tumor upstaging, and survival, are not clear. We hypothesize that NAC will be associated with improved outcomes and survival compared to adjuvant therapy in patients with clinical stage IA/IB PDAC. Methods: The National Cancer Database (NCDB) PUFs (2004-2017) were used to perform a retrospective review of patients with clinical stage IA or IB PDAC undergoing surgery. Treatment groups were selected based on timing of chemotherapy. Patients receiving chemotherapy or surgery alone were excluded. Results: We identified 6,613 patients with clinical stage IA or IB PDAC who underwent surgery. The neoadjuvant therapy group (NAT) included 1,533 patients who received neoadjuvant or perioperative chemotherapy, and the adjuvant therapy group (AT) contained 5080 patients who received chemotherapy after surgery. Patients in the NAT had higher rates of T1 and T2 disease and lower rates of T3 pathology compared to the AT (pT1: 18.7% vs 7.8%; pT2: 20.1 vs 18.6%; pT3: 59.3% vs 72.1%, p

138 Background: HIPEC with MMC is a treatment for gastrointestinal cancers metastatic to the peritoneal cavity. The pharmacokinetics of MMC in plasma, peritoneal fluid, and urine are described. The amount of MMC in intraabdominal tissues after HIPEC are not well described. The aim of this study is to evaluate if MMC concentrated in tissue samples after HIPEC by high performance liquid chromatography (HPLC) from patients with gastrointestinal neoplasms. Methods: HIPEC was performed at 40°C with 40mg of MMC for 90 minutes, after which the peritoneal cavity was flushed, anastomoses created as needed, and the wound closed. Eligible patients were treated at a single institution, ≥18 years old, and underwent HIPEC with MMC. Samples were taken of the omentum, peritoneum, liver core biopsy, tumor, and mesenteric fat before and after HIPEC. All patients signed informed consent. Samples were frozen in liquid nitrogen, minced, and sonicated in 500µL of phosphate buffered saline. The homogenized samples were centrifuged, and the supernatant was analyzed by HPLC for MMC. The HPLC was performed using a Dionex Ultimate 3000. Analysis was performed with a Kinetex - 5µm Biphenyl 100A – 150 x 4.6mm column. MMC was detected with a Diode Array Detector 3000 with fixed UV at 365nm, 280nm, 254nm, and 210nm. The mobile phase used isocratic 40% acetonitrile and 60% water at 0.5 ml/min. The analysis volume was 10µL. Samples were blinded prior to analysis and analyzed in triplicate. Results: Thirteen patients were enrolled, 11 were female, the average age was 57 years (range: 30 – 85). Diagnoses were low-grade appendiceal mucinous neoplasm (7), high-grade appendiceal mucinous neoplasm (1), appendiceal adenocarcinoma (1), colon adenocarcinoma (1), colon mucinous adenocarcinoma (1), peritoneal mesothelioma (1), and small bowel mucinous adenocarcinoma (1). Complete tissue samples were available for 10 patients. Two patients had complete cytoreduction and did not have tumor for analysis after HIPEC. One patient refused liver biopsy. MMC was not detected in any sample prior to HIPEC. After HIPEC, MMC was most often detected in peritoneum (12 of 13 cases) and tumor (9 of 11). MMC was less often detected in omentum (5 of 13), mesenteric fat (2 of 13), or liver (1 of 12). Conclusions: MMC concentrated in 92% of peritoneal samples, 82% of tumor samples, and less often in liver tissue. MMC is hydrophilic which may contribute to the low detection rates in omentum and mesenteric fat. A reliable method to measure MMC concentration in normal and malignant tissues is novel and may have clinical implications. Our next steps are to expand the cohort of patients and evaluate whether tissue concentration is associated with clinical outcomes.