Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses.

Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

RAD51B Harbors Germline Mutations Associated With Pancreatic Ductal Adenocarcinoma.

PURPOSE: Genetic alterations in many components of the homologous recombination, DNA damage response, and repair (HR-DDR) pathway are involved in the hereditary cancer syndromes, including familial pancreatic cancer. HR-DDR genes beyond BRCA1, BRCA2, ATM, and PALB2 may also mutate and confer the HR-DDR deficiency in pancreatic ductal adenocarcinoma (PDAC). METHODS: We conducted a study to examine the genetic alterations using a companion diagnostic 15-gene HR-DDR panel in PDACs. HR-DDR gene mutations were identified and characterized by whole-exome sequencing and whole-genome sequencing. Different HR-DDR gene mutations are associated with variable homologous recombination deficiency (HRD) scores. RESULTS: Eight of 50 PDACs with at least one HR-DDR gene mutation were identified. One tumor with BRCA2 mutations is associated with a high HRD score. However, another tumor with a CHEK2 mutation is associated with a zero HRD score. Notably, four of eight PDACs in this study harbor a RAD51B gene mutation. All four RAD51B gene mutations were germline mutations. However, currently, RAD51B is not the gene panel for germline tests. CONCLUSION: The finding in this study thus supports including RAD51B in the germline test of HR-DDR pathway genes.

Solid Organ Transplantation Is Associated with an Increased Rate of Mismatch Repair Deficiency and PIK3CA Mutations in Colorectal Cancer.

Solid organ transplants are associated with a modestly increased risk of colorectal cancers (CRC). However, the molecular profile of these cancers has not been described. We hypothesized that transplant-related immunosuppression may promote development of more immunogenic tumors as suggested by a high tumor mutation burden or mismatch repair deficiency. We performed an electronic medical record search for patients seen in the Johns Hopkins University Health System (JHHS) between 2017 and 2022 who developed CRC following solid organ transplantation. A comparator cohort of patients treated for CRC at JHHS with molecular profiling data was also identified. In this case, 29 patients were identified that developed post-transplant CRC (renal transplant, n = 18; liver transplant, n = 8; kidney-liver transplantation, n = 3). Compared to the JHHS general population CRC cohort, patients who developed post-transplant CRC had a higher rate of mismatch repair deficiency (41% versus 12%, p-value = 0.0038), and elevated tumor mutation burden (median of 22 mut/Mb versus 3.5 mut/Mb, p-value = 0.033) (range 3.52-53.65). Post-transplant tumors were enriched for PIK3CA mutations (43% versus 24%, p-value = 0.042). Post-Transplant CRCs are associated with clinical and molecular features of immune sensitivity, supporting a potential role for impaired immune surveillance in shaping the landscape of CRCs. These results may help inform the management of patients with post-transplant CRC.

Challenges of the current precision medicine approach for pancreatic cancer: A single institution experience between 2013 and 2017.

Recent research on genomic profiling of pancreatic ductal adenocarcinoma (PDAC) has identified many potentially actionable alterations. However, the feasibility of using genomic profiling to guide routine clinical decision making for PDAC patients remains unclear. We retrospectively reviewed PDAC patients between October 2013 and December 2017, who underwent treatment at the Johns Hopkins Hospital and had clinical tumor next-generation sequencing (NGS) through commercial resources. Ninety-two patients with 93 tumors tested were included. Forty-eight (52%) patients had potentially curative surgeries. The median time from the tissue available to the NGS testing ordered was 229 days (interquartile range 62-415). A total of three (3%) patients had matched targeted therapies based on genomic profiling results. Genomic profiling guided personalized treatment for PDAC patients is feasible, but the percentage of patients who receive targeted therapy is low. The main challenges are ordering NGS testing early in the clinical course of the disease and the limited evidence of using a targeted approach in these patients. A real-time department level genomic testing ordering system in combination with an evidence-based flagging system for potentially actionable alterations could help address these shortcomings.

Multiclonal colorectal cancers with divergent histomorphological features and RAS mutations: one cancer or separate cancers?

Detection of coexisting mutations within the same signal transduction pathway, which are expected to be mutually exclusive, raises a concern of laboratory errors. We have previously confirmed the presence of different RAS (KRAS and NRAS) mutations in the adenoma and/or adenocarcinoma subpopulations of colorectal cancers (CRCs). In this study, multiregional analyses by next-generation sequencing were conducted to elucidate the mechanisms underlying multiple RAS mutations seen in 5 CRC specimens. Multiregional analyses were initially conducted in a single tissue block originally submitted for mutational profiling. In 2 specimens, mutational status of the APC gene was not identical, indicating collisional adenoma and adenocarcinoma. In 3 specimens, the same APC mutation was present in different subpopulations with divergent RAS mutations, indicating a common clonal origin. Subsequent comprehensive multiregional analyses of additional adenoma and adenocarcinoma components revealed multiclonal CRCs with divergent histomorphological features and RAS mutations originating from a common APC-mutated founder lineage of adenoma, but from different RAS-mutated founder lineages of adenocarcinoma. These findings are consistent with the stepwise model of colorectal tumorigenesis along with parallel evolution, which affects RAS genes within the mitogen-activated protein kinase pathway and occurs during the progression from adenomas to adenocarcinomas. Evaluation of tumor subpopulations with divergent histomorphological features by pathologists may help identify multiclonal CRCs. Further studies are warranted to evaluate the incidence of multiclonality in CRCs and its impact on clinical outcomes. Perhaps, multiclonal CRCs originating from the same APC-mutated founder lineage of adenoma but from different RAS-mutated founder lineages of adenocarcinomas should be defined and managed as separate CRCs.

Entinostat Converts Immune-Resistant Breast and Pancreatic Cancers into Checkpoint-Responsive Tumors by Reprogramming Tumor-Infiltrating MDSCs.

Immune-checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TMEs such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs), whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti-PD-1, anti-CTLA-4, or both significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene-expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B-producing CD8+ T effector cells in mice treated with combination therapy. Gene-expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

The Effect of Preservative and Temperature on the Analysis of Circulating Tumor DNA.

Purpose: Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor, and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied, although these may be a critical consideration for assay performance.Experimental Design: To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum with those in plasma. Given the limitations of serum for ctDNA analyses, we evaluated the effects of two plasma processing approaches, K2EDTA and Cell-Free DNA BCT (BCT) tubes, on cfDNA and ctDNA recovery. A total of 45 samples from nine patients with cancer were collected in both tube types. Once collected, blood was processed into plasma immediately or kept at room temperature and processed into plasma at 1, 3, 5, or 7 days.Results: As early as 24 hours after collection, plasma isolated from blood collected in K2EDTA tubes contained an elevated level of cfDNA that increased over time compared with BCT tubes where no significant increase in cfDNA levels was observed. When samples from an additional six patients with cancer, collected in the same manner, were stored at 4°C in K2EDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes. At day 3, there was a trend toward a decrease in ctDNA levels in both tubes that was more pronounced when measuring the mutant allele fraction for cases stored at 4°C in K2EDTA tubes.Conclusions: In summary, methods of blood processing have a strong influence on cfDNA and ctDNA levels and should be a consideration when evaluating ctDNA in peripheral circulation. Clin Cancer Res; 23(10); 2471-7. ©2016 AACR.

Surgery for colorectal liver metastases: The evolution of determining prognosis.

Despite improvements in the multi-modality treatment of colorectal liver metastasis (CRLM), survival after resection remains varied. Determining prognosis after surgical resection has historically been predicated on preoperative clinicopathological factors such as primary tumor stage, carcinoembryonic antigen levels, number of liver metastases, presence of extrahepatic disease, as well as other factors. While scoring systems have been developed by combining certain preoperative factors, these have been inconsistent in accurately determining prognosis. There has been increasing interest in the use of biologic and molecular markers to predict prognosis following CRLM. The role of markers such as KRAS, BRAF, p53, human telomerase reverse transcriptase, thymidylate synthase, Ki-67, and hypoxia inducible factor-1α and their correlation with accurately predicting survival after surgical resection have been supported by several studies. Furthermore, other elements such as pathological response to chemotherapy and the presence of circulating tumor cells have shown promise in accurately determining prognosis after resection for colorectal liver metastasis. We herein review past, present, and possible future markers of prognosis among colorectal cancer patients with liver metastasis undergoing resection with curative intent.