DNA Sequencing and FACS Core Facilities

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Current Surgery Branch studies are focused on the identification and characterization of T cells that recognize neoantigens, the products of somatic mutations expressed in common epithelial cancers that include colorectal, breast and lung cancer and melanoma. For these studies, high throughput sequencing is carried out to identify all somatic mutations in patients' tumors, followed by the generation of minigenes encoding these products and the corresponding mutant peptides that are then tested for their ability to be recognized by autologous TIL. Screening of individual tumor infiltrating lymphocyte (TIL) generated from tumor fragments for their ability to recognize candidate neoantigens have demonstrated that these represent the dominant targets of TIL and can be identified in 60- 80% of patients bearing epithelial cancer types. In a study carried out in a patient with cholangiocarcinoma, analysis of reactivity with a mutated antigen was used to guide the selection of TIL used for treatment of a patient who has demonstrated significant regression of multiple metastatic lesions. Long term complete and substantial partial tumor regressions have been observed in multiple patients with breast and colorectal cancer whose autologous TIL recognizing multiple neoantigens including those encoded by hotspot mutations in the KRAS and TP53 oncogenes. Ongoing efforts are focused on optimizing strategies for identifying neoantigen-reactive T cells, including studies involving the use of MHC binding algorithms to identify the short peptides or epitopes and HLA restriction element used for antigen recognition. For these studies, the DNA sequencing laboratory provides multiple services to members of the Surgery Branch that include construction and analysis of whole exome sequencing (WES) and RNA-seq expression libraries, reverse transcription polymerase chain (RT-PCR) and quantitative RT-PCR reaction studies to complement next generation sequencing results. In addition, the mutant and wild type whole gene products and genes encoding HLA molecules expressed by patients' T cells needed for neoantigen characterization. Bioinformatics analysis of WES and RNA-seq data carried out by the DNA laboratory provides critical information to investigators that leads to the generation of lists of candidate neoantigens for screening with patients' T cells, along with critical information regarding tumor cell chromosomal copy number variations and data regarding hotspot driver mutations. A variety of additional studies are being carried out on tumor samples that include identifying mutations that are expressed in different tumor regions and in a high fraction of tumor cells from these regions in attempt to prioritize immunotherapy targets for immunotherapy. Target antigens derived from multiple mechanisms that include gene fusions and the overexpression of gene products with little or no expression in normal adult tissues are also being evaluated for their ability to be recognized by patients' T cells. Recent findings indicate that patient T cells recognize 2-3% of gene fusion products expressed by patient tumors. Ongoing studies have also focused on evaluating the reactivity of cultured TIL for their ability to recognize autologous tissue culture lines or organoids from colorectal and breast tumor samples that have been sequenced to identify candidate mutant antigens that can be then evaluated for their ability to be recognized by autologous T cells. In some patients, that majority of T cells reactive with autologous organoids do not appear to recognize any of the screened neoantigen targets identified by sequencing of the corresponding tumors, findings that are being vigorously pursued to determine the mechanisms involved with generating these target antigens. In a recent study, autologous TIL derived from a panel of 168 patients with gastrointestinal cancer were evaluated for their ability to recognize candidate neoantigens identified by sequencing autologous tumors. The results demonstrated that cultures with strong reactivity against candidate neoantigen targets could be identified in 95 patients. Adoptive cell transfer (ACT) of autologous TIL was carried out in 73 patients with neoantigen-reactive TIL, 15% of whom demonstrated objective clinical responses. These responses were associated with the number of neoantigen-reactive CD4 T cells and the total number of neoantigens identified by screening TIL. Preliminary results also indicate that recognition of autologous organoid cultures was also associated with response to therapy. Ongoing studies have resulted in the identification of T cell receptors (TCRs) that mediate recognition of widely shared tumor antigens encoded by common driver mutations plus TCRs that mediate recognition of unique neoantigens expressed on patients' tumors. Additional studies have focused on identifying factors that are involved with resistance and tumor escape following ACT. The results of ongoing investigations have indicated that loss of mutations HLA molecules represent important mechanism of tumor escape in patients who progress after achieving objective tumor regression or demonstrate mixed responses consisting of regressing and progressing tumor lesions following adoptive immunotherapy. Ongoing studies being carried out to identify additional mechanisms of resistance to therapy in additional patients have revealed that antigen loss can also contribute to these findings. Overall, these observations demonstrate the importance of studies aimed at increasing the number of ACT target antigens and the frequency of T cells recognizing these antigens in the administered cell products, studies that are currently being conducted by multiple members of the Surgery Branch. The FACS laboratory provides another critical resource for the Surgery Branch, facilitating the analysis of T cell populations that are administered in clinical cancer adoptive immunotherapy trials. In addition, the FACS laboratory is utilized daily for the analysis of the results of in vitro experiments examining the phenotype and function of tumor reactive T cells as well as to carry out the separation of cells based upon their expression of a wide variety of cell surface markers. Phenotypic analyses are being carried out on 2 BD Symphony instrument capable of analyzing up to 26 different fluorophores, as well as a 16 color LSR Fortessa. Cell separations are being carried out on a state-of-the-art BD FACS Symphony S6 instrument capable of carrying out 4-way cell sorts using up to 23 separate fluorophores. Two Sony cell sorters being maintained by the FACS laboratory staff with the capability of carrying out cell sorts using up to 12 fluorophores are critical for pre-clinical studies carried out by individual Surgery Branch investigators. These include the identification and characterization of T cell subsets that are enriched for tumor-reactive T cells and the isolation of T cell receptors that mediate potent tumor reactivity being evaluated in current clinical trials.