DNA Sequencing and FACS Core Facilities

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Current studies have on the identification of novel antigens expressed in melanoma and renal cancers as well as the characterization of T cells that recognize epitopes of previously described antigens. In a series of recent studies, several novel point-mutated genes have been identified as the targets of melanoma-reactive TIL that mediated long terms clinical tumor regression following adoptive transfer. Current investigations are being carried out to develop methods to facilitate the identification of mutated gene products that can be evaluated as targets of tumor-reactive T cells. Recent studies have focused on the use of high throughput sequencing to identify all of the somatic mutations in patient tumors. Minigenes encoding the somatic mutations identified from tumor sequencing have been generated and 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 mutated antigens or neoantigens has resulted in the identification of one or more mutated antigen targets in 80-90% of patients with melanoma, gastrointestinal, breast, lung and ovarian cancers. In recent studies 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. Partial tumor regressions have also been observed in a patient with colon cancer treated with autologous TIL that predominantly recognized a hotspot mutation in the KRAS oncogene and a breast cancer patient whose TIL recognized two neoantigens. Ongoing efforts are focused on the 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 Core facility provides multiple services members of the Surgery Branch that include construction and analysis of whole exome sequencing (WES) and RNA-seq expression libraries, RT-PCR, quantitative RT-PCR, gene cloning and RNA/DNA analysis. In carrying out bioinformatics analysis of WES and RNA-seq data, the DNA Core provides investigators with lists of candidate neoantigens for screening with patient T cells, along with valuable information regarding tumor cell chromosomal copy number variations and data regarding hotspot driver mutations, and evaluate tumor samples for expression of genes involved with tumor antigen processing and presentation. Tumor samples are also being analyzed to identify chromosomal copy number changes and to determine if mutations are shared between different tumor regions in an attempt to identify the optimal targets for immunotherapy. These studies have identified HLA loss as a mechanism of tumor escape in patients who demonstrate mixed responses in which some tumor lesions regress but additional lesions continue to progress following adoptive immunotherapy. Ongoing studies are also being carried out to identify mechanisms of resistance to therapy in additional patients with mixed responses to immunotherapy. The FACS Core Facility is currently being utilized for the analysis of T cell populations that are administered to patients as a part of the analysis of ongoing clinical cancer adoptive immunotherapy trials. In addition, the FACS Core is utilized on a daily basis for the analysis of the results of experiments to analyze the results of in vitro experiments to examine factors that influence 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. For these studies phenotypic analyses are being carried out on a BD FACSCalibur, FACSCantoI, FACSCantoII, as well as a recently acquired LSRFortessa, and cell separations were carried out on a BD FACSAriaII. Recent studies have focused on evaluating the reactivity of T cells against autologous tissue culture lines from colorectal and breast tumor samples. These studies have involved sequencing the cell lines and identifying addition candidate mutant antigens that can be then evaluated for their ability to be recognized by autologous T cells. Initial studies carried out in a breast cancer patient have revealed that unique mutant peptides expressed by the patient tumor cells are recognized by T cells, demonstrating the importance of evaluating these responses, which could aid in the development of effective immunotherapies. The 2 core facilities have also purchased state of the art instrumentation for the in depth analysis of T cell phenotypes that are critical to understanding the factors that influence response to adoptive T cell therapy. These DNA core facility has acquired 2 10X Chromium controllers for carrying of single cell transcriptome analysis, a robotic system for preparing DNA and RNA-seq libraries and is in the process of obtaining an Illumina NovaSeq DNA sequencer that will enhance our sequencing capacity and allow us to provide Surgery Branch investigators with critical sequence information in a more timely manner. The FACS core has also purchased a BD Symphony instrument capable of analyzing up to 26 different fluorophores and is in the process of purchasing a BD FACS Symphony cell sorter capable of carrying out 6-way cell sorts using up to 23 separate fluorophores Additional studies have been carried out to investigate the recognition of tissue culture cell line derived from clinical fresh tumor samples that include tumor organoids grown in suspension cultures and xenograft cell lines passaged in mice. The results of screening assays demonstrate that organoid recognition by autologous T cells can occur in the absence of neoantigen recognition, indicating that non-mutant tumor antigens may be recognized by these T cells. Further investigations are being carried out to determine if normal proteins that are overexpressed in tumor samples represent the targets of these T cells. These antigens, if identified in gastrointestinal cancers, lung cancers or breast cancers could served as additional targets for T cell based immunotheraipes.