Detection of Productively Rearranged TcR-α V-J Sequences in TCGA Exome Files: Implications for Tumor Immunoscoring and Recovery of Antitumor T-cells.

Tumor immunoscoring is rapidly becoming a universal parameter of prognosis, and T-cells isolated from tumor masses are used for ex vivo amplification and readministration to patients to facilitate an antitumor immune response. We recently exploited the cancer genome atlas (TCGA) RNASeq data to assess T-cell receptor (TcR) expression and, in particular, discovered strong correlations between major histocompatibility class II (MHCII) and TcR-α constant region expression levels. In this article, we describe the results of searching TCGA exome files for TcR-α V-regions, followed by searching the V-region datasets for TcR-α-J regions. Both primary and metastatic breast cancer sample files contained recombined TcR-α V-J regions, ranging in read counts from 16-39, at the higher level. Among four such V-J rearrangements, three were productive rearrangements. Rearranged TcR-α V-J regions were also detected in TCGA-bladder cancer, -lung cancer, and -ovarian cancer datasets, as well as exome files representing bladder cancer, in Moffitt Cancer Center patients. These results suggest that a direct search of commonly available, conventional exome files for rearranged TcR segments could play a role in more sophisticated immunoscoring or in identifying particular T-cell clones and TcRs directed against tumor antigens.

Immunoscoring by correlating MHC class II and TCR expression: high level immune functions represented by the KIRP dataset of TCGA.

The Cancer Genome Atlas (TCGA) is primarily oriented towards revealing the status of cancer cells but TCGA RNASeq data have the potential of representing gene expression for a variety of cells that are included during RNA preparation, i.e., cells that cannot be removed from the microenvironment during cancer sample isolation. Thus, we seek to determine whether RNASeq data can be used to pioneer greater precision for immunoscoring. We obtained the RNASeq results for HLA class II genes, the class II transactivator (CIITA) and T-cell receptor (TCR) alpha segments. The data indicated strong degrees of correlation of HLA class II expression with TCR expression. Furthermore, biomarkers of professional antigen-presenting cells also correlated with TCR expression, with the kidney renal papillary cell carcinoma (KIRP) dataset indicative of the highest level, immune function microenvironment. These analyses indicate that an immune function signature, with probable internal HLA class II-TCR verifications, can be obtained from individual TCGA samples; this in turn indicates that such signatures might provide a basis for correlations with prognosis or for convenient indications of therapy options, such as tumor-infiltrating lymphocyte availability for ex-vivo amplification. Although tumor immunoscoring has been proposed, the above analysis represents the first immunoscoring approach that correlates antigen presentation capacity with TCR mRNA expression.

HLA-DR peptide occupancy can be regulated with a wide variety of small molecules.

HLA-DR is the most commonly expressed and likely the most medically important human MHC class II, antigen presenting protein. In a normal immune response, HLA-DR binds to antigenic peptide and the HLA-DR/peptide complex binds to a T-cell receptor, thus contributing to T-cell activation and stimulation of an immune response against the antigen. When foreign antigen is not present, HLA-DR binds endogenous peptide which, under normal conditions does not stimulate an immune response. In most cases, the human peptide is CLIP, but a certain percentage of HLA-DR molecules will be present at the cell surface with other human peptides. We have recently shown that cell surface, CLIP/HLA-DR ratios are a measure of peptide heterogeneity, and in particular, changes in CLIP/HLA-DR ratios represent changes in the occupancy of HLA-DR by other, endogenous peptides. For example, treatment of cells with the HDAC inhibitor, Entinostat, leads to an upregulation of Cathepsin L1 and replacement of Cathepsin L1 senstitive peptides with HLA-DR binding, Cathepsin L1 resistant peptides, an alteration that can be at least partially assessed via assessment of CLIP/HLA-DR cell surface ratios. Here we assay for CLIP/HLA-DR ratios following treatment of immortalized B-cells with a variety of common drugs, almost all of which indicate significant changes in the CLIP/HLA-DR ratios. Furthermore, the CLIP/HLA-DR ratio changes parallel the impact of the drug panoply on cell viability, suggesting that alterations in the HLA-DR peptidome are governed by a variety of mechanisms, rather than exclusively dependent on a dedicated peptide loading process. These results raise questions about how FDA approved drugs may affect the immune response, and whether any of these drugs could be useful as vaccine adjuvants?

Copy number loss or silencing of apoptosis-effector genes in cancer.

Cancer cells undergo a variety of DNA copy number gains and losses (CNV), raising two important questions related to cancer development: (i) Which genes are affected? (ii) And how do CNVs, that do not represent complete deletions but do represent gene-dosage alterations, impact cancer cell functions? Recent studies have indicated that CNVs in cancer can impact genes for regulatory proteins long known to be associated with cancer development, but less is understood about CNVs affecting effector genes. Also, we have recently indicated the likely importance of transcription factor binding site (TFBS) copies in effector genes, in regulating the transition from a proliferative to an apoptotic state. Here we report data-mining analyses that indicate that copies of apoptosis-effector genes are commonly lost in cancer development, in comparison to proliferation-effector genes, and when not, apoptosis effector genes have silenced chromatin structures.