John M. Yavorski

Assessing microenvironment immunogenicity using tumor specimen exomes: Co‐detection of TcR‐α/β V(D)J recombinations correlates with PD‐1 expression

T‐cell receptor (TcR) recombinations can be recovered from tumor specimen, whole exome sequences (WXS) files. However, it is not yet clear how these recombinations represent lymphocytes or an anti‐tumor immune response. Here we report the identification of productive TcR‐β recombinations in WXS files representing primary and metastatic melanoma. The recombinations are identifiable in about 20% of the cancer genome atlas melanoma samples. This frequency of detection is lower than the frequency of TcR‐α VJ recombinations, consistent with the occurrence of biallelic TcR‐α recombinations and possibly consistent with the fact that only one junctional recombination is required for TcR‐α whereas two recombinations are required to form a TcR‐β gene. Nevertheless, the ratio of productive TcR‐β to unproductive TcR‐β samples, in comparison to the ratio of productive to unproductive TcR‐α or TcR‐γ positive‐samples, is very high. This result indicates that detection of a productive TcR‐β VDJ recombination represents a comparatively high standard for potential antigen binding capacity, when employing a tumor specimen exome file for the assessment. Additionally, PD‐1 expression and antigen presentation functions correlated with the co‐detection of TcR‐α and ‐β recombinations (e.g., p < 0.0004), suggesting that co‐detection of TcR‐α and ‐β recombinations represents an anti‐melanoma response that has been blunted by the advent of PD‐1 expression. We further show that the algorithm for detecting the TcR‐β VDJ recombinations is applicable to exome files generated from mouse tissue, thus providing for opportunities to develop empirical paradigms for interpreting the identification of TcR V(D)J recombinations in tissue resident lymphocytes.

TCGA: Increased oncoprotein coding region mutations correlate with a greater expression of apoptosis-effector genes and a positive outcome for stomach adenocarcinoma

ABSTRACT Oncogene mutations are primarily thought to facilitate uncontrolled cell growth. However, overexpression of oncoproteins likely leads to apoptosis in a feed forward mechanism, whereby a certain level of oncoprotein leads to the activation of pro-proliferation effector genes and higher levels lead to activation of pro-apoptotic effector genes. TCGA STAD barcodes having no oncoprotein coding region mutations represented reduced expression of the apoptosis-effector genes compared with barcodes with multiple oncoprotein coding region mutations. Furthermore, STAD barcodes in a “no-subsequent tumor” group, representing 224 samples, and in a “positive outcome” group, had more oncoprotein coding regions mutated, on average, than barcodes of the new tumor and negative outcome groups, respectively. BRAF, CTNNB1, KRAS and MTOR coding region mutations (as a group) had the strongest association with the no-subsequent tumor group. Tumor suppressor coding region mutations were also correlated with no-subsequent tumor. These results are consistent with an oncoprotein-mediated, feed-forward mechanism of apoptosis in patients. Importantly, the no-subsequent tumor group also had more overall mutations. This result leads to considerations of unhealthy cells or cells with more neo-antigens for immune rejection. However, a probabilistic aspect of mutagenesis is also consistent with more oncoprotein and tumor suppressor protein mutations, in cases of more overall mutations, and thus a higher likelihood of activation of feed forward apoptosis pathways.

Immunogenomics: A Negative Prostate Cancer Outcome Associated with TcR-γ/δ Recombinations

We developed a scripted algorithm, based on previous, earlier editions of the algorithm, to mine prostate cancer exome files for T-cell receptor (TcR) recombination reads: Reads representing TcR gene recombinations were identified in 497 prostate cancer exome files from the cancer genome atlas (TCGA). As has been reported for melanoma, co-detection of productive TcR-α and TcR-β recombination reads correlated with an RNA expression signature representing T-cell exhaustion, particularly with high RNA levels for PD-1 and PD-L1, in comparison to several different control sets of samples. Co-detection of TcR-α and TcR-β recombination reads also correlated with high level expression of genes representing antigen presenting functions, further supporting the conclusion that co-detection of TcR-α and TcR-β recombination reads represents an immunologically relevant microenvironment. Finally, detection of unproductive TcR-δ recombinations, and unproductive and productive TcR-γ recombinations, strongly correlated with, and may represent a convenient biomarker for a poor clinical outcome. These results underscore the value of the genomics-based assessment of unproductive TcR recombinations and raise questions about the impact of tumor microenvironment lymphocytes in the absence of antigenicity.

Recovery of Immunoglobulin VJ Recombinations from Pancreatic Cancer Exome Files Strongly Correlates with Reduced Survival

We assessed pancreatic cancer, lymphocyte infiltrates with a computational genomics approach. We took advantage of tumor-specimen exome files available from the cancer genome atlas to mine T- and B-cell immune receptor recombinations, using highly efficient, scripted algorithms established in several previous reports. Surprisingly, the results indicated that pancreatic cancer exomes represent one of the highest level yields for immune receptor recombinations, significantly higher than two comparison cancers used in this study, head and neck and bladder cancer. In particular, pancreatic cancer exomes have very large numbers of immunoglobulin light chain recombinations, both with regard to number of samples characterized by recovery of such recombinations and with regard to numbers of recombination reads per sample. These results were consistent with B-cell biomarkers, which emphasized the Th2 nature of the pancreatic lymphocyte infiltrate. The tumor specimen exomes with B-cell immune receptor recombination reads represented a dramatically poor outcome, a result not detected with either the head and neck or bladder cancer datasets. The results presented here support the potential value of immunotherapies designed to engineer a Th2 to Th1 shift in treating certain forms of pancreatic cancer.

Protected cytoskeletal‑related proteins: Towards a resolution of contradictions regarding the role of the cytoskeleton in cancer

Initial reports of the role of the cytoskeleton in cancer indicated that tumor cells with a more disorganized cytoskeleton were more tumorigenic. These reports were based on stains for the F-actin cytoskeleton, for example, using phalloidin or anti-F-actin antibody reagents, and gave a basic impression of F-actin-based cytoskeletal integrity. Later developments emphasized the significance of the cytoskeletal elements in cell migration, presumably associated with either basement membrane invasion or metastasis, or both, with several specific proteins implicated in the formation of cell invadopodia. With the advent of genomics approaches, it has become clear that cytoskeletal related proteins are indeed common targets of mutagenesis in cancer and commonly rank among the most mutated proteins in cancers, presumably due to large coding region sizes and the significant stochastic component to human mutagenesis. This cytoskeletal genomics result is consistent with the loss of cytoskeleton integrity as a hallmark of tumor development, but raises the question of whether such mutational sensitivity relates to the migration and invadopodia aspects of tumor progression. In the present study, the authors report that it is possible to identify a set of cytoskeletal related proteins protected from mutation, in comparison to the commonly mutated cytoskeleton related proteins in certain, but not all cancer, datasets.

Smoking correlates with increased cytoskeletal protein-related coding region mutations in the lung and head and neck datasets of the cancer genome atlas.

Cancer from smoking tobacco is considered dependent on mutagens, but significant molecular aspects of smoking‐specific, cancer development remain unknown. We defined sets of coding regions for oncoproteins, tumor suppressor proteins, and cytoskeletal‐related proteins that were compared between nonsmokers and smokers, for mutation occurrences, in the lung adenocarcinoma (LUAD), head and neck squamous carcinoma (HNSC), bladder carcinoma (BLCA), and pancreatic adenocarcinoma ( PAAD) datasets from the cancer genome atlas (TCGA). We uncovered significant differences in overall mutation rates, and in mutation rates in cytoskeletal protein‐related coding regions (CPCRs, including extracellular matrix protein coding regions), between nonsmokers and smokers in LUAD and HNSC (P < 0.001), raising the question of whether the CPCR mutation differences lead to different clinical courses for nonsmoker and smoker cancers. Another important question inspired by these results is, whether high smoker cancer mutation rates would facilitate genotoxicity or neoantigen‐based therapies. No significant, mutation‐based differences were found in the BLCA or PAAD datasets, between nonsmokers and smokers. However, a significant difference was uncovered for the average number of overall cancer mutations, in LUAD, for persons who stopped smoking more than 15 years ago, compared with more recent smokers (P < 0.032).

T-cell receptor-β V and J usage, in combination with particular HLA class I and class II alleles, correlates with cancer survival patterns

Class I and class II HLA proteins, respectively, have been associated with subsets of V(D)J usage resulting from recombination of the T-cell receptor (TCR) genes. Additionally, particular HLA alleles, in combination with dominant TCR V(D)J recombinations, have been associated with several autoimmune diseases. The recovery of TCR recombination reads from tumor specimen exome files has allowed rapid and extensive assessments of V(D)J usage, likely for cancer resident T-cells, across relatively large cancer datasets. The results from this approach, in this report, have permitted an extensive alignment of TCR-β VDJ usage and HLA class I and II alleles. Results indicate the correlation of both better and worse cancer survival rates with particular TCR-β, V and J usage-HLA allele combinations, with differences in median survival times ranging from 7 to 130 months, depending on the cancer and the specific TCR-β V and J usage/HLA class allele combination.

Stratifying melanoma and breast cancer TCGA datasets on the basis of the CNV of transcription factor binding sites common to proliferation- and apoptosis-effector genes

Transcription factors that activate both proliferation- and apoptosis-effector genes, along with a number of related observations, have led to a proposal for a feed forward mechanism of activating the two gene classes, whereby a certain concentration of a transcription factor activates the proliferation-effector genes and a higher concentration of the transcription factor activates the apoptosis-effector genes. We reasoned that this paradigm of regulation could lead to, in the cancer setting, a selection for relatively reduced copy numbers of apoptosis-effector gene, transcription factor binding sites (TFBS). Thus, the aim of this investigation was to examine the DNA sequencing read depths of TFBS for a set of proliferation- and apoptosis-effector genes, normalized to the read depths found in matching blood samples, as provided by the cancer genome atlas (TCGA); and thereby document copy number differences among these TFBS. We determined that the melanoma and breast cancer, TCGA datasets could be divided into three categories: (i) no detectable copy number variation for the proliferation- and apoptosis-effector, shared TFBS; (ii) a relative increase in the copy number of proliferation-effector gene TFBS, compared with the copy number of the apoptosis-effector gene TFBS; and (iii) a relative decrease in the number of proliferation-effector gene TFBS. Thus, we conclude that changes in the relative copies of the shared TFBS, for proliferation- and apoptosis-effector genes, have the potential of impacting tumor cell proliferative and apoptotic capacities.

Identification of Sets of Cytoskeletal Related and Adhesion-related Coding Region Mutations in the TCGA Melanoma Dataset that Correlate with a Negative Outcome

Background: Relatively little cancer genome atlas data has been associated with clinically relevant stratifications of individual cancers. Results: Mutations in two subsets of a cytoskeletal related and adhesion-related protein coding region set (CAPCRs) were determined to have strong associations with a negative outcome for melanoma, in-cluding a subset constituted by: DSCAM, FAT3, MUC17 and PCDHGC5 (p < 0.0001). Conclusion: Roles for CAPCR mutations in cancer progression raise a question about the potential dominant negative impact of these mutations for multi-meric subcellular and extra-cellular protein struc-tures.

Elucidating feed‑forward apoptosis signatures in breast cancer datasets: Higher FOS expression associated with a better outcome

Overstimulation of pro-proliferative pathways and high level expression of pro-proliferative transcription factors (TFs) can lead to apoptosis. This is likely due to TF binding sites for pro-proliferative TFs common to pro-proliferative and pro-apoptosis-effector genes. Certain clinical datasets have indicated that molecular markers associated with higher proliferation rates lead to improved outcomes for patients with cancer. These observations have been extensively assessed on a general basis, however there has been little work dissecting feed-forward apoptosis signaling pathways that may represent specific distinctions between a pro-proliferative mechanism and a pro-apoptotic mechanism in samples from patients with cancer. Using The Cancer Genome Atlas datasets and bioinformatic approaches, the present study reports that higher FOS expression levels, along with higher FOS target apoptosis-effector gene expression, is associated with an increased survival, while higher POU2F1 expression is associated with a reduced survival (average difference of 25.9 months survival). In summary, in the datasets examined FOS represents an apoptosis-driver and high POU2F1 represents a driver mechanism for cancer development.