TRB-J1 usage, in combination with the HLA-A*01:01 allele, represents an apparent survival advantage for uterine corpus endometrial carcinoma: Comparisons with microscopic assessments of lymphocyte infiltrates.

The opportunity for the highly efficient recovery of immune receptor recombination data from cancer specimens, including the ready assessment of immune receptor V and J usage, raises the issue of establishing precise values of assessing the immune receptor status as opposed to obtaining basic information regarding lymphocyte infiltration, in the cancer setting. In this report, we obtained the lymphocyte infiltration percentages from the cancer digital slide archive representing uterine corpus endometrial carcinoma (UCEC) and correlated these data with recovery of the immune receptor recombination reads from corresponding UCEC exome files. Results indicated a basic correlation of the recovery of productive T-cell receptor beta (TRB) recombination reads with lymphocyte infiltration percentages. However, the recovery of specific immune receptor recombination reads did not indicate the same survival outcomes as microscope detection of lymphocyte infiltrate percentages. To further exploit the value of recovery of the TRB recombination reads from the UCEC exome files, we determined the survival outcomes for combinations of TRB gene segment usage and HLA class I alleles, with the most important result being that the combination of HLA-A*01:01 and TRB-J1 segment usage reflected a strikingly high survival rate. Overall, this report emphasized the increased value of the knowledge of the immune receptor recombinations, in comparison with basic lymphocyte infiltration percentages, in assessing cancer survival rates.

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.

T-cell receptor-ß 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-ß VDJ usage and HLA class I and II alleles. Results indicate the correlation of both better and worse cancer survival rates with particular TCR-ß, 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-ß V and J usage/HLA class allele combination.

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.

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-ß 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-ß 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-ß 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.

MHC class II associated stomach cancer mutations correlate with lack of subsequent tumor development.

The role of tumor cell expression of major histocompatibility class II (MHCII) has been controversial, with evidence indicating that tumor cell expression of MHCII may lead to an anti-tumor immune response and to tumor cell apoptosis and that MHCII has pro-tumorigenic functions. The cancer genome atlas (TCGA) indicates numerous deleterious mutations for the highly specific, MHCII transcriptional activation proteins, RFX5, RFXAP, RFXANK and CIITA. Also, mutations in the non-polymorphic, human leukocyte antigen (HLA)-DRA gene, which encodes the heavy chain for the most prominent human MHCII molecule, HLA-DR, are common. For many, if not most TCGA cancer datasets, the MHCII specific mutations do not associate with clinical outcomes. However, stomach carcinoma represents an exception, where the data indicate that MHCII-specific mutations are associated with a more favorable outcome. These data raise the question of whether stomach cancer mutations represent effective haploinsufficiency or whether mutations that are associated with a favorable outcome occur with other stomach cancer molecular features that limit the function of the two alleles that represent these MHCII-related proteins.

The human, F-actin-based cytoskeleton as a mutagen sensor.

BACKGROUND: Forty years ago the actin cytoskeleton was determined to be disrupted in fibroblasts from persons with DNA repair-defective, hereditary colon cancer, with no clear connection between the cytoskeleton and DNA repair defects at that time. Recently, the large number of sequenced genomes has indicated that mammalian mutagenesis has a large stochastic component. As a result, large coding regions are large mutagen targets. Cytoskeletal protein-related coding regions (CPCRs), including extra-cellular matrix proteins, are among the largest coding regions in the genome and are indeed very commonly mutated in cancer. METHODS: To determine whether mutagen sensitivity of the actin cytoskeleton could be assessed experimentally, we treated tissue culture cells with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and quantified overall cytoskeleton integrity with rhodamine-phalloidin stains for F-actin. RESULTS: The above approach indicated cytoskeletal degradation with increasing mutagen exposure, consistent with increased mutagenesis of CPCRs in TCGA, smoker samples, where overall mutation rates correlate with CPCR mutation rates (R2 = 0.8694; p < 0.00001). In addition, mutagen exposure correlated with a decreasing cell perimeter to area ratio, raising questions about potential decreasing, intracellular diffusion and concentrations of chemotherapy drugs, with increasing mutagenesis and decreasing cytoskeleton integrity. CONCLUSION: Determination of cytoskeletal integrity may provide the opportunity to assess mutation burdens in nonclonal cell populations, such as in intact tissues, where DNA sequencing for heterogeneous mutation burdens can be challenging.

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.

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.

Assessing microenvironment immunogenicity using tumor specimen exomes: Co-detection of TcR-α/ß 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-ß 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-ß gene. Nevertheless, the ratio of productive TcR-ß to unproductive TcR-ß 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-ß 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 -ß recombinations (e.g., p < 0.0004), suggesting that co-detection of TcR-α and -ß 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-ß 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.

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.

T cell receptor gene recombinations in human tumor specimen exome files: detection of T cell receptor-ß VDJ recombinations associates with a favorable oncologic outcome for bladder cancer.

Understanding tumor-resident T cells is important for cancer prognosis and treatment options. Conventional, solid tumor specimen exome files can be searched directly for recombined T cell receptor (TcR)-α segments; RNASeq files can include TcR-ß VDJ recombinations. To learn whether there are medically relevant uses of exome-based detection of TcR V(D)J recombinations in the tumor microenvironment, we searched cancer genome atlas and Moffitt Cancer Center, tumor specimen exome files for TcR-ß, TcR-γ, and TcR-δ recombinations, for bladder and stomach cancer. We found that bladder cancer exomes with productive TcR-ß recombinations had a significant association with No Subsequent Tumors and a positive response to drug treatments, with p < 0.004, p < 0.05, and p < 0.004, depending on the sample sets examined. We also discovered the opportunity to detect productive TcR-γ and TcR-δ recombinations in the tumor microenvironment, via the tumor specimen exome files.

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

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.

Impact of SNPs on CpG Islands in the MYC and HRAS oncogenes and in a wide variety of tumor suppressor genes: A multi-cancer approach.

Single nucleotide polymorphisms (SNPs) that occur within CpG Islands may lead to increased hypermethylation if a SNP allele has the potential to form a CpG dinucleotide, as well as potentially lead to hypomethylation if a SNP allele eliminates a CpG dinucleotide. We analyzed CpG-related SNP allele frequencies in whole genome sequences (WGS) across 5 TCGA cancer datasets, thereby exploiting a more recent appreciation for signaling pathway degeneracy in cancer. The cancer data sets were analyzed for SNPs in CpG islands associated with the oncogenes, HRAS and MYC, and in the CpG islands associated with the tumor suppressor genes, APC, DCC, and RB1. We determined that one SNP allele (rs3824120) in a CpG island associated with MYC which eliminated a CpG was more common in the cancer datasets than in the 100Genomes databases (p < 0.01). For HRAS, 2 SNP alleles (rs112690925, rs7939028) that created CpG's occurred significantly less frequently in the cancer data sets than in the general SNP databases (e.g., rs7939028, p < 0.0002, in comparison with AllSNPs(142)). Also, one SNP allele (rs4940177) that created a CpG in a CpG island associated with the DCC tumor suppressor gene, was more common in the cancer datasets (p < 0.0007). To understand a broader picture of the potential of SNP alleles to create CpG's in CpG islands of tumor suppressor genes, we developed a scripted algorithm to assess the SNP alleles associated with the CpG islands of 43 tumor suppressor genes. The following tumor suppressor genes have the possibility of significant, percent increases in their CpG counts, depending on which SNP allele(s) is present: VHL, BRCA1, BRCA2, CHEK2, PTEN and RB1.

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).