Yaping N. Tu

Identification of immunoglobulin V(D)J recombinations in solid tumor specimen exome files: Evidence for high level B-cell infiltrates in breast cancer

ABSTRACT It has recently become apparent that it is possible to characterize productively recombined, T-cell receptor (TcR) gene segments in tumor exome files, which presumably include representations of the DNA of other cells in the microenvironment. Similar characterizations have been done for TcR recombinations in tumor specimen RNASeq files. While exome files have been used to characterize immunoglobulin gene segment recombinations for tumors closely related to B-cells, immunoglobulin recombinations have yet to be characterized for putative microenvironment cells for solid tumors. Here we report a novel scripted algorithm that detects productive and unproductive immunoglobulin recombinations in both B-cell related tumor exome files and in solid tumor exome files, with the most important result being the relatively high level B-cell infiltrate in breast cancer. This analysis has the potential of streamlining and dramatically augmenting the knowledge base regarding B-cell infiltrates into solid tumors; and leading to antibody reagents directed against tumor antigens and tissue resident, infectious pathogens.

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.

Lung tumor exome files with T-cell receptor recombinations: a mouse model of T-cell infiltrates reflecting mutation burdens

Tumor exomes and RNASeq data were originally intended for obtaining tumor mutations and gene expression profiles, respectively. However, recent work has determined that tumor exome and RNAseq read files contain reads representing T-cell and B-cell receptor (TcR and BcR) recombinations, presumably due to infiltrating lymphocytes. Furthermore, the recovery of immune receptor recombination reads has demonstrated correlations with specific, previously appreciated aspects of tumor immunology. To further understand the usefulness of recovering TcR and BcR recombinations from tumor exome files, we developed a scripted algorithm for recovery of reads representing these recombinations from a previously described mouse model of lung tumorigenesis. Results indicated that exomes representing lung adenomas reveal significantly more TcR recombinations than do exomes from lung adenocarcinomas; and that exome files representing high mutation adenomas, arising from chemical mutagens, have more TcR recombinations than do exome files from low mutation adenomas arising from an activating Kras mutation. The latter results were also consistent with a similar analysis performed on human lung adenocarcinoma exomes. The mouse and human results for obtaining TcR recombination reads from tumor specimen exomes are consistent with human tumor biology results indicating that adenomas and high mutation cancers are sites of high immune activity. The results indicate hitherto unappreciated opportunities for the use of tumor specimen exome files, particularly from experimental animal models, to study the connection between the adenoma stage of tumorigenesis, or high cancer mutation rates, and high level lymphocyte infiltrates.

TcR-α recombinations in renal cell carcinoma exome files correlate with an intermediate level of T-cell exhaustion biomarkers

Renal cell carcinoma exome-derived, V(D)J recombination reads had an elevated presence and variability, for both TcR-α and -β, when compared to marginal tissue, reflecting an opportunity to assess tumor immunogenicity by comparison with marginal tissue T cells. PD-1, PD-L2, CTLA4 and FOXP3, all of which are implicated in the evasion of an anti-tumor immune response, had a significantly higher expression for samples representing co-detection of productive TcR-α and -β recombination reads. Samples representing tumors with productive TcR-α recombination reads but no detectable, productive TcR-β recombination reads, reflected a 20% survival advantage, and RNASeq data indicated an intermediate level of immune checkpoint gene expression for those samples. These results raise the question of whether relatively high levels of detection of productive TcR-α recombination reads, in comparison with detection of reads representing the TcR-β gene, identify a microenvironment that has not yet entered a T-cell exhaustion phase and may thereby represent conditions for immune enhancements that do not require anti-immune checkpoint therapies.

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.

Identification of specific feed-forward apoptosis mechanisms and associated higher survival rates for low grade glioma and lung squamous cell carcinoma

The mechanisms of cell proliferation due to the overexpression of certain transcription factors (TFs) have been well documented in the cancer setting. However, many of these same TFs have pro-apoptotic effects, particularly when expressed or activated at high levels, a process referred to as feed-forward apoptosis (FFA). To determine whether cancers could be stratified on the basis of specific FFA signatures, RNASeq data representing samples from the cancer genome atlas were analyzed, revealing that high expression of the pro-proliferative TFs, MYC and YY1, is associated with a favorable outcome in low-grade glioma (LGG) and lung squamous cell carcinoma (LUSC), respectively. Analysis of the RNASeq data also led to the identification of specific apoptosis-effector genes whose expression levels correlate with increased survival rates, for both LGG and LUSC. Although FFA has been demonstrated as a general effect in cancer, in this report, for the first time, results identify specific TFs and their responsive effector genes that distinguish subsets of cancer samples undergoing more or less of a FFA process in a way that is associated with distinct patient survival rates.

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.

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.

Germline cytoskeletal and extra-cellular matrix-related single nucleotide variations associated with distinct cancer survival rates

BACKGROUND Human mutagenesis has a large stochastic component. Thus, large coding regions, especially cytoskeletal and extra-cellular matrix protein (CECMP) coding regions are particularly vulnerable to mutations. Recent results have verified a high level of somatic mutations in the CECMP coding regions in the cancer genome atlas (TCGA), and a relatively common occurrence of germline, deleterious mutations in the TCGA breast cancer dataset. METHODS The objective of this study was to determine the correlations of CECMP coding region, germline nucleotide variations with both overall survival (OS) and disease-free survival (DFS). TCGA, tumor and blood variant calling files (VCFs) were intersected to identify germline SNVs. SNVs were then annotated to determine potential consequences for amino acid (AA) residue biochemistry. RESULTS Germline SNVs were matched against somatic tumor SNVs (i.e., tumor mutations) over twenty TCGA datasets to identify 23 germline-somatic matched, deleterious AA substitutions in coding regions for FLG, TTN, MUC4, and MUC17. CONCLUSIONS The germline-somatic matched SNVs, in particular for MUC4, extensively implicated in cancer development, represented highly, statistically significant effects on OS and DFS survival rates. The above results contribute to the establishment of what is potentially a new class of inherited cancer-facilitating genes, namely dominant negative tumor suppressor proteins.