Wei Lue Tong

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.

T cell receptor-β J usage, in combination with particular HLA class II alleles, correlates with better cancer survival rates

T cell receptor (TCR) β V and J usage correlates with either the HLA class I or HLA class II major histocompatibility subtypes, and in both infectious diseases and autoimmune settings, the use of particular TCR-β V and J’s, in persons with specific HLA alleles, represents either better outcomes or certain clinical features. However, the relationship of TCR V and J usage, HLA alleles, and clinical parameters in the cancer setting has been less well studied. Here, we have evaluated the relationship of what is likely dominant TCR-β V and J usage among tissue-resident lymphocytes for lung, head and neck, kidney, stomach, ovarian, and endometrial cancers, with patient HLA class II alleles. The most striking indication is that TCR-β J subgroup usage, in combination with particular patient HLA class II alleles, correlated with either better or worse outcomes for lung cancer. One combination, TCR-β J2 segment usage and the HLA-DRB1*1501 allele, correlated with a better survival rate for both lung and head and neck cancers. These results fill a gap in knowledge regarding the relevance of HLA typing to cancer and indicate that HLA typing, along with an indication of dominant TCR-β J usage among tissue-resident lymphocytes, can be useful for prognosis.

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.

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.

Immune receptor recombinations from breast cancer exome files, independently and in combination with specific HLA alleles, correlate with better survival rates

PurposeImmune characterizations of cancers, including breast cancer, have led to information useful for prognoses and are considered to be important in the future of refining the use of immunotherapies, including immune checkpoint inhibitor therapies. In this study, we sought to extend these characterizations with genomics approaches, particularly with cost-effective employment of exome files.MethodsBy recovery of immune receptor recombination reads from the cancer genome atlas (TCGA) breast cancer dataset, we observed associations of these recombinations with T-cell and B-cell biomarkers and with distinct survival rates.ResultsRecovery of TRD or IGH recombination reads was associated with an improved disease-free survival (p = 0.047 and 0.045, respectively). Determination of the HLA types using the exome files allowed matching of T-cell receptor V- and J-gene segment usage with specific HLA alleles, in turn allowing a refinement of the association of immune receptor recombination read recoveries with survival. For example, the TRBV7, HLA-C*07:01 combination represented a significantly worse, disease-free outcome (p = 0.014) compared to all other breast cancer samples. By direct comparisons of distinct TRB gene segment usage, HLA allele combinations revealed breast cancer subgroups, within the entire TCGA breast cancer dataset with even more dramatic survival distinctions.ConclusionsIn sum, the use of exome files for recovery of adaptive immune receptor recombination reads, and the simultaneous determination of HLA types, has the potential of advancing the use of immunogenomics for immune characterization of breast tumor samples.

Mutant cytoskeletal and ECM peptides sensitive to the ST14 protease are associated with a worse outcome for glioblastoma multiforme

We previously identified a set of the most frequently mutated cytoskeleton- and extracellular matrix-related proteins (CECMPs) in numerous cancer datasets. In this report, we used a bioinformatics approach to assess the impact of amino acid (AA) substitutions on the sensitivity of CECMPs to the ST14 protease (matriptase I), a transmembrane serine protease previously implicated in cancer development. Results indicated that AA substitutions in glioblastoma multiforme (GBM) CECMPs are skewed toward increased resistance to the ST14 protease, in comparison to the wild-type peptide sequence. Furthermore, the protease resistant AA substitutions represent relatively high binding affinities to HLA class I proteins, when assessing the binding specificities using HLA class I alleles matched to the source of the mutant AA. Moreover, samples representing AA substitutions that increased protease sensitivity also represented reduced overall and disease-free survival periods for patients with glioblastoma. To assess tumor specimen immunogenicity, we identified T-cell receptor (TCR) V(D)J recombinations in GBM exome files. The overlap between ST14 protease sensitive mutant barcodes and the TCR V(D)J recombination read positive barcodes represented significantly reduced survival.