Abstract 6254: Detection of anti-CMV TCR CDR3s correlates with increased survival for soft tissue sarcoma and stomach adenocarcinoma

ABSTRACTWhile the role of anti‐cytomegalovirus (CMV) T‐cell responses in the cancer setting is becoming increasingly recognized, the association between CMV and survival outcomes has not been subject to study through more recent immunogenomic approaches. Thus for this study, we extracted T‐cell receptor (TCR) recombination reads from whole exome sequencing (WXS) and RNA sequencing (RNAseq) files of stomach adenocarcinoma (STAD) and soft tissue sarcoma (SARC) samples from the Cancer Genome Atlas (TCGA) and matched the corresponding TCR complementarity determining region‐3 (CDR3) amino acid (AA) sequences represented by these reads to known anti‐CMV CDR3 sequences. Results indicated that the recovery of both TRA and TRB recombination reads from WXS files, from either blood or tumor samples, which corresponded to AA sequences matching anti‐CMV CDR3s, correlated with better survival probabilities for both STAD and SARC. However, TCR CDR3s represented by recombination reads recovered from RNAseq files and matching anti‐CMV TCR CDR3 AA sequences correlated with lower survival probabilities. These results raise the question of whether exposure to CMV prior to onset or progression of cancer may lead to better survival probability whereas a potentially active T‐cell response to CMV during cancer onset or progression could represent an ongoing comorbidity? In conclusion, TCR CDR3s may be an important indicator of prognosis for stomach cancer and soft tissue sarcoma and may support further research into anti‐CMV CDR3s as a tool for development of immunotherapy for these cancers.

Background: Even within patients with high tumor mutational burden (TMB) and/or PD-L1 expression, objective response rates to checkpoint therapies remain below 50% (e.g. Checkmate 227). Identifying predictive biomarkers for immune checkpoint inhibition therapy is an active area of research. Tumor infiltrating lymphocytes (TILs) show potential as a predictive biomarker for immune-oncology therapies. Rearrangement of the T-cell receptor (TCR) complementarity-determining region 3 (CDR3) serves as a marker for T-cell clones, and can be detected by RNA-seq of the region. The standard mechanism of tissue storage, formalin fixation and paraffin embedding (FFPE), degrades nucleic acids and makes sequencing more challenging. We present detection of TCR CDR3 of TILs from FFPE RNA-seq as a potential biomarker. Methods: FFPE Tumors from 65 head and neck squamous cell cancer (HNSCC), 233 colorectal carcinoma (CRC), and 217 non-small cell lung cancer (NSCLC) patients were profiled using deep full-transcriptome RNA-seq. Patients were assigned a TCR score by first aligning RNA-seq reads to TCRα and TCRβ CDR3 sequences, then counting overall CDR3 read support. Gene expression profiles were also used to predict consensus molecular subtype (CMS) and estimate immune cell abundance. To assess tumor sample purity, deep whole genome (N=244) or whole exome (N=271) DNA sequencing was performed on both tumor and normal blood samples from each patient. TMB was calculated directly by counting somatic non-synonymous exonic mutations. Results: Immune populations revealed high correlation (Pearson's r = .84, p = 3.6e-169) between TCR CDR3 counts and independent gene expression-based estimates of T-cell activity. Using the CMS of colon cancer samples, we found that CMS2 patients had a significantly lower average number of reads aligned to CDR3 (46.5 vs 91.6, p = 1.5e-7) which agrees with previous reports of low immune activity in that subtype. We compared CDR3 read count against TMB and found significant correlation (Spearman's rho = 0.34, p = 0.0048) in head and neck tumors where the median TMB was 98, but no association in lung or colon tumors where TMB is higher (median TMB 169 and 132 respectively). Finally, tumor purity estimated via DNA-seq was anti-correlated with CDR3 read count (Spearman's ρ = -0.39, p = 3.3e-21). Conclusions: TCR CDR3 read support from bulk RNA-seq on 515 solid tumor FFPE samples provides a related but distinct measure of T-cell infiltration to gene expression-based estimates. TMB and TCR support are generally independent biomarkers, especially in those tissues with higher average mutation rate. TCR support has potential to complement TMB as a predictive biomarker of immune checkpoint therapy response. Citation Format: Andrew J. Sedgewick, Jacob J. Adashek, Christopher W. Szeto, Charles J. Vaske, Philippe E. Spiess, Stephen C. Benz. T-cell receptor clonotyping and immune infiltrate quantification with whole transcriptome RNA-seq from FFPE [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4980.

Objective: This paper aims to investigate the dynamic changes of the T-cell receptor (TCR) β complementarity-determining region 3 (CDR3) repertoire during cyclophosphamide or Cytoxan (CTX) damage or inhibition of bone marrow hematopoiesis caused by a reduction of peripheral blood white blood cells (WBCs) in BALB/c mice.Methods: We analyze TCR CDR3 repertoire of BALB/c mice including (1) NS control group (2) CTX damage group (3) CTX damage + GM-CSF recovery group (4) CTX damage + auto-recovery group.Results: The number of WBCs in the CTX group is significantly lower than that in the NS group and after GM-CSF injection, the GM-CSF group is higher than that in the NS group. The diversity of the CTX damage group is the highest and there is a significant difference in high-frequency clonal proliferation between the CTX damage group and CTX damage + GM-CSF recovery group compared with the NS control group. In addition, the numbers of unique productive CDR3 overlapping numbers in the four experimental groups are similar.Conclusions: These data reveal that CTX significantly reduced the number of WBCs and ratio of high-frequency TCR CDR3 sequences, and indirectly increased the diversity of the TCR CDR3 repertoire. GM-CSF quickly restored the number of WBCs, and partially restored changes in the TCR CDR3 repertoire induced by CTX. Results from monitoring the dynamic changes of the TCR CDR3 repertoire can be used to assess the effects of CTX and GM-CSF on the function of peripheral blood T cells and to explore the possible underlying mechanisms.

Decision letter: TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs

Editor's evaluation: TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs

A very large and still expanding collection of adaptive immune receptor (IR) recombination reads, representing many diseases, is becoming available for downstream analyses. Among the most productive approaches has been to establish risk stratification parameters via the chemical features of the IR complementarity determining region-3 (CDR3) amino acid (AA) sequences, particularly for large datasets where clinical information is available. Because the IR CDR3 AA sequences often play a large role in antigen binding, the chemistry of these AAs has the likelihood of representing a disease-related fingerprint as well as providing pre-screening information for candidate antigens. To approach this issue in a novel manner, we developed a bladder cancer, case evaluation approach based on CDR3 aromaticity. We developed and applied a simple and efficient algorithm for assessing aromatic, chemical complementarity between T-cell receptor (TCR) CDR3 AA sequences and the cancer specimen mutanome. Results indicated a survival distinction for aromatic CDR3-aromatic mutanome complementary, versus non-complementary, bladder cancer case sets. This result applied to both tumor resident and blood TCR CDR3 AA sequences and was supported by CDR3 AA sequences represented by both exome and RNAseq files. The described aromaticity factor algorithm has the potential of assisting in prognostic assessments and guiding immunotherapies for bladder cancer.

The anti-tumor immune response is considered to be due to the T-cell receptor (TCR) binding to tumor antigens, which can be either wild-type, early stem cell proteins, presumably foreign to a developed immune system; or mutant peptides, foreign to the immune system because of a mutant amino acid (aa) or otherwise somatically altered aa sequence. Recently, very large numbers of TCR complementarity-determining region-3 (CDR3) aa sequences obtained from tumor specimens have become available. We developed a novel algorithm for assessing the complementarity of tumor mutant peptides and TCR CDR3s, based on the retrieval of TCR CDR3 aa sequences from both tumor specimen and patient blood exomes and by using an automated process of assessing CDR3 and mutant aa electrical charges. Results indicated many instances where high electrostatic complementarity was associated with a higher survival rate. In particular, our approach led to the identification of specific genes contributing significantly to the complementary, TCR CDR3-mutant aa. These results suggest a novel approach to tumor immunoscoring and may lead to the identification of high-priority neo-antigen, peptide vaccines; or to the identification of ex vivo stimulants of tumor-infiltrating lymphocytes.

Burkitt lymphoma (BL) has a tight association with Epstein-Barr virus (EBV), especially in sub-Saharan Africa. While the relationship between BL and EBV is well documented, the relationship between the anti-EBV adaptive immune response, particularly in sub-Saharan African cases, and disease course, has not been substantially investigated. An analysis of T-cell receptor (TCR) complementarity determining region-3 (CDR3) sequences, reported here, from EBV-positive, Ugandan BL tumor samples revealed a correlation between the presence of anti-EBV CDR3s and improved overall survival probabilities. Furthermore, chemical complementarity assessments demonstrated higher complementarity for TCR CDR3s and EBV epitopes in the cases where there had been a detection of the anti-EBV CDR3 AA sequence matches in the BL tumor samples. Overall, the results reported here raise the question of whether EBV targeted immunotherapy would lead to better BL outcomes?

Chemical complementarity between tumor resident, T-cell receptor CDR3s and MAGEA3/6 correlates with increased melanoma survival: Potential relevance to MAGE vaccine auto-reactivity

An innovative and affordable quantitative assessment of human TCR repertoire.

Background Stomach adenocarcinoma (STAD) has an extremely high fatality rate worldwide, and survival after metastasis is extremely poor. Cytokine-like protein 1 (CYTL1) has prognostic significance in various tumors. We aimed to explore the impact and underlying molecular mechanisms of CYTL1 in STAD through bioinformatics analysis. Methods We used R software to analyze CYTL1 expression in STAD samples (n = 375) and normal samples (n = 32) in The Cancer Genome Atlas database. Kaplan–Meier analysis was used to verify the relationship between CYTL1 expression and overall survival (OS) and disease-specific survival (DSS) based on the clinical characteristics and subgroups of patients with STAD. Furthermore, univariate and multivariate Cox regression analyses were used to verify the outcome variables of OS and DSS in patients with STAD. Receiver operating characteristic curves were used to test the predictive power of CYTL1. The biological functions and signaling pathways of CYTL1 were determined using gene set enrichment analysis (GSEA), and the immune infiltration patterns of CYTL1 and correlation of immune-related markers were analyzed using single-sample GSEA (ssGSEA) and an estimate algorithm. Results In our research, low CYTL1 expression (tumor vs. normal) was noted in patients with STAD. High CYTL1 expression was detrimental to OS and DSS and had good diagnostic performance (AUC = 0.731). In the subtype analysis of STAD, T3 and T4 stages, N0 and N1 stages, M0 stage, gender (female), and age (≤65 years) showed different performances between OS and DSS. Univariate and multivariate Cox analyses identified CYTL1 as an independent factor, and logistic regression analysis indicated that CYTL1 was associated with M stage (OR = 3.406) and sex (OR = 1.535). GSEA of the differential genes of CYTL1 showed the possible involvement of immunity. ssGSEA and estimation algorithms were used to further evaluate whether immune cells were closely related to CYTL1 expression, and many markers of immune cells also had statistical significance with the expression of CYTL1. Conclusion CYTL1 may, thus, act as an independent prognostic factor for STAD and regulate STAD progression by affecting the immune microenvironment.

Simple SummaryThe chemical complementarity of glioblastoma, tumor-resident T-cell receptors and cancer testis antigens were associated with a worse outcome. Additionally, the high expression of immune marker and low expression of apoptosis genes were associated with a high T-cell receptor–cancer testis antigen chemical complementarity and a worse outcome. In sum, T-cell receptor recombination reads from exome files have the potential to aid in glioblastoma prognoses and may provide opportunities to detect unproductive immune responses. Introduction. Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite a growing understanding of glioblastoma pathology, the prognosis remains poor. Methods. In this study, we used a previously extensively benchmarked algorithm to retrieve immune receptor (IR) recombination reads from GBM exome files available from the cancer genome atlas. The T-cell receptor complementarity determining region-3 (CDR3) amino acid sequences that represent the IR recombination reads were assessed and used for the generation of chemical complementarity scores (CSs) that represent potential binding interactions with cancer testis antigens (CTAs), which is an approach particularly suited to a big data setting. Results. The electrostatic CSs representing the TRA and TRB CDR3s and the CTAs, SPAG9, GAGE12E, and GAGE12F, indicated that an increased electrostatic CS was associated with worse disease-free survival (DFS). We also assessed the RNA expression of immune marker genes, which indicated that a high-level expression of SPHK2 and CIITA genes also correlated with high CSs and worse DFS. Furthermore, apoptosis-related gene expression was revealed to be lower when the TCR CDR3-CTA electrostatic CSs were high. Conclusion. Adaptive IR recombination reads from exome files have the potential to aid in GBM prognoses and may provide opportunities to detect unproductive immune responses.

The liver is a site of immune privilege, compared with the bladder and skin, for example. To study this attenuation of the immune response in the cancer setting, we compared quantities and features of adaptive immune receptor (IR) recombination reads obtained from hepatocellular carcinoma (HCC) and six other cancers. Of these cancers, HCC had the lowest numbers of IR recombination reads and was the only cancer with a greater number immunoglobulin rather than T-cell receptor recombination reads. To better understand the role of adaptive IRs obtained from the tumor microenvironment in shaping the outcome of HCC cases, we quantified the chemical complementarity between HCC tumor TRB and IGH complementarity determining region-3 (CDR3) amino acid (AA) sequences, and known hepatitis B virus (HBV) epitopes. High chemical complementarity between HCC-resident CDR3s and three HBV epitopes correlated with increased survival probabilities, for two sources of CDR3s representing different CDR3 recovery algorithms. These results suggest the potential of CDR3 AA sequences as biomarkers for HCC patient stratification and as guides for future development of therapeutics.

Introduction: About one-third of prostate cancer (PCa) patients will experience rising prostate specific antigen levels, a condition known as biochemical recurrence. INO-5150 is a DNA-based immunotherapy targeting PSA and PSMA previously tested with or without plasmid-encoded cytokine adjuvant IL-12 (INO-9012) in a phase 1/2, open-label, multi-center study. Treatment was well-tolerated and safe, with no treatment-related serious adverse events. Here we sought to characterize the T cell receptor (TCR) repertoire of treated patients before and after INO-5150. Experimental Procedures: 62 post-prostatectomy/radiation therapy patients with rising PSA were enrolled in the trial. INO-5150±INO-9012 was administered IM followed by electroporation with the CELLECTRA® device on day 0, weeks 3, 12 and 24. Peripheral blood mononuclear cells (PBMCs) were collected before and after treatment from a subset of patients (n=19) and stimulated with PSA or PSMA peptides, negative or positive controls. After 5 days the Complementary Determining Region 3 (CDR3) of the TCR beta chain was sequenced. Results: TCR sequences that expanded after PSA or PSMA stimulation only were considered antigen specific sequences. On average, >70% of antigen specific TCR sequences were expanded following treatment with INO-5150 +/- INO-9012, including fold increases of up to 327 fold for PSA and 471 fold for PSMA over pretreatment frequencies. All treated patients had a Shannon's entropy (H) index >3.5 for TCR diversity. Previous examination of these patients using flow cytometry suggested that half of them demonstrated expansion of a PSA/PSMA specific CD8+ T cell subset harboring lytic potential (CD38+PD1+Prf+). TCR analysis performed by grouping patients based on the presence or absence of these cells suggested PSA/PSMA specific TCR expansion occurred for both groups. Deeper analysis suggested differences in the degree of expansion, as 56% of patients with the CD38+PD1+Prf+ phenotype were found to have at least 150 TCRs with a 10 fold expansion over baseline, whereas only 20% of patients without this cell population met this criteria. Conclusions: Treatment with INO-5150 +/- INO-9012 in PCa patients drove the expansion of a diverse pool of antigen specific T cells with a high Shannon's Entropy value suggesting high TCR diversity. Patients with immune reactivity by flow cytometry were more likely to have a higher frequency of PSA/PSMA specific TCRs that expanded >10 fold over their pre-treatment value; however patients without immune reactivity also demonstrated expansion in TCRs. These data suggest that INO-5150 induced PSA/PSMA specific T cells and that TCR sequencing is a valuable tool for assessment of immune responses induced by immunotherapy that does not bias towards a specific T cell function. Citation Format: Kimberly A. Kraynyak, Matthew P. Morrow, Albert J. Sylvester, Snehal Wani, Jean Boyer, Kamal Bhatt, Trevor McMullan, Jessica Lee, Brian Sacchetta, Samantha Rosencranz, Jeffrey Skolnik, Neal Shore. Immunotherapy targeting PSA and PSMA in patients with biochemically recurrent prostate cancer expands antigen-specific T cell receptor repertoire in a PhI/II trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-144.

10042 Background: Neuroblastoma has variable outcomes across different risk groups. In children with stage 4 neuroblastoma, five-year overall remains around 50% in high-risk children despite the emergence of anti-GD2 antibodies. T- and NK-cell infiltration is prognostic in therapy-resistant neuroblastoma, and higher HLA class I expression is linked to better overall survival (OS). In other cancers, specific HLA alleles and T-cell receptor (TCR) V, J- gene segments have been associated with survival. Thus, we conducted a retrospective study in stage 4S neuroblastoma patients to assess whether specific HLA allele, TCR V- and J-gene segment usage combinations correlated with OS in NBL. Among combinations that were associated with OS, we also identified changes in expression of immune marker genes. Methods: We obtained HLA allele data from exome files of the TARGET-NBL dataset using the xHLA software. The TCR recombination reads were obtained from the TARGET-NBL RNAseq files representing tumor specimens from 99 cases, utilizing a high-stringency search algorithm. The TCR recombination reads were translated, and the complementarity determining region-3 (CDR3) amino acid sequences were obtained. HLA and TCR datasets were integrated to assess OS probabilities, comparing cases with and without specific HLA allele, TCR V- or J-gene usage combinations. Significance was determined only if independent HLA allele or V- and J-gene usage assessments were not statistically significant, but significant in the corresponding HLA allele, TCR V- or J-gene segment usage combinations. HLA allele and TCR usage combinations were grouped by association with better or worse OS probabilities, and immune marker gene expression correlations were assessed via Student’s t-test and Mann-Whitney U test with a Bonferroni-corrected threshold of p = 0.00114. Results: We identified 73 HLA allele, TCR V- and J-gene usage combinations with significant OS distinctions: 20 associated with improved OS and 53 with worse OS. For example, 20 TARGET-NBL cases with the HLA-DQB1*04:02 and TRAJ29 usage combination did not reach the median compared to the 1319-day OS median for all remaining cases (log-rank p = 0.009). Among the cases with at least one HLA allele, TCR V- or J-gene segment usage combination with improved OS, we found that the RNAseq values for the immune markers CD4, CD22, CD38, RPH1 , TNFRSF17 , and TNFRSF13B were upregulated, as assessed via a Mann-Whitney U analysis. Conclusions: Identifying specific HLA allele, TCR V- and J- gene segment usage combinations associated with survival may further indicate patients who could benefit from immunologic-boosting treatments. Studies employing functional assays, immunogenomic profiling, and targeted immune pathway analyses may advance immunotherapeutic strategies and predictive biomarkers for neuroblastoma, particularly in high-risk patients.