Detection of Anti‐CMV TCR CDR3s via Blood and Tumor Exome Files Correlates With Increased Survival for Stomach Adenocarcinoma and Soft Tissue Sarcoma

Introduction: While 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. This study aimed to investigate the association between anti-CMV T-cell receptor (TCR) sequences and improved survival outcomes in two types of cancers where the role of CMV remains currently underexplored, stomach adenocarcinoma (STAD) and soft tissue sarcoma (SARC). The significance of this study is that, to our knowledge, it is the first to identify a potential prognostic role of CMV in both STAD and SARC and highlights the potential for future development of anti-CMV T-cell therapies. Methods: We extracted TCR recombination reads from whole exome sequencing (WXS) and RNA sequencing (RNASeq) files of STAD and SARC samples from the Cancer Genome Atlas (TCGA) and matched the corresponding TCR complementarity determining region-3 (CDR3) AA sequences represented by these reads to known anti-CMV CDR3 sequences. Results: Results indicated that, in STAD cases, the recovery of both TRA and TRB recombination reads from WXS files, from either normal blood or tumor samples, which corresponded to AA sequences matching anti-CMV CDR3s, correlated with improved disease-free survival (DFS) (logrank p=0.025) and improved overall (p=0.026), progression-free (p=0.015), and disease specific (p=0.026) survival on an STP comparison (i.e., a comparison at 45 months). Within the SARC WXS dataset, these were associated with better overall survival (OS) (logrank p=0.021) and disease-specific survival (DSS) (logrank p=0.008). However, TCR CDR3s represented by recombination reads recovered from RNAseq files and matching anti-CMV TCR CDR3 AA sequences correlated with lower DFS probabilities (i.e., STAD STP comparison at 45 months, p=0.035, SARC STP comparison at 65 months, p=0.032). Conclusion: This study reveals a novel association between anti-CMV TCR sequences and survival probability in STAD and SARC. This suggests that TCR CDR3s may be possible indicator of prognosis for STAD and SARC and supports further research into anti-CMV CDR3s as a tool for the development of immunotherapy for these cancers. Additionally, the contrasting results between WXS and RNASeq-derived sequences warrant further investigation into the timing and nature of anti-CMV immune responses in cancer progression. One explanation for these results include the possibility that exposure to CMV prior to onset or progression of cancer may lead to better survival probability, while a potentially active T-cell response to CMV during cancer onset or progression could represent an ongoing comorbidity. Citation Format: Utsav Kapoor, Michael T. Aboujaoude, Konrad Cios, Andrea Chobrutskiy, Boris I. Chobrutskiy, George Blanck. Detection of anti-CMV TCR CDR3s correlates with increased survival for soft tissue sarcoma and stomach adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6254.

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

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.

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.

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.

Overexpression of the secretory protein renalase-1 negatively impacts the survival of melanoma and pancreatic cancer patients, while inhibition of renalase-1 signaling drives tumor rejection by promoting T-cell activation. Thus, we investigated the chemical complementarity between melanoma-resident, T-cell receptor (TCR) complementarity-determining region 3 (CDR3) amino acid sequences (AAs) and the renalase-1 protein. Increasing complementarity of TCR CDR3s to renalase-1 AAs, as assessed by a chemical complementarity scoring algorithm, was associated with improved overall survival (OS) in melanoma patients. The expression levels of several immune signature genes were significantly, positively correlated with increasing TCR CDR3-renalase-1 complementarity scores. Additionally, the survival association observed with high complementarity of TCR CDR3s to renalase-1 AAs was more robust in cases with low renalase-1 gene expression levels. Mapping of TCR CDR3-renalase-1 in silico interaction sites identified major epitope candidates including RP220, the signaling module of the renalase-1 protein, consistent with the fact that a monoclonal antibody to RP220 is a potent inhibitor of melanoma growth. These findings indicate that renalase-1 is a potential antigen for TCR recognition in melanoma and could be considered as a target for immunotherapy.

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

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

T cells play a critical role in antitumor immunity and are the primary targets in many immunotherapies. However, the study of tumor-infiltrating T cells has been complicated by the extremely diverse repertoire of T cell receptors, characterized by the complementarity determining region 3 (CDR3). Here using our newly developed algorithm, we studied the CDR3 sequences of tumor infiltrating T cells in 9,142 RNA-seq samples across 29 diseases. In total, we assembled over 600 thousands CDR3 sequences, covering the α, β, γ, δ chains. TRBV genes usage, CDR3 sequence length and conservation of infiltrating T cells resembled those in the peripheral blood of healthy donors in many tumors, except brain and kidney cancers. About 20% of the CDR3 calls were shared in multiple individuals. T cell clonotypes carrying these CDR3 sequences are known as the ‘public T cells’. In this work, we found that public T cell CDR3 amino acid (AA) sequences are shorter compared to the private ones and the middle 3 positions of the private CDR3 sequences were enriched for hydrophobic AAs. This finding potentially suggests that private T cell clonotypes in the tumors have greater potential to recognize neoantigens. We defined clonotype per kilo-reads (CPK) to measure the diversity of T cell repertoire and identified a strong positive association between CPK and tumor mutation load, implying that neoantigens may diversify the infiltrating T cell population. In addition, we predicted SPAG5 and TSSK6 as putative immunogenic cancer/testis antigens in multiple cancers based on their strong associations with CPK. Finally, we identified 3 potential immunogenic somatic mutations based on their co-occurrence with CDR3 sequences. One of them, PRAMEF4 F300V, was predicted to bind strongly to both MHC-I and MHC-II, with matched HLA types in its carriers. Our analyses have the potential to simultaneously identify immunogenic neoantigen and the tumor-reactive T-cell clonotype. Citation Format: Bo Li, Taiwen Liu, Binbin Wang, Jinzeng Wang, Sachet Shukla, Ruoxu Dou, Stephen Hodi, Catherine Wu, Nir Hacohen, Jun Liu, X. Shirley Liu. Landscape of tumor-infiltrating T-cell repertoire of human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-264.

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?

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.

Wilms' tumors are pediatric renal tumors that generally have a good prognosis and outcomes. Viral illnesses have been linked to development of neoplasms and should be considered as a factor that could modulate overall survival. We considered recently developed adaptive immune receptor, genomics and bioinformatics approaches to assess the potential impact of cytomegalovirus (CMV) infections in Wilms' tumor. T-cell receptor (TCR) complementarity determining region-3 (CDR3) amino acid sequences from Wilms' tumor specimens represented by the Therapeutically Applicable Research to Generate Effective Treatments dataset were compared with known anti-CMV TCR CDR3s, indicating that cases representing the anti-CMV TCR CDR3s had worse outcomes. Then, a chemical complementarity scoring approach for the Wilms' tumor, TCR CDR3s and a series of CMV antigens further indicated that cases representing a higher chemical complementarity to the CMV antigens had worse outcomes. Overall, we present a potentially novel method to assess CMV infections and identify patients who could benefit from therapies that address such infections.

Background: Patients with advanced soft tissue sarcomas (STS) have a dismal prognosis with few effective therapeutic options. A defect in the homologous recombination repair (HRR) pathway can accumulate DNA repair errors and gene mutations, which can lead to tumorigenesis. BRCAness describes tumors with an HRR deficiency (HRD) in the absence of a germline BRCA1/2 mutation. However, the characteristics of BRCAness in STS remain largely unknown. Thus, this study aimed to explore the genomic and molecular landscape of BRCAness using whole exome sequencing (WES) in STS, aiming to find a potential target for STS treatment.Methods: WES was performed in 22 STS samples from the First Affiliated Hospital of Sun Yat-sen University to reveal the possible genomic and molecular characteristics. The characteristics were then validated using data of 224 STS samples from The Cancer Genome Atlas (TCGA) database and in vitro data. The analysis of the potential biomarker for BRCAness was performed. Targeted drug susceptibility and combination therapy screening of chemotherapeutics for STS were evaluated in STS cell lines, cell-line-derived xenografts (CDX), and patient-derived xenografts (PDX).Results: Compared with 30 somatic mutation signatures of cancers, high cosine-similarity (0.75) was identified for HRD signatures in the 22 STS samples using nonnegative matrix factorization. Single nucleotide polymorphism indicated a low mutation rate of BRCA1/2 in the 22 STS samples (11.76% and 5.88%, respectively). However, copy number variation analyses demonstrated widespread chromosomal instability; furthermore, 54.55% of STS samples (12/22) carried BRCAness traits. Subsequently, similar genomic and molecular characteristics were also detected in the 224 STS samples from TCGA and in vitro. Poly (ADP-ribose) polymerases (PARP)-1 could be a promising reflection of HRD and therapeutic response. Furthermore, the level of PAR formation was found to be correlated with PARP-1. Subsequently, STS cell lines were determined to be sensitive to PARP inhibitor (PARPi), niraparib. Moreover, based on the screening test of the five common PARPis and combination test among doxorubicin, ifosfamide, dacarbazine, and temozolomide (TMZ), niraparib and TMZ were the most synergistic in STS cell lines. The synergistic effect and safety of niraparib and TMZ combination were also shown in CDX and PDX.Conclusions: BRCAness might be the common genomic and molecular characteristics of majority of STS cases. PARP-1 and PAR could be potential proper and feasible theranostic biomarkers for assessing HRD in patients. STSs were sensitive to PARPi. Moreover, the combination of niraparib and TMZ showed synergistic effect. Niraparib and TMZ could be a promising targeted therapeutic strategy for patients with STS.

Acute Respiratory Distress Syndrome (ARDS) is an illness that typically develops in people who are significantly ill or have serious injuries. ARDS is characterized by fluid build-up that occurs in the alveoli. T-cells are implicated as playing a role in the modulation of the aberrant response leading to excessive tissue damage and, eventually, ARDS. Complementarity Determining Region 3 (CDR3) sequences derived from T-cells are key players in the adaptive immune response. This response is governed by an elaborate specificity for distinct molecules and the ability to recognize and vigorously respond to repeated exposures to the same molecules. Most of the diversity in T-cell receptors (TCRs) is contained in the CDR3 regions of the heterodimeric cell-surface receptors. For this study, we employed the novel technology of immune sequencing to assess lung edema fluid. Our goal was to explore the landscape of CDR3 clonal sequences found within these samples. We obtained more than 3615 CDR3 sequences across samples in the study. Our data demonstrate that: (1) CDR3 sequences from lung edema fluid exhibit distinct clonal populations, and (2) CDR3 sequences can be further characterized based on biochemical features. Analysis of these CDR3 sequences offers insight into the CDR3-driven T-cell repertoire of ARDS. These findings represent the first step towards applications of this technology with these types of biological samples in the context of ARDS.