Dynamics and survival associations of T cell receptor clusters in patients with pleural mesothelioma treated with immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden and no robust predictors of survival with ICI. Since ICIs enable adaptive antitumor immune responses, we investigated T-cell receptor (TCR) associations with survival in participants from two clinical trials treated with ICI. METHODS: We included patients with pleural mesothelioma who were treated with nivolumab (NivoMes, NCT02497508) or nivolumab and ipilimumab (INITIATE, NCT03048474) after first-line therapy. TCR sequencing was performed with the ImmunoSEQ assay in 49 and 39 pretreatment and post-treatment patient peripheral blood mononuclear cell (PBMC) samples. These data were integrated with TCR sequences found in bulk RNAseq data by TRUST4 program in 45 and 35 pretreatment and post-treatment tumor biopsy samples and TCR sequences from over 600 healthy controls. The TCR sequences were clustered into groups of shared antigen specificity using GIANA. Associations of TCR clusters with overall survival were determined by cox proportional hazard analysis. RESULTS: We identified 4.2 million and 12 thousand complementarity-determining region 3 (CDR3) sequences from PBMCs and tumors, respectively, in patients treated with ICI. These CDR3 sequences were integrated with 2.1 million publically available CDR3 sequences from healthy controls and clustered. ICI-enhanced T-cell infiltration and expanded T cell diversity in tumors. Cases with TCR clones in the top tertile in the pretreatment tissue or in circulation had significantly better survival than the bottom two tertiles (p<0.04). Furthermore, a high number of shared TCR clones between pretreatment tissue and in circulation was associated with improved survival (p=0.01). To potentially select antitumor clusters, we filtered for clusters that were (1) not found in healthy controls, (2) recurrent in multiple patients with mesothelioma, and (3) more prevalent in post-treatment than pretreatment samples. The detection of two-specific TCR clusters provided significant survival benefit compared with detection of 1 cluster (HR<0.001, p=0.026) or the detection of no TCR clusters (HR=0.10, p=0.002). These two clusters were not found in bulk tissue RNA-seq data and have not been reported in public CDR3 databases. CONCLUSIONS: We identified two unique TCR clusters that were associated with survival on treatment with ICI in patients with pleural mesothelioma. These clusters may enable approaches for antigen discovery and inform future targets for design of adoptive T cell therapies.

Epigenomics may begin to explain in vitro differential response to hypomethylating agents in MMR-D hypermethylated endometrial cancer.

This work examines differences in chromatin accessibility, methylation, and response to DNA hypomethylating agents between mismatch repair-deficient and non-mismatch repair-deficient endometrial cancer. Next-generation sequencing of a stage 1B, grade 2 endometrioid endometrial cancer tumor revealed microsatellite instability and a variant of unknown significance in POLE along with global and MLH1 hypermethylation. Inhibition of viability by decitabine in the study and comparison tumors was minimal, as shown by an inhibitory effect of 0 and 17.9, respectively. Conversely, the inhibitory effect of azacitidine on the study tumor was more pronounced, at 72.8 versus 41.2. In vitro, mismatch repair-deficient endometrial cancer with MLH1 hypermethylation respond better to DNA methyltransferase inhibition by azacytidine (DNA/RNA inhibition), than to decitabine (DNA-only inhibition). Additional large studies are needed to substantiate our findings.

Renin Production by Juxtaglomerular Cell Tumors and Clear Cell Renal Cell Carcinoma and the Role of Angiotensin Signaling Inhibitors.

OBJECTIVE: To profile juxtaglomerular cell tumors (JXG) and histologic mimics by analyzing renin expression; to identify non-JXG renin-producing tumors in The Cancer Genome Atlas (TCGA) data sets; and to define the prevalence of hypertension (HTN) and patient outcomes with angiotensin signaling inhibitor (ASI) use in tumors of interest. PATIENTS AND METHODS: Thirteen JXGs and 10 glomus tumors (GTs), a histologic mimic, were evaluated for clinicopathologic features; TCGA data were analyzed to identify non-JXG renin-overexpressing tumors. An institutional registry was queried to determine the incidence of HTN, the use of ASIs in hypertensive patients, and the impact of ASIs on outcomes including progression-free survival (PFS) in a tumor type with high renin expression (clear cell renal cell carcinoma [CC-RCC] diagnosed between January 1, 2005, and December 31, 2012). RESULTS: We found an association between renin production and HTN in JXG compared with GT. Analysis of TCGA data found that a subset of CC-RCCs overexpress renin relative to 29 other tumor types. Furthermore, analysis of our institutional registry revealed a high prevalence (64%) of HTN among 1203 patients treated with radical or partial nephrectomy for nonmetastatic CC-RCC. On multivariable Cox regression, patients with HTN treated with ASIs (34%) had improved PFS (hazard ratio, 0.76; 95% CI, 0.57 to 1.00; P=.05) compared with patients with HTN not treated with ASIs (30%). CONCLUSION: The identification of renin expression in a subset of CC-RCC may provide a biologic rationale for the high prevalence of HTN and improved PFS with ASI use in hypertensive patients with nonmetastatic CC-RCC.

Tumor Junction Burden and Antigen Presentation as Predictors of Survival in Mesothelioma Treated With Immune Checkpoint Inhibitors.

INTRODUCTION: The favorable outcomes with immunotherapy for mesothelioma were somewhat unexpected because this tumor has a low tumor mutation burden which has been associated with benefit in other cancers. Because chromosomal rearrangements are common in mesothelioma and have neoantigenic potential, we sought to determine whether they are associated with survival in patients treated with immunotherapy. METHODS: Pleural biopsies of mesothelioma after at least one line of therapy were obtained from patients (n = 44) before treatment with nivolumab alone (NCT29908324) or in combination with ipilimumab (NCT30660511). RNA and whole-genome sequencing were performed to identify the junctions resulting from chromosomal rearrangements and antigen processing and presentation gene set expression. Associations with overall survival (OS) were estimated using Cox models. An OS cutoff of 1.5 years was used to distinguish patients with and without durable benefit for use in receiving operating characteristic curves. RESULTS: Although tumor junction burdens were not predictive of OS, we identified significant interactions between the junction burdens and multiple antigen processing and presentation gene sets. The "regulation of antigen processing and presentation of peptide antigen" gene set revealed an interaction with tumor junction burden and was predictive of OS. This interaction also predicted 1.5-year or greater survival with an area under the receiving operating characteristic curve of 0.83. This interaction was not predictive of survival in a separate cohort of patients with mesothelioma who did not receive immune checkpoint inhibitors. CONCLUSIONS: Analysis of structural variants and antigen presentation gene set expression may facilitate patient selection for immune checkpoint inhibitors.

Personalized tumor-specific DNA junctions to detect circulating tumor in patients with endometrial cancer.

INTRODUCTION: There are no reliable blood biomarkers for monitoring endometrial cancer patients in the current clinical practice. Circulating tumor DNA (ctDNA) is emerging as a promising non-invasive method to measure tumor burden, define prognosis and monitor disease status in many solid cancers. In this pilot study, we investigated if unique tumor-specific DNA junctions can be used to detect ctDNA levels in patients with endometrial cancer. METHODS: Chromosomal rearrangements in primary tumors of eleven patients with high-grade or advanced stage endometrial cancer were determined by whole-genome Mate-Pair sequencing. Identified unique tumor-specific junctions were evaluated in pre- and six-week post-surgery patient plasma using individualized quantitative polymerase chain reaction (qPCR) assays. The relationship between clinicopathological features and detection of ctDNA was investigated. RESULTS: CtDNA was detected in 60% (6/10) of cases pre-surgery and in 27% (3/11) post-surgery. The detection of ctDNA pre-surgery was consistent with clinical indicators of aggressive disease such as advanced stage (80% - 4/5), lymphatic spread of disease (100% - 3/3), serous histology (80% - 4/5), deep myometrial invasion (100% - 3/3), lympho-vascular space invasion (75% - 3/4). All patients in which ctDNA was detected post-surgically had type II endometrial cancer. DISCUSSION: This pilot study demonstrates the feasibility of using personalized tumor-specific junction panels for detecting ctDNA in the plasma of endometrial cancer patients. Larger studies and longer follow-up are needed to validate the potential association between pre-surgical ctDNA detection and the presence of cancers with aggressive pathologic tumor characteristics or advanced stage observed in this study.

Theragnostic chromosomal rearrangements in treatment-naive pancreatic ductal adenocarcinomas obtained via endoscopic ultrasound.

A crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment-naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome-wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome-wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors.

DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Optimizing clinical cytology touch preparations for next generation sequencing.

Intraoperative diagnosis is routinely performed on cytology touch preparations (TPs) from core needle biopsies (CNBs). Current interest promotes their utility as an important source of patient tissue for clinical genomic testing. Herein we present whole genome structural variant analysis (SVA) from mate-pair sequencing (MPseq) and whole exome sequencing (WES) mutation calling in DNA directly whole genome amplified (WGA) from TPs. Chromosomal copy changes and somatic DNA junction detection from MPseq of TPs were highly consistent with associated CNBs and bulk resected tissues in all cases. While increased frequency coverage noise from limitations of amplification of limited sample input was significant, this was effectively compensated by natural tumor enrichment during the TP process, which also enhanced variant detection and loss of heterozygosity evaluations from WES. This novel TP methodology enables expanded utility of frequently limited CNB for both clinical and research genomic testing.

Using Genomics to Differentiate Multiple Primaries From Metastatic Lung Cancer.

INTRODUCTION: Genomic technologies present a promising mechanism of resolving the clinical dilemma of distinguishing independent primary tumors from intrapulmonary metastases in NSCLC. We evaluated the utility of discordant mapping somatic junctions from chromosomal rearrangements in diagnosing metastatic disease compared to the current standard histologic review. MATERIAL AND METHODS: Mate-pair sequencing was performed on DNA extracted from 76 distinct tumors from 37 cases of multiple lung cancers. Discordant mapping junctions and chromosomal copy levels were assessed for each tumor. Blood-derived DNA was available on 22 of these cases for germline assessments. A lung cancer next-generation sequencing panel was additionally performed on tumor pairs from 17 patients. RESULTS: Whereas mate-pair sequencing was able to classify lineage in all tumor pairs, histologic review appeared to misclassify lineage in 9 of 33 (27%) same-histology tumor pair comparisons. Based on disagreement between the reviewing pathologists, histopathologic lineage was classified as indeterminate in seven cases. In two cases where pathologists agreed on a metastatic call, no shared junctions were found suggesting independent primaries. Although germline junctions passing algorithmic filters were common, on average less than three were present and all had predictable structures of small focal rearrangements or transposons. Evaluation of shared chromosomal copy changes and driver mutations through a lung cancer next-generation sequencing panel, while informative, were nondefinitive in calling lineage in all cases. CONCLUSIONS: The highly unique nature and prevalence of chromosomal rearrangement in lung cancers provide a useful and definitive technique for calling lineage in multifocal lung cancer.

Neoantigenic Potential of Complex Chromosomal Rearrangements in Mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Whereas other carcinogen-related tumors are associated with a high tumor mutation burden, mesothelioma is not. We sought to resolve this discrepancy. METHODS: We used mate-pair (n = 22), RNA (n = 28), and T cell receptor sequencing along with in silico predictions and immunologic assays to understand how structural variants of chromosomes affect the transcriptome. RESULTS: We observed that inter- or intrachromosomal rearrangements were present in every specimen and were frequently in a pattern of chromoanagenesis such as chromoplexy or chromothripsis. Transcription of rearrangement-related junctions was predicted to result in many potential neoantigens, some of which were proven to bind patient-specific major histocompatibility complex molecules and to expand intratumoral T cell clones. T cells responsive to these predicted neoantigens were also present in a patient's circulating T cell repertoire. Analysis of genomic array data from the mesothelioma cohort in The Cancer Genome Atlas suggested that multiple chromothriptic-like events negatively impact survival. CONCLUSIONS: Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel immunotherapeutic strategies and the selection of patients to receive immunotherapies.