Clinical Research (Excluding Clinical Trials)最新文献

{"title":"Abstract 363: Investigating the potential clinical predictive value of virus genotype, menopausal status and mutational landscape in cervical cancer tissue using a NGS based human papillomavirus (HPV) assay and whole exome sequencing (WES)","authors":"Jean-Christophe Pignon, Heidi Giese, D. Foernzler, L. McDaniel, G. Bartha, J. Scheuenpflug, Zheng Feng","doi":"10.1158/1538-7445.AM2021-363","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-363","url":null,"abstract":"Purpose: Persistent HPV infections are associated with nearly all cervical cancers and some head & neck and genitourinary cancers. In cervical cancer, 14 high risk HPV genotypes have been previously identified with varying geographical prevalence and carcinogenicity. HPV16 and 18 account for >70% of cervical cancer incidence worldwide. There is an unmet need to explore HPV-associated cancer pathology, HPV infection status and molecular profiles to detect virus load and identify predictive gene signatures. We investigated the association between HPV genotypes and clinicopathological features and analyzed the mutational landscape using the ImmunoID NeXT Platform®. Methods: Early stage cervical cancer FFPE tissue samples (n=38) were analyzed. We detected and quantified oncoviral DNA, identified likely somatic mutations, and quantified genome-wide mutational burden. Any sample with at least one filtered read of HPV evidence was considered positive; zero such evidential reads were reported across a negative control cohort of > 439 non-cervical cancer samples. Oncovirus DNA counts and HPV genotype status were then evaluated against covariates (age; menopausal status) for statistical significance. Finally, mutation frequency was assessed in genes comprising major cervical cancer driver pathways from the KEGG and Biocarta databases. Results: Oncoviral HPV infections were detected using a NGS based approach in all tumor tissue with demonstrated robust assay performance. A statistically significant difference in viral DNA counts was observed between high risk genotypes HPV16 and 18 (n=27) and all other genotypes (n=10), (p=0.028, Student9s t). Genotypes were also significantly associated with menopausal status (p=0.01, Fisher9s Exact Test) and age at surgical resection (p=0.011, Student9s t). Among the pathways considered, TGF-β and MAP-kinase were the most frequently mutated across 38 samples and multiple pathway annotations. Conclusions: We applied a powerful NGS based WES approach that enables accurate detection of HPV genotypes with quantification of HPV viral DNA. This quantitative approach represents a possible promising clinical benefit over existing qualitative HPV genotyping assays and may have the potential for further development to define cancer type and treatment specific cutoff values for cancer prognosis. Furthermore, our results demonstrate that a WES based approach provides clinically valuable insights into the constitutive molecular mechanisms of cervical cancer. The association of HPV genotypes with clinical patient characteristics and/or mutational profiles has the potential to identify prognostic and predictive clinical biomarkers and to provide critical information for treatment defining strategies. Citation Format: Jean-Christophe Pignon, Heidi Giese, Dorothee Foernzler, Lee D. McDaniel, Gabor Bartha, Juergen Scheuenpflug, Zheng Feng. Investigating the potential clinical predictive value of virus genotype, menopausal status and mutat","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"108 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87604016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 548: Urine biopsy as dynamic marker to enhance clinical staging of urothelial carcinoma","authors":"Uttam Satyal, David Liu, M. Slifker, R. Alpaugh, C. Lallas, Eduoard J. Trabulsi, J. Hoffman-Censits, K. Mouw, Ilsiya Ibragimova, P. Cairns, E. Allen, E. Plimack, A. Kutikov, Philip H. Abbosh","doi":"10.1158/1538-7445.AM2021-548","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-548","url":null,"abstract":"Introduction: Neoadjuvant chemotherapy results in 30-40% complete response rates in patients undergoing radical cystectomy (RC) for bladder cancer. There is significant interest in identifying complete responders prior to RC in order to potentially avoid removal of a pathologically benign bladder. However, clinical restaging after neoadjuvant chemotherapy (NAC) with cystoscopy and imaging techniques is highly inaccurate. Since, bladder tumors are known to shed DNA into the urine, we hypothesized that mutations from tumor tissue (MT) would be detectable in urine DNA (uDNA) using NGS methods and that the presence or absence of urinary mutations (MU) after NAC would correlate with presence or absence of residual disease at the time of RC, respectively. Results: MU were benchmarked against MT by comparing the mutation profiles from 15 patients enrolled in clinical trial NCT01611662 (‘The AMVAC trial9; NCT01031420) who underwent whole exome sequencing of their pre-NAC tumor tissue and had available urine supernatant. We were able to detect 46 of 76 (61%) MT as MU that included 6 of 14 (43%) subclonal MT being detected as MU. Residual MU status correlated with residual disease status based on mutations present in pre-RC urine supernatant from 22 patients in this trial (13/13 patients with residual MU had residual disease; 9/9 patients with absence of MU achieved pathological complete response). Residual MU status correlated with residual disease status (p 20% of samples were nondiagnostic because of low DNA quantity/quality and/or poor library composition/sequencing. We validated the correlation between mutation persistence and residual disease in pre-RC urine pellets from clinical trial NCT02968732 (‘The pT0 trial9). DNA from Urine pellets yielded a much lower nondiagnostic rate ( Conclusions: MU are representative of MT and thus can be used to enhance clinical staging of urothelial carcinoma. Urine biopsy may be a reliable tool that can be further developed to identify complete response to neoadjuvant chemotherapy. This prospect would facilitate safe radical cystectomy avoidance and could also be used as a surveillance tool to detect recurrence. The optimal urine compartment for accuracy appears to be the supernatant, however, there is a high nondiagnostic rate with these samples. Further refinements to DNA isolation protocols or library preparation may decrease the nondiagnostic rate.*PHA has applied patent based on this technology. Citation Format: Uttam Satyal, David Liu, Michael Slifker, R K. Alpaugh, Costas D. Lallas, Eduoard Trabulsi, Jean H. Hoffman-Censits, Kent W. Mouw, Ilsiya Ibragimova, Paul Cairns, Eliezer M. Van Allen, Elizabeth R. Plimack, Alexander Kutikov, Philip Abbosh. Urine biopsy as dynamic marker to enhance clinical staging of urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abst","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87761775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 418: Lower survival among HIV-infected head and neck cancer patients","authors":"Syim Salahuddin, Margaret C. Wu, J. P. Irizarry, Teresita Vega, N. Isaeva, K. Schalper, W. Yarbrough, B. Emu","doi":"10.1158/1538-7445.AM2021-418","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-418","url":null,"abstract":"","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83876699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 336: Gut microbiome predicts response to CDK4/6 inhibitor and immune check point inhibitor combination in patients with hormone receptor positive metastatic breast cancer","authors":"C. Wong, S. Yost, J. S. Lee, S. Highlander, Yuan Yuan","doi":"10.1158/1538-7445.AM2021-336","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-336","url":null,"abstract":"Background: CDK4/6 inhibitors (CDK4/6i) have been shown to modulate immune responses in the preclinical setting. Palbociclib is a front-line therapy for hormone receptor positive (HR+) metastatic breast cancer (MBC), and the combination with immune check point inhibitors (ICI) is increasingly being studied. An ongoing phase I trial was designed to test the safety and efficacy of palbociclib, pembrolizumab, and letrozole in postmenopausal women with HR+ HER2- MBC (NCT02778685). Stool microbiome was collected and analyzed. The aim of this study is to determine the association of gut microbiota and response using metagenome sequencing data through a machine learning model. Methods: Postmenopausal women with histologically proven stage IV HR+ HER2- MBC were enrolled. Stool samples were collected at baseline and during treatment for analysis using metagenome sequencing. Response per RECIST 1.1 were grouped into: responders (complete response or partial response) and non-responders (stable disease or progressive disease). Using metagenomic relative abundance data, a gradient-boosted tree model was developed with leave-one-patient-out cross validations to predict patient response at baseline. Kruskal-Wallis tests were used to assess the differences of the most important microbiota relative abundance (generated from the machine learning model) between responders and non-responders. Results: Forty-seven stool samples from 11 patients were collected at baseline and during treatment, and metagenome sequencing was performed. For predictive modeling, the validation Area Under the Receiver Operating Characteristic Curve (AUROC) and Area Under the Precision Recall Curve (AUPRC) were 0.71 and 0.83, respectively. Among the top 5 features from the model, patients with a larger relative abundance of Gemmiger formicillis have increased probability of responding to therapy (p Citation Format: Chi Wah Wong, Susan E. Yost, Jin Sun Lee, Sarah K. Highlander, Yuan Yuan. Gut microbiome predicts response to CDK4/6 inhibitor and immune check point inhibitor combination in patients with hormone receptor positive metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 336.","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"64 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91517274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 600: Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin","authors":"Jennifer Chow, A. A. Tevis, Edward C. Lo, Alisa C Clein, D. Sabath, Arturo B. Ramirez, E. Kaldjian, Tad George","doi":"10.1158/1538-7445.AM2021-600","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-600","url":null,"abstract":"Protein expression of synaptophysin (SYP) is characteristic of neuroendocrine subtype tumors. In prostate cancer, neuroendocrine differentiation is correlated with disease progression, poor prognosis, and treatment resistance. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and patient stratification. Here we describe the validation of a biomarker IF assay for enumeration of CTCs and characterization of their SYP expression. The 0920-VB SYP CTC assay workflow includes processing blood samples to slides (AccuCyte® Sample Preparation System), staining slides with a panel of fluorescent markers (RarePlex® Staining Kit and Ventana® DISCOVERY® ULTRA immunostaining system), and multiparameter imaging and analysis (CyteFinder® Instrument). The panel consists of a nuclear dye, and antibodies against cytokeratins and EpCAM (to identify epithelial CTCs), CD45 (to exclude white blood cells) and SYP. Both clinical and spike-in samples were used for assay validation. The model CTCs used to generate spike-in samples included 22Rv1 (prostate carcinoma, SYP positive) and BT-474 (breast carcinoma, SYP negative). Performance metrics for the assay included accuracy, sensitivity, specificity, repeatability, and intermediate precision of SYP detection and CTC enumeration. Single-cell SYP mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. An MFI threshold for SYP positivity was established by maximizing the classification accuracy of the positive and negative cell lines. Using this threshold, 98.7% of 22Rv1 cells were correctly classified as SYP-positive, and 99.7% of BT474 cells were correctly classified as SYP-negative with an inter-run coefficient of variation of 13.9% for the 22Rv1 cell line. In clinical prostate, breast and colon cancer patient samples, subsets of CTCs were found to be SYP positive with the expected cytoplasmic localization of the marker. In summary, this liquid biopsy assay provides an analytically sensitive and specific method for CTC enumeration and SYP biomarker expression analysis, allowing non-invasive detection of neuroendocrine differentiation. Citation Format: Jennifer Chow, A Anders Tevis, Edward Lo, Alisa Clein, Daniel E. Sabath, Arturo B. Ramirez, Eric P. Kaldjian, Tad George. Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 600.","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90197047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract LB053: Clinical evaluation of NGS-based liquid biopsy genotyping in non-small cell lung cancer (NSCLC) patients","authors":"Hillary S Sloane, P. Sathyanarayan, D. Edelstein, F. Jones, J. Preston, Sam Wu, Jenna G. Los, Lara Duchstein, J. Fredebohm, K. Wichner, D. Heim, F. Holtrup, H. Quinn, D. Feller-Kopman","doi":"10.1158/1538-7445.AM2021-LB053","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-LB053","url":null,"abstract":"Background: Access to molecular testing for metastatic NSCLC (mNSCLC) patients has been improved by circulating tumor DNA (ctDNA) based liquid biopsies (LB), which can obviate invasive procedures, expedite results, and enable serial testing. Emerging clinical applications for LB include therapeutic efficacy monitoring and minimal residual disease (MRD) detection, which demand a ctDNA assay with high sensitivity, specificity, and appropriate genomic coverage. Here we demonstrate that SafeSEQ next-generation sequencing (NGS) LB delivers equivalent performance to OncoBEAM digital PCR, a sensitive ctDNA approach that has been extensively clinically validated in pivotal trials (e.g., AURA, TIGER-X). Importantly, SafeSEQ delivers significantly expanded genomic coverage to address the need for expanding targeted therapy indications, monitoring treatment response, and detecting MRD. Methods: Whole blood samples (n=176) were collected from mNSCLC patients prior to/during treatment or at disease progression and transported to a CLIA laboratory for OncoBEAM analysis to detect mutations in EGFR (exon 19 del, L858R, T790M, C797S), KRAS (codons 12, 13, 61) and BRAF V600E. Replicate plasma aliquots were analyzed with SafeSEQ to interrogate clinically relevant regions in BRAF, EGFR, ERBB2, KRAS, MET, NRAS, PIK3CA, and TP53. Mutation level concordance between the methods was assessed, where only genomic alterations interrogated by both platforms were considered. For mutations detected by both methods, correlation analysis of mutant allelic frequency (MAF) was performed. Results: Concordance analysis of the mutation results from OncoBEAM and SafeSEQ testing of 176 replicate patient samples demonstrated an overall percent agreement (OPA) of 99.6%, with a positive percent agreement (PPA) of 78.1% and a negative percent agreement (NPA) of 99.9%. The mean MAF for discordant mutations (n=16) was 0.06% (range: 0.04-0.12%). When considering mutations with MAF >0.1%, PPA and OPA increased to 96.0% and 99.9%, respectively. MAF levels for mutations detected by both methods demonstrated a strong linear correlation (R2=0.98). Of 124 patient samples having no mutation detected by OncoBEAM, 75 (60%) showed ≥1 alteration with SafeSEQ. Panel-wide, 76% and 30% of all mutations were detected at Citation Format: Hillary Sloane, Priya Sathyanarayan, Daniel Edelstein, Frederick Jones, Jennifer Preston, Sam Wu, Jenna Los, Lara Duchstein, Johannes Fredebohm, Katharina Wichner, Denise Heim, Frank Holtrup, Hannah Quinn, David Feller-Kopman. Clinical evaluation of NGS-based liquid biopsy genotyping in non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB053.","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"51 48","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91420667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 496: Humanized SIRPA and double humanized SIRPA/CD47 B-NDG mouse models are promising tools for anti-SIRPα antibodyin vivoefficacy studies","authors":"Yuting Hu, Lei Zhao, Yujie Liu, L. Hui, Zhaoxue Yu","doi":"10.1158/1538-7445.AM2021-496","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-496","url":null,"abstract":"","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83121169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 512: Tetramer and pentamer assays for antigen-specific lymphocytes: Implementation in clinical trials","authors":"C. Xue, Weiguo Zhong, Yeji Cho, J. Cuomo, C. Swenson, T. M. Closkey","doi":"10.1158/1538-7445.AM2021-512","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-512","url":null,"abstract":"The success of inhibiting PD1 on T cells for cancer immunotherapy initiated strong interest in studying therapeutic antibodies to alter the adaptive immune system. As the knowledge of neoantigens, oncogenes, and autoantigens is rapidly accelerating, the search for antigen-specific lymphocytes for better treatment outcomes is increasing. A tetramer or pentamer assay is an essential tool to immunophenotype and enumerate antigen-specific lymphocytes. These assays offer a powerful technique able to monitor immune status, using peripheral whole blood as a matrix, in contrast to invasive tissue biopsies. However, the assays have a number of obstacles to overcome in clinical research. Over several years, we have developed and validated tetramer and pentamer assays with both successes and failures. Rare events and precision: Clinical studies require robust reproducibility and high precision. As a standard in judging the acceptability of a flow cytometric biomarker test, the majority of tests are set up to have 20-30% CV between results. However, if a biomarker is expressed in less than 1% of a target population, the criteria can fail. The tetramer assay requires precision analyses that are even more stringent. These cells are rare events among millions of hematopoietic cells. The precision requirements may be at one hundredth percent or lower. A successful sign for the assay is that over a one-year period during the course of the clinical trial, some biomarkers remain at a stable level. Increasing signals and reduction of background noise: Unlike single marker based antibodies, however, tetramers and pentamers are highly sensitive. It is critical, therefore, to increase signals and reduce background noise. A combination of 7AAD (to remove dead cells) and incorporation of a dump channel (to remove non-target populations) significantly improves the precision of the assays. Data analyses and management: With new and powerful flow cytometers, a single sample can be labelled with multiple surface markers along with fluorescent tetramer or pentamers. The file size can reach megabytes when two to five million cells are tagged. This creates a bottleneck for data analysis and analytical software, especially in an assay with a panel of multiple tubes. The solution is to cut non-essential cell numbers including those with high side-scatter (SSC) numbers in half and to re-export data from cytometers. Controls setup: The most challenging part of these assays is to have strict controls, both for reagents and cells. Customized reagents and a source for characterized specific peripheral blood cells (PBMC) with HLA-A markers are necessary for developing and validating these complex assays. In conclusion, tetramer and pentamer assays targeting antigen-specific lymphocytes are deliverables for cancer immunotherapy research, cancer vaccine development and for use in studying other autoimmune diseases, such as type 1 diabetes. Citation Format: Chengsen Xue, Weiguo Zhong, Yej","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"139 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89361998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing","authors":"Michelle A. Toro, G. Lowman, Loni Pickle, Stephanie Ostresh, M. Andersen, S. Sarda, Chenchen Yang","doi":"10.1158/1538-7445.AM2021-511","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-511","url":null,"abstract":"Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90783592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract 493: A novel humanized B-hPD-1/hPD-L1/hTIGIT mouse model reveals enhanced efficacy of combined TIGIT and PD-L1 blockade against cancer","authors":"Chonghui Liu, Zhou Xiaofei, Yanan Guo, E. Lin, L. Yu, Yuelei Shen","doi":"10.1158/1538-7445.AM2021-493","DOIUrl":"https://doi.org/10.1158/1538-7445.AM2021-493","url":null,"abstract":"","PeriodicalId":10518,"journal":{"name":"Clinical Research (Excluding Clinical Trials)","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91070527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}