Tumor Next-generation Sequencing Research Articles

Clinical and molecular features and survival in thyroid cancer with brain metastases.

2023 Background: Brain metastases (BM) in advanced thyroid cancer are rare; however, they may be underreported as staging brain imaging is not routine. Prognostic features and molecular alterations for this group are poorly described. We characterized the clinical and molecular features of thyroid cancer with BM and evaluated differences in survival. Methods: In this single-center retrospective analysis, patients with metastatic radioactive iodine refractory well-differentiated, poorly-differentiated, or medullary thyroid carcinoma seen at the Princess Margaret Cancer Center from 2007-2022 were included. Electronic medical records were reviewed to collect clinicopathologic and treatment data. All patients had tumor next-generation sequencing (NGS) by a targeted gene panel. Overall survival (OS) from initial medical oncology consultation was analyzed using the Kaplan-Meier method and univariate and multivariate Cox proportional hazards models. Results: Of 248 patients with advanced thyroid cancer, 41(17%) were diagnosed with BM. The median interval from thyroid cancer diagnosis to BM development was 6.49 years (y) (IQR 3.51- 14.35 y). The median age at the time of BM diagnosis was 63.6 y and 23 (56%) were male. Most (76%) were asymptomatic at the diagnosis of BM and 80% had an ECOG of 0-1. Among patients with well-differentiated histology, 27 (18%) developed BM, while 7 (12%) with poorly-differentiated and 7 (19%) with medullary thyroid cancer developed BM. The most common molecular alterations included BRAFV600E (n=19), TERT (n=11), RAS (n=9), RET (n=8), and FANCA (n=3). Patients were treated with surgery (n=5), stereotactic radiation (n=21), whole brain radiation therapy (n=12), or a combination of both (n=2). OS was similar in patients with BM compared to those without (6.05 vs 6.45 y). There were no differences in histology or molecular alterations between patients that developed BM compared to those who did not. The median survival from the time of BM was 3.23 y. Patients with BM with ≥4 lesions had worse survival (0.52 vs 5.09 y, p=0.01). There were no differences in survival based on histology, symptoms, treatment or molecular alterations among those with BM. Conclusions: This is one of the largest reported thyroid BM cohorts and the first with complete NGS. The prevalence of BM in our cohort was higher than previously described, often diagnosed incidentally. Screening for BM in advanced thyroid cancer may detect higher rates, offering prognostic insights. While the number of BM was predictive of survival, molecular alterations did not predict BM development or survival and larger studies are needed.

Circulating metabolic profiling as a biomarker for immune checkpoint blockade efficacy.

2564 Background: Immune checkpoint blockade (ICB) has changed the treatment landscape of non-small cell lung cancer (NSCLC). Despite being the mainstay of treatment for advanced NSCLC, the development of resistance to ICB is common. Hence, identifying biomarkers that can be used to select patients (pts) who will benefit from ICB is crucial. Here, we interrogate the circulating metabolome to identify the metabolic parameters associated with ICB effectiveness. Methods: This single-center retrospective analysis used a mass-spectrometry (MS) targeted metabolomics approach to assess the relative abundance of 115 metabolites in baseline (pre-ICB) plasma of 55 pts with advanced NSCLC. Pts treated with ICB at the Princess Margaret Cancer Centre from 2018-2020 were included. Electronic medical records were reviewed to collect clinicopathological and treatment data. All pts had tumour next-generation sequencing (NGS) by a targeted gene panel. Descriptive statistics were used to summarize the patient characteristics, treatment modalities, and outcomes. Time-to-event outcomes were analyzed using the Kaplan-Meier method. Progression-free survival (rwPFS) and overall survival (rwOS) were defined as the time from the first treatment dose to radiographic or clinical progression or death from any cause and time from the first dose of treatment to death from any cause, respectively. The response rate was assessed using RECIST 1.1. Statistical significance was determined as a p-value <0.05. Results: Amongst the 55 pts profiled, 42 (76.3%) had adenocarcinoma. The most common molecular alterations included KRAS (n=15), BRAF non-V600 (n=5), and METex14 (n=3). The median PD-L1 score was 65% (IQR 22.5 – 59%). All pts were treated with a single-agent PD-1 inhibitor, and 67.2% of pts received ICB as the first line of treatment. Metabolomic analysis of the pre-ICB initiation plasma samples identified 8 metabolites whose relative abundance significantly differed between responding (CR/PR) and non-responding pts (SD/PD). Amongst these metabolites was the endogenous danger signal Glucosylceramide (d 18:1/24:0), whose abundance was increased in the plasma of the responding pts. When we stratified pts by circulating levels of glucosylceramide, we found a statistically significant higher rwPFS (8.5 vs. 1.6 months, p=0.0001) and rwOS (13.5 vs. 6.1 months, p=0.0001) in pts who had high baseline plasma levels of Glucosylceramide (d 18:1/24:0) (top 50%) versus those with lower levels (bottom 50%). Conclusions: Utilizing a targeted metabolomics approach, we identified the metabolite and endogenous danger signal glucosylceramide (d 18:1/24:0) as a potential metabolic biomarker of response to ICB therapy. Future work will aim to validate this finding in a larger cohort and to understand the biological mechanism underpinning this correlation.

Association between socioeconomic factors and utilization of next-generation sequencing in metastatic breast cancer.

e13700 Background: Targeted therapies such as PI3K inhibitors have revolutionized the treatment of patients with metastatic breast cancer (MBC). Their selective use is enabled by next-generation sequencing (NGS) approaches, which help identify actionable tumor alterations. There may be socioeconomic disparities in NGS testing and NGS-guided treatment selection. This study aimed to investigate the role of socioeconomic status (SES) (race and area deprivation index [ADI]) among patients with MBC undergoing NGS. Methods: Our retrospective study included 418 adult patients (pts) with MBC treated at the Magee Women’s Cancer Center in Pittsburgh, PA between September 2016 and December 2022. Demographic information, including race and ADI, clinicopathologic information, and NGS results were collected. ADI based on zip code was derived utilizing Neighborhood Atlas. Scores range from 0-10 (higher score = higher deprivation), representing the census-based ranking of the ZIP code at the state level based on the composite of several factors, including income, education, employment, and housing quality. Descriptive statistics, chi-squared, and independent sample t tests were used. Multivariate logistic regression analysis was done to evaluate potential associations with receipt of NGS and NGS-directed therapy. Results: 89% of pts were non-Hispanic white (NHW); 11% were non-Hispanic black (NHB). A greater proportion of NHB pts were from higher ADI (6-10) neighborhoods (p=0.00001), suggesting lower SES. NHW pts were more likely to have hormone receptor (HR)+/human epidermal growth factor receptor (HER)2- tumors compared with NHB pts (p=0.022). 219 (52.4%) total pts had NGS tumor testing performed: 53% of NHW and 47.8% of NHB pts (p=0.511). 55.1% (102/185) of pts living in higher SES neighborhoods (ADI 1-5) and 50.2% (117/233) living in lower SES neighborhoods (ADI 6-10) received NGS testing (p=0.317). Of the 219 pts who got NGS testing, 62.6% (137) had a clinically actionable alteration identified, and of those, 46% (63) received corresponding targeted therapy. Of the 63 pts who received NGS-guided therapy, 88.9% were NHW and 11.1% were NHB (p=0.849), and 50.8% were from neighborhoods with ADI 1-5 and 49.2% were from neighborhoods with ADI 6-10 (p=0.571). In multivariate analysis including race, ADI, insurance status, year of MBC diagnosis, and tumor subtype, only tumor subtype (non-HR+/HER2- tumors) was significantly associated with a lower likelihood of both receiving NGS testing (p=0.032) and receiving NGS-directed therapy (p=0.001). Conclusions: There was a higher numerical rate of NGS testing and NGS-guided therapy initiation in NHW pts and pts from lower ADI (1-5 vs. 6-10) neighborhoods, but this did not reach statistical significance. Overall, in this single institution cohort limited by small numbers, there were no differences in NGS tumor testing based on race and SES.

Comparative analysis of actionable gene reporting in targeted panels versus comprehensive NGS testing for solid tumor samples.

11185 Background: The landscape of actionable genes significantly influences clinical decisions in cancer diagnosis, prognosis, and treatment planning. In a clinical context, the selection of NGS panels hinges on striking a balance among informative data, insurance coverage, and the preferences of patients and healthcare providers. Molecular pathology reference laboratories commonly employ large-scale next-generation sequencing (NGS) testing, curating smaller, disease-specific targeted panels. This study investigates the prevalence of unreported actionable genes in real-world clinical samples subjected to disease-specific panels. Methods: We analyzed SNV/InDel DNA variants in 795 clinical solid tumor samples using the Illumina TSO 500 platform for comprehensive NGS panel testing of 517 genes. Comprehensive NGS panel-tested samples were intersected with disease-specific targeted NGS panels for breast (54 genes), brain (62 genes), colorectal (36 genes), and lung (44 genes). An artificial filter was applied to align targeted panels with patient disease. Variants were classified based on pathogenicity, and benign variants were filtered out. Detected variants were assessed for actionable mutations as defined by the FDA recognized OncoKB actionable gene database criteria. Additionally, 1484 lung patient samples tested with a lung-specific targeted panel were de-identified and unmasked to the comprehensive NGS tumor profiling to investigate the prevalence of actionable alteration beyond those covered by the disease-specific panel. Results: The study revealed that 3.2% of breast, 0% of brain, 66.2% of colorectal, and 2.7% of lung samples had additional actionable mutations undisclosed by the targeted panels. The number of actionable genes beyond those included in the targeted panels were 14 for breast, 2 for brain, 25 for lung, and 41 for colorectal cancer. This underscores the importance of comprehensive testing to fully capture each patient tumor’s actionable mutation profile. Conclusions: Our findings detect masked actionable mutations in down-sampled targeted panels and highlight the need for comprehensive testing to accurately identify actionable mutations in various cancer types. Reporting actionable mutations can significantly influence clinical decision-making, emphasizing the importance of NGS test selection in routine molecular diagnostics as well as the impact of integrating comprehensive NGS testing.

1238 Next Generation Sequencing of Gliomas to Identify Predictors of Time to Postoperative Epilepsy

INTRODUCTION: Seizures during the course of disease for patients with diffuse gliomas is common. As genetic characterization of gliomas has improved, how a tumors mutational profile influences the timing of postoperative seizures, and if postoperative seizures relate to survival and tumor progression, is poorly understood. METHODS: A retrospective review of consecutive adults from 2017 to 2020 who underwent resection of gliomas with next generation sequencing of tumors at a single institution was performed. Cox proportional hazards regression was performed to identify factors associated with time to postoperative seizures. Seizures within 72 hours of surgery were excluded. RESULTS: 363 patients with newly diagnosed or recurrent tumor tissue genotyped via next generation sequencing were included. Median follow-up time was 37.4 months, median time to seizure after surgery was 39.2 months (95% CI 34.6 – 60.8), and IDH-mutant tumors comprised 38.3% of the cohort. Patients with IDH-mutant tumors were more likely to have non-immediate postoperative seizures (p = 0.031) and more persistent postoperative seizures (p = 0.004) compared to IDH-wildtype tumors. PDGFRA mutation was associated with shorter time to seizure in IDH-mutant gliomas (p = 0.033), with no such genetic factors in IDH-wildtype tumors. Among IDH-mutant and wildtype tumors, patients with shorter seizure freedom had significantly shorter overall and progression-free survival. CONCLUSIONS: PDGFRA mutation is associated with earlier seizures after resection of IDH-mutant diffuse gliomas. Earlier first non-immediate postoperative seizure after resection is associated with worse overall and progression-free survival.

Abstract 970: Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling

Abstract Background: The expanding role of next-generation sequencing (NGS) in precision oncology has led to a paradigm shift towards genotype-targeted therapies, with liquid biopsies (LBs) offering a minimally invasive means of comprehensive genomic profiling. Methods: This is an observational LB-informed precision oncology clinical trial with primary endpoints to assess feasibility and determine the prevalence of actionable mutations in LB informing genotype-targeted therapeutic recommendations by the Johns Hopkins Molecular Tumor Board (JH MTB; NCT05585684). Secondary endpoints are progression-free and overall survival, ctDNA dynamics assessment, and concordance between plasma and tumor NGS. Target enrollment is 150 patients with advanced solid tumors who have progressed on at least one line of systemic therapy. Serial LB-derived genotypes are obtained at baseline, early on therapy, and upon progression using a CLIA ultra-sensitive 33-gene panel NGS assay (Elio Plasma Resolve, Personal Genome Diagnostics/Labcorp). Variant actionability is assessed by an ensemble knowledgebase, registry, and computational characterization approach, and results are reviewed by the JH MTB, followed by treatment recommendations. Results: The trial’s pre-specified interim analysis included 30 enrolled patients with evaluable LBs; 30 baseline, 13 on-therapy, and 6 progression LBs were analyzed. Twenty-five patients had non-small cell lung cancer among which 9 were EGFR-mutated, 1 patient had small cell lung cancer, 3 patients had gastro-esophageal cancer, and 1 patient had a neuroendocrine tumor. In total, 74 variants were identified across all time points; these were cross-referenced in somatic and germline registries, variant knowledgebases, and the biomedical literature, together with computational characterization of functional consequence to assign mutation actionability. Tumor-confirmed variants comprised 36% of baseline and 42% of both on-therapy and progression variants. At baseline, 20% of the variants were putatively clonal hematopoiesis-derived with similar findings at the on-therapy timepoint. Thirty percent of baseline variants and 33% of variants at progression were deemed actionable. Upon JH MTB review, 29% of patients received a genotype-matched therapeutic recommendation based on LB results, with MTB recommendations followed in 38% of the patients. In analyzing ctDNA dynamics, of the 13 patients with on-therapy samples, 23% showed ctDNA clearance, 31% showed persistence, while 46% had undetectable ctDNA at both time points. Among patients with LBs at the time of progression, a third showed ctDNA emergence, a third had ctDNA persistence, and the remainder had undetectable ctDNA throughout the follow-up period. Conclusion: Our findings highlight the clinical relevance of liquid biopsies in guiding treatment selection and monitoring. Citation Format: Maria Fatteh, Michael Conroy, Timsy Wanchoo, Selina Shiqing Teh, Jaime Wehr, Archana Balan, Rachel Karchin, Ellen Verner, Katerina Karaindrou, Rena Xian, Christopher Gocke, Ming-Tseh Lin, Peggy Fitzpatrick, Christine L. Hann, Joseph C. Murray, Vincent K. Lam, Josephine L. Feliciano, Julie R. Brahmer, Kristen A. Marrone, Patrick M. Forde, Katie Fiallos, Dana Petry, Taxiarchis Botsis, Mark Sausen, the Johns Hopkins Molecular Tumor Board Investigators, Jenna Canzoniero, Jessica Tao, Valsamo Anagnostou. Liquid biopsy-informed precision oncology clinical trial to evaluate the utility of ctDNA comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 970.

Abstract 2498: Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue

Abstract Introduction Tumor profiling including RAS, BRAF, HER2, and MSI/MMR status, is required to determine the treatment for patients with metastatic colorectal cancer (mCRC) at the time of diagnosis. While comprehensive tumor profiling using tissue-based next-generation sequencing (NGS) is increasingly adopted for initial profiling in mCRC, the role of circulating tumor DNA (ctDNA) NGS as initial testing in patients with sufficient tumor tissue is not clearly understood. We assessed the clinical value of ctDNA sequencing compared to tumor NGS in patients with newly diagnosed mCRC who have sufficient tumor specimens. Methods We prospectively enrolled consecutive patients with newly diagnosed mCRC at the National Cancer Center Korea. As per the institutional protocol for mCRC, initial tumor profiling was performed on primary tumor tissue using an in-house NGS panel (NCC PCP ver.3), which included 525 genes. For ctDNA sequencing, patients were evaluated using the AlphaLiquid®100 comprehensive cancer panel (IMBdx, Inc.), which included 118 genes, before the initiation of chemotherapy. Additionally, immunohistochemical (IHC) testing for HER2 and polymerase chain reaction (PCR)/IHC testing for MSI and/or MMR were performed to assess the accuracy of HER2 and MSI/MMR status. Results A total of 188 patients were enrolled. In 139 eligible patients, 275 potentially actionable mutations were found in 12 selected CRC-related genes (APC, TP53, KRAS, NRAS, BRAF, FBXW7, PIK3CA, ERBB2, SMAD4, NF1, EGFR, MET). Of these, 32% were found both in ctDNA and tissue; 54% were found in ctDNA only; 12% in tissue only, and 2% were discordant. For RAS/BRAF mutations, which are required for anti-EGFR treatment decisions, the concordance rate between ctDNA and tissue NGS was 83.1%, and the concordance was higher in patients with higher ctDNA concentrations. For 9 patients with potentially actionable copy number variations (CNV) in EGFR, HER2, MET, and FGFR1, 3 cases were found by both assays; 4 were found by ctDNA only, and 2 were found by tissue only. ctDNA NGS correctly predicted MSI/MMR status in 2 out of 4 patients with MSI-H/dMMR; in the 2 other patients, the MSI and MMR statuses were different, suggesting potential false positivity. In addition, the fold changes in ctDNA dynamics during treatment significantly correlated with changes in tumor size and CEA levels, as well as with droplet digital PCR copy number fold changes. In a patient with MET amplification, ctDNA NGS identified MET Y1230H as a potential acquired resistance mutation after crizotinib treatment, which responded to cabozantinib but not to capmatinib. Conclusions Initial tumor profiling using ctDNA NGS yielded outcomes comparable to those of tumor tissue NGS in guiding treatment for patients with newly diagnosed mCRC, thereby suggesting its utility as an initial profiling method in mCRC. Citation Format: Yongjun Cha, Bun Kim, Dong Woon Lee, Hyun Yang Yeo, Chang Won Hong, Kyung Su Han, Byung Chang Kim, Hee Jin Chang. Assessing the clinical value of ctDNA sequencing for initial tumor profiling in metastatic colorectal cancer patients with sufficient tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2498.

Abstract 6570: HLA genotyping and HLA-based clinical trial matching using MSK-IMPACT, a targeted next-generation sequencing assay

Abstract Purpose: Performing tumor next-generation sequencing (NGS) and human leukocyte antigen (HLA) typing in a single test can maximize the likelihood of matching patients with cancer to clinical trials. We investigated the utility of a clinical-grade NGS assay for this purpose. Methods: HLA class I genotypes were determined on an investigational basis by MSK-IMPACT (FDA-authorized hybrid-capture, tumor and matched normal DNA-based NGS) using POLYSOLVER for patients with cancers of any histology between March 2014 and September 2023. Patient HLA genotype matches for phase I clinical trials were identified; for a subset of patients, concordance between HLA genotyping was compared to a CLIA-certified laboratory test. Results: 60,565 patients successfully underwent complete class I HLA genotyping (two alleles each of HLA-A, HLA-B, and HLA-C). The median age at diagnosis was 64 years old and most patients were female (55%) and white (76%). The most common genotypes were HLA-A*02:01 (42%, n=25,457), HLA-C*04:01 (28%, n=17,274), HLA-A*01:01 (25%, n=14,867), HLA-C*07:01 (24%, n=14,538), HLA-A*03:01 (21%, n=12,668), HLA-C*07:02 (21%, n=12,493), and HLA-B*07:02 (16%, n=9,787). Most patients (96%, n=58,287) were HLA genotype eligible [HLA-A*02:01 (n=28), HLA-A*11:01 (n=3), HLA-A*01:01 (n=1), and HLA-C*07:02 (n=1)] for ≥1 of 33 active early phase adoptive T cell therapy or T cell receptor bispecific trials on Clinicialtrials.gov. 280 patients with HLA class I matches were screened for phase I trial candidacy by the MSK phase I group. 117 were candidates for confirmatory HLA and neoantigen testing, of whom 43 patients were enrolled on a trial after additional screening. 72 patients had HLA genotyping both by MSK-IMPACT and an outside CLIA-certified laboratory; 100% (72/72) of these subjects had concordant genotypes. Conclusions: MSK-IMPACT successfully determined class I HLA genotypes in a large, multi-ethnic population of patients. A substantial proportion of patients were identified as potential candidates for HLA-matched trials, supporting local trial enrollment. Citation Format: Monica F. Chen, Michael V. Gormally, Anne Marie Noronha, Kristalla Panageas, Maggie Reynolds, Kaelyn Kohlasch, Bryan Novak, Tatiana Kee-Velez, Nikeysha Clarke, Chrisann Kyi, Claire Friedman, Sandra D'Angelo, Mark G. Kris, Christopher A. Klebanoff, Ronak Shah, Michael Berger, Chaitanya Bandlamudi, Alexander Drilon, Mark T. Donoghue. HLA genotyping and HLA-based clinical trial matching using MSK-IMPACT, a targeted next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6570.

Abstract B013: Folate receptor alpha (FRa) expression and correlation with other molecular alterations in high grade serous endometrial cancer (EC)

Abstract Background: Advanced and recurrent EC remains a therapeutic challenge with rising mortality rates and few options beyond first line platinum-based chemotherapy and immunotherapy. Mirvetuximab soravtansine (MIRV), an antibody drug conjugate (ADC) comprising a FRα-binding antibody, cleavable linker, and maytansinoid payload DM4, is FDA-approved for patients with FRa-high platinum resistant ovarian cancer (PROC). FRa is also expressed in EC and is associated with poor prognosis. Here, we evaluate FRa expression levels and associations with other molecular alterations, including in HER2, MYC and CCNE1, in patients with advanced/recurrent serous EC. Methods: Paraffin fixed tumor tissue from 110 patients who signed prescreening consent for a phase 2 study testing the combination of MIRV with pembrolizumab in recurrent/persistent serous EC (NCT03835819) were analyzed for FRα expression by IHC (Ventana, Inc). All patients had advanced or recurrent MSS serous EC and had received 1-3 prior lines of therapy. FRα positivity was defined as ≥50% of cells with ≥2+ membrane staining based on results from a phase 1b study of mirv and pembrolizumab in PROC (Matulonis U, SGO 2018 abstract 74). HER2/neu expression was assessed by independent pathology review at the treating institution. Tumor mutational burden (TMB), amplification of CCNE1, MYC and ERBB2 and other somatic mutations were determined in tumors with next generation sequencing (NGS). Results: 37 of 110 tumors were determined to be FRa positive (FRa+) with FRa ≥50% for an overall positivity rate of 33.63%. The median membrane positivity was 20.5% (IQR: 3-51.25). Within a cohort of 44 patients with available tumor NGS, the average TMB was 6.23 (SD: 3.34) and did not differ between FRα+ and -negative (FRα-) tumors at 5.74 and 6.45, respectively (p=0.73). Of 32 tumors with HER2 IHC performed, 5 (16%) were both FRα+ and HER2+ (2+ or 3+); 7 (22%) were FRα+ and HER2-, 5 (16%) were FRα- and HER2+, and 15 (47%) had negative expression for both. CCNE1, MYC and ERBB2 amplifications were present in 1 (9%), 0 and 3 (27%) of FRα+ tumors, and 6 (23%), (8) 31%, and 2 (8%) and of FRα- tumors, respectively. There was no association between expression of FRα and HER2 (Fisher exact test, p=0.44), nor with amplification of CCNE1 (p=0.65), MYC (p=0.08) and ERBB2 (p=0.14). The most common co-occurring somatic mutations were in PIK3CA, with similar frequency in FRα+ (55%) and FRα- (42%) tumors. Conclusions: An FRα positivity rate of 33.6% in this large cohort of serous EC tumors supports further clinical development of MIRV in this high-risk subtype of EC. Within a limited cohort subset, it does not appear that FRα expression correlates with TMB or HER2 expression, or with CCNE1, MYC or ERBB2 amplifications. This data highlights the importance of comprehensive testing of EC tumors for all relevant biomarkers that may guide selection of targeted therapies. Additional studies are needed to explore FRα expression in other molecular subsets of EC and to determine the longitudinal stability of ADC target expression. Citation Format: Rebecca L. Porter, Sandy Elias, Niya Xiong, Madeline Polak, Kayla Fahey, Nabihah Tayob, Courtney Qi, Carina Feeney, Swati Narayan, Jennifer Curtis, Susana Campos, Carolyn Krasner, Elizabeth Lee, Elizabeth Stover, Alexi A. Wright, Joyce F. Liu, Ursula A. Matulonis, Panagiotis Konstantinopoulos. Folate receptor alpha (FRa) expression and correlation with other molecular alterations in high grade serous endometrial cancer (EC) [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B013.

Machine Learning Methods for Gene Selection in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy with a limited five-year survival for metastatic patients. Limited therapeutic treatments are currently available for metastatic disease, even if the genomics of this tumor has been deeply studied using next-generation sequencing (NGS) and functional experiments. The profound knowledge of the molecular features that characterize this tumor has not led to the development of efficacious therapies, and the survival of metastatic patients has not changed for decades. Several bioinformatics methods have been applied to mine NGS tumor data in order to unveil tumor biology and detect possible molecular targets for new therapies. Each application can be single domain based while others are more focused on data integration from multiple genomics domains (as gene expression and methylation data). Examples of single domain approaches include differentially expressed gene (DEG) analysis on gene expression data with statistical methods such as SAM (significance analysis of microarray) or gene prioritization with complex algorithms such as deep learning. Data fusion or integration methods merge multiple domains of information to define new clusters of patients or to detect relevant genes, according to multiple NGS data. In this work, we compare different strategies to detect relevant genes for metastatic disease prediction in the TCGA uveal melanoma (UVM) dataset. Detected targets are validated with multi-gene score analysis on a larger UM microarray dataset.

An analysis pipeline for understanding 6-thioguanine effects on a mouse tumour genome

Mouse tumour models are extensively used as a pre-clinical research tool in the field of oncology, playing an important role in anticancer drugs discovery. Accordingly, in cancer genomics research, the demand for next-generation sequencing (NGS) is increasing, and consequently, the need for data analysis pipelines is likewise growing. Most NGS data analysis solutions to date do not support mouse data or require highly specific configuration for their use. Here, we present a genome analysis pipeline for mouse tumour NGS data including the whole-genome sequence (WGS) data analysis flow for somatic variant discovery, and the RNA-seq data flow for differential expression, functional analysis and neoantigen prediction. The pipeline is based on standards and best practices and integrates mouse genome references and annotations. In a recent study, the pipeline was applied to demonstrate the efficacy of low dose 6-thioguanine (6TG) treatment on low-mutation melanoma in a pre-clinical mouse model. Here, we further this study and describe in detail the pipeline and the results obtained in terms of tumour mutational burden (TMB) and number of predicted neoantigens, and correlate these with 6TG effects on tumour volume. Our pipeline was expanded to include a neoantigen analysis, resulting in neopeptide prediction and MHC class I antigen presentation evaluation. We observed that the number of predicted neoepitopes were more accurate indicators of tumour immune control than TMB. In conclusion, this study demonstrates the usability of the proposed pipeline, and suggests it could be an essential robust genome analysis platform for future mouse genomic analysis.

Benchmarking mismatch repair testing for patients with cancer receiving immunotherapy.

179 Background: Mismatch repair deficiency (MMR-D) serves as a theranostic marker for directing immune checkpoint inhibitor (ICI) treatment across various cancer stages and tumor types. The College of American Pathologists endorses immunohistochemistry (IHC) as the primary approach for assessing mismatch repair (MMR) testing in colorectal (CRC) and endometrial cancer (EC) patients. An alternative method involves genetic evaluation of microsatellite instability (MSI). Previous studies have shown that resistance to immune checkpoint inhibitors (ICIs) may occur due to inaccurate MSI or MMR IHC status determination. Accurate assessment of MMR status is critical for treatment decision-making. Methods: We hypothesized that tumor-only next generation sequencing (NGS) for MMR mutation signature assessment for identification of MMR-D tumors is more sensitive than MMR IHC testing. Using a standardized, clinically validated, tumor-only NGS mutation signature pipeline in place in the Oncopanel assay at DFCI/BWH, we compared the concordance in MMR determination between NGS and IHC. We then evaluated a subset of ICI-treated patients to assess the clinical implications of these findings. Results: 1655 colorectal cancer (CRC) and endometrial carcinoma (EC) patients who had both NGS MSI (NGSd = deficient or NGSp = proficient) and MMR IHC (IHC- = absent or IHC+ = intact) assays performed were assessed. Discordant NGSd/IHC+ results were identified in 1% of CRC and 5% of EC cases that were IHC+. Importantly, patients with discordant NGSd/IHC+ (median OS: not reached [NR], 95% CI, 9.9 months-NR; median TTF: 44.4 months, 95% CI, 4.1-NR) and concordant NGSd/IHC- results, showed a similar overall survival and time to treatment failure with ICI treatment (p=non-significant) which contrasted with results for patients with concordant NGSp/IHC+ results (median OS: 14.9 months, 95% CI, 7.3-24.6, p-value 0.0025 and < 0.0001; median TTF: 5.0 months, 95% CI, 3.2-7.3; p-value 0.0014 and < 0.0001). Patients with EC or CRC and discordant NGSd/IHC+ results had better survival when treated with ICI-based regimens, compared to those who received non-ICI treatments (p= 0.054 and 0.017). Conclusions: In 2022, 151,030 new CRC and 65,950 new EC cases were diagnosed in the USA. Extrapolating our findings to this larger population, use of NGS mutational signature analysis instead of IHC would amount to about 1510 additional MMR-D CRC cases and 3297 additional MMR-D endometrial cases identified each year for whom first-line ICI therapy is preferred. Our findings support revisiting guideline recommendations for diagnostic testing of MMR to incorporate both IHC and targeted NGS tumor panel testing using sensitive and specific mutation signature calling algorithms. Larger prospective studies are warranted to confirm these findings in patients with mCRC and mEC treated with ICIs. Evaluation of this approach in other cancer types is also warranted.

Abstract B007: Concordance between tumor tissue and plasma genotyping in NCI-MATCH Trial (EAY131)

Abstract Background: Utilization of liquid biopsy has become increasingly prevalent in the field of cancer therapeutics over the past decade. However, there remain unanswered questions that need to be addressed including better understanding of concordance between tumor tissue and plasma-derived circulating tumor DNA (ctDNA) from the same patient. In this study, we present the results of plasma ctDNA analysis by next-generation sequencing (NGS) in patients participating in selected arms of the NCI-MATCH Trial, with a focus on concordance between tumor tissue and plasma findings. Methods: Our study included patients treated on the NCI-MATCH Trial (NCT02465060) who had their plasma samples collected within 90 days before commencing treatment. 16 out of 39 treatment subprotocols of NCI-MATCH Trial were included in this plasma analysis protocol. Plasma samples were tested using the Guardant360 NGS assay (Guardant Health) and tumor tissue NGS results were obtained through central testing by the Oncomine assay (ThermoFisher). Concordance was defined as the detection of the same gene alteration leading to patient enrollment in NCI-MATCH trial, in both tissue and plasma. Results: Our study included 334 patients with a variety of tumor types who had baseline plasma samples. Plasma sequencing data were available in 328 patients; of whom 326 (99.4%) had a total of 2,779 alterations in plasma (2,055 somatic [74%] and 724 presumed germline [26%]). The tissue alteration of interest was detected in plasma of 196 (76.9%) of 255 patients enrolled based on central lab testing who had contemporaneous plasma and tissue samples. The concordance rate was higher in patients with tissue biopsies obtained from metastatic sites compared to patients with tissue biopsies obtained from primary tumor (79.9% [n=183] vs 45.5% [n=10], p<0.001). Concordance was highest for copy number variations (82.5%, n=52), single nucleotide variants (79.5%, n=124) and insertions/deletions (75%, n=12), while lower concordance rates were found for gene fusions (40%, n=8). A lower concordance rate was observed in 73 patients enrolled based on outside designated lab tissue testing (43.8% [n=32]). Notably, these patients had a median interval between plasma and tissue samples of 239 days (range; 38-1917). However, concordance was also higher in this group with samples collected from metastatic sites compared to primary tumors (62.5% vs 26.5%, p=0.003). Conclusions: In the NCI-MATCH Trial, the tumor tissue alteration of interest was detected in plasma of 76.9% of patients who had contemporaneous tissue and plasma samples, suggesting a potential role for liquid biopsy in patients’ enrollment. Concordance rates varied according to type of alteration of interest, and site of tissue sample (primary versus metastatic tumor), as well as the interval between sampling of tissue and plasma. Citation Format: Mohamed A Gouda, Filip Janku, Ying Yuan, Leylah Drusbosky, Alice P Chen, Keyur Patel, Stanley R Hamilton, Mark Routbort, P. Mickey Williams, John Iafrate, Jeffrey Sklar, Brent Coffey, Richard F Little, Carlos L Arteaga, Jeffrey S Abrams, Peter J O’Dwyer, Keith T Flaherty, Lyndsay N Harris, Funda Meric-Bernstam. Concordance between tumor tissue and plasma genotyping in NCI-MATCH Trial (EAY131) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B007.

Abstract B169: Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A

Abstract Background: Lunresertib (RP-6306) is a first-in-class membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) inhibitor that disrupts the G2/M checkpoint leading to premature mitosis and catastrophic DNA damage in cells harboring synthetic lethal genomic alterations. The safety and tolerability of lunresertib alone and in combination with camonsertib (RP-3500) is being investigated in the Phase 1 MYTHIC study in patients with advanced solid tumors with either CCNE1 amplifications (amp) or deleterious alterations in FBXW7 or PPP2R1A (NCT04855656). Here, we present a comprehensive retrospective biomarker analysis aimed at understanding concordance between local vs. central and tissue- vs. plasma-based next generation sequencing (NGS). The correlation between CCNE1 copy number assessment by NGS and fluorescence in situ hybridization (FISH), as well as the relationship between CCNE1 amp and Cyclin E1 protein levels, was investigated. Methods: Patients were enrolled based on results from sponsor-approved local tumor NGS and plasma circulating tumor DNA (ctDNA) tests. To ensure molecular eligibility criteria were met, local NGS reports were centrally reviewed and functionally annotated prior to enrollment. Tumor tissue and plasma samples collected at screening were retrospectively analyzed by SNiPDx™ and Tempus xF(+), respectively. Tissue samples were further evaluated by FISH and immunohistochemistry. Results: As of June 1, 2023, 145 patients were screened with uterine (23%), colorectal (CRC, 15%), ovarian (13%), and upper GI (6%) as the most frequent cancers. Ninety-four percent (137/145) of patients screened had local NGS results available (98% tissue, 2% plasma). CCNE1 amp (40.1%) were most frequently observed in ovarian, uterine, and upper GI malignancies. FBXW7 (39.4%) and PPP2R1A (16.0%) alterations were enriched in CRC and uterine cancer, respectively. TP53 alterations were frequent across all biomarker groups (76% CCNE1, 54% FBXW7, and 88% PPP2R1A). Central tissue NGS confirmed 94% (47/50) of the enrollment alterations. Central plasma ctDNA confirmed 83.0% (39/47) of FBXW7 and PPP2R1A mutations and had high positive predictive value (100%, 17/17) but low sensitivity (42.5%; 17/40) for CCNE1 amp. CCNE1 amp call and copy number estimation by tissue NGS and FISH were highly correlated (Spearman ρ=0.65, p=0.006). Seventy-five percent of tumors with CCNE1 amp had Cyclin E1 overexpression (H-score ≥100; mean=168; range [0, 300]). Conclusion: In this study, local NGS tests were an efficient and robust strategy for directing patients into a molecularly defined Phase 1 trial. Plasma ctDNA reliably detected alterations in all 3 genes, but caution should be taken when interpreting CCNE1 amplification negative results due to a high false negative rate. FISH and genomics are appropriate methods to estimate CCNE1 amp copy number. These retrospective analyses enable the testing of the synthetic lethal hypothesis of lunresertib and CCNE1, PPP2R1A and FBXW7, assuring reliable interpretation of the clinical data. Citation Format: Elia Aguado-Fraile, Sunantha Sethuraman, Adam Petrone, Emeline Bacque, Alison Schram, Stephanie Lheureux, Elizabeth K Lee, Maria Koehler, Ian M Silverman, Victoria Rimkunas, Timothy A Yap. Retrospective baseline biomarker analyses in a first-in-human Phase 1 trial of the PKMYT1 inhibitor lunresertib (RP-6306) in pts with advanced solid tumors harboring CCNE1 amplification and/or deleterious alterations in FBXW7 or PPP2R1A [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B169.

FindDNAFusion: An Analytical Pipeline with Multiple Software Tools Improves Detection of Cancer-Associated Gene Fusions from Genomic DNA

Detection of cancer-associated gene fusions is crucial for diagnosis, prognosis, and treatment selection. Many bioinformatics tools are available for the detection of fusion transcripts by RNA sequencing, but there are fewer well-validated software tools for DNA next-generation sequencing (NGS). A 542-gene solid tumor NGS panel was designed, with exonic probes supplemented with intronic bait probes against genes commonly involved in oncogenic fusions, with a focus on lung cancer. Three software tools for the detecting gene fusions in this DNA-NGS panel were selected and evaluated. The performance of these tools was compared after a pilot study, and each was configured for optimal batch analysis and detection rate. A blacklist for filtering common tool-specific artifacts, and criteria for selecting clinically reportable fusions, were established. Visualization tools for annotating and confirming somatic fusions were applied. Subsequently, a full clinical validation was used for comparing the results to those from in situ hybridization and/or RNA sequencing. With JuLI, Factera, and GeneFuse, 94.1%, 88.2%, and 66.7% of expected fusions were detected, respectively. With a combinatorial pipeline (termed FindDNAFusion), developed by integrating fusion-calling tools with methods for fusion filtering, annotating, and flagging reportable calls, the accuracy of detection of intron-tiled genes was improved to 98.0%. FindDNAFusion is an accurate and efficient tool in detecting somatic fusions in DNA-NGS panels with intron-tiled bait probes when RNA is unavailable.

Targeted Molecular Profiling of Circulating Cell-Free DNA in Patients With Advanced Hepatocellular Carcinoma.

Next-generation sequencing (NGS) of tumor-derived, circulating cell-free DNA (cfDNA) may aid in diagnosis, prognostication, and treatment of patients with hepatocellular carcinoma (HCC). The operating characteristics of cfDNA mutational profiling must be determined before routine clinical implementation. This was a single-center, retrospective study with the primary objective of defining genomic alterations in circulating cfDNA along with plasma-tissue genotype agreement between NGS of matched tumor samples in patients with advanced HCC. cfDNA was analyzed using a clinically validated 129-gene NGS assay; matched tissue-based NGS was analyzed with a US Food and Drug Administration-authorized NGS tumor assay. Fifty-three plasma samples from 51 patients with histologically confirmed HCC underwent NGS-based cfDNA analysis. Genomic alterations were detected in 92.2% of patients, with the most commonly mutated genes including TERT promoter (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%), and TSC2 (14%). In total, 37 (73%) patients underwent paired tumor NGS, and concordance was high for mutations observed in patient-matched plasma samples: TERT (83%), TP53 (94%), CTNNB1 (92%), ARID1A (100%), and TSC2 (71%). In 10 (27%) of 37 tumor-plasma samples, alterations were detected by cfDNA analysis that were not detected in the patient-matched tumors. Potentially actionable mutations were identified in 37% of all cases including oncogenic/likely oncogenic alterations in TSC1/2 (18%), BRCA1/2 (8%), and PIK3CA (8%). Higher average variant allele fraction was associated with elevated alpha-fetoprotein, increased tumor volume, and no previous systemic therapy, but did not correlate with overall survival in treatment-naïve patients. Tumor mutation profiling of cfDNA in HCC represents an alternative to tissue-based genomic profiling, given the high degree of tumor-plasma NGS concordance; however, genotyping of both blood and tumor may be required to detect all clinically actionable genomic alterations.

Machine learning models-based on integration of next-generation sequencing testing and tumor cell sizes improve subtype classification of mature B-cell neoplasms.

Next-generation sequencing (NGS) panels for mature B-cell neoplasms (MBNs) are widely applied clinically but have yet to be routinely used in a manner that is suitable for subtype differential diagnosis. This study retrospectively investigated newly diagnosed cases of MBNs from our laboratory to investigate mutation landscapes in Chinese patients with MBNs and to combine mutational information and machine learning (ML) into clinical applications for MBNs, especially for subtype classification. Samples from the Catalogue Of Somatic Mutations In Cancer (COSMIC) database were collected for ML model construction and cases from our laboratory were used for ML model validation. Five repeats of 10-fold cross-validation Random Forest algorithm was used for ML model construction. Mutation detection was performed by NGS and tumor cell size was confirmed by cell morphology and/or flow cytometry in our laboratory. Totally 849 newly diagnosed MBN cases from our laboratory were retrospectively identified and included in mutational landscape analyses. Patterns of gene mutations in a variety of MBN subtypes were found, important to investigate tumorigenesis in MBNs. A long list of novel mutations was revealed, valuable to both functional studies and clinical applications. By combining gene mutation information revealed by NGS and ML, we established ML models that provide valuable information for MBN subtype classification. In total, 8895 cases of 8 subtypes of MBNs in the COSMIC database were collected and utilized for ML model construction, and the models were validated on the 849 MBN cases from our laboratory. A series of ML models was constructed in this study, and the most efficient model, with an accuracy of 0.87, was based on integration of NGS testing and tumor cell sizes. The ML models were of great significance in the differential diagnosis of all cases and different MBN subtypes. Additionally, using NGS results to assist in subtype classification of MBNs by method of ML has positive clinical potential.

Molecular residual disease detection in resected, muscle-invasive urothelial cancer with a tissue-based comprehensive genomic profiling-informed personalized monitoring assay.

Circulating tumor DNA (ctDNA) detection postoperatively may identify patients with urothelial cancer at a high risk of relapse. Pragmatic tools building off clinical tumor next-generation sequencing (NGS) platforms could have the potential to increase assay accessibility. We evaluated the widely available Foundation Medicine comprehensive genomic profiling (CGP) platform as a source of variants for tracking of ctDNA when analyzing residual samples from IMvigor010 (ClinicalTrials.gov identifier NCT02450331), a randomized adjuvant study comparing atezolizumab with observation after bladder cancer surgery. Current methods often involve germline sampling, which is not always feasible or practical. Rather than performing white blood cell sequencing to filter germline and clonal hematopoiesis (CH) variants, we applied a bioinformatic approach to select tumor (non-germline/CH) variants for molecular residual disease detection. Tissue-informed personalized multiplex polymerase chain reaction-NGS assay was used to detect ctDNA postsurgically (Natera). Across 396 analyzed patients, prevalence of potentially actionable alterations was comparable with the expected prevalence in advanced disease (13% FGFR2/3, 20% PIK3CA, 13% ERBB2, and 37% with elevated tumor mutational burden ≥10 mutations/megabase). In the observation arm, 66 of the 184 (36%) ctDNA-positive patients had shorter disease-free survival [DFS; hazard ratio (HR) = 5.77; 95% confidence interval (CI), 3.84-8.67; P < 0.0001] and overall survival (OS; HR = 5.81; 95% CI, 3.41-9.91; P < 0.0001) compared with ctDNA-negative patients. ctDNA-positive patients had improved DFS and OS with atezolizumab compared with those in observation (DFS HR = 0.56; 95% CI, 0.38-0.83; P = 0.003; OS HR = 0.66; 95% CI, 0.42-1.05). Clinical sensitivity and specificity for detection of postsurgical recurrence were 58% (60/103) and 93% (75/81), respectively. We present a personalized ctDNA monitoring assay utilizing tissue-based FoundationOne® CDx CGP, which is a pragmatic and potentially clinically scalable method that can detect low levels of residual ctDNA in patients with resected, muscle-invasive bladder cancer without germline sampling.

Pan-Cancer Pharmacogenomic Analysis of Patient-Derived Tumor Cells Using Clinically Relevant Drug Exposures.

As a result of tumor heterogeneity and solid cancers harboring multiple molecular defects, precision medicine platforms in oncology are most effective when both genetic and pharmacologic determinants of a tumor are evaluated. Expandable patient-derived xenograft (PDX) mouse tumor and corresponding PDX culture (PDXC) models recapitulate many of the biological and genetic characteristics of the original patient tumor, allowing for a comprehensive pharmacogenomic analysis. Here, the somatic mutations of 23 matched patient tumor and PDX samples encompassing four cancers were first evaluated using next-generation sequencing (NGS). 19 antitumor agents were evaluated across 78 patient-derived tumor cultures using clinically relevant drug exposures. A binarization threshold sensitivity classification determined in culture (PDXC) was used to identify tumors that best respond to drug in vivo (PDX). Using this sensitivity classification, logic models of DNA mutations were developed for 19 antitumor agents to predict drug response. We determined that the concordance of somatic mutations across patient and corresponding PDX samples increased as variant allele frequency increased. Notable individual PDXC responses to specific drugs, as well as lineage-specific drug responses were identified. Robust responses identified in PDXC were recapitulated in vivo in PDX-bearing mice and logic modeling determined somatic gene mutation(s) defining response to specific antitumor agents. In conclusion, combining NGS of primary patient tumors, high-throughput drug screen using clinically relevant doses, and logic modeling, can provide a platform for understanding response to therapeutic drugs targeting cancer.

Review article: new treatments for advanced differentiated thyroid cancers and potential mechanisms of drug resistance.

The treatment of advanced, radioiodine refractory, differentiated thyroid cancers (RR-DTCs) has undergone major advancements in the last decade, causing a paradigm shift in the management and prognosis of these patients. Better understanding of the molecular drivers of tumorigenesis and access to next generation sequencing of tumors have led to the development and Food and Drug Administration (FDA)-approval of numerous targeted therapies for RR-DTCs, including antiangiogenic multikinase inhibitors, and more recently, fusion-specific kinase inhibitors such as RET inhibitors and NTRK inhibitors. BRAF + MEK inhibitors have also been approved for BRAF-mutated solid tumors and are routinely used in RR-DTCs in many centers. However, none of the currently available treatments are curative, and most patients will ultimately show progression. Current research efforts are therefore focused on identifying resistance mechanisms to tyrosine kinase inhibitors and ways to overcome them. Various novel treatment strategies are under investigation, including immunotherapy, redifferentiation therapy, and second-generation kinase inhibitors. In this review, we will discuss currently available drugs for advanced RR-DTCs, potential mechanisms of drug resistance and future therapeutic avenues.