Tumor-only Sequencing Research Articles

Clinical Utility of Tumor Next-Generation Sequencing Panel Testing to Inform Treatment Decisions for Patients With Advanced Solid Tumors in a Tertiary Care Center.

There is limited information about the clinical utility of targeted next-generation sequencing (NGS) panel testing to inform decision making for patients with advanced solid tumors. The Ontario-wide Cancer Targeted Nucleic Acid Evaluation (OCTANE) is a prospective study that enrolled more than 4,500 patients with solid tumor for NGS panel testing. We performed a retrospective survey of medical oncologists to evaluate the impact of NGS testing on treatment decisions. Patients and treating oncologists were identified at the Princess Margaret Cancer Center between 2016 and 2021. Tumor-only sequencing was performed using a gene panel of either 555 or 161 cancer genes. Oncologists were asked to review testing results and complete a survey indicating whether NGS testing affected treatment decisions. The primary outcome of this study was rate of treatment change on the basis of mutation results. Patient, test, and physician factors were evaluated for association with treatment changes using univariate analyses and a mixed-effects model. Of the 582 surveys sent, 394 (67.7%) were completed. We found that 188 (47.7%) patients had testing results classified as actionable by the oncologist and 62 (15.7%) patients were matched to treatment, of whom 37 (60%) were enrolled in a clinical trial, 13 (21%) received an approved drug, four (6%) were prescribed off-label therapy, and eight (13%) avoided ineffective treatment. Patient, test, and physician characteristics were not significantly associated with treatment change. There was no difference in overall survival between patients who received matched treatment versus those who did not (P = .55, median survival not reached). OCTANE testing led to a change in drug treatment in 15.7% of patients, supporting the clinical utility of NGS panel testing for patients with advanced solid tumors.

Limited Independent Follow-Up with Germline Testing of Variants Detected in BRCA1 and BRCA2 by Tumor-Only Sequencing.

Genomic profiling is performed in patients with advanced or metastatic cancer, in order to direct cancer treatment, often sequencing tumor-only, without a matched germline comparator. However, because many of the genes analyzed on tumor profiling overlap with those known to be associated with hereditary cancer predisposition syndromes (HCPS), tumor-only profiling can unknowingly uncover germline pathogenic (P) and likely pathogenic variants (LPV). In this study, we evaluated the number of patients with P/LPVs identified in BRCA1 and BRCA2 (BRCA1/2) via tumor-only profiling, then determined the germline testing outcomes for those patients. A retrospective chart review was performed to identify patients with BRCA1/2 variants on tumor-only genomic profiling, and whether they had germline testing. This study found that of 2923 patients with 36 tumor types who underwent tumor-only testing, 554 had a variant in BRCA1/2 (19.0%); 119 of the 554 patients (21.5%) had a P/LP BRCA1/2 variant, representing 4.1% of the overall population who underwent genomic profiling. Seventy-three (61.3%) of 119 patients with BRCA1/2 P/LPV on tumor-only testing did not undergo germline testing, 34 (28.6%) had already had germline testing before tumor-only testing, and 12 (10.1%) underwent germline testing after tumor-only testing. Twenty-eight germline BRCA1/2 P/LPVs were detected, 24 in those who had prior germline testing, and 4 among the 12 patients who had germline testing after tumor-only testing. Tumor-only testing is likely to identify P/LPVs in BRCA1/2. Efforts to improve follow-up germline testing is needed to improve identification of germline BRCA1/2 alterations.

Prevalence of BRCA1, BRCA2, and PALB2 genomic alterations among 924 Taiwanese breast cancer assays with tumor-only targeted sequencing: extended data analysis from the VGH-TAYLOR study

BackgroundThe homologous recombination (HR) repair pathway for DNA damage, particularly the BRCA1 and BRCA2 genes, has become a target for cancer therapy, with poly ADP-ribose polymerase (PARP) inhibitors showing significant outcomes in treating germline BRCA1/2 (gBRCA1/2) mutated breast cancer. Recent studies suggest that some patients with somatic BRCA1/2 (sBRCA1/2) mutation or mutations in HR-related genes other than BRCA1/2 may benefit from PARP inhibitors as well, particularly those with PALB2 mutations. The current analysis aims to evaluate the prevalence of genetic alterations specific to BRCA1, BRCA2, and PALB2 in a large cohort of Taiwanese breast cancer patients through tumor-targeted sequencing.MethodsA total of 924 consecutive assays from 879 Taiwanese breast cancer patients underwent tumor-targeted sequencing (Thermo Fisher Oncomine Comprehensive Assay v3). We evaluated BRCA1, BRCA2, and PALB2 mutational profiles, with variants annotated and curated by the ClinVAR, the Oncomine™ Knowledgebase Reporter, and the OncoKB™. We also conducted reflex germline testing using either whole exome sequencing (WES) or whole genome sequencing (WGS), which is ongoing.ResultsAmong the 879 patients analyzed (924 assays), 130 had positive mutations in BRCA1 (3.1%), BRCA2 (8.6%), and PALB2 (5.2%), with a total of 14.8% having genetic alterations. Co-occurrence was noted between BRCA1/BRCA2, BRCA1/PALB2, and BRCA2/PALB2 mutations. In BRCA1-mutated samples, only p.K654fs was observed in three patients, while other variants were observed no more than twice. For BRCA2, p.N372H was the most common (26 patients), followed by p.S2186fs, p.V2466A, and p.X159_splice (5 times each). For PALB2, p.I887fs was the most common mutation (30 patients). This study identified 176 amino acid changes; 60.2% (106) were not documented in either ClinVAR or the Oncomine™ Knowledgebase Reporter. Using the OncoKB™ for annotation, 171 (97.2%) were found to have clinical implications. For the result of reflex germline testing, three variants (BRCA1 c.1969_1970del, BRCA1 c.3629_3630del, BRCA2 c.8755-1G > C) were annotated as Pathogenic/Likely pathogenic (P/LP) variants by ClinVar and as likely loss-of-function or likely oncogenic by OncoKB; while one variant (PALB2 c.448C > T) was not found in ClinVar but was annotated as likely loss-of-function or likely oncogenic by OncoKB.ConclusionOur study depicted the mutational patterns of BRCA1, BRCA2, and PALB2 in Taiwanese breast cancer patients through tumor-only sequencing. This highlights the growing importance of BRCA1/2 and PALB2 alterations in breast cancer susceptibility risk and the treatment of index patients. We also emphasized the need to meticulously annotate variants in cancer-driver genes as well as actionable mutations across multiple databases.

Somatic Mutation Screening in Adult Patients with Myeloid Malignancies Provides Insights into Likely Germline Variants

Introduction Emerging evidence has shed light on the prevalence of germline variants (GV) that increase the risk of myeloid neoplasms in adults. Despite the use of next-generation sequencing (NGS) in the diagnostic workup of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), establishing a standard-of-care approach to screening patients for GV has been difficult due to a rapidly evolving list of candidate variants and inconsistent referral patterns for confirmatory testing. Here we describe our efforts to characterize molecular features at diagnosis and in remission based on tumor-only sequencing for patients with possible germline predisposition syndromes to myeloid neoplasms. Methods We retrospectively analyzed molecular pathology records of adult patients treated at the University of Pennsylvania who were diagnosed with AML and/or MDS between 2013 to 2022, had somatic NGS performed with diagnostic bone marrow biopsy, and had identified variants in genes affiliated with known or emerging MDS/AML germline predisposition syndromes. These variants (Fig. 1A) were based on those defined in ELN 2022 as well curated from the hereditary hematologic malignancy panel at the Children's Hospital of Philadelphia . Analysis focused on pathogenic and likely pathogenic variants. Results We reviewed records from 364 patients with a diagnosis of AML (n=311), MDS or MDS/MPN overlap (n=48), or other myeloid neoplasm (n=5). Median age of diagnosis was 66 years (range 21-92 years) with 11% <40 years and 15% <50 years; 56% were male, and 44% were female. Data on race and ethnicity were collected for 331 patients, which reflected demographics typically seen at our center: 86% White, 13% Black, 1% Asian/Pacific Islander, and 4% identified as Hispanic/Latino. Family history data were collected for 316 patients: 13% with hematologic malignancy and/or unexplained cytopenias, 50% with solid tumors, and 2% with a previously known germline predisposition syndrome. 31% of patients had a prior personal history of a non-myeloid cancer. Possible germline-associated variants in our cohort were most frequently seen in TP53 (n=93), RUNX1 (n=49), KRAS (n=48), and PTPN11 (n=47). Only 5 patients had undergone confirmatory exome sequencing via fibroblast culture (2 DDX41, 1 GATA2, 1 TP53, 1 CBL), so we considered patients with a baseline variant allele frequency (VAF) of 30-60% (n=161) on diagnostic studies to have possible GV (PGV) (Fig. 1A). Of these patients, 125 received frontline treatment with intensive chemotherapy (49%), HMA/venetoclax (29%), or HMA or other low-intensity therapy (23%). NGS was performed in remission for 53 patients, of whom 22 (42%) patients had persistence of their previously identified variant, which we classified as likely GV (LGV); 20/22 patients with LGV had persistence of germline-associated VAF >40% in remission. Approximately 6% of patients were <40 years of age in the PGV and LGV groups. The most frequent LGV were DDX41 (18%), CEBPA (14%), NF1 (9%), BRCA1 (9%), and RUNX1 (9%). Within the LGV cohort, 31% of patients had complex cytogenetics; 61% of patients were classified with ELN 2022 adverse risk or IPSS-R high/very high-risk disease; 64% of LGV patients had a family or personal history of cancer or unexplained cytopenias. Median OS was 18 months for both the PGV and LGV groups (Fig. 1B). When examining co-occurring somatic mutational patterns in the LGV group, we most frequently observed alterations in canonical AML/MDS-associated genes, including ASXL1, FLT3, IDH1/2, NPM1, TET2, and splicing factors. Conclusions As the acknowledgement of germline predisposition syndromes increases, it is imperative that suspected GV are recognized by laboratories and clinicians so patients can appropriately be referred for genetic counselling and confirmatory testing. Although confirmatory sequencing in unaffected tissue is considered the gold-standard approach for diagnosis, use of other factors like VAF and persistence of mutational burden in remission can help identify patients with LGV. Indeed, in our cohort, 6% of patients were considered to have LGV, of which 64% had a family or personal history of cancer or unexplained cytopenias. Limitations include the evolution of our somatic sequencing panel throughout the time course evaluated, inability to exclude somatic reversion due to somatic mutational screening only, and exclusion of variants of uncertain significance.

Pathogenic Germline Variants in BRCA1/2 and p53 Identified by Real-world Comprehensive Cancer Genome Profiling Tests in Asian Patients.

We analyzed real-world data from over 23,000 patients in Japan, revealing 4.1% harbored PGVs, particularly in BRCA1/2 and TP53, in CSGs. It highlights the prevalence of PGVs in Asian populations and supports the broader adoption of tumor-normal sequencing CGP tests for PGV evaluation.

Genomic analysis of advanced breast cancer tumors from talazoparib-treated gBRCA1/2mut carriers in the ABRAZO study

These analyses explore the impact of homologous recombination repair gene mutations, including BRCA1/2 mutations and homologous recombination deficiency (HRD), on the efficacy of the poly(ADP-ribose) polymerase (PARP) inhibitor talazoparib in the open-label, two-cohort, Phase 2 ABRAZO trial in germline BRCA1/2-mutation carriers. In the evaluable intent-to-treat population (N = 60), 58 (97%) patients harbor ≥1 BRCA1/2 mutation(s) in tumor sequencing, with 95% (53/56) concordance between germline and tumor mutations, and 85% (40/47) of evaluable patients have BRCA locus loss of heterozygosity indicating HRD. The most prevalent non-BRCA tumor mutations are TP53 in patients with BRCA1 mutations and PIK3CA in patients with BRCA2 mutations. BRCA1- or BRCA2-mutated tumors show comparable clinical benefit within cohorts. While low patient numbers preclude correlations between HRD and efficacy, germline BRCA1/2 mutation detection from tumor-only sequencing shows high sensitivity and non-BRCA genetic/genomic events do not appear to influence talazoparib sensitivity in the ABRAZO trial.ClinicalTrials.gov identifier: NCT02034916.

Loss of heterozygosity does not occur in BRCA1/2 mutant pediatric solid and central nervous system tumors.

Utilization of tumor-only sequencing has expanded in pediatric cancer patients, which can lead to identification of pathogenic variants in genes that may be germline and/or have uncertain relevance to the tumor in question, such as the homologous recombination (HR) pathway genes BRCA1/2. We identified patients with pathogenic BRCA1/2 mutations from somatic tumor sequencing, and performed additional germline sequencing to assess for the presence of loss of heterozygosity (LOH). Of seven patients identified, four (57.1%) mutations were found in the germline and none had associated LOH. Our data suggest that BRCA1/2 mutations identified in this context are likely incidental findings.

Germline Variants Incidentally Detected via Tumor-Only Genomic Profiling of Patients With Mesothelioma

Patients with mesothelioma often have next-generation sequencing (NGS) of their tumor performed; tumor-only NGS may incidentally identify germline pathogenic or likely pathogenic (P/LP) variants despite not being designed for this purpose. It is unknown how frequently patients with mesothelioma have germline P/LP variants incidentally detected via tumor-only NGS. To determine the prevalence of incidental germline P/LP variants detected via tumor-only NGS of mesothelioma. A series of 161 unrelated patients with mesothelioma from a high-volume mesothelioma program had tumor-only and germline NGS performed during April 2016 to October 2021. Follow-up ranged from 18 months to 7 years. Tumor and germline assays were compared to determine which P/LP variants identified via tumor-only NGS were of germline origin. Data were analyzed from January to March 2023. The proportion of patients with mesothelioma who had P/LP germline variants incidentally detected via tumor-only NGS. Of 161 patients with mesothelioma, 105 were male (65%), the mean (SD) age was 64.7 (11.2) years, and 156 patients (97%) self-identified as non-Hispanic White. Most (126 patients [78%]) had at least 1 potentially incidental P/LP germline variant. The positive predictive value of a potentially incidental germline P/LP variant on tumor-only NGS was 20%. Overall, 26 patients (16%) carried a P/LP germline variant. Germline P/LP variants were identified in ATM, ATR, BAP1, CHEK2, DDX41, FANCM, HAX1, MRE11A, MSH6, MUTYH, NF1, SAMD9L, and TMEM127. In this case series of 161 patients with mesothelioma, 16% had confirmed germline P/LP variants. Given the implications of a hereditary cancer syndrome diagnosis for preventive care and familial counseling, clinical approaches for addressing incidental P/LP germline variants in tumor-only NGS are needed. Tumor-only sequencing should not replace dedicated germline testing. Universal germline testing is likely needed for patients with mesothelioma.

Alternative allele frequency recallibration after NGS data analysis.

3077 Background: With the spread of NGS in routine clinical practice data analysis methods actively evolve to increase and refine information yield. Not much attention is given to variant allele fraction (VAF) estimation though it impacts clinical insignificance of subclonal somatic variants and identification of variant origin. Methods: Real-world sequencing data of 2379 samples (including 781 tumor-only and 1598 blood-only sequencing datasets) obtained via amplicon-based sequencing was used for the retrospective analysis of variant allele frequency estimation of identified genetic variants employing different methods including ITVC, SiNVICT, Mutect2 and SGA. Original method (AODvAF) utilizing alignment-free algorithm based on wild-type and alternative genome subsequence abundance estimation was developed and compared with the original output from NGS data analysis software. Anticipated VAF estimation was based on analyte type and database annotation. Results: Across 3447 identified potentially germline variations (PGV) VAF estimations provided by ITVC, SiNVICT and SGA were highly correlated (Pearson correlation coefficient [PCC] 1.00), while Mutect2 and AODvAF demonstrated discordant results (Mutect2/AODvAF vs ITVC PCC 0.87/0.82). VAF miscalculation defined by absolute difference between estimated and anticipated VAF of 10% and more was noted for 25%, 26%, 22%, 15% and 7% for ITVC, SiNVICT, SGA, Mutect2 and AODvAF respectively. Across 308 pathogenic BRCA1, BRCA2, PALB2 mutations identified in blood-only sequencing 25, 15, 16, 10 and 5 were characterized by VAF of 30% and lower based on ITVC, SiNVICT, SGA, Mutect2 and AODvAF estimations respectively (54, 49, 33, 51 and 9 for VAF 40% and lower), which may lead to misinterpretation of these variants as post-zygotic. Automatic in-silico annotation of variant origin (germline vs somatic) based on ISOWN was correct for 89%, 91%, 94%, 93% and 98% of these variants. Variant origin for 55 pathogenic variants of genes associated with hereditary cancer syndromes identified in tumor-only sequencing was verified by Sanger sequencing. Cohen's kappa coefficient for variant origin discrimination (Sanger vs in-silico prediction) was 0.91, 0.90, 0.91, 0.94 and 0.97 based on VAF provided by ITVC, SiNVICT, SGA, Mutect2 and AODvAF respectively. Conclusions: Accurate VAF calculation allows precise variant origin identification and impacts interpretation of variants. Developed method allows correction of VAF after NGS data core analysis.

Impact of pathogenic germline BRCA1/2 and PALB2 mutations and tumor aneuploidy in patients with HR+/HER2- metastatic breast cancer treated with CDK4/6 inhibitors.

1075 Background: CDK4/6 inhibitors lack established predictors of efficacy in HR+/HER2- mBC. Pathogenic germline BRCA2 variants, somatic homologous recombination deficiency (HRD) and APOBEC signatures conferred worse survival in small series. Broader tumor DNA panels enable the detection of these and other parameters such as aneuploidy and tumor mutational burden. Methods: Clinical and molecular data from a single institution cohort of CDK4/6i-treated HR+/HER2- mBC pts were analyzed. ECOG PS was 0-1. Clinical variables were collected retrospectively. Primary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor-only sequencing was performed on clinical samples with 161-gene OCAv3, 523-gene TSO500 or 555-gene lab-developed NGS panels. HRD was assessed with the Signature Multivariate Analysis (SigMA) tool using a likelihood-based measure to estimate single base substitution signature 3. Aneuploidy scores were defined as the fraction of covered arms harboring arm-level somatic copy number alterations as determined by ASCETS v.1.1. Results: 153 pts were available for analysis. Median age was 54 y (29–88). 74% were postmenopausal, 28% de novo metastatic and 67% had visceral disease. Palbociclib was used in 80%, ribociclib in 17% and abemaciclib in 3%, mostly as early lines (1L 65%, 2L 22%). Median follow up was 46 mo (7–84) and median time from sample collection to CDK4/6i was 0.6 y (-18.5–+2.6). 76% of samples have been sequenced. The median number of mutations (muts) was 3 (1–32), most commonly in PIK3CA (37%), GATA3 (26%) and TP53 (20%). ESR1, KRAS and ARID1A, but not RB1 muts were numerically enriched in post–CDK4/6i mets (n=8). TP53 (HR 1.85 CI 1.02–3.33) and GATA3 (HR 0.48, CI 0.28–0.83) muts were associated with PFS. Germline ( g) testing was available in 68 (44%) pts, with 21 pts carrying pathogenic variants including 11 BRCA2, 2 BRCA1 and 2 PALB2. Cox regression showed a strong negative impact of gBRCA1/2-PALB2 on PFS (HR 4.0 CI 2.0–7.9) and OS (HR 3.5 CI 1.6–7.7) adjusted for age, visceral disease and prior chemo. In 1L, mPFS/OS was 9.9/28.1 mo for gBRCA1/2-PALB2 carriers, 26.8/NR mo for non-carriers and 23.7/49.2 mo for untested (Cox HR PFS 5.1 CI 2.1-14.0; HR OS 4.0 CI 1.4-11.2). Pts with gATM/ CHEK2/ BRIP1 had a 1L PFS/OS>15/45 mo. Among 36 pts assessed for HRD, 22 were HRDlow and 14 HRDhigh with no significant survival association. 3/4 known gBRCA1/2 carriers were HRDhigh and 7/8 gWT HRDlow. In these pts, higher aneuploidy was associated with shorter PFS and OS (Cox HR PFS 2.0 CI 0.9-4.3, p=0.07; HR OS 2.6 CI 1.0-7.1; p=0.05). Conclusions: Pathogenic gBRCA1/2- PALB2 and higher aneuploidy were associated with shorter survival. Profiling of aneuploidy, HRD, TMB and APOBEC in all pts is ongoing including 55 baseline ctDNA samples and 361 samples from the GENIE BPC cohort. Results will account for FDR, left truncation and optimal cutoffs.

Homologous recombination pathway gene variants identified by tumor-only sequencing assays in lung carcinoma patients.

The homologous recombination (HR) repair pathway plays a key role in double-stranded DNA break repair, and germline HR pathway gene variants are associated with increased risk of several cancers, including breast and ovarian cancer. HR deficiency is also a therapeutically targetable phenotype. Somatic (tumour-only) sequencing was performed on 1,109 cases of lung tumors, and the pathological data were reviewed to filter for lung primary carcinomas. Cases were filtered for variants (disease-associated or of uncertain significance) in 14 HR pathway genes, including BRCA1, BRCA2, and ATM. The clinical, pathological and molecular data were reviewed. Sixty-one HR pathway gene variants in 56 patients with primary lung cancer were identified. Further filtering by variant allele fraction (VAF) of ≥30% identified 17 HR pathway gene variants in 17 patients. ATM gene variants were most the commonly identified (9/17), including two patients with c.7271T>G (p.V2424G), a variant in the germline that is associated with increased familial cancer risk. Four (4/17) patients had a family history of lung cancer, among which three patients had ATM gene variants suspected to be germline in origin. In three other patients with BRCA1/2 or PALB2 gene variants who had undergone germline testing, the variants were confirmed to be germline; lung cancer was the sentinel cancer in two of these patients with a BRCA1 or PALB2 variant. Genomic variants in the HR repair pathway identified in tumor-only sequencing and occurring at higher VAFs (i.e., ≥30%) may suggest a germline origin. Correlating with personal and family history, a subset of these variants is also suggested to be associated with familial cancer risks. Patient age, smoking history and driver mutation status are expected to be a poor screening tool in identifying these patients. Finally, the relative enrichment for ATM variants in our cohort suggests a possible association between ATM mutation and lung cancer risk.

Genomic alterations and self-reported Agent Orange exposure in United States veterans with metastatic prostate cancer.

5068 Background: Agent Orange has been posited as an environmental risk factor for prostate cancer among United States Veterans. Prior studies have shown that Veterans with Agent Orange exposure are diagnosed at a younger age and at more advanced clinical stages. It is unknown if there are differences in somatic alterations among Veterans exposed to Agent Orange. Methods: Genomic alterations were identified from clinical tumor testing conducted from 2019 – 2022 in a large cohort of Veterans with metastatic prostate cancer diagnosed from 2001 – 2022. Primary prostate and metastatic carcinoma tissue specimens were submitted for Foundation Medicine tumor-only sequencing. Baseline demographics, clinical, and genomic alterations data were stratified by Veteran self-reported exposure to Agent Orange in accordance with an institutional review board approved protocol. Results: Of the 2673 Veterans with metastatic prostate cancer who underwent tumor-only genomic testing, 629 reported exposure to Agent Orange and 2044 were not exposed. After verification of military service records of Veterans with self-reported Agent Orange exposure to include only Veterans who served in the Vietnam War between 1962 – 1975 and Korean War between 1967 – 1971, 603 patients with self-reported Agent Orange exposure (22.8%) and 2044 without exposure (77.2%) were included in the analysis. On univariate analysis, TMPRSS2-ERG fusions were significantly more frequent in men who reported exposure to Agent Orange (35.3% vs 28.8%, p<0.01) after multiple hypothesis testing with Benjamini-Hochberg. Agent Orange exposure also correlated to higher rates of androgen receptor ( AR) alterations (12.4% vs 9.5%, p=0.04) and FGFR1/2/3/4 alterations (3.2% vs 1.7%, p=0.02). There were no significant differences in alterations by Agent Orange exposure in TP53, CDK12, ERBB2, EGFR, BRCA, and genes implicated in the DNA damage response and repair (DDR), mismatch repair (MMR), and PTEN/PI3K/AKT pathways. On multivariable analysis controlling for race, age at diagnosis, smoking, secondary malignancies, and environment, only alterations in AR (OR 1.43, p=0.02) remained more frequent in Veterans exposed to Agent Orange. As expected, age at diagnosis (OR 0.98, p <0.001) and Black/African-American race (OR 0.33, p<0.001) were associated with decreased odds of TMPRSS2-ERG alterations. Conclusions: Agent Orange exposure may contribute to tumor somatic alterations in Veterans with prostate cancer. These findings hold potential prognostic and therapeutic implications for U.S. Veterans with metastatic prostate cancer. [Table: see text]

PD21-09 GENOMIC ALTERATIONS IN UNITED STATES VETERANS WITH METASTATIC PROSTATE CANCER AND SELF-REPORTED AGENT ORANGE EXPOSURE

You have accessJournal of UrologyCME1 Apr 2023PD21-09 GENOMIC ALTERATIONS IN UNITED STATES VETERANS WITH METASTATIC PROSTATE CANCER AND SELF-REPORTED AGENT ORANGE EXPOSURE JJ Zhang, Adam Weiner, Lorna Kwan, Sara Ahmed, Michael Kelley, Karim Chamie, Kara Maxwell, and Isla Garraway JJ ZhangJJ Zhang More articles by this author , Adam WeinerAdam Weiner More articles by this author , Lorna KwanLorna Kwan More articles by this author , Sara AhmedSara Ahmed More articles by this author , Michael KelleyMichael Kelley More articles by this author , Karim ChamieKarim Chamie More articles by this author , Kara MaxwellKara Maxwell More articles by this author , and Isla GarrawayIsla Garraway More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003287.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Agent Orange has been posited as an environmental risk factor for prostate cancer among United States Veterans. Prior studies have shown that Veterans with Agent Orange exposure are diagnosed at a younger age and at more advanced clinical stages. It is unknown if there are differences in somatic alterations among Veterans exposed to Agent Orange. METHODS: Genomic alterations were identified from clinical tumor testing conducted from 2019–2022 in a large cohort of Veterans with metastatic prostate cancer diagnosed from 2001–2022. Primary prostate and metastatic carcinoma tissue specimens were submitted for Foundation Medicine tumor-only sequencing. Baseline demographics, clinical, and genomic alterations data were stratified by Veteran self-reported exposure to Agent Orange in accordance with an Institutional Review Board approved protocol. RESULTS: Of the 2673 Veterans with metastatic prostate cancer who underwent tumor-only genomic testing, 629 reported exposure to Agent Orange (23.5%) and 2044 were not exposed. TMPRSS2-ERG fusions were more frequent in men who reported exposure to Agent Orange (35.1% vs 28.8%, p<0.01). Agent Orange exposure also correlated to higher rates of androgen receptor (AR) alterations (12.4% vs 9.5%, p=0.04) and FGFR1/2/3/4 alterations (3.3% vs 1.7%, p=0.01). On multivariable analysis, alterations in AR (OR 1.43, p=0.02), and FGFR1/2/3/4 (OR 1.84, p=0.03) remained more frequent in Veterans exposed to Agent Orange. As expected, age at prostate cancer diagnosis (OR 0.98, p<0.001) and Black/African-American race (OR 0.33, p<0.001) were associated with decreased odds of TMPRSS2-ERG alterations. In addition, current smoking status (OR 1.43, p=0.01) and rural environment (OR 1.33, p<0.01) were also associated with increased odds of a TMPRSS2-ERG fusion. There were no significant differences in alterations by Agent Orange exposure in TP53, CDK12, ERBB2, EGFR, BRCA, and genes implicated in the DNA damage response and repair (DDR), mismatch repair (MMR), and PTEN/PI3K/AKT pathways. CONCLUSIONS: Agent Orange exposure may contribute to tumor genomic alterations in AR and FGFR in prostate cancer. These findings hold potential prognostic and therapeutic implications for U.S. Veterans with metastatic prostate cancer. Source of Funding: NIH (K08CA215312, KNM; 5P50CA092131, R01 PAR-20-077, IPG); US DOD (W81XWH211075, IPG) Prostate Cancer Foundation (PCF17CHAL04, IPG; 20YOUNG02, KNM), Burroughs Wellcome Foundation (1017184, KNM); Basser Center for BRCA at UPENN (KNM); Jean Perkins Foundation (IPG), STOP Cancer Foundation (IPG) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e593 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information JJ Zhang More articles by this author Adam Weiner More articles by this author Lorna Kwan More articles by this author Sara Ahmed More articles by this author Michael Kelley More articles by this author Karim Chamie More articles by this author Kara Maxwell More articles by this author Isla Garraway More articles by this author Expand All Advertisement PDF downloadLoading ...

Probabilistic Mixture Models Improve Calibration of Panel-derived Tumor Mutational Burden in the Context of both Tumor-normal and Tumor-only Sequencing.

Tumor mutational burden (TMB) has been investigated as a biomarker for immune checkpoint blockade (ICB) therapy. Increasingly, TMB is being estimated with gene panel-based assays (as opposed to full exome sequencing) and different gene panels cover overlapping but distinct genomic coordinates, making comparisons across panels difficult. Previous studies have suggested that standardization and calibration to exome-derived TMB be done for each panel to ensure comparability. With TMB cutoffs being developed from panel-based assays, there is a need to understand how to properly estimate exomic TMB values from different panel-based assays. Our approach to calibration of panel-derived TMB to exomic TMB proposes the use of probabilistic mixture models that allow for nonlinear relationships along with heteroscedastic error. We examined various inputs including nonsynonymous, synonymous, and hotspot counts along with genetic ancestry. Using The Cancer Genome Atlas cohort, we generated a tumor-only version of the panel-restricted data by reintroducing private germline variants. We were able to model more accurately the distribution of both tumor-normal and tumor-only data using the proposed probabilistic mixture models as compared with linear regression. Applying a model trained on tumor-normal data to tumor-only input results in biased TMB predictions. Including synonymous mutations resulted in better regression metrics across both data types, but ultimately a model able to dynamically weight the various input mutation types exhibited optimal performance. Including genetic ancestry improved model performance only in the context of tumor-only data, wherein private germline variants are observed. A probabilistic mixture model better models the nonlinearity and heteroscedasticity of the data as compared with linear regression. Tumor-only panel data are needed to properly calibrate tumor-only panels to exomic TMB. Leveraging the uncertainty of point estimates from these models better informs cohort stratification in terms of TMB.

Detection of Biallelic Loss of DNA Repair Genes in Formalin-Fixed, Paraffin-Embedded Tumor Samples Using a Novel Tumor-Only Sequencing Panel

Patient selection for synthetic lethal-based cancer therapy may be improved by assessment of gene-specific loss of heterozygosity (LOH) and biallelic loss of function (LOF). This report describes SyNthetic lethal Interactions for Precision Diagnostics (SNiPDx), a targeted next-generation sequencing (NGS) panel for detection of LOH and biallelic LOF alterations in 26 target genes focused on DNA damage response pathways, in tumor-only formalin-fixed, paraffin-embedded (FFPE) samples. NGS was performedacross all exons of these 26 genes and encompassed a total of 7632 genome-wide single-nucleotide polymorphisms on genomic DNA from 80 FFPE solid tumor samples. The Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing algorithm was optimized to assess tumor purity and copy number based on heterozygous single-nucleotide polymorphisms. SNiPDx demonstrated high sensitivity (95%) and specificity (91%) for LOH detection compared with whole genome sequencing. Positive agreement with local NGS-based testing in the detection of genetic alterations was 95%. SNiPDx detected 93% of biallelic ATM LOF mutations, 100% of ATM single-nucleotide variants and small insertions/deletions, and 100% of all ATM LOH status events identified by orthogonal NGS-based testing. SNiPDx is a novel, clinically feasible test for analysis of allelic status in FFPE tumor samples, which demonstrated high accuracy when compared with other NGS-based approaches in clinical use.

Concordance Between Genomic Alterations Detected by Tumor and Germline Sequencing: Results from a Tertiary Care Academic Center Molecular Tumor Board.

The majority of tumor sequencing currently performed on cancer patients does not include a matched normal control, and in cases where germline testing is performed, it is usually run independently of tumor testing. The rates of concordance between variants identified via germline and tumor testing in this context are poorly understood. We compared tumor and germline sequencing results in patients with breast, ovarian, pancreatic, and prostate cancer who were found to harbor alterations in genes associated with homologous recombination deficiency (HRD) and increased hereditary cancer risk. We then evaluated the potential for a computational somatic-germline-zygosity (SGZ) modeling algorithm to predict germline status based on tumor-only comprehensive genomic profiling (CGP) results. A retrospective chart review was performed using an academic cancer center's databases of somatic and germline sequencing tests, and concordance between tumor and germline results was assessed. SGZ modeling from tumor-only CGP was compared to germline results to assess this method's accuracy in determining germline mutation status. A total of 115 patients with 146 total alterations were identified. Concordance rates between somatic and germline alterations ranged from 0% to 85.7% depending on the gene and variant classification. After correcting for differences in variant classification and filtering practices, SGZ modeling was found to have 97.2% sensitivity and 90.3% specificity for the prediction of somatic versus germline origin. Mutations in HRD genes identified by tumor-only sequencing are frequently germline. Providers should be aware that technical differences related to assay design, variant filtering, and variant classification can contribute to discordance between tumor-only and germline sequencing test results. In addition, SGZ modeling had high predictive power to distinguish between mutations of somatic and germline origin without the need for a matched normal control, and could potentially be considered to inform clinical decision-making.

Expanded genetic testing of GIST patients identifies high proportion of non-syndromic patients with germline alterations

Traditional genetic testing for patients with gastrointestinal stromal tumors (GISTs) focus on those with syndromic features. To assess whether expanded genetic testing of GIST patients could identify hereditary cancer predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs over a 6-year period. Germline pathogenic/likely pathogenic (P/LP) variants in GIST-associated genes (SDHA, SDHB, SDHC, NF1, KIT) were identified in 69% of patients with KIT/PDGFRA-wildtype GISTs, 63% of whom did not have any personal or family history of syndromic features. To evaluate the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed 499 de-identified tumor-normal pairs. P/LP variants in certain genes (e.g., BRCA1/2, SDHB) identified in tumor-only sequencing of GISTs were almost exclusively germline in origin. Our results provide guidance for genetic testing of GIST patients and indicate that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs regardless of their history of syndromic features.

Abstract A006: Increased germline mutational burden in individuals of African ancestry: Implications for interpretation of tumor mutation burden

Abstract Introduction: Tumor mutation burden (TMB), defined as the number of somatic gene mutations per megabase in a tumor genome, is used clinically to identify cancer patients that may respond to immune checkpoint inhibitors. Recent studies suggest that patient ancestry can influence TMB, where individuals of African ancestry were found to have elevated TMB values based on tumor-only sequencing analysis. However, the impact of patient ancestry on germline mutational burden and implications for interpretation of tumor-only mutational burden data is largely unexplored. Methods: We examined the influence of patient ancestry on germline and tumor mutation burden using tumor-normal whole exome sequencing (WES) data from a pan-cancer cohort of 1228 individuals from a single institution. Genetic ancestry was estimated from constitutional WES data using single nucleotide polymorphism weights from external reference panels. Variant calling was performed using constitutional, tumor-only, and paired tumor-normal workflows. Total and loss-of-function burden scores were calculated for each participant from each workflow based on the total number of mutations detected in each sample and the total number of predicted loss-of-function mutations, based on snpEff annotations, respectively. Results: Genetic ancestry analysis found that one-third of this pan-cancer cohort was of non-European ancestry. 9.4% of individuals were of Eastern Asian ancestry, 5.3% of African ancestry, 1.0% of South Asian ancestry, 0.2% of Native American ancestry, and 17.1% of admixed ancestry, predominantly European and Native American admixed ancestry (15.6%). Total and loss-of-function germline burden scores varied across ancestral groups, with individuals of African ancestry showing significantly increased germline burden scores compared to other ancestral groups (p&amp;lt;0.0001). Tumor-only analysis also showed increased mutation burden for individuals of African ancestry. However, when private and rare germline variation was taken into account using paired tumor-normal analysis, tumor mutation burden did not differ based on patient ancestry, suggesting the differences seen in tumor-only analysis are due to germline variation rather than differences in true somatic mutation burden. Conclusion: Our study reveals that the paucity of knowledge of ancestry-specific reference genomes leads to unacceptable health disparities in cancer, specifically in patients with African ancestry. We show that germline mutational burden varies by ancestral background, with individuals of African ancestry displaying increased genetic variation compared to other ancestral populations. These results suggest that patient ancestry should be considered when interpreting tumor mutational burden values, particularly from tumor-only analysis. Approaches utilizing paired tumor-normal analysis or ancestry-specific reference genomes can aid in more accurate assessment of TMB, thereby avoiding futile treatments targeted at presumed high mutational burden tumors specifically in African American populations. Citation Format: Janith Don, Sara A. Byron, Guangfa Zhang, Tyler Izatt, Jiaming Zhang, Bethany Davis, Bryce Turner, Jonathan J. Keats, Jeffrey M. Trent, Lorna Rodriguez-Rodriguez, Nicholas J. Schork. Increased germline mutational burden in individuals of African ancestry: Implications for interpretation of tumor mutation burden [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A006.

Systematic assessment and optimizing algorithm of tumor mutational burden calculation and their implications in clinical decision-making.

Tumor mutation burden (TMB) has been validated as a biomarker to predict the response of immune checkpoint inhibitors (ICIs) treatment in various cancers. However, the effects of different sequencing platforms, cancer types, and calculation algorithms on TMB as well as its cut-off value for predicting immunotherapy efficacy in the East Asian population still need to be further investigated. In this study, the data of 4126 samples generated by targeted panel sequencing or whole-exome sequencing (WES) in different platforms and public sequencing data from 3680 samples that contained targeted panel sequencing, WES and whole-genome sequencing (WGS) were obtained. The impact of different sequencing platforms and methods on TMB calculation was assessed. No significant bias was found in TMB calculated by different platforms. However, TMB calculated from WGS was significantly lower than those calculated from targeted panel sequencing and WES. The distribution of TMB at different sequencing depths and tumor purity were analyzed. There was no significant difference in the distribution of TMB when the sequencing depth was greater than 500, the tumor purity estimated by hematoxylin-eosin (HE) staining was between 0.1-1.0 or estimated by next-generation sequencing (NGS) was greater than 0.4. In addition, the somatic-germline-zygosity (SGZ) algorithm was optimized to calculate TMB from tumor-only sequencing samples in the East Asian population. The correlation coefficient of TMB calculated with the optimized SGZ algorithm and paired normal-tumor sequencing is 0.951. Furthermore, the optimal cut-off value of TMB in East Asian lung cancer patients treated with ICIs was determined to be 7 mut/Mb instead of 10 mut/Mb through the ROC curve and Log-rank analysis in the training cohort and validated in the test cohort. Patients with TMB ≥ 7 mut/Mb had better outcomes than patients with TMB<7 mut/Mb. In conclusion, this study systematically analyzed the factors that influenced the TMB calculation and optimized the SGZ algorithm to calculate TMB from tumor-only sequencing samples in the East Asian population. More importantly, the cut-off value of TMB for predicting immunotherapy efficacy was determined to be 7 mut/Mb instead of 10 mut/Mb in East Asian lung cancer patients, which can help in clinical decision-making.

The importance of ancestry to understanding tumor mutation burden in cancer.

In the current issue of Cancer Cell, Nassar and colleagues find that in solid tumors, tumor-only sequencing leads to an overestimate of the biomarker tumor mutation burden (TMB), particularly in patients of African or Asian ancestry. Correction of the TMB estimate improves the correlation between TMB and response to immunotherapy.