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10589 Background: Germline and somatic genetic testing are established tools for the management of cancer patients. Somatic testing is primarily used to inform therapy and germline testing is used to diagnose hereditary cancer predisposition syndromes. While somatic testing can detect germline variants, the interpretation and reporting algorithms are optimized to predict therapeutic efficacy. As a result, germline variants may be missed or only interpreted in context of their potential to act as a therapeutic target. We retrospectively reviewed a series of patients who received both germline and somatic testing to examine the opportunities for concurrent germline testing to improve somatic reporting. Methods: Our study reviewed data from 43 patients with solid cancer diagnoses who were otherwise unselected and underwent testing with a 435-gene somatic genetic test and an 83-gene germline test. The most frequent cancers were pancreatic (18), ovarian (8), and prostate (7). Results: Out of the 43 patients, 7 (16%) harbored a pathogenic or likely pathogenic germline variant (PGV) in a cancer susceptibility gene. PGVs were identified in MLH1, MSH6, CHEK2, PALB2, CDKN2A, NBN, and MUTYH. Notably, 3 of these genes ( CHEK2, PALB2, MUTYH) were not considered therapeutic targets, and therefore were only included as ancillary findings near the end of the preliminary somatic test reports (generated prior to integration of germline test results). In addition, 40 of 43 (93%) patients had at least one variant detected by somatic testing in at least one of the germline panel genes (mean number variant genes = 4.1, maximum = 10); all of these variants were within the reportable range of the germline assay, and therefore germline test results were able to resolve their germline versus somatic origins. The genes that most frequently had somatic variants identified were TP53 (79% of patients), CDKN2A (37%), SMAD4 (30%), and FLCN (21%). Conclusions: Due to the size of commonly ordered somatic gene panels, there is a high probability of detecting variants in hereditary cancer predisposition genes (> 90% of patients in this study) that can provide either therapy or cancer risk information or both. Given that a significant proportion (16% in this study) of cancer patients harbor PGVs (which can further inform treatment, disease surveillance, preventive measures, and risk assessment for family members), it is crucial to resolve the somatic versus germline origin of these variants. Since interpretation and reporting algorithms for somatic testing are optimized for therapy prediction, and variables such as specimen tumor purity, tumor ploidy, and variant allele fraction render estimates of variant origin unreliable for diagnostic purposes, it is important to take advantage of germline testing concurrently in patients receiving somatic testing to glean this critical information.

Importance Both germline genetic testing and tumor DNA sequencing are increasingly used in cancer care. The indications for testing and utility of these 2 tests differ, and guidelines recommend that germline analysis follow tumor sequencing in certain patients to determine whether particular variants are of somatic or germline origin. Broad clinical experience with such follow-up testing has not yet been thoroughly described. Objective To examine the yield and utility of germline testing following tumor DNA sequencing in a large, diverse patient population. Design, Setting, and Participants A retrospective cohort study examined germline testing through a laboratory supporting multiple academic and community clinics. Participants included 2023 patients with cancer who received germline testing and previously underwent tumor DNA sequencing. These patients received germline testing between January 5, 2015, and January 31, 2020, although most (81% of patients) received testing between January 2, 2018, and January 31, 2020. Main Outcomes and Measures The prevalence of pathogenic germline variants (PGVs) was calculated by gene, cancer type, and age at diagnosis. Potential actionability of these findings was determined based on current management guidelines, precision therapy labels, and clinical trial eligibility criteria. Patient records were reviewed to determine whether germline follow-up testing would have been recommended by current guidelines. Results Among 2023 eligible patients, 1085 were female (53.6%), and the median age at cancer diagnosis was 56 (range, 0-92) years. Pathogenic germline variants were detected in 617 patients (30.5%; 95% CI, 28.5%-32.6%) and were prevalent across patient ages (1-85 years) and cancer types, including cancers known to be strongly associated with germline variance (eg, breast, colorectal) as well as others (eg, renal, lung, and bladder). Many patients (78%-82%) with PGVs met criteria for germline follow-up testing, and 8.1% of PGVs were missed by tumor sequencing. Among those with germline-positive findings, 69 patients (11.2%) had PGVs identified only after presenting with a second primary cancer that possibly could have been detected earlier or prevented given current gene-specific surveillance and risk-reduction recommendations. Conclusions and Relevance The findings of this study suggest that germline analysis following tumor sequencing often produces findings that may impact patient care by influencing systemic therapy choices, surgical decisions, additional cancer screening, and genetic counseling in families. Current guidelines and tumor testing approaches appear to capture many, but not all, of these germline findings, reinforcing the utility of both expanded germline follow-up testing as well as germline analysis independent of tumor sequencing in appropriate patients.

1591 Background: NCCN guidelines recommend germline testing for patients with localized or advanced prostate cancer meeting family history or clinical/pathologic criteria. However, the guidelines for somatic molecular analysis generally consider advanced disease only, primarily to inform therapy. As the analytical and clinical specifications of both testing modalities differ accordingly, we examined the results of germline testing following prior somatic testing. Methods: We reviewed somatic and germline variants in an otherwise unselected consecutive series of patients who: (a) had a current or previous diagnosis of prostate cancer; (b) had undergone tumor sequencing; and (c) were referred for germline testing. Indications for germline testing included: potential germline origin of somatic test result, treatment or surgical planning, personal or family history, and patient concern. Results: 208 patients met study criteria of whom 81 (39%) harbored a pathogenic germline variant (PGV) in a cancer predisposition gene. Certain genes were more likely to harbor germline variants, and 98% (81) of PGVs were potentially actionable (Table). 9.6% of PGVs were not reported by somatic testing, reflecting analytical limitations of the somatic testing. Of note, 11 patients (14%) had PGVs identified after diagnosis of a subsequent primary malignancy. Conclusions: The high PGV rate of 39% was unexpected, given reported rates of 11.8% in patients with metastatic prostate cancer and 6% in high-risk localized disease (NCCN)--even considering potential clinician ascertainment bias. This finding, the potential clinical utility of 98% of PGVs identified, the significant proportion unreported by somatic testing, and the fraction of patients diagnosed with a PGV after a subsequent malignancy all suggest that germline testing is an underutilized tool in the care of prostate cancer patients and their families. [Table: see text]

e17615 Background: Increasing evidence suggests criteria for germline testing of patients with prostate cancer (PCa) may miss potentially actionable pathogenic variants. A sponsored testing program with significantly broader criteria was initiated to increase access. Methods: We analyzed de-identified data from 2,252 men with PCa who received at least an 84 gene panel. Inclusion criteria were clinical stage ≥T2 and willingness of the patient and physician to participate. Positive results were categorized as: pathogenic (P), likely pathogenic (LP), or increased risk alleles (IRAs). Results: Clinical stage was available in 1825 men (81%): 541 (30%) were stage II, 228 (12%) stage III, and 1,056 (58%) stage IV (Demographics Table). Among all men, 297 (13%) had positive results, including 286 P/LP variants in 38 genes and 31 IRAs in APC and HOXB13. There was no statistically significant association between stage and positive rate. Of men with positive variants, only 30% reported a family history of prostate, breast, ovarian or pancreatic cancer. The most common positive variants were in BRCA1 (9) , BRCA2 (43) , PALB2 (11) , ATM (36) , CHEK2 (58) , HOXB13 (20) , and mismatch repair genes (8). With respect to ethnicity, the highest positive rate was observed in Ashkenazi Jews (23.8%) and the lowest in African Americans (5.4%). Overall, 190 (70%) of men had P/LP variants conferring eligibility for gene-specific precision therapies or clinical trials based on variants in homologous repair (182), or mismatch repair (8) genes. Conclusions: In a broad PCa patient population, we found overall germline positive rate of 13%, with no statistical difference in this rate with respect to stage. Overall, these data suggest that broader testing criteria and more access to testing could lead to better informed care for many additional patients and their families. [Table: see text]

e16089 Background: Practice guidelines support genomic and biomarker testing of colorectal cancer (CRC) to inform treatment options in advanced disease and to screen for MMR deficiency/MSI/Lynch syndrome. As yet, guidelines for follow-up germline testing are not well established. In 2019 ESMO recommended reflex germline testing when pathogenic variants (PV) were found in any of 27 cancer susceptibility genes (CSG) in tumor samples (PMID 31050713). The CSGs were chosen because PV in these genes were clinically actionable and had “germline conversion rates” (GCR) of > 10% (proportion of tumor-detected PV that were germline). We examined the utility of germline testing in a cohort of CRC patients who had prior tumor sequencing. Methods: We reviewed somatic & germline PV in an unselected consecutive series of CRC patients who were referred for germline testing and had prior tumor testing. We determined the GCR for genes with pathogenic germline variants (PGV) and compared our results to ESMO data. Results: 66 of 238 (27%) patients harbored PGV. 15% of PGV were not reported in the tumor assay. High GCR for BRCA1/2 (30%), MMR genes (40%), PALB2 (57%), and BRIP1 (67%) were in keeping with ESMO. Low GCR for APC (1.4%) and TP53 (3%) were similar to ESMO, and reflective of high somatic mutation rates in these genes. We noted high GCR for CHEK2 (75%) and ATM (24%), two genes not included in ESMO guidelines due to their intermediate penetrance. Conclusions: ESMO guidelines provide a key starting point for when reflex germline testing should be pursued after tumor testing. In our study 27% (65/238) of patients had PGV that conferred potential eligibility for targeted therapy or clinical trials. For CRC, finding PV in MMR genes, BRCA1/2, and other relevant CSGs should prompt follow-up germline testing. In cases with APC PV in tumor, ESMO recommended reflex germline testing only in patients < 30 yo, where the GCR was > 10%. Our study suggests that reflex germline testing after tumor sequencing yields clinically actionable results. [Table: see text]

e16783 Background: PARP inhibitor (PARPi) treatment was recently approved for pancreatic cancer (PaCa) patients with germline mutations in 2 DNA damage repair (DDR) genes. Despite criteria recommending germline multigene panel testing for all PaCa patients, barriers to testing remain, including among underserved populations, which limit access to precision therapies. We initiated a sponsored testing program that increases access to germline genetic testing for PaCa in two ways: 1) offering a comprehensive multigene panel, and 2) removing the barrier of cost. Here we present initial results from this program, including the diagnostic yield in patients across stages of PaCa and clinical utility of the findings. Methods: We retrospectively analyzed de-identified data from 966 PaCa patients tested on an 84 gene panel as part of the program to date. The only inclusion criterion was a willingness to participate in the sponsored program by the patient and the provider who ordered the testing. Data included likely pathogenic (LP) and pathogenic (P) mutations, disease stage and ethnicity. Results: In total, 166 (17%) patients were positive for P/LP germline mutations in 30 genes. Mutation rate by ethnicity was: Caucasian 17%, African American 12%, Hispanic 16%, Ashkenazi Jewish 20%, Asian 3%. Only 25% of patients with P/LP variants reported a family history of cancer. There was no statistical difference in mutation rates by stage (p = 0.11) [Table]. In positive patients, 83 (78%) had mutations conferring potential eligibility for DDR gene-specific precision therapies or clinical treatment trials. 28 (26%) were potentially eligible for olaparib due to BRCA1/2 mutations, 8 (7%) were potentially eligible for pembrolizumab, and 47 (44%) for PARPi clinical trials. Conclusions: This study found 8.5% of all PaCa patients tested are potentially eligible for germline-based precision therapies and/or clinical treatment trials. Of mutation positive patients, 75% did not report a family history of cancer. The positive rate was not statistically different between patients with stage I and stage IV PaCa, underscoring the recommendation to test all patients with PaCa. This program had a 1.5% increased relative uptake among African American patients compared to a standard insurance reimbursement delivery model. These data suggest reducing barriers improves PaCa patient access to genetic information that enables precision therapy. [Table: see text]

1092 Background: Germline genetic testing is recommended for breast cancer patients with specific presentations or family histories. Separately, tumor DNA sequencing is increasingly used to inform therapy, most often in patients with advanced disease. Recent NCCN and ESMO guidelines recommend germline testing following somatic testing, under specific circumstances and for specific genes. We examined the utility of germline findings in patients referred for both test modalities. Methods: We reviewed somatic and germline mutations in a consecutive series of patients who: (a) had a current or previous breast cancer diagnosis, (b) were referred for germline testing, and (c) previously received tumor sequencing. Diverse reasons for germline testing included: a tumor finding of potential germline origin, treatment or surgical planning, personal or family history, and patient concern. Results: 227 patients met study criteria of whom 88 (39%) harbored a pathogenic germline variant (PGV) in a high or moderate risk cancer predisposition gene. Mutations in certain genes were most likely to be of germline origin, and most PGVs were potentially actionable (Table). 13% of PGVs were not reported by tumor tests as either germline or somatic findings, usually a result of tumor test limitations. Of note, 27 of the patients with PGVs (31%) had these variants uncovered only after presenting with a second, possibly preventable, malignancy. Conclusions: Germline testing following tumor sequencing often yielded findings that may impact care. Indeed, the 39% PGV rate we observed suggests that such testing may be underutilized. We observed actionable PGVs missed by somatic tests, PGVs uncovered in patients’ second malignancies, and PGVs not within germline reflex testing criteria. These results reinforce the utility of germline testing separate from somatic testing in appropriate patients. [Table: see text]

About 20% of the population suffers from “functional syndromes”. Since these syndromes overlap greatly in terms of co-morbidity, pathophysiology (including aberrant autonomic activity) and treatment responses, common predisposing genetic factors have been postulated. We had previously showed that two common mitochondrial DNA (mtDNA) polymorphisms at positions 16519 and 3010 are statistically associated with the functional syndromes of migraine, cyclic vomiting syndrome and non-specific abdominal pain. Herein, among individuals with mtDNA haplogroup H (HgH), the presence of these two mtDNA polymorphisms were ascertained in additional functional syndromes: chronic fatigue syndrome, complex regional pain syndrome, sudden infant death syndrome, and major depressive disorder. Polymorphic prevalence rates were compared between disease and control groups, and within each disease group in participants with and without specific clinical findings. In all four conditions, one or both of the polymorphisms was significantly associated with the respective condition and/or co-morbid functional symptomatology. Thus, we conclude that these two mtDNA polymorphisms likely modify risk for the development of multiple functional syndromes, likely constituting a proportion of the postulated common genetic factor, at least among individuals with HgH. Pathophysiology likely involves broad effects on the autonomic nervous system.

Purpose To identify the variables and factors that affect the quantity and quality of nucleic acid yields from imaging-guided core needle biopsy. Materials and Methods This study was approved by the institutional review board and compliant with HIPAA. The authors prospectively obtained 232 biopsy specimens from 74 patients (177 ex vivo biopsy samples from surgically resected masses were obtained from 49 patients and 55 in vivo lung biopsy samples from computed tomographic [CT]-guided lung biopsies were obtained from 25 patients) and quantitatively measured DNA and RNA yields with respect to needle gauge, number of needle passes, and percentage of the needle core. RNA quality was also assessed. Significance of correlations among variables was assessed with analysis of variance followed by linear regression. Conditional probabilities were calculated for projected sample yields. Results The total nucleic acid yield increased with an increase in the number of needle passes or a decrease in needle gauge (two-way analysis of variance, P.0001 for both). However, contrary to calculated differences in volume yields, the effect of needle gauge was markedly greater than the number of passes. For example, the use of an 18-gauge versus a 20-gauge biopsy needle resulted in a 4.8-5.7 times greater yield, whereas a double versus a single pass resulted in a 2.4-2.8 times greater yield for 18- versus 20-gauge needles, respectively. Ninety-eight of 184 samples (53%) had an RNA integrity number of at least 7 (out of a possible score of 10). Conclusion With regard to optimizing nucleic acid yields in CT-guided lung core needle biopsies used for genomic analysis, there should be a preference for using lower gauge needles over higher gauge needles with more passes.

DNA mismatch-repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′-UTRs and 3′-UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI-H (microsatellite instability-high) tumors. The microsomal prostaglandin E synthase-1 (mPGES-1) gene product, mPGES-1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES-1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES-1 gene 3′ untranslated region (3′-UTR), and analyzed its mutation frequencies in MSI-H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI-H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES-1 mRNA levels in MSI-H and MSS colorectal cancer cell lines. Three of four previously designated “MSI-H” cell lines showed higher mPGES-1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES-1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES-1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.