Multigene Testing Research Articles

Guideline-Based, Multi-Gene Panel Germline Genetic Testing for at-Risk Patients with Breast Cancer.

Genetic testing for at-risk patients with breast cancer should be routinely offered. Knowledge generated may influence both treatment decisions and cancer prevention strategies among the patients themselves and their relatives. In this study, we report on the prevalence and patterns of germline mutations, using commercially available next-generation sequencing (NGS)-based multi-gene panels (MGP). Consecutive at-risk breast cancer patients, as determined by international guidelines, were offered germline genetic testing using a 20-gene NGS-based panel at a reference lab. Samples of peripheral blood were obtained for DNA extraction and genetic variants were classified as benign/likely benign (negative), pathogenic/likely pathogenic (positive) or variants of uncertain significance (VUS). A total of 1310 patients, median age (range) 43 (19-82) years, were enrolled. Age ≤45 years (n = 800, 61.1%) was the most common indication for testing. Positive family history of breast, ovarian, pancreatic or prostate cancers, and triple-negative disease were among the common indications. Among the whole group, 184 (14.0%) patients had pathogenic/likely pathogenic variants; only 90 (48.9%) were in BRCA1 or BRCA2, while 94 (51.9%) others had pathogenic variants in other genes; mostly in APC, TP53, CHEK2 and PALB2. Mutation rates were significantly higher among patients with positive family history (p = 0.009); especially if they were 50 years or younger at the time of breast cancer diagnosis (p < 0.001). Patients with triple-negative disease had relatively higher rate (17.5%), and mostly in BRCA1/2 genes (71.4%). Variants of uncertain significance (VUS) were reported in 559 (42.7%) patients; majority (90.7%) were in genes other than BRCA1 or BRCA2. Pathogenic mutations in genes other than BRCA1/2 are relatively common and could have been missed if genetic testing was restricted to BRCA1/2. The significantly high rate of VUS associated with multi-gene panel testing can be disturbing.

CRAC (Clinical Relevance of Alterations in Cancer): a Knowledge Base for the Selection of Molecularly Matched Therapy for Solid Tumors.

Multigene testing using NGS (next-generation sequencing) provides a large amount of information and can detect multiple molecular alterations. Subsequent clinical interpretation is a time-consuming process necessary to select a treatment strategy. Existing databases often contain inconsistent information and are not regularly updated. The use of ESCAT levels of evidence requires a deep understanding of the nature of alterations and does not answer the question of which therapy option to select when multiple biomarkers with the same level of evidence are detected. To address these issues, we created the Clinical Relevance of Alterations in Cancer (CRAC) database on the relevance of detected alterations in specific genes, which are often analyzed as part of NGS panels. The team of oncologists and biologists assigned a CRAC score from 1 to 10 to each biomarker (a type of genomic alteration characteristic of specific genes) for 15 malignancies; an average score was entered into the database. CRAC scores are a numerical reflection of the following factors: therapy availability and the prospects of drug treatment with experimental drugs for patients with a particular type of tumor. A total of 134 genes and 15 of the most common tumor types have been selected for CRAC. The biomarker-nosology associations with CRAC scores in the range of 1-3 are the most frequent (n=2719 out of 3495; 77.8%), the least frequent ones (n=52 out of 3495; 1.5%) are with the highest CRAC scores 9 and 10. To estimate the practical effectiveness of the CRAC database, 208 reports on comprehensive molecular profiling were retrospectively analyzed; the applicability of CRAC was compared with the ESCAT level of evidence system. The highest CRAC scores corresponded to the ESCAT maximum levels of evidence: the range of scores 8-10 corresponded to evidence levels I and II. No biomarker within the same level of evidence was represented by the same CRAC score; the largest range of CRAC scores was observed for biomarkers of levels evidence IIIA and IV - from 2 to 10 and from 1 to 9, respectively. The use of CRAC scores allowed to identify additional 95 alterations with CRAC scores of 1-5 in the studied patients. The developed database is available at: https://crac.oncoatlas.ru/.

RF21 | PSUN07 Germline Genetic Testing in a Cohort of Adults with Adrenocortical Carcinoma : Insights From a Clinical Care Setting

Abstract Context About 5-10% of adrenocortical cancer (ACC) arise in patients with genetic predisposition syndromes, including Li-Fraumeni, Lynch, MEN1, or Familial Adenomatous Polyposis (APC). Recent European (2018) and American guidelines (2020) recommend that all adults with ACC should be offered clinical genetic counseling. There is a paucity of data regarding systematic germline testing in adults with ACC and the extent of the genetic evaluation is unclear. Objective To describe the germline genetic characteristics in a cohort of adult patients with ACC evaluated in a tertiary clinical care center. Methods Data including demographics, personal and family history of neoplasia, pathology reports, clinical features and genetic testing were retrospectively collected from charts of patients treated at Centre hospitalier de l'Université de Montréal (CHUM). After genetic counseling, genetic testing was proposed to patients with a pathologic diagnosis of ACC. From 2005 to 2015, TP53 gene analysis was performed using direct sequencing and MLPA. Since 2016, multigene testing was performed for a progressively increasing number of oncogenic genes using a custom next-generation panel for germline leucocyte DNA including at least the TP53, MSH2, MSH6, MLH1,PMS2, EPCAM, MEN1, BRCA1 and BRCA2 genes (Invitae, CA). An extended panel, based on past medical and familial history, was performed at the discretion of the geneticist. Patient data was retrospectively investigated. Results We analyzed data from 44 patients with available germline genetic results. Median age of the patients was 46 years (ranging from 22 to 79 years), including 11 males (25.0%) and 33 females (75.0%). Ten patients (22.7%) underwent only TP53 gene analysis and 32 patients (72.7%) were studied using the larger oncogenic genetic panel. Germline pathogenic or likely pathogenic variants were identified in 5 of the 44 patients (11.4%) while genetic variants of unknown significance (VUS) were found in 7 of the 44 patients (15.9%). Among patients with pathogenic variants, one had a family history and known germline mutation in the BRCA2 gene (8765delAG, p.Glu2846GlyfsX23). One patient had a personal and familial medical history suggesting a MEN1 syndrome that was confirmed with the finding of a germline MEN1 mutation (c.1556delC, p.Pro519Leu fs40). Unsuspected germline pathogenic variants were found in three patients: 1) TP53 (c.425delC, p.Pro142fs), 2) MUTYH (c.536A&amp;gt;G, p.Tyr179Cys) and 3) MSH6 (c.3649-3655, p.Arg1217Leu fs9) genes. Variants of unknown significance (VUS) were found in the following genes: POT1, MSH3, PALB2, RAD51d, APC, ATM and BRCA2. Conclusions Germline pathogenic variants were found in 11.4% of our cohort of patients with ACC. VUS were found in 15.9% of patients but their significance remains to be determined. Genetic counseling and germline genetic testing should be offered to all patients with ACC; however the optimal use and extent of oncogenic gene panels need to be better defined. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Sunday, June 12, 2022 12:48 p.m. - 12:53 p.m.

263 (PB-087) Poster - Comparison of ER, PR, HER2 and Ki67 expression by MammaTyper® RT-qPCR and immunohistochemistry (IHC) on needle core biopsies of breast cancer

Background: Breast cancer molecular subtyping is traditionally defined by the immunohistochemical expression of ER, PR, HER2 and Ki67. There is limited data on the role of multigene tests and their correlation with IHC particularly on conventional core biopsies. MammaTyper® is a quantitative CE marked in vitro diagnostic RT-qPCR test for assessment of mRNA expression of these four biomarkers (ESR1, PGR, ERBB2, MKI67), classifying breast cancer into five subtypes. We evaluated the concordance of MammaTyper® with ER, PR, HER2 and Ki67 by IHC on core needle biopsy samples of breast cancer.

Integrative Genomic Tests in Clinical Oncology.

Many clinical decisions in oncology practice rely on the presence or absence of an alteration in a single genetic locus, be it a pathogenic variant in a hereditary cancer gene or activating mutation in a drug target. In addition, there are integrative tests that produce continuous variables and evaluate complex characteristics of the entire tumor genome. Microsatellite instability (MSI) analysis identifies tumors with the accumulation of mutations in short repetitive nucleotide sequences. This procedure is utilized in Lynch syndrome diagnostic pipelines and for the selection of patients for immunotherapy. MSI analysis is well-established for colorectal malignancies, but its applications in other cancer types lack standardization and require additional research. Homologous repair deficiency (HRD) indicates tumor sensitivity to PARP inhibitors and some cytotoxic drugs. HRD-related "genomic scars" are manifested by a characteristic pattern of allelic imbalances, accumulation of deletions with flanking homology, and specific mutation signatures. The detection of the genetic consequences of HRD is particularly sophisticated and expensive, as it involves either whole genome sequencing (WGS) or the utilization of large next-generation sequencing (NGS) panels. Tumor mutation burden (TMB) can be determined by whole exome sequencing (WES) or middle-throughput NGS multigene testing. Although TMB is regarded as an agnostic indicator of tumor sensitivity to immunotherapy, the clinical utility of this test is proven only for a few cancer types.

Diagnostic value of multigene sequencing for inherited thrombocytopenia.

Multigene testing for inherited thrombocytopenia can detect an unexpected diagnosis as demonstrated in the case report from Ming-Ping et al., where the mode of inheritance and clinical phenotype did not point towards dominant Schlafen family member 14 (SLFN14) moderate thrombocytopenia. They describe two siblings with very severe thrombocytopenia due a pathogenic SLFN14 variant and gonosomal mosaicism in their healthy mother. In their paper Ming-Ping et al. describe two siblings with severe thrombocytopenia (platelets counts <15 × 109/l) and bleeding symptoms, born from healthy non-consanguineous parents.1 The first child died at the age of 5 months due to intracranial haemorrhage. This child also had larger platelets, while this was not found for his affected sister. She has received regular platelet transfusions since birth. Neonatal and immune thrombocytopenia were ruled out. Their diagnosis was made after using whole-exome sequencing with the detection of a heterozygous pathogenic SLFN14 variant that is responsible for dominant thrombocytopenia. Interestingly, gonosomal mosaicism for this variant was detected in the mother who has normal platelet counts. This case report nicely demonstrates the power of multigene sequencing for the diagnosis of inherited thrombocytopenia, which is an extremely heterogenous disorder with already 40 diagnostic-grade genes as potential underlying cause (Figure 1).2, 3 Using only textbook knowledge, the diagnosis of SLFN14-related thrombocytopenia could probably never have been made for these patients. Based on the clinical phenotypes present in the family members, a recessive form of inherited (macro)thrombocytopenia was expected as parents have a normal platelet count. In addition, SLFN14-related thrombocytopenia is an ultrarare disorder and clinical and laboratory data have only been described for four unrelated pedigrees.4 All patients with pathogenic SLFN14 variants have platelet counts >65 × 109/l (moderate thrombocytopenia) and highly variable degrees of bleeding symptoms and International Society of Thrombosis and Haemostasis (ISTH) bleeding scores ranging between 2 and 20. The children in this case report have a much more severe phenotype and still they carry the same genetic variant as previously described.4 In the past, several diagnostic algorithms have been proposed to assist clinicians in the differential diagnosis of inherited thrombocytopenia. The classification of inherited thrombocytopenia was typically driven by the presence or absence of other clinical features besides a platelet defect (syndromic versus non-syndromic), the mode of inheritance (recessive, dominant and X-linked) and the platelet size (macro, micro and normal size platelets) (Figure 1).5 Applying such an algorithm was very useful, as the numbers of genes implicated in inherited thrombocytopenia was less than half of the genes we know today. However, this algorithm would have missed the diagnosis in the family. Since the discovery of many novel genes for inherited thrombocytopenia using next-generation sequencing approaches over the last decade, phenotype–genotype associations for this disorder have become very complex.6 In addition, the mode of inheritance has expanded for well-known genes (e.g., dominant mild Bernard–Soulier syndrome versus the classical recessive form and recessive Fli-1 proto-oncogene, ETS transcription factor [FLI1] and growth factor-independent 1B transcriptional repressor [GFI1B] variants versus the known dominant forms of this type of thrombocytopenia). To date, multigene panel testing is typically used to diagnose inherited thrombocytopenia by investigating all the known genes involved in this pathology in a single analysis.7 Providing detailed clinical and laboratory information to genetic laboratories remains very important for the interpretation of this test as it assists in the variant classification to decide if a variant is pathogenic or not. The major drawbacks of performing a multigene panel test for inherited thrombocytopenia are the detection of many variants of unknown significance than cannot be used for clinical management and unsolicited findings. If for example pathogenic variants are detected in either RUNX family transcription factor 1 (RUNX1), ETS variant transcription factor 6 (ETV6) or ankyrin repeat domain-containing 26 (ANKRD26) in a patient with thrombocytopenia without a family history of leukaemia, such variants can be considered as an unsolicited finding if the patient was not aware about these genetic risk factors for leukaemia before initiating the multigene test. On the other hand, a multigene test allows the detection of ‘the unexpected diagnosis’. This case report provides such an example as a SLFN14 defect was not really expected based on the known disease phenotypes associated with pathogenic variants in this gene.

39P Clinical relevance of alterations in cancer (CRAC): A DB for selecting biomarkers for molecularly matched therapy in cancer patients

Multigene testing via NGS can detect multiple genomic alterations (GA). The use of current levels of evidence (LOE) requires understanding the functional consequences of the GA and an extensive literature search and have less practical value for narrowing down therapy recommendations if multiple GAs with the same LOE are found.

Increased Co-Occurrence of Pathogenic Variants in Hereditary Breast and Ovarian Cancer and Lynch Syndromes: A Consequence of Multigene Panel Genetic Testing?

The probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes for hereditary breast and ovarian cancer and lynch syndromes in the same patient is uncommon, except in populations where founder effects exist. Two breast cancer women that are double heterozygotes (DH) for both BRCA1/BRCA2, one ovarian cancer case DH for BRCA1/RAD51C, and another breast and colorectal cancer who is DH for BRCA2/PMS2 were identified in our cohort. Ages at diagnosis and severity of disease in BRCA1/BRCA2 DH resembled BRCA1 single-carrier features. Similarly, the co-existence of the BRCA2 and PMS2 mutations prompted the development of breast and colorectal cancer in the same patient. The first BRCA1/BRCA2 DH was identified by HA-based and Sanger sequencing (1 of 623 families with BRCA PVs). However, this ratio has increased up to 2.9% (1 DH carrier vs. 103 single PV carriers) since using a custom 35-cancer gene on-demand panel. The type of cancer developed in each DH patient was consistent with the independently inherited condition, and the clinical outcome was no worse than in patients with single BRCA1 mutations. Therefore, the clinical impact, especially in patients with two hereditary syndromes, lies in genetic counseling tailor-made for each family based on the clinical guidelines for each syndrome. The number of DH is expected to be increased in the future as a result of next generation sequencing routines.

TPMT and NUDT15 testing for thiopurine therapy: A major tertiary hospital experience and lessons learned.

Variants in thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) are associated with an accumulation of cytotoxic metabolites leading to increased risk of drug-related toxicity with standard doses of thiopurine drugs. We established TPMT and NUDT15 genetic testing for clinical use and evaluated the utilization, service outcomes and potential value of multi-gene PGx testing for 210 patients that underwent pharmacogenetics (PGx) testing for thiopurine therapy with the aim to optimize service delivery for future prescribing. The test was most commonly ordered for Gastroenterology (40.0%) and Neurology (31.4%), with an average turnaround time of 2 days. Following testing, 24.3% patients were identified as intermediate or poor metabolizers, resulting in 51 recommendations for a drug or dose change in thiopurine therapy, which were implemented in 28 (54.9%) patients. In the remaining patients, 14 were not adjusted and 9 had no data available. Focusing on drug gene interactions available for testing in our laboratory, multi-gene PGx results would present opportunities for treatment optimization for at least 33.8% of these patients who were on 2 or more concurrent medications with actionable PGx guidance. However, the use of PGx panel testing in clinical practice will require the development of guidelines and education as revealed by a survey with the test providers. The evaluation demonstrated successful implementation of single gene PGx testing and this experience guides the transition to a pre-emptive multi-gene testing approach that provides the opportunity to improve clinical care.

Clinical Utility of Universal Germline Genetic Testing for Patients With Breast Cancer

National Comprehensive Cancer Network guidelines currently recommend germline testing for high-risk genes in selected patients with breast cancer. The clinical utility of recommending testing all patients with breast cancer with multigene panels is currently under consideration. To examine the implications of universal testing of patients with breast cancer with respect to clinical decision-making. Patients from a previously reported cohort were assessed as in-criteria or out-of-criteria according to the 2017 guidelines and underwent testing with a multigene germline panel between 2017 to 2018. Patients were women and men aged 18 to 90 years, with a new and/or previous diagnosis of breast cancer who had not undergone either single or multigene testing. Clinicians from 20 community and academic sites documented patient clinical information and changes to clinical recommendations made according to test findings. Association between prevalence of pathogenic or likely pathogenic germline variants and previously unreported clinical features, including scores generated by the BRCAPRO statistical model, was determined. Data were analyzed from April 2020 to May 2022. New and/or previous diagnosis of breast cancer. Disease management recommendations that were changed as a result of genetic testing results are reported. Clinicians were asked to assess changes to clinical management as a result of germline genetic testing for 952 patients. Informative clinician-reported recommendations were provided for 939 (467 in-criteria and 472 out-of-criteria) of the patients with breast cancer (936 [99.7%] female; 702 [74.8%] White; mean [SD] age at initial diagnosis, 57.6 [11.5] years). One or more changes were reported for 31 of 37 (83.8%) in-criteria patients and 23 of 34 (67.6%) out-of-criteria patients with a pathogenic or likely pathogenic variant. Recommendations were changed as a result of testing results for 14 of 22 (63.6%) out-of-criteria patients who had a variant in a breast cancer predisposition gene. Clinicians considered testing beneficial for two-thirds of patients with pathogenic or likely pathogenic variants and for one-third of patients with either negative results or variants of uncertain significance. There was no difference in variant rate between patients meeting the BRCAPRO threshold (≥10%) and those who did not (P = .86, Fisher exact test). No changes to clinical recommendations were made for most patients with negative results (345 of 349 patients [98.9%]) or variants of uncertain significance (492 of 509 patients [96.7%]). In this cohort study, germline genetic testing was used by clinicians to direct treatment for most out-of-criteria patients with breast cancer with pathogenic or likely pathogenic germline variants, including those with moderate-risk variants. Universal germline testing informs clinical decision-making and provides access to targeted treatments and clinical trials for all patients with breast cancer.

Does the 21-gene recurrence score have clinical utility in HR+/HER2+ breast cancer?

The 21-gene recurrence score assay has been validated as a predictive biomarker in early-stage HR+ and HER2-breast cancer. It is not indicated for use in HER2+ disease based on national guidelines. In this study, we assessed the value of 21-gene recurrence score (RS), or OncotypeDX (ODX), testing in HR+/HER2+ breast cancer.We used the National Cancer Database to identify patients with stages I-II, HR+/HER2+ breast cancer who received multi-gene testing with ODX. We then explored the prognostic and predictive value of this biomarker through various forms of survival modeling.ODX testing was performed in n = 5,280 patients. N = 2,678 patients (50.7%) had a RS < 26, while n = 2,602 (49.3%) had a RS ≥26. In Kaplan-Meier survival modeling for patients with recurrence scores <26, there was no significant difference in overall survival (p = 0.445) between patients receiving different systemic treatment regimens. However, when recurrence scores were ≥26, there was a statistically-significant difference in overall survival between systemic treatment regimens (p < 0.001). 5-year overall survival was highest (97.4%) for patients receiving triple therapy (anti-HER2 with chemotherapy and endocrine therapy), followed by those receiving dual therapy with endocrine and anti-HER2 (96.7%), and endocrine with chemotherapy (94.9%). Patients receiving endocrine therapy alone exhibited the lowest 5-year overall survival (88.5%). ResultsAnalysis from this large national cancer registry suggests that multigene testing may have predictive value in treatment selection for patients with early-stage, HR+/HER2+ breast cancer. Prospective trials are warranted to identify subgroups of patients with HR+/HER2+ breast cancer who can be spared anti-HER2 treatments and cytotoxic chemotherapy.

Update Breast Cancer 2022 Part 3 - Early-Stage Breast Cancer.

This review summarizes recent developments in the prevention and treatment of patients with early-stage breast cancer. The individual disease risk for different molecular subtypes was investigated in a large epidemiological study. With regard to treatment, new data are available from long-term follow-up of the Aphinity study, as well as new data on neoadjuvant therapy with atezolizumab in HER2-positive patients. Biomarkers, such as residual cancer burden, were investigated in the context of pembrolizumab therapy. A Genomic Grade Index study in elderly patients is one of a group of studies investigating the use of modern multigene tests to identify patients with an excellent prognosis in whom chemotherapy may be avoided. These and other aspects of the latest developments in the diagnosis and treatment of breast cancer are described in this review.

Detection of BRCA1, BRCA2, and ATM Alterations in Matched Tumor Tissue and Circulating Tumor DNA in Patients with Prostate Cancer Screened in PROfound.

Not all patients with metastatic castration-resistant prostate cancer (mCRPC) have sufficient tumor tissue available for multigene molecular testing. Furthermore, samples may fail because of difficulties within the testing procedure. Optimization of screening techniques may reduce failure rates; however, a need remains for additional testing methods to detect cancers with alterations in homologous recombination repair genes. We evaluated the utility of plasma-derived circulating tumor DNA (ctDNA) in identifying deleterious BRCA1, BRCA2 (BRCA), and ATM alterations in screened patients with mCRPC from the phase III PROfound study. Tumor tissue samples were sequenced prospectively at Foundation Medicine, Inc. (FMI) using an investigational next-generation sequencing (NGS) assay based on FoundationOne®CDx to inform trial eligibility. Matched ctDNA samples were retrospectively sequenced at FMI, using an investigational assay based on FoundationOne®Liquid CDx. 81% (503/619) of ctDNA samples yielded an NGS result, of which 491 had a tumor tissue result. BRCA and ATM status in tissue compared with ctDNA showed 81% positive percentage agreement and 92% negative percentage agreement, using tissue as reference. At variant-subtype level, using tissue as reference, concordance was high for nonsense (93%), splice (87%), and frameshift (86%) alterations but lower for large rearrangements (63%) and homozygous deletions (27%), with low ctDNA fraction being a limiting factor. We demonstrate that ctDNA can greatly complement tissue testing in identifying patients with mCRPC and BRCA or ATM alterations who are potentially suitable for receiving targeted PARP inhibitor treatments, particularly patients with no or insufficient tissue for genomic analyses.

Molecular Subtyping of Invasive Breast Cancer Using a PAM50-Based Multigene Expression Test-Comparison with Molecular-Like Subtyping by Tumor Grade/Immunohistochemistry and Influence on Oncologist's Decision on Systemic Therapy in a Real-World Setting.

In intermediate risk hormone receptor (HR) positive, HER2 negative breast cancer (BC), the decision regarding adjuvant chemotherapy might be facilitated by multigene expression tests. In all, 142 intermediate risk BCs were investigated using the PAM50-based multigene expression test Prosigna® in a prospective multicentric study. In 119/142 cases, Prosigna® molecular subtyping was compared with local and two central (C1 and C6) molecular-like subtypes relying on both immunohistochemistry (IHC; HRs, HER2, Ki-67) and IHC + tumor grade (IHC+G) subtyping. According to local IHC, 35.4% were Luminal A-like and 64.6% Luminal B-like subtypes (local IHC+G subtype: 31.9% Luminal A-like; 68.1% Luminal B-like). In contrast to local and C1 subtyping, C6 classified >2/3 of cases as Luminal A-like. Pairwise agreement between Prosigna® subtyping and molecular-like subtypes was fair to moderate depending on molecular-like subtyping method and center. The best agreement was observed between Prosigna® (53.8% Luminal A; 44.5% Luminal B) and C1 surrogate subtyping (Cohen’s kappa = 0.455). Adjuvant chemotherapy was suggested to 44.2% and 88.6% of Prosigna® Luminal A and Luminal B cases, respectively. Out of all Luminal A-like cases (locally IHC/IHC+G subtyping), adjuvant chemotherapy was recommended if Prosigna® testing classified as Prosigna® Luminal A at high / intermediate risk or upgraded to Prosigna® Luminal B.

Associations between genomic instability score (GIS), molecular characterization, and clinical outcomes in high grade serous ovarian cancers (HGSOC).

e17596 Background: To describe the molecular profiles of tumors with a positive GIS status (GI+) in patients with newly diagnosed BRCA wild-type HGSOC and to examine possible associations between genomic mutations and survival outcomes in these patients. Methods: Genetic testing results of patients who underwent both somatic BRCA1/BRCA2 and germline multigene panel testing between 1/1/2015 and 12/1/2020 were prospectively collected. Patients had multigene panel germline testing in tandem with Myriad somatic BRCA1/2 testing and GIS assay. Expanded somatic NGS testing (108 gene panel) was then performed on GIS+ (GIS ≥42) tumor samples from women without somatic or germline BRCA mutations. The primary outcomes were the type and frequency of genomic mutations, which were grouped according to the following pathways: DNA-repair related (separated into homologous recombination (HR), Fanconi anemia (FA), other DNA repair related pathway) or non-DNA repair related. GIS and molecular profiles of tumors from patients who recurred versus those who did not recur were compared. Results: 143 women with HGSOC underwent both germline and somatic testing. 18/143 (12.5%) tumors with negative germline multigene panel and negative somatic BRCA1/2 testing were GIS+. 17/18 (94.4%) GIS+ tumors had pathogenic TP53 mutations. While all samples had at least one DNA repair related variant, only 22.2% had an HR-associated variant, and 16.7% had an FA variant. At time of analysis, 10 patients had recurred and 8 patients had not recurred. Median PFS of those who recurred was 15.8 months (range 4.8-30.1 months). Patients who recurred had lower GIS (median 47.5 vs. 69.5, p = 0.04), but they had no appreciable differences in gene or pathway alterations when compared to those who did not recur. Conclusions: Known pathogenic alterations in DNA repair pathways commonly associated with HR were rare in this cohort of BRCA negative HGSOC patients with GIS+ tumors. However, there was a high frequency of other DNA repair pathway alterations. Further investigation of the molecular signatures associated with recurrence are needed to identify potential novel targets. Lower GIS in this cohort were associated with higher risk of recurrence, although this finding must be validated in the setting of larger studies. [Table: see text]

Effective biomarker testing rates in a large U.S. community practice.

e21093 Background: Molecular testing is an integral part of care for patients with non-small cell lung cancer (NSCLC). Professional guidelines advise broad molecular profiling to identify driver mutations that have either FDA approved therapies or available clinical trials. Testing is recommended for at least 8 biomarkers plus PD-L1 immunohistochemistry and obtaining biomarker testing results back before initiation of treatment is critical. However, many patients with NSCLC remain untested or receive inadequate treatment prior to obtaining a test result. We performed a retrospective chart review to assess real world effective biomarker testing rates and practice patterns to understand the current biomarker testing scenario in a large US community practice, Florida Cancer Specialists (FCS). Methods: Advanced NSCLC patients (Stage IV; n = 361) who received care in the FCS network and were diagnosed or received first-line treatment on or after 1/1/2020 were included in this retrospective observational study. Data was extracted from the electronic medical records in participating practices as well as via abstraction of patient records when available. Patient-level data was anonymized and reported in aggregate. All data collected for this analysis was evaluated in accordance with regulatory requirements and applicable guidance for electronic records. We determined the percentage of biomarkers tested, orders placed and received before treatment, test ordered after treatment, and the overall effective testing rate, defined as orders placed and result received before treatment start/stage IV patients treated. Results: The median patient age was 70 years (range, 41-90), 188 (52%) were male, and 277 (77%) were Caucasian. 270 (75%), had adenocarcinoma histology, and 273 (76%) had an ECOG status of 0 or 1. Biomarker testing was ordered for 285 patients (79%). EGFR tests were more likely to be ordered before the start of treatment than other biomarkers. The median time between biomarker test ordering to treatment start was 15 days. 217 patients received test results before treatment start (effective testing rate = 60%). 6% of patients had tests ordered later in their workup, while 13% had tests ordered early, but received test results after treatment start. 21% of patients were not tested for any biomarker. 85% of stage IV patients tested received multi-gene tests. 71% of patients had PD-L1 testing and less than 2% received PD-L1 testing alone. Conclusions: This real-world study showed that 85% stage IV NSCLC patients in a large US community practice network underwent multigene biomarker testing and 60% received their test results prior to treatment start. The median time between biomarker test ordering to treatment start was 15 days. Strategies to improve time to biomarker test order, enhance percentage of patients tested, and improvement of percentage of patients with test results prior to initiation of therapy are ongoing.

Blood based multigene testing using next generation sequencing to guide management in solid tumors: Experience from Augusta-Georgia.

e15023 Background: The pool of somatic alterations with prognostic and predictive significance in management of solid tumors is rapidly expanding. Multigene panel testing using next generation sequencing (NGS) technology from peripheral blood specimen is convenient and readily available in most oncology practices. Widespread adoption is lagging, in part due to anticipation of low probability of finding an actionable information. We share our experience of real-world utilization of this technology. Methods: Blood based next generation testing ordered by providers in Georgia (outside Atlanta) between January 1, 2015, and December 31, 2021, via Guardant platform for all solid tumors was included in this analysis. Guardant360 and Guardant360CDx assays sequenced circulating cell-free DNA to detect somatic alterations in up to 83 cancer associated genes. Data was obtained from Guardant360 on site of cancer, comprehensive genomic profile, and therapeutic options at the time of report. Results: 25 providers in Georgia from 11 institutions sent 335 blood samples from solid cancer patients. There were 192 males and 143 females. Most common cancers were lung and bronchus (45%), prostate (13%), breast (11%), pancreaticobiliary (8%), colorectal (4%), melanoma (3%) and kidney (3%). Any somatic mutations were detected in 85% (285/335) patients. A total 1524 alterations comprising of 977 unique alterations involving 83 genes were detected, with an average of 5.3 unique genomic alterations per patient. The genes that were altered at least 2% of times were TP53 (20%), ATM (5.5%), PI3KCA (4.3%), KRAS (3.8%), APC (3.5%), AR (2.8%), PDGFRA (2.8%), ARIDIA (2.5%) and BRAF (2.2%). Of all alterations, 11% (170/1524) were synonymous and 31% (474/1524) were variant of unknown significance. 85 samples had tumor mutational burden (TMB) results available, with 55% (47/85) having TMB &gt; 10 mut/Mb. The average TMB was 14.9 mutations/Mb. Out of 252 specimens evaluated for microsatellite instability (MSI), 1 had high MSI. An FDA approved drug targeting the alteration/pathway was available for the same cancer in 4% (77/1524) and for other cancers in 19% (291/1524) of alterations. An approved drug matched to the alteration was available for the same cancer in 16% (54/335) patients and for other cancers in 45% (153/335) patients. 12.5% (42/335) patients were predicted not to benefit from certain drugs. 74% (249/335) had a clinical trial available for the mutations present. Conclusions: Eighty five percent solid tumor patients had a positive result in blood based NGS. Seventy four percent patients had at least one therapeutically applicable (suggestion for a drug to target the alteration approved for the same/different cancers or lack of benefit due to the alteration) information. Thus, blood based NGS yields clinically relevant and actionable information to guide management in solid tumors.

Molecular profiling of dedicated lung cancer biopsy tissue sample collected at time of diagnostic bronchoscopy.

e20587 Background: The combined use of molecular biomarker testing and targeted precision therapeutics has led to improved survival in lung cancer. Broadening access to national guideline recommended comprehensive molecular testing requires overcoming the challenges of inadequate tissue biopsies, which can lead to the need for additional procedures and ultimately, delays in initiation of care. We show Percepta Genomic Atlas identifies key molecular alterations in transbronchial needle aspirate (TBNA) and transbronchial biopsy (TBB) samples of lung or lymph node collected during the initial diagnostic bronchoscopy. Methods: Percepta Genomic Atlas combines the whole exome TruSeq RNA Exome and targeted AmpliSeq Focus DNA assays (Illumina) for a comprehensive gene panel including ALK, RET, ROS1, NTRK1/3, MET, EGFR, BRAF, KRAS and HER2. TruSight Oncology 500 DNA and AmpliSeq Focus RNA assays (Illumina) were used as reference assays. DNA and RNA were extracted from samples with the AllPrep Micro kit (Qiagen) and analyzed by Percepta Genomic Atlas and reference assays. 94 biopsy samples (73 TBNA and 21 TBB from 71 patients undergoing a diagnostic bronchoscopy for suspected lung cancer were collected into RNAprotect (Qiagen) under an IRB approved protocol. Local molecular testing results from FFPE biopsy samples taken during the same bronchoscopy procedure were collected. Results: RNA and DNA in sufficient amounts to run the Percepta Genomic Atlas and reference assays was obtained from 85 of 94 lung biopsies from 63 of 71 patients. Percepta Genomic Atlas identified pathogenic variants in 29 bronchoscopy biopsy samples from 23 patients including single nucleotide variants in EGFR, KRAS, BRAF and PIK3CA, an EGFR exon 19 deletion/insertion and copy number amplications in AR, EGFR, CDK4, CCND1, MYC, MYCN and PIK3CA. No fusions were identified. This results in a 100% sensitivity for detecting pathogenic alterations when compared to reference assay results. When comparing Percepta Genomic Atlas results to local molecular testing of bronchoscopy samples performed as part of routine clinical care, we found 100% concordance with 7 of 7 alterations detected in 7 patients, including mutations in EGFR, KRAS and BRAF. In a further 4 patients, Percepta Genomic Atlas and local multi-gene NGS testing results agreed, with neither assay identifying guideline recommended alterations. Conclusions: Using a combination of whole exome RNA and targeted DNA sequencing, Percepta Genomic Atlas detects the clinically actionable mutations in patients with non-small cell lung cancer using fresh tissue specimens collected during bronchoscopic tissue sampling, with high concordance to standard of care testing. By initiating broad molecular testing at the time of the bronchoscopy, Percepta Genomic Atlas may provide timelier results for patients with lung cancer.

A real-world (rw) evidence study quantifying the clinical value of multi-gene testing in early-stage lung adenocarcinoma (LUAD).

8525 Background: Resected early-stage NSCLC has high risk of recurrence, and tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICI), each requiring testing for precision biomarkers, have recently been approved in the adjuvant (adj) setting. We assessed the potential value of multi-gene testing in early NSCLC via enabling timely 1L therapy selection at recurrence and through avoidance of adj ICI in pts with driver alterations unlikely to benefit per current guidelines and the label for atezolizumab in first line (1L) NSCLC. Methods: Using the nationwide (̃280 US cancer clinics) de-identified EHR-derived rw Flatiron Health- Foundation Medicine clinico-genomic database, we selected 6,725 evaluable pts with LUAD who underwent tissue comprehensive genomic profiling (CGP) as part of routine cancer care (01/2011 – 06/2021). We focused on alterations in 4 drivers ( EGFR, ALK, ROS1, RET) and studied prevalence in early-stage specimens versus advanced (adv) specimens, as well as the rate of timely delivery of 1L therapy at recurrence for pts receiving CGP in early disease. We estimated the cost implications of adj ICI in pts with PD-L1+ LUAD and an ALK, ROS1, or RET fusion with a probabilistic decision tree. Results: CGP was performed on 1,490 specimens collected prior to adv disease (stage I 36%, II 27%, IIIA 37%) and ordered prior to adv diagnosis for 981 pts (15% of total, median 10 weeks after initial diagnosis). In specimens collected in early (n = 1,490) or adv (n = 5,130) stage LUAD, CGP identified drivers in EGFR (early/adv: 13%/16%), ALK (2.0%/4.2%), ROS1 (0.7%/1.1%), RET (1.0%/1.1%), and prevalence was similar when limiting to PD-L1+ cases. Studying 596 pts with recurrence and CGP on samples collected prior to recurrence, pts with CGP results obtained before (n = 196) vs after (n = 400) recurrence had less time between recurrence and start of any 1L therapy (median 3.1 vs 5.9 weeks, p &lt; 0.001). In the subset with a targetable driver detected, 32/42 (76%) with CGP before recurrence initiated matched 1L TKI while 43/67 (64%) with CGP after recurrence received matched 1L TKI (p = 0.2). Through avoidance of ICI in PD-L1+ early LUAD with an ALK/ ROS1/ RET driver, we estimate universal CGP (compared to EGFR single-gene testing) could reduce per pt expected costs by $875 (an average incremental $4,050 treatment cost reduction compared to a $3,175 increase in diagnostic cost per pt). Conclusions: CGP of early-stage LUAD can identify EGFR, ALK, ROS1 and RET drivers and enable appropriate selection of precision therapies and timely use of effective 1L therapy at recurrence. Assuming adj ICI maintains the lack of activity in RET/ ROS1 as seen for ALK, CGP could represent a cost-effective approach for avoiding futile adj ICI and reducing the risk of subsequent TKI-associated toxicity. Additional analysis of driver+ pts receiving adj ICI is needed to help balance the risk/benefit for these high risk pts.

How does re-classification of variants of unknown significance (VUS) impact the management of patients at risk for hereditary breast cancer?

BackgroundThe popularity of multigene testing increases the probability of identifying variants of uncertain significance (VUS). While accurate variant interpretation enables clinicians to be better informed of the genetic risk of their patients, currently, there is a lack of consensus management guidelines for clinicians on VUS.MethodsAmong the BRCA1 and BRCA2 mutations screening in 3,544 subjects, 236 unique variants (BRCA1: 86; BRCA2: 150) identified in 459 patients were being reviewed. These variants consist of 231 VUS and 5 likely benign variants at the initial classification.ResultsThe variants in 31.8% (146/459) patients were reclassified during the review, which involved 26 unique variants (11.0%). Also, 31 probands (6.8%) and their family members were offered high-risk surveillance and related management after these variants were reclassified to pathogenic or likely pathogenic. At the same time, 69 probands (15%) had their VUS downgraded to cancer risk equivalent to the general population level.ConclusionA review of archival variants from BRCA1 and BRCA2 genetic testing changed the management for 31.8% of the families due to increased or reduced risk. We encourage regular updates of variant databases, reference to normal population and collaboration between research laboratories on functional studies to define the clinical significances of VUS better.