Kathleen S. Hruska

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing

Purpose:Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance.Methods:Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history.Results:Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2.Conclusion:The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823–832.

Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders

Purpose:Mendelian disorders are most commonly caused by mutations identifiable by DNA sequencing. Exonic deletions and duplications can go undetected by sequencing, and their frequency in most Mendelian disorders is unknown.Methods:We designed an array comparative genomic hybridization (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demonstrate for the first time the utility of exon-level array CGH in a large clinical cohort by testing for 136 autosomal dominant, 53 autosomal recessive, and 30 X-linked disorders.Results:Overall, 98 deletions and two duplications were identified in 53 genes, corresponding to a detection rate of 3.3%. Approximately 40% of positive findings were deletions of only one or two exons. A high frequency of deletions was observed for several autosomal dominant disorders, with a detection rate of 2.9%. For autosomal recessive disorders, array CGH was usually performed after a single mutation was identified by sequencing. Among 138 individuals tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or duplication.Conclusion:Our results demonstrate that exon-level array CGH provides a robust option for intragenic copy number analysis and should routinely supplement sequence analysis for Mendelian disorders.Genet Med 2012:14(6):594–603

MSH6 and PMS2 germ-line pathogenic variants implicated in Lynch syndrome are associated with breast cancer

PurposeAn association of Lynch syndrome (LS) with breast cancer has been long suspected; however, there have been insufficient data to address this question for each of the LS genes individually.MethodsWe conducted a retrospective review of personal and family history in 423 women with pathogenic or likely pathogenic germ-line variants in MLH1 (N = 65), MSH2 (N = 94), MSH6 (N = 140), or PMS2 (N = 124) identified via clinical multigene hereditary cancer testing. Standard incidence ratios (SIRs) of breast cancer were calculated by comparing breast cancer frequencies in our study population with those in the general population (Surveillance, Epidemiology, and End Results 18 data).ResultsWhen evaluating by gene, the age-standardized breast cancer risks for MSH6 (SIR = 2.11; 95% confidence interval (CI), 1.56–2.86) and PMS2 (SIR = 2.92; 95% CI, 2.17–3.92) were associated with a statistically significant risk for breast cancer whereas no association was observed for MLH1 (SIR = 0.87; 95% CI, 0.42–1.83) or MSH2 (SIR = 1.22; 95% CI, 0.72–2.06).ConclusionOur data demonstrate that two LS genes, MSH6 and PMS2, are associated with an increased risk for breast cancer and should be considered when ordering genetic testing for individuals who have a personal and/or family history of breast cancer.

Gene-specific criteria for PTEN variant curation: Recommendations from the ClinGen PTEN Expert Panel

The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN‐specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic (PATH/LPATH), uncertain significance (VUS), and conflicting (CONF) ClinVar assertions. Utilizing these rules, classifications concordant with ClinVar assertions were achieved for 14/15 (93.3%) BEN/LBEN and 16/16 (100%) PATH/LPATH ClinVar consensus variants for an overall concordance of 96.8% (30/31). The variant where agreement was not reached was a synonymous variant near a splice donor with noncanonical sequence for which in silico models cannot predict the native site. Applying these rules to six VUS and five CONF variants, adding shared internal laboratory data enabled one VUS to be classified as LBEN and two CONF variants to be as classified as PATH and LPATH. This study highlights the benefit of gene‐specific criteria and the value of sharing internal laboratory data for variant interpretation. Our PTEN‐specific criteria and expertly reviewed assertions should prove helpful for laboratories and others curating PTEN variants.

Germline pathogenic variants identified in women with ovarian tumors

OBJECTIVE The recognition of genes implicated in ovarian cancer risk beyond BRCA1, BRCA2, and the Lynch syndrome genes has increased the variety of testing options available to providers and patients. We report the frequency of pathogenic variants identified among individuals with ovarian cancer undergoing clinical genetic testing via a multi-gene hereditary cancer panel. METHODS Genetic testing of up to 32 genes using a hereditary cancer panel was performed on 4439 ovarian cancer cases, and results were analyzed for frequency of pathogenic variants. Statistical comparisons were made using t-tests and Fishers exact tests. RESULTS The positive yield was 13.2%. While BRCA1/2 pathogenic variants were most frequent, one third (33.7%) of positive findings were in other homologous recombination genes, and accounted for over 40.0% of findings in endometrioid and clear cell cases. Women with a personal history of breast cancer (22.1%), who reported a family history of ovarian cancer (17.7%), and/or serous histology (14.7%) were most likely to harbor a pathogenic variant. Those with very early onset (<30 years) and late onset (≥70 years) ovarian cancer had low positive yields. CONCLUSIONS Our study highlights the genetic heterogeneity of ovarian cancer, showing that a large proportion of cases are not due to BRCA1/2 and the Lynch syndrome genes, but still have an identifiable hereditary basis. These findings substantiate the utility of multi-gene panel testing in ovarian cancer care regardless of age at diagnosis, family history, or histologic subtype, providing evidence for testing beyond BRCA1/2 and the Lynch syndrome genes.

Early onset renal cell carcinoma in an adolescent girl with germline FLCN exon 5 deletion

Birt-Hogg-Dube (BHD) disease is an autosomal dominant cancer syndrome characterized by benign skin tumors, renal cancer and spontaneous pneumothorax and is caused by mutations in the Folliculin (FLCN) gene. Benign skin tumors and pneumothorax occur in the majority of patients affected by BHD syndrome, but only 30–45% of them develop renal cell carcinoma (RCC) with a median age of diagnosis at 48. The earliest onset of RCC in a BHD patient has been reported at age 20. Here we report a case of a 14 year-old patient with germline FLCN mutation leading to an early-onset bulky RCC that could not be classified strictly according to existing histological types. Germline genetic testing revealed a deletion at FLCN exon 5. The father of the patient was identified as the asymptomatic carrier. We report the youngest patient with BHD-related RCC. This early onset presentation supports genetic testing of at-risk patients and initiation of imaging surveillance for RCC in early adolescence. In addition, future studies are necessary to understand the determinants of reduced penetrance in BHD disease.

Abstract PD1-06: MosaicTP53variants in women with breast cancer

Background: Germline TP53 pathogenic variants are indicative of Li-Fraumeni syndrome (LFS), a dominantly inherited hereditary cancer syndrome with high lifetime risks for female breast and other cancers. Genetic testing for LFS may reveal mosaicism, indicating a variant is present in some, but not all, of the cells tested. While these findings may represent a true constitutional event, mosaicism for TP53 variants also has been reported as a somatic event in lymphoblastoid cells from individuals with hematologic malignancy, previous chemotherapy exposure, or due to age-related clonal hematopoietic expansion. Understanding if a mosaic variant is constitutional can influence the patient9s management and impact familial risk assessment. We present clinical history and follow-up testing from a series of female breast cancer patients with blood or oral rinse testing revealing a mosaic TP53 pathogenic or likely pathogenic variant (collectively, PV). Methods: We retrospectively reviewed clinical history and genetic testing results to identify women with a personal history of breast cancer and mosaicism for one or more TP53 PV identified on multi-gene hereditary cancer testing at our clinical diagnostic laboratory. Descriptive statistics were employed. Results: Forty-eight women with breast cancer were identified as having a mosaic TP53 PV, defined as an allelic fraction of 40 years of age and three had other primary cancer diagnoses, including one with sarcoma. In one patient with early-onset and HER2-positive breast cancer, testing of fibroblasts also identified mosaicism for the TP53 PV, indicating constitutional mosaicism. Conclusions: For individuals with a mosaic TP53 PV, identification of the variant in a second tissue is necessary to confirm constitutional mosaicism and heightened risk for other LFS-associated cancers. In this series, additional testing confirmed one of six patients with mosaic TP53 PV pursuing fibroblast testing to have constitutional mosaicism. This individual9s breast cancer was HER2-positive and her age at diagnosis was younger than those whose mosaic PV was not identified in fibroblasts. Additional follow-up testing data are needed to understand whether confirmatory testing in a second tissue is indicated for any breast cancer patient with a mosaic TP53 PV, or would be most likely to reveal constitutional mosaicism in individuals whose breast cancers are HER2-positive or early-onset. Citation Format: Mester JL, Postula K, Bissonnette J, Klein RT, Hruska K. Mosaic TP53 variants in women with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-06.

Abstract P2-09-17: Frequency of large rearrangements in BRCA1, BRCA2, ATM, CHEK2, and PALB2 in hereditary cancer testing

Objectives Large deletions and duplications are recognized as a cause of many inherited cancer syndromes; however, few studies have examined the frequency. In one study, large rearrangements accounted for 11-14% of pathogenic variants in BRCA1 and 1-3% of pathogenic variants in BRCA2 (Judkins 2012). Additionally, in a study of invasive ovarian cancer patients, large deletions and duplications accounted for 6% of BRCA1 and 3% of BRCA2 pathogenic variants (Zhang 2011). The frequency of large deletions and duplications in moderate penetrance genes, such as ATM, CHEK2, and PALB2, has not been carefully studied. We therefore sought to determine the frequency of pathogenic and likely pathogenic (P/LP) large deletions and duplications in BRCA1, BRCA2, ATM, CHEK2, and PALB2. Methods We analyzed a consecutive case series of patients who had BRCA1/2 and cancer panel testing. The data were analyzed to determine the percentage of deletions or duplications of greater than 250 bp identified on array comparative genome hybridization as a percentage of all pathogenic variants (PVs)/likely pathogenic variants (LPVs) in each of BRCA1, BRCA2, ATM, CHEK2, and PALB2. Results Large deletions/duplications accounted for 10.1% (55/545) of cases with a BRCA1 PV/LPV and 1.8% (10/556) of cases with a BRCA2 PV/LPV. By ethnicity, BRCA1/2 large deletions/duplications accounted for a higher fraction of PV/LPVs in Asians (11.3%) and Hispanics (8.1%). In Hispanic individuals, all of the large deletions/duplications were identified in BRCA1, accounting for 14.6% of all BRCA1 PV/LPV in this group. In the moderate penetrance genes, large deletions/duplications accounted for 4.6% (9/195), 5.1% (22/436), and 9.9% (13/132) of cases with a PV/LPV in ATM, CHEK2, and PALB2, respectively. There were two recurrent deletions in our cohort: the CHEK2 founder Slavic deletion of exons 9-10 observed in 16 of our cases, and a deletion of exon 11 in PALB2 observed in 6 cases. Conclusions Large deletions/duplications account for a substantial proportion of clinically significant variants in hereditary cancer genes. The proportion of large deletions/duplications in BRCA1 and BRCA2 reported here is similar to previous data. However, our cohort suggests that Asian individuals may have a higher proportion of mutations due to large deletions/duplications than previously reported. Among moderate risk cancer genes, PALB2 has the highest proportion of large deletions/duplications. Citation Format: Bissonnette J, Doonanco K, Xu Z, Klein RT, Hruska KS. Frequency of large rearrangements in BRCA1, BRCA2, ATM, CHEK2, and PALB2 in hereditary cancer testing. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-17.

Abstract P4-12-05: Individuals with more than one pathogenic variant: Rationale for considering multi-gene panel testing for cancer susceptibility

Introduction: Expansion of genetic testing technologies has brought multi-gene panels for cancer susceptibility into the clinic; however, the clinical utility of these next-generation sequencing (NGS) panels is largely unknown. Hypothesis: We hypothesized that the use of multi-gene panels would yield a significant number of cases with more than one pathogenic variant. Methods: We retrospectively queried oncology tests reported at GeneDx from August 2013 to April 2014 for individuals with more than one pathogenic variant. Next, we calculated the proportion of individuals with more than one pathogenic variant among all positive reports, excluding familial and stand-alone BRCA1/2 tests. We then extracted personal and family histories, including available segregation data, to categorize the phenotypes. Results: Of 406 unique, unrelated individuals with pathogenic or likely pathogenic findings, 11 (2.7%) had more than one pathogenic variant. This total includes nine individuals with a mutation in more than one gene, as well as two individuals with two mutations in trans in the same gene. Ten of these 11 individuals were identified by multi-gene panel tests, one individual by step-wise (tiered) testing. Seven of 11 individuals were positive for a pathogenic variant in a traditional, highly-penetrant cancer susceptibility gene and another pathogenic variant in a gene with moderate cancer susceptibility, such as CHEK2 and ATM. Three of 11 probands had more than one primary tumor. Several of the families were significant for bilineal cancer risk. Conclusions: Our data suggest that a traditional single gene approach to cancer testing may fail to identify all pathogenic variants related to the clinical presentation. The identification of a second risk factor for inherited susceptibility to cancer allows for appropriate testing and management considerations for family members. In conclusion, we provide early evidence for the consideration of multi-gene panel testing in a clinical oncology setting. Citation Format: Rachel Nusbaum, Lisa Susswein, Kathleen Hruska, Melanie Hussong, Windy Berkofsky-Fessler, Mingjuan Liao, Erica Rinella, Nina Sanapareddy, Joaquin Villar, Haiyan Wan, Zhixiong Xu, Rebecca Y Bassett, Elisabeth McKeen, Constance Murphy, Deborah Pencarinha, Jessica Booker, Maria L Cremona, Patricia Murphy, Rachel T Klein. Individuals with more than one pathogenic variant: Rationale for considering multi-gene panel testing for cancer susceptibility [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-05.

Abstract P4-12-01: Characterizing the clinical presentation of individuals with pathogenic variants in a breast/ovarian cancer gene panel

Background: Hereditary breast and ovarian cancer (HBOC) is a common indication for referral to cancer genetic counselors. Next generation sequencing panels allow for the efficient evaluation of many genes associated with increased risk of these cancers. The purpose of this study is to compare clinical histories of those with pathogenic variants in high risk versus low/moderate risk genes in order to determine which patients might benefit from more extensive testing by a panel approach. Methods: We queried the results of patients tested at GeneDx for a panel of 21 genes causing increased breast and/or ovarian cancer risk. Data regarding personal and family history of cancer provided on the test requisition forms were analyzed and classified according NCCN guidelines for testing criteria for HBOC syndrome. Results: Of 1709 individuals referred for testing, 146 (8.5%) tested positive for a pathogenic variant. Of these, 33% percent were found to carry a pathogenic BRCA1/2 variant while 67% tested positive for a pathogenic variant in a gene other than BRCA1/2 (CHEK2: 17%; ATM: 12%; PALB2: 10%; BRIP1: 7%; BARD1: 3%; PTEN: 3%; each of FANCC, MSH2, MSH6, NBN, PMS2, RAD51C, RAD51D: 2%; MLH1: 1%). Eighty-six percent of these individuals were affected with cancer. In the probands with a pathogenic BRCA1/2 variant, 66% were diagnosed with breast cancer and 22% with ovarian cancer compared to the probands with a pathogenic variant in a gene other than BRCA1/2 of whom 84% had a history of breast cancer and 17% had ovarian cancer. The highest number of breast cancer diagnoses were found, in decreasing order, in association with pathogenic variants in BRCA1 (20), CHEK2 (20), ATM (17), PALB2 (14), BRCA2 (13), and BRIP1 (12). The greatest number of ovarian cancers were identified, in decreasing order, in association with pathogenic variants in BRCA2 (8), BRCA1 (3), CHEK2 (3), and ATM (3). Furthermore, all of the individuals with pathogenic variants met NCCN guidelines for HBOC. Conclusion: A high yield of pathogenic variants were found in genes other than BRCA1/2. Data analysis also shows that individuals with a pathogenic variant in genes other than BRCA1/2 did not have a notably less severe clinical history than those pathogenic variants in BRCA1/2. As all individuals tested meet NCCN guidelines for HBOC testing, panel testing should be considered in this population. Citation Format: Erica Vaccari, Lauren Yackowski, Melanie Hussong, Patricia Murphy, Maria L Cremona, Jessica Booker, Kathleen Hruska. Characterizing the clinical presentation of individuals with pathogenic variants in a breast/ovarian cancer gene panel [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-01.