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PURPOSE To determine the contribution of germline pathogenic variants (PVs) in hereditary cancer testing panel genes to invasive lobular carcinoma (ILC) of the breast. MATERIALS AND METHODS The study included 2,999 women with ILC from a population-based cohort and 3,796 women with ILC undergoing clinical multigene panel testing (clinical cohort). Frequencies of germline PVs in breast cancer predisposition genes ( ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, and TP53) were compared between women with ILC and unaffected female controls and between women with ILC and infiltrating ductal carcinoma (IDC). RESULTS The frequency of PVs in breast cancer predisposition genes among women with ILC was 6.5% in the clinical cohort and 5.2% in the population-based cohort. In case-control analysis, CDH1 and BRCA2 PVs were associated with high risks of ILC (odds ratio [OR] > 4) and CHEK2, ATM, and PALB2 PVs were associated with moderate (OR = 2-4) risks. BRCA1 PVs and CHEK2 p.Ile157Thr were not associated with clinically relevant risks (OR < 2) of ILC. Compared with IDC, CDH1 PVs were > 10-fold enriched, whereas PVs in BRCA1 were substantially reduced in ILC. CONCLUSION The study establishes that PVs in ATM, BRCA2, CDH1, CHEK2, and PALB2 are associated with an increased risk of ILC, whereas BRCA1 PVs are not. The similar overall PV frequencies for ILC and IDC suggest that cancer histology should not influence the decision to proceed with genetic testing. Similar to IDC, multigene panel testing may be appropriate for women with ILC, but CDH1 should be specifically discussed because of low prevalence and gastric cancer risk.

PURPOSE The prevalence of germline pathogenic variants (PVs) in established breast cancer predisposition genes in women in the general population over age 65 years is not well-defined. However, testing guidelines suggest that women diagnosed with breast cancer over age 65 years might have < 2.5% likelihood of a PV in a high-penetrance gene. This study aimed to establish the frequency of PVs and remaining risks of breast cancer for each gene in women over age 65 years. METHODS A total of 26,707 women over age 65 years from population-based studies (51.5% with breast cancer and 48.5% unaffected) were tested for PVs in germline predisposition gene. Frequencies of PVs and associations between PVs in each gene and breast cancer were assessed, and remaining lifetime breast cancer risks were estimated for non-Hispanic White women with PVs. RESULTS The frequency of PVs in predisposition genes was 3.18% for women with breast cancer and 1.48% for unaffected women over age 65 years. PVs in BRCA1, BRCA2, and PALB2 were found in 3.42% of women diagnosed with estrogen receptor (ER)–negative, 1.0% with ER-positive, and 3.01% with triple-negative breast cancer. Frequencies of PVs were lower among women with no first-degree relatives with breast cancer. PVs in CHEK2, PALB2, BRCA2, and BRCA1 were associated with increased risks (odds ratio = 2.9-4.0) of breast cancer. Remaining lifetime risks of breast cancer were ≥ 15% for those with PVs in BRCA1, BRCA2, and PALB2. CONCLUSION This study suggests that all women diagnosed with triple-negative breast cancer or ER-negative breast cancer should receive genetic testing and that women over age 65 years with BRCA1 and BRCA2 PVs and perhaps with PALB2 and CHEK2 PVs should be considered for magnetic resonance imaging screening.

Background: DNA genetic testing is commonly used to inform treatment decisions for breast cancer patients. In recent years, RNA genetic testing has shown promise for increasing the detection of disease-causing variants and decreasing inconclusive results. Here we describe the impact of concurrent DNA and RNA genetic testing on identifying breast cancer patients with germline cancer predisposition who may have been missed by DNA-only testing. Methods: We performed a retrospective review of breast cancer patients who received concurrent DNA and RNA hereditary cancer panel testing between March 2019 and February 2020 at Ambry Genetics. Patients underwent DNA and RNA genetic testing of up to 18 hereditary cancer genes at the discretion of the ordering healthcare provider (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2 exons 1-10, PTEN, RAD51C, RAD51D, and TP53). Breast cancer patients with positive results, defined as the presence of a pathogenic or likely pathogenic variant in any of these 18 genes, were selected for inclusion in this study (n=946). Results: Concurrent DNA and RNA genetic testing led to the identification of 23 breast cancer patients with positive results who would have otherwise received inconclusive or negative results with DNA-only testing. These cases represented 2.4% of all positive results reported in the 18 genes studied (n=23/946). The majority of RNA-related positive results occurred in either ATM (n=14) or BRCA1/2 (n=7). The remaining two cases involved alterations in NF1 and PMS2. Guidelines for risk-reducing breast surgery (BRCA1/2) and breast imaging surveillance (ATM, BRCA1/2, NF1) were relevant for 30.4% and 95.7% of patients with RNA-dependent positive results, respectively. In addition, treatment options such as PARP inhibitors or clinical trial eligibility were potentially implicated for 91.3% of RNA-dependent positives (BRCA1/2, ATM). In 16 of the 23 cases, variants would have been detected by DNA-only testing but would have remained inconclusive without supporting RNA data. In the remaining 7 cases, abnormal RNA results led to the identification of pathogenic/likely pathogenic intronic variants beyond the analytical range of DNA testing. Thus, these seven patients would have received negative results from DNA-only testing. Four of these cases involved pathogenic/likely pathogenic intronic variants in ATM and three involved pathogenic intronic variants in BRCA1. Conclusions: One in 41 breast cancer patients who test positive on concurrent DNA and RNA genetic testing would have received negative or inconclusive results from DNA-only testing. These findings demonstrate the impact of a comprehensive diagnostic testing approach that includes concurrent DNA and RNA analysis and highlights the important implications for the personalized management of these breast cancer patients including potential missed opportunities for early detection and prevention of additional cancer and familial testing in the absence of RGT. Citation Format: Holly LaDuca, Lily Hoang, Carolyn Horton, Jessica Profato, Tameron Harvell, Jill S Dolinsky, Tina Pesaran, Rachid Karam. Rna genetic testing improves detection of patients with hereditary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-07.

BACKGROUND: Germline genetic testing is routinely incorporated into clinical care for breast cancer patients to inform management decisions and reduce risk for developing subsequent cancers. While the diagnostic yield of cancer genetic testing has increased over the years due to adoption of multigene panels, a substantial portion of breast cancer patients remain without a molecular diagnosis yet are suspected to have a genetic mutation that could not be detected and/or classified with standard DNA testing techniques. We assessed the ability of a novel genetic testing approach involving simultaneous DNA and RNA analysis to increase the diagnostic yield and decrease the number of variants of unknown significance (VUS). METHODS: Women with a personal history of breast cancer were ascertained from a larger cohort of patients referred for concurrent RNA sequencing alongside DNA hereditary cancer panel testing by ordering clinicians from 18 collaborating medical centers across the United States. Test result classifications were evaluated for women whose testing included sixteen clinically-actionable hereditary breast and/or ovarian cancer (HBOC) genes (ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53). RESULTS: In this cohort of 746 breast cancer patients, the addition of RNA sequencing increased the pathogenic variant detection rate from 8% to 9% across sixteen HBOC genes. These RNA-related positive results included two pathogenic variants in BRCA1 occurring outside the standard analytical range of DNA testing and three VUS (one each in ATM, BRCA2, and PMS2) that were reclassified as likely pathogenic as a result of additional information provided by RNA sequencing. In addition, two VUS were reclassified to benign/likely benign (one each in MSH2 and BRCA2). Together, these five variant reclassifications contributed to a 3% relative decrease in the number of unique VUS classifications (reduced from 182 to 177 unique VUS). In addition, 31 previously-tested patients received reclassification reports. CONCLUSIONS: Concurrent DNA and RNA genetic testing has shown immediate promise in this pilot study, leading to the identification of five breast cancer patients with mutations in clinically actionable genes that would otherwise have received inconclusive or negative results with DNA testing alone. By increasing the detection of germline pathogenic variants and reducing VUS classifications, concurrent DNA and RNA genetic testing increases the diagnostic yield and clinical impact of hereditary cancer testing for breast cancer patients. Citation Format: Holly LaDuca, Lily Hoang, Jill Dolinsky, Jessica Profato, Amal Yussuf, Carolyn Horton, Cara Dresbold, Cassie Garcia, Catherine Koptiuch, Danielle Dondanville, Danielle McKenna, Danielle Menashe, Deborah Wham, Deepika Nathan, Diane Samad, Elizabeth Hoodfar, Gayle Patel, Jen Moore, Jennifer Geurts, John Lee, Kara Milliron, Khateriaa Pyrtel, Meagan Farmer, Meredith Seidel, Morgan Depas, Nichole Morman, Olivia Tan, Rebekah Krukenberg, Rob Pilarski, Samantha Stachowiak, Sandra Jenkinson, Sara Pirzadeh-Miller, Shraddha Gaonkar, Tiffani Demarco, Brigette Tippin Davis, Elizabeth C Chao, Rachid Karam. Concurrent DNA and RNA genetic testing identifies more patients with hereditary breast cancer than DNA testing alone [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-08.

BackgroundThe clinical phenotype of CDH1 pathogenic variant carriers has mostly been studied in families that fulfil criteria of hereditary diffuse gastric cancer (HDGC). We aimed at determining cancer phenotype and cancer risk estimation among families with CDH1 pathogenic variants not selected by HDGC clinical criteria.MethodsPatients were all consecutively identified CDH1 pathogenic variant carriers from a clinical laboratory tested with multigene panel testing and from an academic cancer genetics programme. Clinical and demographic features, cancer phenotypes and genotype–phenotype correlations were determined among CDH1 families. Age-specific cumulative cancer risks (penetrance) were calculated based on 38 families with available pedigrees.ResultsWithin the 113 CDH1 pathogenic variant probands and 476 relatives, 113 had gastric cancer, 177 breast cancer and 196 other cancers. Mean age at diagnosis was 47 for gastric and 54 for breast cancer. Forty-six per cent fulfilled criteria of HDGC. While 36% of families had both gastric and breast cancers, 36% had breast but no gastric cancers and 16% had gastric but not breast cancers. Cumulative risk of cancer by age 80 was 37.2% for gastric and 42.9% for breast cancer.ConclusionIn unselected CDH1 pathogenic variant carrier families, gastric cancer risks were lower and age at diagnosis higher than previously reported in families pre-selected for HDGC criteria. A substantial proportion of families did not present with any gastric cancers and their cancers were limited to breast. Thus, clinical criteria for CDH1 testing should be widened, including breast cancer families only, and a consideration for delayed prophylactic gastrectomy/surveillance should be evaluated.

The identification of the substrate of idiopathic ventricular fibrillation (VF) in the absence of structural heart disease or electrocardiographic changes remains a challenge for patient management and family counseling. The availability of expanded genetic testing panels and clinical exome screening has not led to a significant increase in the identification of genetic causes of these events, as shown by recent reports.1, 2 Indeed, the majority of studies detected only a small number of genetic variants1, 2 that could explain a sudden cardiac arrest event. Additionally, most variants still remain classified as “of unknown significance” and do not provide information useful for mechanistic understanding and cascade screening. Variants of unknown significance (VUS) of particular complex adjudication are those located at, and possibly altering, RNA splicing sites. RNA testing is thus critical in characterizing splicing alterations and could provide more precise insights compared to in silico prediction algorithms alone. We present here a case of idiopathic VF where RNA studies on the patient’s blood cells helped revise the adjudication of a VUS, providing valuable insights and implications for cascade screening.

Purpose Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. Methods The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. Results DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. Conclusion DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

BackgroundThe nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families).MethodsWhole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts.ResultsThe previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: c.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3–18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity.ConclusionWe report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants.

Purpose Mutations in the CDH1 gene confer up to an 80% lifetime risk of diffuse gastric cancer and up to a 60% lifetime risk of lobular breast cancer. Testing for CDH1 mutations is recommended for individuals who meet the International Gastric Cancer Linkage Consortium (IGCLC) guidelines. However, the interpretation of unexpected CDH1 mutations identified in patients who do not meet IGCLC criteria or do not have phenotypes suggestive of hereditary diffuse gastric cancer is clinically challenging. This study aims to describe phenotypes of CDH1 mutation carriers identified through multigene panel testing (MGPT) and to offer informed recommendations for medical management. Patients and Methods This cross-sectional prevalence study included all patients who underwent MGPT between March 2012 and September 2014 from a commercial laboratory (n = 26,936) and an academic medical center cancer genetics clinic (n = 318) to estimate CDH1 mutation prevalence and associated clinical phenotypes. CDH1 mutation carriers were classified as IGCLC positive (met criteria), IGCLC partial phenotype, and IGCLC negative. Results In the laboratory cohort, 16 (0.06%) of 26,936 patients were identified as having a pathogenic CDH1 mutation. In the clinic cohort, four (1.26%) of 318 had a pathogenic CDH1 mutation. Overall, 65% of mutation carriers did not meet the revised testing criteria published in 2015. All three CDH1 mutation carriers who had risk-reducing gastrectomy had pathologic evidence of diffuse gastric cancer despite not having met IGCLC criteria. Conclusion The majority of CDH1 mutations identified on MGPT are unexpected and found in individuals who do not fit the accepted diagnostic testing criteria. These test results alter the medical management of CDH1-positive patients and families and provide opportunities for early detection and risk reduction.

Purpose PTEN mutations are associated with breast, colon, endometrial, kidney, and thyroid cancers. Most PTEN promoter alterations, however, are characterized as variants of unknown significance, and their contribution to cancer risk is unclear. Materials and Methods Personal and family histories of 88,333 patients undergoing PTEN analysis as part of multigene panel testing (MGPT) were retrospectively reviewed. Cases (n = 59,784) were individuals with personal history of PTEN-related cancer. Controls (n = 28,549) had no personal history of cancer. Individuals were categorized as positive for one or more mutations (PATHO), without mutations but carrying one or more promoter variant (PROM), or negative for alterations (WT). Multivariable logistic regression was used to assess PTEN associations with phenotypes, adjusted for race/ethnicity, age, sex, and MGPT. Results Overall, 79 (0.09%) patients were PATHO and 791 (0.9%) were PROM carriers. Compared with WT, PATHOs were 2.30 (95% CI, 1.19 to 4.72) times as likely to have breast, 7.23 (95% CI, 2.74 to 19.14) times as likely to have bilateral/multiple primary breast, and 7.56 (95% CI, 1.97 to 23.98) times as likely to have uterine/endometrial cancer. PROMs were not significantly more likely than WT to have cancer (all 0.84 < odds ratio < 1.15; P > .05). Conclusion PTEN promoter variants were not associated with cancer. These results do not support the inclusion of PTEN promoter sequencing in MGPT.

Hormonal therapy targeting androgen receptor (AR) is initially effective to treat prostate cancer (PCa), but it eventually fails. It has been hypothesized that cellular heterogeneity of PCa, consisting of AR(+) luminal tumor cells and AR(−) neuroendocrine (NE) tumor cells, may contribute to therapy failure. Here, we describe the successful purification of NE cells from primary fresh human prostate adenocarcinoma based on the cell surface receptor C-X-C motif chemokine receptor 2 (CXCR2). Functional studies revealed CXCR2 to be a driver of the NE phenotype, including loss of AR expression, lineage plasticity, and resistance to hormonal therapy. CXCR2-driven NE cells were critical for the tumor microenvironment by providing a survival niche for the AR(+) luminal cells. We demonstrate that the combination of CXCR2 inhibition and AR targeting is an effective treatment strategy in mouse xenograft models. Such a strategy has the potential to overcome therapy resistance caused by tumor cell heterogeneity.

Importance Performing DNA genetic testing (DGT) for hereditary cancer genes is now a well-accepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians’ ability to accurately apply strategies for cancer risk reduction and treatment. Objective To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition. Design, Setting, and Participants Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018. Main Outcomes and Measures Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach. Results In total, 93 if 909 eligible families (10.2%) submitted samples for RGT. Evidence from RGT clarified the interpretation of 49 of 56 inconclusive cases (88%) studied; 26 (47%) were reclassified as clinically actionable and 23 (41%) were clarified as benign. Variant reclassifications based on RGT results changed clinical management recommendations for 8 of 18 patients (44%) and 14 of 18 families (78%), based on responses from 18 of 45 clinicians (40%) surveyed. A total of 7265 of 307 812 patients who underwent DGT had likely pathogenic variants or variants of uncertain significance potentially affecting splicing, indicating that approximately 1 in 43 individuals could benefit from RGT. Conclusions and Relevance In this diagnostic study, conducting RNA testing resolved a substantial proportion of variants of uncertain significance in a cohort of individuals previously tested for cancer predisposition by DGT. Performing RGT might change the diagnostic outcome of at least 1 in 43 patients if performed in all individuals undergoing genetic evaluation for hereditary cancer.

An appreciable number of patients with gastric cancer have an underlying hereditary cancer susceptibility syndrome as the cause of their gastric cancer, particularly those with early onset gastric cancer or a family history of gastric or other cancers. Pathogenic germline variants in specific genes account for the known gastric cancer predisposition syndromes. Germline genetic testing can identify individuals and their family members who carry inherited pathogenic gene variants, and thus have increased risk of developing gastric or other cancers. Ideally, germline pathogenic variants can be identified in family members before the onset of disease, when early detection or prevention strategies can be implemented most effectively to decrease gastric cancer- related morbidity and mortality. This article reviews some of the currently known gastric cancer predisposition syndromes and their associated cancer risks. We also discuss current research and advances in the field of genetic gastric cancer susceptibility.

Inherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1/2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2, MUTYH, and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA-negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.

Reticular dysgenesis is a form of severe combined immunodeficiency (SCID) caused by biallelic pathogenic variants in AK2. Here we present the case of a boy diagnosed with SCID following a positive newborn screen (NBS). Genetic testing revealed a homozygous variant: AK2 c.330 + 5G > A. In silico analyses predicted weakened native donor splice site. However, this variant was initially classified as a variant of uncertain significance (VUS) given lack of direct evidence. To determine the impact on splicing, we analyzed RNA from the proband and his parents, using massively parallel RNA-seq of cloned RT-PCR products. Analysis showed that c.330 + 5G > A results in exon 3 skipping, which encodes a critical region of the AK2 protein. With these results, the variant was upgraded to pathogenic, and the patient was given a diagnosis of reticular dysgenesis. Interpretation of VUS at noncanonical splice site nucleotides presents a challenge. RNA sequencing provides an ideal platform to perform qualitative and quantitative assessment of intronic VUS, which can lead to reclassification if a significant impact on mRNA is observed. Genetic disorders of hematopoiesis and immunity represent fruitful areas to apply RNA-based analysis for variant interpretation given the high expression of RNA in blood.

The variant curation guidelines published in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provided the genetics community with a framework to assess variant pathogenicity; however, these rules are not gene-specific. Germline pathogenic variants in the CDH1 gene cause hereditary diffuse gastric cancer and lobular breast cancer, a clinically challenging cancer predisposition syndrome that often requires a multidisciplinary team of experts to be properly managed. Given this challenge, the Clinical Genome Resource (ClinGen) Hereditary Cancer Domain prioritized the development of the CDH1 Variant Curation Expert Panel (VCEP) to develop and implement rules for CDH1 variant classifications. Here we describe the CDH1 specifications of the ACMG/AMP guidelines, which were developed and validated after a systematic evaluation of variants obtained from a cohort of clinical laboratory data encompassing ~827,000 CDH1 sequenced alleles. Comparing previously reported germline variants that were classified using the 2015 ACMG/AMP guidelines to the CDH1 VCEP recommendations resulted in reduced variants of uncertain significance and facilitated resolution of variants with conflicted assertions in ClinVar. Overall, the ClinGen CDH1 VCEP recommends the use of these CDH1-specific guidelines for the assessment and classification of variants identified in this clinically actionable gene.

Genetic testing for hereditary breast cancer is an integral part of individualized care in the new era of precision medicine. The accuracy of an assay is reliant on not only the technology and bioinformatics analysis utilized but also the experience and infrastructure required to correctly classify genetic variants as disease-causing. Interpreting the clinical significance of germline variants identified by hereditary cancer testing is complex and has a significant impact on the management of patients who are at increased cancer risk. In this review we give an overview of our clinical laboratory’s integrated approach to variant assessment. We discuss some of the nuances that should be considered in the assessment of genomic variants. In addition, we highlight lines of evidence such as functional assays and structural analysis that can be useful in the assessment of rare and complex variants.

Most truncating cadherin 1 (CDH1) pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer (DGC) and lobular breast cancer (LBC). However, transcripts containing carboxy-terminal premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing because of a personal history of invasive LBC diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.Glu836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most carboxy-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 carboxy-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other carboxy-terminal truncating variants as either pathogenic or benign, a fundamental step to offering presymptomatic screening and prophylactic procedures to the appropriate patients.

Background & Aims Hereditary diffuse gastric cancer (HDGC) families carry CDH1 heterozygous germline mutations; their tumors acquire complete CDH1 inactivation through "2nd-hit" mechanisms. Most frequently, this occurs via promoter hypermethylation (epigenetic modification), and less frequently via CDH1 mutations and loss of heterozygosity (LOH). We quantified the different 2nd hits in CDH1 occurring in neoplastic lesions from HDGC patients. Methods Samples were collected from 16 primary tumors and 12 metastases from 17 patients among 15 HDGC families; CDH1 mutations, LOH, and promoter hypermethylation were analyzed. E-cadherin protein expression and localization were determined by immunohistochemistry. Results Somatic CDH1 epigenetic and genetic alterations were detected in lesions from 80% of HDGC families and in 75% of all lesions analyzed (21/28). Of the 28 neoplastic lesions analyzed, promoter hypermethylation was found in 32.1%, LOH in 25%, both alterations in 17.9%, and no alterations in 25%. Half of the CDH1 2nd hits in primary tumors were epigenetic modifications, whereas a significantly greater percentage of 2nd hits in metastases were LOH (58.3%; P = .0274). Different neoplastic lesions from the same patient frequently displayed distinct 2nd-hit mechanisms. Different 2nd-hit mechanisms were also detected in the same tumor sample. Conclusion The 2nd hit in CDH1 frequently occurs via epigenetic changes in HDGC primary tumors and LOH in metastases. Because of the concomitance and heterogeneity of these alterations in neoplastic lesions and the plasticity of hypermethylated promoters during tumor initiation and progression, drugs targeting only epigenetic alterations might not be effective, particularly in patients with metastatic HDGC.

We report the association of CDH1/E‐cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in‐frame deletion, removing the extracellular cadherin repeat domains involved in cell‐cell adhesion. Such transcripts might encode mutant proteins with trans‐dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E‐cadherin pathway can contribute to human clefting.

Human papillomavirus (HPV) DNA has been detected in breast carcinoma by different laboratorial techniques, suggesting the virus could play a role in the pathogenesis of this tumor. The aim of the present study is to investigate the presence of HPV in patients with breast carcinoma and the correlation of the viral infection with prognostic factors for the disease outcome. Between June 2001 and July 2002, 101 paraffin embedded breast carcinoma specimens were analyzed through polymerase chain reaction (PCR) and sequencing of HPV-E6 gene. Twenty specimens of reduction mammoplasty and 21 specimens of fibroadenomas were also studied as a non-malignant control group. Two different specific primer sets targeting E6 region of the HPVs 16 and 18 were used for the analysis. The HPV DNA was detected in 25 breast carcinomas (24.75%), but in none of the benign breast specimens ( p < 0.001). Out of the 25 positive cases, 14 were HPV-16 positive (56%) and 10 were HPV-18 positive (40%). An original finding was the detection of both HPV-16 and -18 in a single tumor (4%). The amplified viral sequences confirmed the presence of HPV-16 and -18. No correlation between the presence of HPV DNA and specific prognostic predictors for the disease outcome was observed. Our results suggest that the presence in the breast of either HPV-16 or -18 might be related to development of the malignant phenotype. Further studies are warranted.

Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5’ donor and 3’ acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2. Citation Format: Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2017-1450

Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are, to our knowledge, the first known unique molecular signatures of pancreatic ASC.

Recent studies have reported germline CDH1 mutations in cases of lobular breast cancer (LBC) not associated with the classical hereditary diffuse gastric cancer syndrome. A multidisciplinary workgroup discussed genetic susceptibility, pathophysiology and clinical management of hereditary LBC (HLBC). The team has established the clinical criteria for CDH1 screening and results’ interpretation, and created consensus guidelines regarding genetic counselling, breast surveillance and imaging techniques, clinicopathological findings, psychological and decisional support, as well as prophylactic surgery and plastic reconstruction. Based on a review of current evidence for the identification of HLBC cases/families, CDH1 genetic testing is recommended in patients fulfilling the following criteria: (A) bilateral LBC with or without family history of LBC, with age at onset CDH1 asymptomatic mutant carriers, breast surveillance with clinical examination, yearly mammography, contrast-enhanced breast MRI and breast ultrasonography (US) with 6-month interval between the US and the MRI should be implemented as a first approach. In selected cases with personal history, family history of LBC and CDH1 mutations, prophylactic mastectomy could be discussed with an integrative group of clinical experts. Psychodecisional support also plays a pivotal role in the management of individuals with or without CDH1 germline alterations. Ultimately, the definition of a specific protocol for CDH1 genetic screening and ongoing coordinated management of patients with HLBC is crucial for the effective surveillance and early detection of LBC.