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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.

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.

Liquid biopsy represents a powerful clinical tool that makes use of the detection of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes in peripheral blood. Each different analysis is complementary to each other and can give additional valuable clinical information. These biomarkers are susceptible to quick degradation, presenting a challenge in a clinical environment and may require different blood collection tubes. The aim of this CANCER-ID study was to analyze the influence of different blood tubes on quantity and quality of CTCs, ctDNA and total and exosomal miRNA isolated from the same blood tube. After confirming that plasma removal does not influence CTC recovery rates, peripheral blood from 20 cases of metastatic melanoma patients was collected in EDTA, Streck and Transfix tubes. CTC isolation from the PBMC fraction was performed by the ClearCell device (Clearbridge BioMedics). CTCs were identified by immunofluorescence staining. CtDNA was extracted from Streck and EDTA plasma samples, quantity (Qubit) and quality (Tapestation) measurements were performed before analyzing 86 hot-spot mutations in 13 genes by the UltraSEEK chemistry (Agena Bioscience). Ten of the 20 metastatic melanoma patients and 5 healthy donors were chosen for miRNA analysis. MiRNAs were extracted with miRNeasy Serum/Plasma Advanced Kit and miRNeasy Serum/Plasma Kit (QIAGEN) from total plasma and from extracellular vesicles (EVs), respectively. EVs were isolated by ultracentrifugation. QIAseq miRNA libraries were produced and sequenced (Illumina NextSeq 550). The reads were mapped to miRBase and normalized (geNorm). Volcano plots of fold change versus p-value were used to display the miRNAs that are significantly regulated. The CTC enrichment results showed that 3/20 of the EDTA blood samples were positive, whereas samples from Streck and Transfix tubes were negative. The quantity or quality of the ctDNA did not significantly differ between the EDTA and Streck tubes. Mutation analysis of ctDNA performed so far showed similar detection sensitivities between the two tubes. The morphology, particle concentration and size distribution of EVs did not either differ between the two tubes. MiRNA-NGS analyses from plasma revealed that seven (EDTA) and four (Streck) miRNAs are significantly differentially expressed in patients compared with healthy donors. No overlap of these miRNAs was found between the two tubes. In the EV fraction 24 and 31 miRNAs were found significantly differentially expressed in the EDTA and Streck tubes, respectively. Here, one EV miRNA is up-regulated and six are down-regulated in both tubes. In conclusion, the CTC recovery rates and minimal overlap of circulating miRNAs indicate that the different tubes affect CTC, ctDNA and miRNA results. Thus, the outcome of liquid biopsy analyses strongly depends on the choice of blood collection tubes as important pre-analytical variable. Citation Format: Svenja Schneegans, Lelia Lück, Leonie Bluhm, Janina Staub, Rüdiger Greinert, Beate Volkmer, Alexander Sartori, Darryl Irwin, Taija af Hallstrom, Melanie Hussong, Jonathan Shaffer, Markus Sprenger-Haussels, Stefan W. Schneider, Peter Mohr, Klaus Pantel, Harriet Wikman. Impact of blood collection tubes on CTC-, ctDNA- and miRNA recoveries in malignant melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5596.

RNA Sequencing (RNA-Seq) uses the capabilities of Next Generation high-throughput sequencing (NGS) methods to provide insight into the transcriptome of a cell as it generates millions of reads. Whole transcriptome sequencing can be used to quantify gene expression on a transcriptome-wide scale, identify splice variants, quantify allele specific expression, and characterize fusion transcripts. Development of a highly reproducible and sensitive targeted quantitative sequencing method would aid in facilitating a deeper understanding and characterization of the roles of a specific set of genes, while enabling much higher sample throughput and significant cost savings relative to whole-transcriptome sequencing. In this study, we report a targeted RNA-Seq technology, QIAseq RNA, which makes use of several methodologies to deliver an extremely flexible, highly precise tool for characterizing gene expression. QIASeq RNA incorporates 12-base random molecular barcodes into each unique target strand which benefits quantifying gene expression in a given multiplexed sample. Counting the number of molecular tags allows one to determine the sequence coverage per target and adjust experimental conditions to use the read budget of any sequencing platform most efficiently. Using either the Illumina or Ion Torrent platforms, users can choose to multiplex up to 96 RNA samples from 12 to 1000-plex expression panels. No mRNA selection or rRNA removal or blocking is required. The entire protocol, from cDNA synthesis to finished library, which is ready for sequencing, can be accomplished in under one day. Custom assays for a specific target site can add the ability to distinguish between isoforms or identify allele specific expression. We explore the capabilities of this system by profiling large numbers of genes in a cell model system's response to small molecule treatment. Changes in gene expression by these treatments were measured by targeted RNA NGS, and fold-changes in gene expression due to chemical perturbation were characterized. Complex gene relationships in perturbed pathways were mapped using QIAGEN's Ingenuity Pathway Analysis (IPA) tool. The IPA tool also facilitated the elucidation of the impact of gene expression changes in the context of biological processes, molecular interactions, cellular phenotypes and disease. This article will provide application data for GRRC including a discussion of technical challenges faced when profiling large numbers of genes in a large cohort. Citation Format: Eric Lader, Melanie Hussong, Matthew Fosbrink. Development of a quantitative targeted RNA-Seq methodology for use in differential gene expression analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1845.