Abstract PS16-02: Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests
Authors: Angeloni Taraneh E , Bhattacharya Anindya , Cheng Linda L , Chong Hansook K , Conlan Kelli M , Elzinga Christopher D , Gerasimova Anna , Grover David , Grupe Andrew , Hua Michael , Hodko Domagoj , Kazmierkiewicz Krista , Nakles Rebecca E , Nery Camille R , Owen Renius , Goos-Root Dana M , Rowland Charles M , Smolgovsky Alla , Weltmer Elaine C , Zhang Ke , Lacbawan Felicitas L 
Published: February 2021

Abstract
Introduction
Germline genetic testing is increasingly relevant to breast and gynecologic (GYN) cancer clinicians for monitoring and managing high-risk patients. In particular, multi-gene panels (MGPs) can identify unsuspected cancer syndromes and variants that may become clinically significant. Effective MGPs must be comprehensive and up-to-date. For the current study, we used a probe panel designed for targeted enrichment of 4,500 genes associated with various inherited diseases to develop and validate a 66-gene comprehensive hereditary cancer panel, including subsets of genes associated with breast and GYN cancers.
Materials and Methods
Genomic DNA was extracted and taken through next generation sequencing (NGS) library preparation to be sequenced on an Illumina NovaSeq instrument. Targeted capture-based enrichment with a long-range PCR (LR-PCR) component was used to interrogate all protein-coding exons, intron-exon splice sites (+/-10bp), as well as clinically relevant deep intronic, 5’UTR, and 3’UTR regions for single nucleotide variants (SNVs) and insertions/deletions (indels) of all genes of interest. Copy number variations (CNVs) were also interrogated for all applicable regions. Data analysis was performed using a proprietary in-house bioinformatics variant analysis pipeline.
For validation, samples from the Coriell Repository and more than 100 unique de-identified genomic DNA specimens from whole blood and saliva were analyzed for 17,911 variants in 508 genes. Variants were previously identified by orthogonal methods (in-house Sanger sequencing, CLIA validated NGS assays, and microarray). The well-characterized Genome in a Bottle (GIAB) NA12878 and Ashkenazim Trio samples (NA24149, NA24385, and NA24143) were also included. The analytic sensitivity (Positive Percent Agreement, %PPA) and specificity (Technical Positive Predictive Value, %TPPV and Negative Percent Agreement, %NPA) were determined for each variant type (SNV, indel, and CNV).
The 66-gene hereditary cancer panel includes genes that confer ≥2-fold increased risk or 5% lifetime risk for developing cancer (APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN1B, CDKN2A (p16, p14), CHEK2, DICER1, EGFR, EPCAM, FANCA, FANCC, FANCM, FH, FLCN, GALNT12, GREM1, HOXB13, MAX, MEN1, MET, MITF, MLH1, MRE11 (MRE11A), MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NTHL1, PALB2, PMS2, POLD1, POLE, POT1, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RECQL, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMARCA4, SMAD4, STK11, SUFU, TMEM127, TP53, TSC1, TSC2, VHL, and XRCC2). Of these, 30 genes increase the lifetime risk of breast and/or GYN cancers and are also distributed among smaller phenotype-specific panels.
Results
The analytical sensitivity (%PPA) for SNVs and indels was 100.0% and 97.8% for CNVs. The overall specificity for SNVs, indels, and CNVs was &gt;99.0%. The %TPPV for SNVs, Indels, and CNVs was 100.0%, 99.3%, and 100.0%, respectively. The %NPA for SNVs, Indels, and CNVs was 100.0%. The %PPA, %TPPV, and %NPA for LR-PCR was 100.0%.
Conclusion
Validation of the 66-gene hereditary cancer panel demonstrated high analytical sensitivity and specificity. As additional gene-cancer associations are established, using an already designed and developed comprehensive 4,500 gene panel will expedite the process of updating panel tests to include relevant candidate genes, allowing clinicians and patients to benefit from up-to-date and comprehensive testing.
Citation Format: Taraneh E Angeloni, Anindya Bhattacharya, Linda L Cheng, Hansook K Chong, Kelli M Conlan, Christopher D Elzinga, Anna Gerasimova, David Grover, Andrew Grupe, Michael Hua, Domagoj Hodko, Krista Kazmierkiewicz, Rebecca E Nakles, Camille R Nery, Renius Owen, Dana M Goos-Root, Charles M Rowland, Alla Smolgovsky, Elaine C Weltmer, Ke Zhang, Felicitas L Lacbawan. Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests [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 PS16-02.