Cassandra Gulden

ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing

Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits.

Tumor genome analysis includes germline genome: Are we ready for surprises?

We sought to describe the spectrum of potential and confirmed germline genomic events incidentally identified during routine medium‐throughput somatic tumor DNA sequencing, and to provide a framework for pre‐ and post‐test consent and counseling for patients and families. Targeted tumor‐only next‐generation sequencing (NGS) had been used to evaluate for possible druggable genomic events obtained from consecutive new patients with metastatic gastroesophageal, hepatobiliary or colorectal cancer seen at the University of Chicago. A panel of medical oncologists, cancer geneticists and genetic counselors retrospectively grouped these patients (N = 111) based on probability of possessing a potentially inherited mutation in a cancer susceptibility gene, both prior to and after incorporating tumor‐only NGS results. High‐risk patients (determined from NGS results) were contacted and counseled in person by a genetic counselor (N = 21). When possible and indicated, germline genetic testing was offered. Of 8 evaluable high‐risk patients, 7 underwent germline testing. Three (37.5%) had confirmed actionable germline mutations (all in the BRCA2 gene). NGS offers promise, but poses significant challenges for oncologists who are ill prepared to handle incidental findings that have clinical implications for at risk family members. In this relatively small cohort of patients undergoing tumor genomic testing for gastrointestinal malignancies, we incidentally identified 3 BRCA2 mutations carriers. This report underscores the need for oncologists to develop a framework for pre‐ and post‐test communication of risks to patients undergoing routine tumor‐only sequencing.

Development of a tiered and binned genetic counseling model for informed consent in the era of multiplex testing for cancer susceptibility

Purpose:Multiplex genetic testing, including both moderate- and high-penetrance genes for cancer susceptibility, is associated with greater uncertainty than traditional testing, presenting challenges to informed consent and genetic counseling. We sought to develop a new model for informed consent and genetic counseling for four ongoing studies. Methods:Drawing from professional guidelines, literature, conceptual frameworks, and clinical experience, a multidisciplinary group developed a tiered-binned genetic counseling approach proposed to facilitate informed consent and improve outcomes of cancer susceptibility multiplex testing.Results:In this model, tier 1 “indispensable” information is presented to all patients. More specific tier 2 information is provided to support variable informational needs among diverse patient populations. Clinically relevant information is “binned” into groups to minimize information overload, support informed decision making, and facilitate adaptive responses to testing. Seven essential elements of informed consent are provided to address the unique limitations, risks, and uncertainties of multiplex testing.Conclusion:A tiered-binned model for informed consent and genetic counseling has the potential to address the challenges of multiplex testing for cancer susceptibility and to support informed decision making and adaptive responses to testing. Future prospective studies including patient-reported outcomes are needed to inform how to best incorporate multiplex testing for cancer susceptibility into clinical practice.Genet Med 17 6, 485–492.

Performance of Lynch Syndrome Predictive Models in a Multi-Center US Referral Population

OBJECTIVES:Lynch syndrome is the most common cause of inherited colorectal cancer (CRC) and is due to germline mutations in mismatch repair (MMR) genes. Early Lynch syndrome diagnosis and appropriate CRC surveillance improves mortality. Traditional qualitative clinical criteria including Amsterdam and Bethesda guidelines may miss mutation carriers. Recently, quantitative predictive models including MMRPredict, PREMM(1,2,6), and MMRPro were developed to facilitate diagnosis. However, these models remain to be externally validated in the United States. Therefore, we evaluated the test characteristics of Lynch syndrome predictive models in a tertiary referral group at two US academic centers.METHODS:We retrospectively collected data on 230 consecutive individuals who underwent genetic testing for MMR gene mutations at the University of Chicago and University of California at San Franciscos Cancer Risk Clinics. Each individuals risk of mutation was examined using MMRPredict, PREMM(1,2,6), and MMRPro. Amsterdam and Bethesda criteria were also determined. Testing characteristics were calculated for each of the models.RESULTS:We included 230 individuals in the combined cohort. In all, 113 (49%) probands were MMR mutation carriers. Areas under the receiver operator characteristic curves were 0.76, 0.78, and 0.82 for MMRPredict, PREMM(1,2,6), and MMRPro, respectively. While similar in overall performance, our study highlights unique test characteristics of these three quantitative models including comparisons of sensitivity and specificity. Moreover, we identify characteristics of mutation carriers who were missed by each model.CONCLUSIONS:Overall, all three Lynch syndrome predictive models performed comparably in our multi-center US referral population. These results suggest that Lynch syndrome predictive models can be used to screen for MMR mutation carriers and can provide improved test characteristics compared with traditional clinical criteria. Identification of MMR mutation carriers is paramount as appropriate screening can prevent CRC mortality in this high-risk group.

Risk Assessment and Genetic Testing for Ovarian Cancer

OBJECTIVE Prevention of ovarian cancer starts with identifying women at high risk for this deadly disease. Once identified, these women can be offered risk-reducing options such as oral contraceptives and bilateral salpingo-oophorectomy. In this review, strategies for identifying high-risk individuals and the clinical utility of genetic testing for BRCA1 and BRCA2 will be discussed. CONCLUSION To offer potentially life-saving interventions for at-risk family members, we propose that every woman newly diagnosed with ovarian cancer be offered genetic testing for BRCA1 and BRCA2 genes because mutations in these genes are the strongest known predictors of ovarian cancer risk.

Randomized Noninferiority Trial of Telephone vs In-Person Disclosure of Germline Cancer Genetic Test Results

Background Germline genetic testing is standard practice in oncology. Outcomes of telephone disclosure of a wide range of cancer genetic test results, including multigene panel testing (MGPT) are unknown. Methods Patients undergoing cancer genetic testing were recruited to a multicenter, randomized, noninferiority trial (NCT01736345) comparing telephone disclosure (TD) of genetic test results with usual care, in-person disclosure (IPD) after tiered-binned in-person pretest counseling. Primary noninferiority outcomes included change in knowledge, state anxiety, and general anxiety. Secondary outcomes included cancer-specific distress, depression, uncertainty, satisfaction, and screening and risk-reducing surgery intentions. To declare noninferiority, we calculated the 98.3% one-sided confidence interval of the standardized effect; t tests were used for secondary subgroup analyses. Only noninferiority tests were one-sided, others were two-sided. Results A total of 1178 patients enrolled in the study. Two hundred eight (17.7%) participants declined random assignment due to a preference for in-person disclosure; 473 participants were randomly assigned to TD and 497 to IPD; 291 (30.0%) had MGPT. TD was noninferior to IPD for general and state anxiety and all secondary outcomes immediately postdisclosure. TD did not meet the noninferiority threshold for knowledge in the primary analysis, but it did meet the threshold in the multiple imputation analysis. In secondary analyses, there were no statistically significant differences between arms in screening and risk-reducing surgery intentions, and no statistically significant differences in outcomes by arm among those who had MGPT. In subgroup analyses, patients with a positive result had statistically significantly greater decreases in general anxiety with telephone disclosure (TD -0.37 vs IPD +0.87, P = .02). Conclusions Even in the era of multigene panel testing, these data suggest that telephone disclosure of cancer genetic test results is as an alternative to in-person disclosure for interested patients after in-person pretest counseling with a genetic counselor.

Abstract 07: Challenges of applying tumor genome analysis to the germline: Examples from GI oncology

Purpose: To describe the spectrum of potential and confirmed germline genomic events identified incidentally during routine medium throughput somatic tumor DNA sequencing, and to provide a framework for pre- and post-test consent and counseling for these patients and families. Patients and Methods: Targeted next generation sequencing was used to evaluate for possible actionable genomic events in tumor tissue obtained from consecutive new patients seen at the University of Chicago Gastrointestinal Medical Oncology clinic between 9/2012 and 9/2013. Patients had a diagnosis of either metastatic gastroesophageal, hepatobiliary or colorectal cancer. A panel consisting of medical oncologists, an oncology fellow, cancer geneticists and genetic counselors reviewed the results of each case (N=112). Patients were grouped based on their post-test probability of possessing a potentially inherited mutation in a cancer susceptibility gene. The patients within the high-risk group included those with a somatic mutation in one or more of the genes within accepted panels that test for inherited cancer syndromes. Patient age and family history were also taken into account. The high-risk patients were then contacted and formally evaluated and counseled by our cancer risk specialists. When possible and indicated, germline genetic testing was obtained. Results: One hundred and twelve cases were analyzed. Of these, 23 cases (21%) were identified to have somatic, and potentially germline, mutations in BRCA1, BRCA2, PTEN, CDH1, STK11 or MLH1/MSH6 via routine NGS of tumor samples. Seven patients ultimately underwent germline testing of which three (43%) had confirmed germline mutations - interestingly, all were BRCA2 . Most identified cases would not meet current criteria to refer for genetic counseling. Variants of unknown significance in germline BRCA2 posed a challenge with respect to counseling and recommendations. There was one positive control patient with a known clinical diagnosis of Gorlin syndrome, in whom routine NGS of his gastric tumor sample identified a PTCH1 gene mutation (previously unknown). Conclusions: Next generation sequencing technology offers much promise, as we enter the age of individualized medicine, but also poses many challenges for both germline and somatic DNA testing. We identified, incidentally, several cases (∼20%) with a potentially inherited cancer susceptibility gene via routine somatic tumor DNA sequencing. Of patients available and willing to undergo further testing, several (43%, 3/7) of these cases were confirmed germline events. Many of these patients would not have been referred for counseling without the somatic sequencing information. This report raises awareness of the challenges facing NGS tumor analyses, and provides a framework for pre- and post-test consent and counseling of patients undergoing routine tumor sequencing. Citation Format: Andrea Amico, Sarah Nielsen, Daniel Geynisman, Brittany Rambo, George Ben Carey, Cassandra Gulden, Jim Fackenthal, Olufunmilayo Olopade, Daniel Catenacci. Challenges of applying tumor genome analysis to the germline: Examples from GI oncology. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 07. doi:10.1158/1538-7445.CANSUSC14-07

Racial differences in a high-risk colorectal cancer referral population: a single-center experience.

Background Multiple studies have shown that colorectal cancer (CRC) disparities exist between African Americans and Caucasians. African Americans have higher CRC incidence and mortality than Caucasians [1,2]. Furthermore, African Americans present with more advanced CRC [3] and at younger ages [4]. However, little is known about the impact of hereditary CRC syndromes in African Americans on these disparities. In the University of Chicago Cancer Risk Clinic, we see a diverse referral population for evaluation of hereditary syndromes based on personal or family history of CRC. We sought to compare clinical characteristics of our African American and Caucasian patients to determine if differences exist in a high-risk CRC referral population. Method We retrospectively collected data on self-reported African American and Caucasian individuals who were referred for a personal or family history of CRC from 1992-2010. Available clinical characteristics including number of individuals, number of families (probands with family members who followed-up in our clinic), personal or family history of CRC, age at cancer onset, and number of individuals who underwent genetic testing for APC or mismatch repair genes were collected. Pedigrees were reviewed to determine Revised Bethesda, Amsterdam II and PREMM1,2 scores. Statistical analysis included Student’s t-test for continuous variables and Pearson’ sc hi-square for categorical variables. Comparison of independent proportions was done using z-ratios. Results