Complexities of Next-Generation Sequencing in Solid Tumors: Case Studies.

With the promise and potential of clinical next-generation sequencing for tumor and germline testing to impact treatment and outcomes of patients with cancer, there are also risks of oversimplification, misinterpretation, and missed opportunities. These issues risk limiting clinical benefit and, at worst, perpetuating false conclusions that could lead to inappropriate treatment selection, avoidable toxicity, and harm to patients. This report presents 5 case studies illustrating challenges and opportunities in clinical next-generation sequencing interpretation and clinical application in solid tumor oncologic care. First is a case that dissects the origin of an ATM mutation as originating from a hematopoietic clone rather than the tumor. Second is a case illustrating the potential for tumor sequencing to suggest germline variants associated with a hereditary cancer syndrome. Third are 2 cases showing the potential for variant reclassification of a germline variant of uncertain significance when considered alongside family history and tumor sequencing results. Finally, we describe a case illustrating challenges with using microsatellite instability for predicting tumor response to immune checkpoint inhibitors. The common theme of the case studies is the importance of examining clinical context alongside expert review and interpretation, which together highlight an expanding role for contextual examination and multidisciplinary expert review through molecular tumor boards.

Exploring relatives' perceptions of participation, ethics, and communication in a patient-driven study for hereditary cancer variant reclassification.

Effective communication of genetic information within families depends on several factors. Few studies explore intra-familial communication of variant of uncertain significance (VUS) results or active collaboration between family members to classify VUS. Our qualitative study aimed to describe the experiences of individuals asked by family members to participate in the FindMyVariant study, a patient-driven family study which aimed to reclassify a clinically identified familial VUS in a hereditary cancer gene. We collected feedback from 56 individuals from 21 different families through phone interviews and written correspondence, transcribed the interviews, and performed thematic analysis on all text. We describe themes from three main topics: participation, ethical considerations, and study impacts. Participation in the FindMyVariant study, defined as returning a sample for targeted genotyping, was motivated by convenience and a desire to help the family, oneself, and science. Relatives were generally responsive to invitations to participate in FindMyVariant from another family member. Those who declined to participate did so due to concerns about research program confidentiality rather than family dynamics. No major ethical issues arose in response to the patient-driven study structure, and no major changes in stress and anxiety, medical care, or behavior occurred. Participation in patient-driven familial VUS classification studies has a neutral or positive impact on family health communication. While it is important to design studies to minimize familial coercion, intra-familial confidentiality breaches, and misinterpretation of genetic results, these were not major concerns among relatives in this study. Clinicians and laboratories may consider encouraging familial communication about genetic variants using family members as liaisons.

Exploring the effect of ascertainment bias on genetic studies that use clinical pedigrees.

Recent studies have reported novel cancer risk associations with incidentally tested genes on cancer risk panels using clinically ascertained cohorts. Clinically ascertained pedigrees may have unknown ascertainment biases for both patients and relatives. We used a method to assess gene and variant risk and ascertainment bias based on comparing the number of observed disease instances in a pedigree given the sex and ages of individuals with those expected given established population incidence. We assessed the performance characteristics of the method by simulating families with varying genetic risk and proportion of individuals genotyped. We implemented this method using SEER cancer incidence data to assess clinical ascertainment bias in a set of 42 pedigrees with clinical testing ordered for either breast/ovarian cancer or colorectal/endometrial cancer at the University of Washington and negative sequencing results. In addition to expected biases consistent with the stated testing purpose, there were trends suggesting increased colorectal and endometrial cancer in pedigrees tested for breast cancer risk and trends suggesting increased breast cancer in families tested for colon cancer risk. There was no observed selection bias for prostate cancer in this set of families. This analysis illustrates that clinically ascertained data sets may have subtle biases. In the future, researchers seeking to explore risk associations with clinical data sets could assess potential ascertainment bias by comparing incidence of disease in families that test negative under given ordering criteria to expected population disease frequencies. Failure to assess for ascertainment bias increases the risk of false genetic associations.

Patient goals, motivations, and attitudes in a patient-driven variant reclassification study.

Family studies to reclassify clinically ascertained variants of uncertain significance (VUS) can impact risk assessment, medical management, and psychological outcomes for patients and their families. There are limited avenues for patients and their families to actively participate in VUS reclassification, and access to family studies at most commercial laboratories is restricted by multiple factors. To explore patient attitudes about participation in family studies for VUS reclassification, we conducted semistructured pre- and post-participation telephone interviews with 38 participants in a family-based VUS reclassification study that utilized a patient-driven approach for family ascertainment and recruitment. Participants had VUS from multigene panel testing performed at multiple clinical laboratories for cancer or other disease risk. Inductive thematic analysis of transcribed interviews highlighted four major themes: (a) Participants' study goals were driven by the desire to resolve uncertainty related to the VUS, (b) Participants had mixed reactions to the VUS reclassification outcomes of the study, (c) Personal, public, and familial knowledge increased through study participation and (d) Participants used study participation to actively cope with the uncertainty of a VUS. As personalized genomic medicine becomes more prevalent, clinicians, clinical laboratories, and researchers could consider creating more opportunities for active partnership with patients and families, who are motivated to contribute data to familial VUS studies.

Comparison of CDH1 Penetrance Estimates in Clinically Ascertained Families vs Families Ascertained for Multiple Gastric Cancers.

IMPORTANCE: CDH1 pathogenic variants have been estimated to confer a 40% to 70% and 56% to 83% lifetime risk for gastric cancer in men and women, respectively. These are likely to be overestimates owing to ascertainment of families with multiple cases of gastric cancer. To our knowledge, there are no penetrance estimates for CDH1 without this ascertainment bias. OBJECTIVE: To estimate CDH1 penetrance in a patient cohort not exclusively ascertained based on strict hereditary diffuse gastric cancer (HDGC) criteria. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of 75 families found to have pathogenic variants in CDH1 through clinical ascertainment and multigene panel testing at a large commercial diagnostic laboratory from August 5, 2013, to June 30, 2018. CDH1 pathogenic variants were identified in 238 individuals from 75 families. Pedigrees from those families included cancer status for 1679 relatives. Penetrance estimates are based on 41 families for which completed pedigrees were available. MAIN OUTCOMES AND MEASURES: Gastric cancer standardized incidence ratio estimates relative to Surveillance, Epidemiology, and End Results (SEER) Program incidence for pathogenic CDH1 variants from families ascertained without regard to HDGC criteria. RESULTS: Among the 238 individuals with a CDH1 pathogenic variant, mean (SD) age was 49.3 (18.1) years and 63.4% were female. Ethnicity was reported for 67 of 75 (89%) families; of these 67 families, 51 (76%) reported European ancestry, whereas Asian, African, Latino, and 2 or more ancestries were reported for 4 families (6%) each. Standardized incidence ratios for gastric and breast cancer were significantly elevated above SEER incidence. Extrapolated cumulative incidence of gastric cancer at age 80 years was 42% (95% CI, 30%-56%) for men and 33% (95% CI, 21%-43%) for women with pathogenic variants in CDH1, whereas cumulative incidence of female breast cancer was estimated at 55% (95% CI, 39%-68%). International Gastric Cancer Linkage Consortium criteria were met in 25 of the 75 (33%) families; however, dispensing with the requirement of confirmation of HDGC histologic subtype, 43 (57%) would meet criteria. CONCLUSIONS AND RELEVANCE: The cumulative incidence of gastric cancer for individuals with pathogenic variants in CDH1 is significantly lower than previously described. Because prophylactic gastrectomy can have bearing upon both physical and psychological health, further discussion is warranted to assess whether this surgical recommendation is appropriate for all individuals with pathogenic variants in CDH1.

Outcomes of 92 patient-driven family studies for reclassification of variants of uncertain significance.

PURPOSE: Family studies are an important but underreported source of information for reclassification of variants of uncertain significance (VUS). We evaluated outcomes of a patient-driven framework that offered familial VUS reclassification analysis to any adult with any clinically ascertained VUS from any laboratory in the United States. METHODS: With guidance from FindMyVariant.org, participants recruited their own relatives for study participation. We genotyped relatives, calculated quantitative cosegregation likelihood ratios, and evaluated variant classifications using Tavtigian's unified framework for Bayesian analysis with American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. We report participation and VUS reclassification rates from the 50 families enrolled for at least one year and reclassification results for 112 variants from the larger 92-family cohort. RESULTS: For the 50-family cohort, 6.7 relatives per family were invited to participate and 67% of relatives returned samples for genotyping. Sixty-one percent of VUS were reclassified, 84% of which were classified as benign or likely benign. Genotyping relatives identified a de novo variant, phase variants, and relatives with phenotypes highly specific for or incompatible with specific classifications. CONCLUSIONS: Motivated families can contribute to successful VUS reclassification at substantially higher rates than those previously published. Clinical laboratories could consider offering family studies to all patients with VUS.

Attitudes Towards Prenatal Genetic Counseling, Prenatal Genetic Testing, and Termination of Pregnancy among Southeast and East Asian Women in the United States.

Recognizing the heterogeneity of the Asian population with regards to acculturation, education, health awareness, and cultural values is vital for tailoring culturally sensitive and appropriate care. Prior studies show that cultural values influence perceptions of genetics within Asian populations. The reputation of the family unit factors into decisions such as pregnancy termination and disclosure of family medical history, and the nondirective model of American genetic counseling may conflict with the historical Asian model of paternalistic health care. Previous studies also provide conflicting evidence regarding correlations between education, acculturation, age, and awareness and perceptions of genetic testing. The aims of this study were to describe attitudes towards prenatal genetics among Southeast and East Asian women living in the United States for varying amounts of time and to explore sociocultural factors influencing those attitudes. Twenty-three Asian women who were members of Asian cultural organizations in the United States were interviewed via telephone about their attitudes towards prenatal genetic counseling, prenatal genetic testing, and termination of pregnancy. Responses were transcribed and coded for common themes using a thematic analysis approach. Four major themes emerged. In general, participants: (1) had diverse expectations for genetic counselors; (2) tended to weigh risks and benefits with regards to genetic testing decisions; (3) had mixed views on termination for lethal and non-lethal genetic conditions; and (4) identified cultural factors which influenced testing and termination such as lack of available resources, societal shame and stigma, and family pressure. These findings may allow prenatal genetic counselors to gain a richer, more nuanced understanding of their Asian patients and to offer culturally tailored prenatal genetic counseling.

Can an Internet-based health risk assessment highlight problems of heart disease risk factor awareness? A cross-sectional analysis.

BACKGROUND: Health risk assessments are becoming more popular as a tool to conveniently and effectively reach community-dwelling adults who may be at risk for serious chronic conditions such as coronary heart disease (CHD). The use of such instruments to improve adults' risk factor awareness and concordance with clinically measured risk factor values could be an opportunity to advance public health knowledge and build effective interventions. OBJECTIVE: The objective of this study was to determine if an Internet-based health risk assessment can highlight important aspects of agreement between respondents' self-reported and clinically measured CHD risk factors for community-dwelling adults who may be at risk for CHD. METHODS: Data from an Internet-based cardiovascular health risk assessment (Heart Aware) administered to community-dwelling adults at 127 clinical sites were analyzed. Respondents were recruited through individual hospital marketing campaigns, such as media advertising and print media, found throughout inpatient and outpatient facilities. CHD risk factors from the Framingham Heart Study were examined. Weighted kappa statistics were calculated to measure interrater agreement between respondents' self-reported and clinically measured CHD risk factors. Weighted kappa statistics were then calculated for each sample by strata of overall 10-year CHD risk. Three samples were drawn based on strategies for treating missing data: a listwise deleted sample, a pairwise deleted sample, and a multiple imputation (MI) sample. RESULTS: The MI sample (n=16,879) was most appropriate for addressing missing data. No CHD risk factor had better than marginal interrater agreement (κ>.60). High-density lipoprotein cholesterol (HDL-C) exhibited suboptimal interrater agreement that deteriorated (eg, κ<.30) as overall CHD risk increased. Conversely, low-density lipoprotein cholesterol (LDL-C) interrater agreement improved (eg, up to κ=.25) as overall CHD risk increased. Overall CHD risk of the sample was lower than comparative population-based CHD risk (ie, no more than 15% risk of CHD for the sample vs up to a 30% chance of CHD for the population). CONCLUSIONS: Interventions are needed to improve knowledge of CHD risk factors. Specific interventions should address perceptions of HDL-C and LCL-C. Internet-based health risk assessments such as Heart Aware may contribute to public health surveillance, but they must address selection bias of Internet-based recruitment methods.