The Evolution of Genetic Testing from Focused Testing to Panel Testing and from Patient Focused to Population Testing: Are We There Yet?

The field of cancer genetics has evolved significantly over the past 30 years. Genetic testing has become less expensive and more comprehensive which has changed practice patterns. It is no longer necessary to restrict testing to those with the highest likelihood of testing positive. In addition, we have learned that the criteria developed to determine who has the highest likelihood of testing positive are neither sensitive nor specific. As a result, the field is moving from testing only the highest risk patients identified based on testing criteria to testing all cancer patients. This requires new service delivery models where testing can be mainstreamed into oncology clinics and posttest genetic counseling can be provided to individuals who test positive and those with concerning personal or family histories who test negative. The use of videos, testing kiosks, chatbots, and genetic counseling assistants have been employed to help facilitate testing at a larger scale and have good patient uptake and satisfaction. While testing is important for cancer patients as it may impact their treatment, future cancer risks, and family member's cancer risks, it is unfortunate that their cancer could not be prevented in the first place. Population testing for all adults would be a strategy to identify individuals with adult-onset diseases before they develop cancer in an attempt to prevent it entirely. A few research studies (Healthy Nevada and MyCode) have offered population testing for the three Centers for Disease Control and Prevention Tier 1 conditions: hereditary breast and ovarian cancer syndrome, Lynch syndrome, and familial hypercholesterolemia finding a prevalence of 1 in 70 individuals in the general population. We anticipate that testing for all cancer patients and the general population will continue to increase over the next 20 years and the genetics community needs to help lead the way to ensure this happens in a responsible manner.

Preparing for the unexpected: Recommendations for returning secondary findings in late-stage cancer care.

PURPOSE: We conducted qualitative interviews with patients with cancer and providers to identify gaps in clinical care and highlight care delivery solutions for the return of secondary germline findings. METHODS: Twelve patients and 19 cancer providers from the United States were interviewed between January 2019 and May 2021. Interviews elicited feedback about patient information needs, emotional responses to secondary findings, and recommendations for improving pre-test education. RESULTS: Patients' responses ranged from gratitude to regret, depending on how much pre-test counseling they received before tumor testing. Providers cited insufficient clinic time as a major barrier to pretest education, favoring online support tools and standardized pre-test education models. Providers had differing perspectives on how pre-test education should be integrated into clinical workflows but agreed that it should include the differences between somatic and germline testing, the likelihood of medically actionable findings, and the possibility of being referred to a genetics provider. CONCLUSION: The spectrum of participants' responses to their secondary findings underscores the importance of adequate pre-test discussions before somatic sequencing. Although educational interventions could address patients' information needs and augment traditional pre-test counseling, health care systems, labs, and genetic providers may be called on to play greater roles in pre-test education.

Clinical and laboratory genetic counselor attitudes on the reporting of variants of uncertain significance for multigene cancer panels.

Research suggests variants of uncertain significance (VUSs) present a variety of challenges for genetic counselors (GCs), nongenetics clinicians, and patients. Multigene cancer panels reveal more VUSs than single gene testing as a result of the increase in the number of genes being tested. This study surveyed 87 clinical cancer GCs involved with direct patient care and 19 laboratory GCs who provide guidance to clinicians regarding genetic test results about their attitudes on various options for the reporting of VUSs by laboratories for broad multigene cancer panels. Independent samples t-tests were utilized to compare the two groups. Based on a six-point Likert-type scale (1 = Strongly Disagree to 6 = Strongly Agree), clinical cancer GCs (M = 5.4; SD = 0.8) and laboratory GCs (M = 5.2; SD = 0.9) agreed overall that VUSs should be reported (p = 0.44; Cohen's d = 0.21). When asked about specific reporting options, both clinical cancer GCs (M = 1.9; SD = 1.1) and laboratory GCs (M = 2.1; SD = 1.4) disagreed that VUSs should be reported only for genes related to the indication for testing (p = 0.50; Cohen's d = 0.17). Overall, most GCs felt clinicians should not choose whether VUSs should be reported on genetic test results, with clinical cancer GCs (M = 1.9; SD = 1.3) feeling more strongly against it than laboratory GCs (M = 3.1; SD = 1.4; p = 0.002; Cohen's d = 0.88). Generally, GCs were more in favor of VUSs not being reported for population-based screening, with laboratory GCs (M = 4.7; SD = 0.8) agreeing more with that practice than clinical cancer GCs (M = 3.7; SD = 1.4; p = 0.001; Cohen's d = 0.80). Both clinical cancer GCs (M = 4.1; SD = 1.2) and laboratory GCs (M = 3.9; SD = 1.2) agreed additional guidelines on how to approach VUSs in clinical practice should be developed (p = 0.54; Cohen's d = 0.17). While most GCs supported the reporting of VUSs overall, our analyses suggest clinical cancer and laboratory GCs may have different attitudes toward specific VUS-related reporting options. Further research is needed to elucidate GC preferences to help inform best practices for the reporting of VUSs. The development of additional standardized guidelines on how to approach VUSs would further support clinical practice.

Integration of Universal Germline Genetic Testing for All New Breast Cancer Patients.

BACKGROUND: The American Society of Breast Surgeons recommends genetic testing (GT) for all women with breast cancer (BC), but implementation and uptake of GT has not been well-described. METHODS: A retrospective chart review was performed for newly diagnosed BC patients or patients with a newly identified recurrence of BC seen in a multidisciplinary clinic (MDBC) who were offered genetic counseling (GC) and GT. RESULTS: The 138 women attending the MDBC had a median age of 54 years and comprised non-Hispanic whites (46%), Asians (28%), Hispanics (17%), blacks (4%), and other (5%). Of the 105 (76%) patients without prior GT, 100 (95%) accepted GC, with 93 (93%) of these 100 patients undergoing GT. The patients meeting the National Comprehensive Cancer Network (NCCN) guidelines for GT were more likely to undergo GT. Testing was performed with a 67- to 84-gene panel, together with an 8- to 9-gene STAT panel if needed. Among 120 patients with reports available, including 33 patients previously tested, 15 (12%) were positive (1 BLM, 1 BRCA1, 3 BRCA2, 1 BRIP1, 1 CFTR, 1 CHEK2, 1 MUTYH, 1 PALB2, 1 PRSS1, 1 RAD50, 1 RET, and 2 TP53), 44 (37%) were negative, and 61 (51%) had an uncertain variant. The median time to STAT results (n = 50) was 8 days. The STAT results were available before surgery for 47 (98%) of the 48 STAT patients undergoing surgery. CONCLUSIONS: New BC patients attending the MDBC demonstrated high rates of acceptance of GC and GT. The combination of GC and GT can offer timely information critical to patient risk assessment and treatment planning.

Recall of Genomic Testing Results Among Patients with Cancer.

BACKGROUND: Genomic testing of somatic and germline DNA has transformed cancer care. However, low genetic knowledge among patients may compromise care and health outcomes. Given the rise in genomic testing, we sought to understand patients' knowledge of their genetic test results. MATERIALS AND METHODS: We conducted a survey-based study with 85 patients at a comprehensive cancer center. We compared self-reported recall of (a) having had somatic/germline testing and (b) their specific somatic/germline results to the genomic test results documented in the medical record. RESULTS: Approximately 30% of patients did not recall having had testing. Of those who recalled having testing, 44% of patients with pathogenic/likely pathogenic germline mutations and 57% of patients with reported somatic alterations did not accurately recall their specific gene or variant-level results. CONCLUSION: Given significant knowledge gaps in patients' recall of genomic testing, there is a critical need to improve patient-directed education and return-of-results strategies.

Engaging Patients in Precision Oncology: Development and Usability of a Web-Based Patient-Facing Genomic Sequencing Report.

PURPOSE: Evidence-based somatic and germline sequencing has transformed cancer care and improves patient outcomes. However, patients' low genetic literacy and misunderstanding of their own genomic results poses a threat to the realization of precision oncology. To optimize patient genomic comprehension, we developed a Web-based, patient-directed, genomic sequencing education and return-of-results tool, HOPE-Genomics. METHODS: The HOPE-Genomics prototype included somatic and germline sequencing results, embedded multimedia genomic education, and interactive features (eg, request for genetic counseling). Between January and April 2018, we elicited feedback on tool usability and comprehensiveness through participant surveys, 4 focus groups of patients with cancer and their family members, and 3 provider focus groups (comprising 8 patients, 5 family members, and 19 providers). RESULTS: We identified themes in patient/family tool-related responses, including the desire to view a patient-friendly report, a desire to receive multiple types of genomic information (eg, prognostic and uncertain), high acceptability of report content, and interest in tool-enabled access to genetic counseling. Major themes from the clinician focus groups included believing the tool could help patients formulate questions and facilitate patients' communication of results to family members. However, there were diverse responses from all participants in terms of tool implementation (ie, timing and nature of report release). Some participants preferred report release before meeting with the provider, and others preferred it during the appointment. Additionally, some clinicians were concerned about providing prognostic and treatment information through the tool. CONCLUSION: There was high acceptability and interest from patients, family members, and providers in a patient-directed genomics report. Future work will determine whether direct-to-patient reporting of genomic results improves patient knowledge, care engagement, and compliance with genomically guided interventions.

Experience Gained from the Development and Execution of a Multidisciplinary Multi-syndrome Hereditary Colon Cancer Family Conference.

Genetic healthcare professionals provide genetic cancer risk assessment and follow-up care for patients facing hereditary cancers. To meet the needs of those affected by hereditary colorectal cancer, City of Hope and the Hereditary Colon Cancer Foundation collaborated to develop a "Family Day" conference. We describe the development of our conference based upon the Hereditary Colon Cancer Foundation's "Family Day" program model, with refinements completed using the Participatory Action Research theoretical framework, which incorporated input from conference participants and researchers. Thirty-one participants attended the conference, representing patients with, or families, friends, and caregivers of those with, multiple colorectal cancer predisposition syndromes, including Lynch, familial adenomatous polyposis, and juvenile polyposis. Participants who completed the feedback surveys (n = 22) were highly satisfied with the presentation content, ranking the keynote lecture on family communication the highest of the conference events. Participants also provided feedback regarding how to improve future conferences. In conclusion, we share our experience and provide guidance for developing a successful hereditary colon cancer predisposition patient and family conference.

Prospective Study of Cancer Genetic Variants: Variation in Rate of Reclassification by Ancestry.

Background: In germline genetic testing, variants from understudied ancestries have been disproportionately classified as being of uncertain significance. We hypothesized that the rate of variant reclassification likewise differs by ancestry. Methods: Nonbenign variants in actionable genes were collected from consenting subjects undergoing genetic testing at two Southern California sites from September 1996 through December 2016. Variant reclassifications were recorded as they were received, until February 2017 or reclassification to benign. Excluding duplicate variants (same ancestry, laboratory, classification), generalized linear models for the hereditary breast cancer genes (BRCA1/2) and other variants investigated whether rate of reclassification differed for seven categories of ancestry compared with non-Hispanic European. Models took into account laboratory, year, gene, sex, and current classification (handled as a time-dependent covariate) and were adjusted for multiple hypothesis testing. Results: Among 1483 nonbenign variants, 693 (46.7%) involved BRCA1/2. Overall, 268 (18.1%) variants were reclassified at least once. Few (9.7%) reclassified variants underwent a net upgrade in pathogenicity. For BRCA1/2 variants, reclassification rates varied by ancestry and increased over time, more steeply for ancestries with lower initial rates (African, Ashkenazi, Chinese) than for ancestries whose initial rates were high (Middle Eastern) or similar to non-Hispanic European (non-Chinese Asian, Native American, Hispanic). In contrast, reclassification rates of non-BRCA1/2 variants did not vary over time but were elevated for most minority ancestries except non-Chinese Asian and Native American. Conclusions: For nonbenign variants in cancer-related genes, the rates at which reclassifications are issued vary by ancestry in ways that differ between BRCA1/2 and other genes.

Somatic TP53 variants frequently confound germ-line testing results.

PURPOSE: Blood/saliva DNA is thought to represent the germ line in genetic cancer-risk assessment. Cases with pathogenic TP53 variants detected by multigene panel testing are often discordant with Li-Fraumeni syndrome, raising concern about misinterpretation of acquired aberrant clonal expansions (ACEs) with TP53 variants as germ-line results. METHODS: Pathogenic TP53 variants with abnormal next-generation sequencing metrics (e.g., decreased ratio (<25%) of mutant to wild-type allele, more than two detected alleles) were selected from a CLIA laboratory testing cohort. Alternate tissues and/or close relatives were tested to distinguish between ACE and germ-line status. Clinical data and Li-Fraumeni syndrome testing criteria were examined. RESULTS: Among 114,630 multigene panel tests and 1,454 TP53 gene-specific analyses, abnormal next-generation sequencing metrics were observed in 20% of 353 TP53-positive results, and ACE was confirmed for 91% of cases with ancillary materials, most of these due to clonal hematopoiesis. Only four met Chompret criteria. Individuals with ACE were older (50 years vs. 33.7; P = 0.02) and were identified more frequently in multigene panel tests (66/285; 23.2%) than in TP53 gene-specific tests (6/68; 8.8%, P = 0.005). CONCLUSION: ACE confounds germ-line diagnosis, may portend hematologic malignancy, and may provoke unwarranted clinical interventions. Ancillary testing to confirm germ-line status should precede Li-Fraumeni syndrome management.

The spectrum of genetic variants in hereditary pancreatic cancer includes Fanconi anemia genes.

Approximately 5-10% of all pancreatic cancer patients carry a predisposing mutation in a known susceptibility gene. Since >90% of patients present with late stage disease, it is crucial to identify high risk individuals who may be amenable to early detection or other prevention. To explore the spectrum of hereditary pancreatic cancer susceptibility, we evaluated germline DNA from pancreatic cancer participants (n = 53) from a large hereditary cancer registry. For those without a known predisposition mutation gene (n = 49), germline next generation sequencing was completed using targeted capture for 706 candidate genes. We identified 16 of 53 participants (30%) with a pathogenic (P) or likely pathogenic (LP) variant that may be related to their hereditary pancreatic cancer predisposition; seven had mutations in genes associated with well-known cancer syndromes (13%) [ATM (2), BRCA2 (3), MSH2 (1), MSH6 (1)]. Many had mutations in Fanconi anemia complex genes [BRCA2 (3 participants), FANCF, FANCM]. Eight participants had rare protein truncating variants of uncertain significance with no other P or LP variants. Earlier age of pancreatic cancer diagnosis (57.5 vs 64.8 years) was indicative of possessing a P or LP variant, as was cancer family history (p values <0.0001). Our multigene panel approach for identifying known cancer predisposing genetic susceptibility in those at risk for hereditary pancreatic cancer may have direct applicability to clinical practice in cases with mutations in actionable genes. Future pancreatic cancer predisposition studies should include evaluation of the Fanconi anemia genes.

Genetic Gastric Cancer Susceptibility in the International Clinical Cancer Genomics Community Research Network.

Few susceptibility genes for gastric cancer have been identified. We sought to identify germline susceptibility genes from participants with gastric cancer from an international hereditary cancer research network. Adults with gastric cancer of any histology, and with a germline DNA sample (n = 51), were retrospectively selected. For those without previously identified germline mutations (n = 43), sequencing was performed for 706 candidate genes. Twenty pathogenic or likely pathogenic variants were identified among 18 participants. Eight of the 18 participants had previous positive clinical testing, including six with CDH1 pathogenic or likely pathogenic variants, and two with pathogenic MSH2 and TP53 variants. Of the remaining 10, six were in BRCA1 DNA damage response pathway genes (ATM, ATR, BRCA2, BRIP1, FANCC, TP53), other variants were identified in CTNNA1, FLCN, SBDS, and GNAS. Participants identified with pathogenic or likely pathogenic variants were younger at gastric cancer diagnosis than those without, 39.1 versus 48.0 years, and over 50% had a close family member with gastric cancer (p-values < 0.0001). In conclusion, many participants were identified with mutations in clinically-actionable genes. Age of onset and family history of gastric cancer were mutation status predictors. Our findings support multigene panels in identifying gastric cancer predisposition.

Spectrum of mismatch repair gene mutations and clinical presentation of Hispanic individuals with Lynch syndrome.

Lynch syndrome (LS), the most common hereditary colorectal cancer syndrome, is caused by mismatch repair (MMR) gene mutations. However, data about MMR mutations in Hispanics are limited. This study aims to describe the spectrum of MMR mutations in Hispanics with LS and explore ancestral origins. This case series involved an IRB-approved retrospective chart review of self-identified Hispanic patients (n = 397) seen for genetic cancer risk assessment at four collaborating academic institutions in California, Texas, and Puerto Rico who were evaluated by MMR genotyping and/or tumor analysis. A literature review was conducted for all mutations identified. Of those who underwent clinical genetic testing (n = 176), 71 had MMR gene mutations. Nine mutations were observed more than once. One third (3/9) of recurrent mutations and two additional mutations (seen only once) were previously reported in Spain, confirming the influence of Spanish ancestry on MMR mutations in Hispanic populations. The recurrent mutations identified (n = 9) included both previously reported mutations as well as unique mutations not in the literature. This is the largest report of Hispanic MMR mutations in North America; however, a larger sample and haplotype analyses are needed to better understand recurrent MMR mutations in Hispanic populations.

Lynch Syndrome Limbo: Patient Understanding of Variants of Uncertain Significance.

Providers and patients encounter challenges related to the management of Variants of Unknown Significance (VUS). A VUS introduces new counseling dilemmas for the understanding and psychosocial impact of uncertain genetic test results. This descriptive study uses Mishel's theory of uncertainty in illness to explore the experience of individuals who have received a VUS as part of the genetic testing process. Semi-structured interviews were conducted with 27 adult individuals who received a VUS for Lynch syndrome mismatch repair genes between 2002 and 2013. The interviews were transcribed and analyzed. Most individuals recalled their result and perceived various types of uncertainty associated with their VUS. Half of the participants appraised their variant as a danger and implemented coping strategies to reduce the threat of developing cancer. Mobilizing strategies to reduce their risk included vigilant cancer surveillance, information seeking and notifying relatives. The majority of participants were unaware of the possibility of a VUS before receiving their result and expected reclassification over time. These results provide insight into the ways healthcare providers can support patients who receive VUS for Lynch syndrome. Findings also provide direction for future work that can further explicate the impact of receiving a VUS.

Corrigendum: Clinical Application of Multigene Panels: Challenges of Next-Generation Counseling and Cancer Risk Management.

[This corrects the article on p. 208 in vol. 5, PMID: 26484312.].

Next-Generation Testing for Cancer Risk: Perceptions, Experiences, and Needs Among Early Adopters in Community Healthcare Settings.

BACKGROUND: Advances in next-generation sequencing (NGS) technologies are driving a shift from single-gene to multigene panel testing for clinical genetic cancer risk assessment (GCRA). This study explored perceptions, experiences, and challenges with NGS testing for GCRA among U.S. community-based clinicians. METHODS: Surveys delivered at initial and 8-month time points, and 12-month tracking of cases presented in a multidisciplinary web-based case conference series, were conducted with GCRA providers who participated in a 235-member nationwide community of practice. RESULTS: The proportion of respondents ordering panel tests rose from 29% at initial survey (27/94) to 44% (46/107) within 8 months. Respondents reported significantly less confidence about interpreting and counseling about NGS compared with single-gene test results (p < 0.0001 for all comparisons). The most cited reasons for not ordering NGS tests included concerns about clinical utility, interpreting and communicating results, and lack of knowledge/skills. Multigene panels were used in 204/668 cases presented during 2013, yielding 37 (18%) deleterious (7% in low/moderate-penetrance genes), 88 (43%) with ≥1 variant of uncertain significance, 77 (38%) uninformative negative, and 2 (1%) inconclusive results. CONCLUSIONS: Despite concerns about utility and ability to interpret/counsel about NGS results, a rapidly increasing uptake of NGS testing among community clinicians was documented. Challenges identified in case discussions point to the need for ongoing education, practice-based support, and opportunities to partner in research that contributes to characterization of lesser known genes.

Clinical Application of Multigene Panels: Challenges of Next-Generation Counseling and Cancer Risk Management.

BACKGROUND: Multigene panels can be a cost- and time-effective alternative to sequentially testing multiple genes, especially with a mixed family cancer phenotype. However, moving beyond our single-gene testing paradigm has unveiled many new challenges to the clinician. The purpose of this article is to familiarize the reader with some of the challenges, as well as potential opportunities, of expanded hereditary cancer panel testing. METHODS: We include results from 348 commercial multigene panel tests ordered from January 1, 2014, through October 1, 2014, by clinicians associated with the City of Hope's Clinical Cancer Genetics Community of Practice. We also discuss specific challenging cases that arose during this period involving abnormalities in the genes: CDH1, TP53, PMS2, PALB2, CHEK2, NBN, and RAD51C. RESULTS: If historically high risk genes only were included in the panels (BRCA1, BRCA2, MSH6, PMS2, TP53, APC, CDH1), the results would have been positive only 6.2% of the time, instead of 17%. Results returned with variants of uncertain significance (VUS) 42% of the time. CONCLUSION: These figures and cases stress the importance of adequate pre-test counseling in anticipation of higher percentages of positive, VUS, unexpected, and ambiguous test results. Test result ambiguity can be limited by the use of phenotype-specific panels; if found, multiple resources (the literature, reference laboratory, colleagues, national experts, and research efforts) can be accessed to better clarify counseling and management for the patient and family. For pathogenic variants in low and moderate risk genes, empiric risk modeling based on the patient's personal and family history of cancer may supersede gene-specific risk. Commercial laboratory and patient contributions to public databases and research efforts will be needed to better classify variants and reduce clinical ambiguity of multigene panels.

Mutation spectrum and risk of colorectal cancer in African American families with Lynch syndrome.

BACKGROUND & AIMS: African Americans (AAs) have the highest incidence of and mortality resulting from colorectal cancer (CRC) in the United States. Few data are available on genetic and nongenetic risk factors for CRC among AAs. Little is known about cancer risks and mutations in mismatch repair (MMR) genes in AAs with the most common inherited CRC condition, Lynch syndrome. We aimed to characterize phenotype, mutation spectrum, and risk of CRC in AAs with Lynch syndrome. METHODS: We performed a retrospective study of AAs with mutations in MMR genes (MLH1, MSH2, MSH6, and PMS2) using databases from 13 US referral centers. We analyzed data on personal and family histories of cancer. Modified segregation analysis conditioned on ascertainment criteria was used to estimate age- and sex-specific CRC cumulative risk, studying members of the mutation-carrying families. RESULTS: We identified 51 AA families with deleterious mutations that disrupt function of the MMR gene product: 31 in MLH1 (61%), 11 in MSH2 (21%), 3 in MSH6 (6%), and 6 in PMS2 (12%); 8 mutations were detected in more than 1 individual, and 11 have not been previously reported. In the 920 members of the 51 families with deleterious mutations, the cumulative risks of CRC at 80 years of age were estimated to be 36.2% (95% confidence interval [CI], 10.5%-83.9%) for men and 29.7% (95% CI, 8.31%-76.1%) for women. CRC risk was significantly higher among individuals with mutations in MLH1 or MSH2 (hazard ratio, 13.9; 95% CI, 3.44-56.5). CONCLUSIONS: We estimate the cumulative risk for CRC in AAs with MMR gene mutations to be similar to that of individuals of European descent with Lynch syndrome. Two-thirds of mutations were found in MLH1, some of which were found in multiple individuals and some that have not been previously reported. Differences in mutation spectrum are likely to reflect the genetic diversity of this population.

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.