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

Prospective Study of Cancer Genetic Variants: Variation in Rate of Reclassification by Ancestry Abstract 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. Genetic testing for hereditary cancer susceptibility has dramatically improved delivery of precision cancer prevention or treatment to individuals who harbor pathogenic variants in cancer-associated genes. Increasingly, individuals undergoing a hereditary cancer risk assessment are tested with commercial multigene panels consisting of 25 genes or more (1). Laboratories classify genetic variants along a continuum of clinical significance (ie, pathogenic, likely pathogenic, variant of uncertain significance [VUS], likely benign, or benign) (2). As genomic knowledge evolves, however, variants can be reclassified from one category to another, often with clinical implications for the recipient. Because time to reclassification is highly variable and can exceed a decade or more (3), tested individuals often make health-related decisions based on initial rather than definitive interpretations of their genomic data. Despite the fact that variant reclassification may have profound implications for patient care and medical decision-making, little is known about the factors associated with variant reclassification. One factor that may be important in variant reclassification is ancestry. When classifying or reclassifying variants, labs rely heavily on reference databases of human genetic variation. However, because persons of European descent continue to be disproportionately overrepresented within reference genetic databases (4–6), individuals of non-European ancestry are more likely to receive uncertain test results (7–9). Given potential ethnic disparities in the initial classification of genetic variants, we hypothesized that the rate of reclassification of nonbenign variants likewise differs by ancestry, independent of potential confounding factors such as time, gene, laboratory, and initial classification. To address this hypothesis, we analyzed a prospective cohort of pathogenic, likely pathogenic, VUS, and likely benign variants identified during two decades of a tertiary care–based program for hereditary cancer risk assessment among an ethnically diverse population. Recognizing broad differences in the history and scale of efforts to research and reclassify variants of hereditary breast cancer genes (BRCA1/2) and other genes, we analyzed those two groups of variants separately. As secondary aims, we examined time to reclassification by initial classification and identified characteristics that distinguish reclassified variants that were upgraded. Methods Study Population The study was approved by the Institutional Review Board at City of Hope. Data were obtained from individuals who were referred for cancer genetic risk assessment and underwent genetic testing at one of two Southern California sites (City of Hope and Olive View Medical Center) from September 1996 through December 2016 and who gave informed consent to participate in the Clinical Cancer Genomics Community Research Network registry. Eligible for study were variants of actionable and commonly evaluated genes in hereditary cancer predisposition. In particular, because benign variants were not subject to reclassification, only nonbenign variants (pathogenic, likely pathogenic, VUS, likely benign) were studied. The number of genetic variants studied per person was unrestricted. Characteristics collected per variant were gene, mutation, laboratory, self-reported maternal and paternal ancestry, sex, and dates and results of initial classification and any subsequent reclassifications. Terminology for classifying variants was standardized across laboratories per Richards et al. (2). Variants listed as requiring special interpretation were grouped in the same category as VUS. Self-reported ancestry (defined as at least one grandparent) was assigned to mutually exclusive categories in the following sequence: any African, any Native American, any Chinese, including Taiwanese, any other Asian (Filipino, Japanese, Korean, Indian, Pakistani, Indonesian, Thai, Cambodian, Vietnamese, Nepali, Samoan), any Ashkenazi, any Hispanic, any Middle Eastern (Iranian, Armenian, Syrian, Lebanese, Egyptian, Turkish), non-Hispanic European. Within these categories, individuals were noted as having mixed ancestry if they reported ancestry from at least one additional category. A single laboratory (referred to as “Lab A”) issued most initial classifications and reclassifications under study. Corresponding data from the other 33 testing laboratories were sparse, necessitating their being grouped together (“Other Labs”) for analysis. Variants were reclassified by the commercial laboratory that had performed the original classification. Throughout the study period, variant reclassifications were recorded in the study database as they were received, assigned to the date on which the amended electronic or paper report was received from the laboratory. Prospective follow-up for reclassification was terminated either by study closure at the end of February 2017, by reclassification of the genetic variant to benign, or by receipt of a third reclassification (after which no further reclassification was observed). Follow-up was not terminated by the death of the individual who had been tested, because laboratories issue variant reclassifications regardless of the individual’s vital status. Statistical Analysis Duplicate variants were excluded from the study at both the family and the ancestry group levels. First, when members of a family were tested by the same laboratory for the same variant (“within-family duplicates”), only the first such variant from the family was retained for study. Then retained variants were put into random order, and any nonfamily duplicate variants (same ancestry, laboratory, and classification) were excluded. Rate of reclassification was defined as the number of reclassifications divided by total observation time. All statistical tests were two-sided. The primary hypothesis was that rate of reclassification differed between individual minority ancestries and the referent category, non-Hispanic European ancestry. This hypothesis was tested separately for variants from BRCA1/2 and non-BRCA1/2 genes using generalized linear modeling (log-linked, with Poisson distribution) of reclassification rate. Each model used a generalized estimating equation to take into account intralaboratory correlation and considered as potential confounding factors laboratory, individual genes with at least 50 observations apiece, classification of variant (handled as a time-dependent variable that changed at the time of each reclassification, initiating a new observation), year of current classification, and sex. A covariate or interaction term was retained in the model if it improved the fit to the observed data. When the model retained year of current classification, a quadratic term (year squared) and an interaction term for year by ancestry were considered also. The study’s overall type I error was limited to 5% by evaluating for statistical significance only those associations related to the primary hypothesis, namely the main effects of seven individual ancestries (relative to the referent ancestry, non-Hispanic European) and, when year of current classification was retained in the model, the seven potential interactions between individual nonreferent ancestries and year. Statistical testing was adjusted for multiple (n = 21) hypotheses using Holm-Bonferroni adjustment (10). For the first of two secondary aims, time to first reclassification was plotted graphically using the Kaplan-Meier method, by category of initial classification. For the other secondary aim, variants that underwent reclassification were categorized by net change (from original classification to most recent reclassification), with upgrade defined as any shift toward greater pathogenicity. Among variants at risk of upgrade (ie, not already pathogenic), multivariable logistic regression was used to test the following characteristics for independent association with net upgrade: original classification, type of mutation (splice site vs all others), gene (BRCA1/2 vs non-BRCA1/2), year of original testing, ancestry, and sex. For this secondary analysis, P values were unadjusted for multiple hypothesis testing. Results Not counting within-family duplicate variants, the study enrolled 1816 nonbenign variants. Those represented 96.6% of all eligible, nonbenign variants (n = 1880) identified at the participating hereditary cancer risk assessment sites during the study recruitment period. Further eliminating nonfamily duplicate variants yielded the study sample (n = 1483), comprised of 1282 unique variants in 42 actionable genes (Supplementary Table 1, available online) and another 201 variants that differed in ancestry, laboratory, or initial classification from otherwise similar variants. Discordant classification of variants between laboratories was rare, occurring in four (0.3%) variants (one each within BRCA1, BRCA2, CHEK2, and CDKN2A). Observation time per study variant ranged from 63 days to 20.2 years (median = 3.55 years). During follow-up, reclassification was observed for 268 (18.1%) variants, of which 40/268 (14.9%) were reclassified more than once (Supplementary Figure 1, available online). Overall, 693 (46.7%) variants involved BRCA1/2 (Figure 1). Nearly all BRCA1/2 variants (87.6%) were tested by Lab A, except in the final two years of the study, when Other Labs tested most (79.6%) BRCA1/2 variants. Overall, Other Labs also tested most (89.7%) variants of PTEN and all variants of another 18 genes; variants in the remaining 21 genes were tested approximately equally by Lab A and Other Labs (Supplementary Table 1, available online). Figure 1. View largeDownload slide Nonbenign variants (n = 1483) accrued per year, by class of gene. The volume of BRCA1/2 variants (hatched bars) fluctuated moderately from year to year. In contrast, the volume of non-BRCA1/2 variants (solid bars) went from negligible to very large in the final years of the study. We interpret the latter finding as consistent with the recent increase in use of multigene panel testing (1,11). Figure 1. View largeDownload slide Nonbenign variants (n = 1483) accrued per year, by class of gene. The volume of BRCA1/2 variants (hatched bars) fluctuated moderately from year to year. In contrast, the volume of non-BRCA1/2 variants (solid bars) went from negligible to very large in the final years of the study. We interpret the latter finding as consistent with the recent increase in use of multigene panel testing (1,11). Table 1 presents additional characteristics of BRCA1/2 and non-BRCA1/2 variants. In both groups, reclassification was more common among variants tested by Lab A and rare among pathogenic variants (Table 1). As illustrated in Figure 2, variants initially classified as pathogenic were almost never reclassified, but other classes of nonbenign variants were commonly reclassified and had similar times to first reclassification. Table 1. Variants (total and reclassified), by class of gene Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Table 1. Variants (total and reclassified), by class of gene Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Figure 2. View largeDownload slide Time to first reclassification of the variant, by initial classification. For ease of interpretation, this Kaplan-Meier plot illustrates follow-up of the 1483 variants through their first reclassification only. Variants are distinguished by their initial classification: likely benign (dotted line), likely pathogenic (dash-dot line), pathogenic (dashed line), variant of uncertain significance (solid line). Numbers of unreclassified variants remaining in follow-up are shown below the plot. As the plot indicates, variants initially classified as pathogenic were almost never reclassified. In contrast, the other three classes of variants were often reclassified, with a shared pattern of time to first reclassification that suggests that almost all nonpathogenic, nonbenign variants will be reclassified eventually. LB = likely benign; LP = likely pathogenic; P = pathogenic; VUS = variant of uncertain significance. Figure 2. View largeDownload slide Time to first reclassification of the variant, by initial classification. For ease of interpretation, this Kaplan-Meier plot illustrates follow-up of the 1483 variants through their first reclassification only. Variants are distinguished by their initial classification: likely benign (dotted line), likely pathogenic (dash-dot line), pathogenic (dashed line), variant of uncertain significance (solid line). Numbers of unreclassified variants remaining in follow-up are shown below the plot. As the plot indicates, variants initially classified as pathogenic were almost never reclassified. In contrast, the other three classes of variants were often reclassified, with a shared pattern of time to first reclassification that suggests that almost all nonpathogenic, nonbenign variants will be reclassified eventually. LB = likely benign; LP = likely pathogenic; P = pathogenic; VUS = variant of uncertain significance. Multivariable analyses of reclassification rate were conducted separately for BRCA1/2 and non-BRCA1/2 variants (Table 2). At the start of observation, BRCA1/2 variants from three ancestries (African, Ashkenazi, and Chinese) had reduced rates of reclassification; those from non-Chinese Asian, Hispanic, and Native American ancestries had rates no different than the non-Hispanic European referent category; and those from Middle Eastern ancestry had a higher rate. Over time, rate of reclassification increased in all ancestral categories of BRCA1/2, but it did so faster in each of the three ancestries initially associated with reduced rates (Table 2, Figure 3A). After a period of increase, each ancestry’s rate of BRCA1/2 reclassification began to decline toward its initial rate (Figure 3A), a pattern indicated by the quadratic term in the model (Table 2). For simplicity, Figure 3A ignores initial variation in reclassification rate among ancestries. Figure 3B, on the other hand, applies each ancestry’s initial rate to its rate over time, plotting relative rates of reclassification by year for each ancestry. In this way, it can be seen that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the annual rate of reclassification eventually equaled or surpassed the non-Hispanic European rate (Figure 3B). Table 2. Multivariable models: Association between ancestry and rate of variant reclassification Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* * To control error, statistical testing was limited to the primary hypothesis that the rates of BRCA1/2 and non-BRCA1/2 reclassification (initially and over time) varied for individual ancestries relative to the referent category, Non-Hispanic European. Covariates shown were retained in the models when they improved the fit to the observed data. † These covariates and the interaction of ancestry with time did not improve the non-BRCA1/2 model’s fit, so they were omitted. ‡ Terms for non-BRCA1/2 genes that had at least 50 observations (APC, ATM, CHEK2, MLH1, MSH2, MSH6) did not improve this model’s fit, so they were omitted. § The nonpathogenic category included the classifications of likely pathogenic, variant of uncertain significance, and likely benign. Their individual associations with reclassification were similar, so for efficiency, they were combined in the final model. Table 2. Multivariable models: Association between ancestry and rate of variant reclassification Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* * To control error, statistical testing was limited to the primary hypothesis that the rates of BRCA1/2 and non-BRCA1/2 reclassification (initially and over time) varied for individual ancestries relative to the referent category, Non-Hispanic European. Covariates shown were retained in the models when they improved the fit to the observed data. † These covariates and the interaction of ancestry with time did not improve the non-BRCA1/2 model’s fit, so they were omitted. ‡ Terms for non-BRCA1/2 genes that had at least 50 observations (APC, ATM, CHEK2, MLH1, MSH2, MSH6) did not improve this model’s fit, so they were omitted. § The nonpathogenic category included the classifications of likely pathogenic, variant of uncertain significance, and likely benign. Their individual associations with reclassification were similar, so for efficiency, they were combined in the final model. Figure 3. View largeDownload slide Model-derived estimates of annual growth in rate of reclassification of BRCA1 variants, by ancestry. These panels illustrate two ways of plotting the ancestry-specific variation in reclassification of BRCA1/2 variants as reported in Table 2. For clarity, these plots use estimates for that model’s referent category, specifically nonpathogenic BRCA1 variants from females tested by Lab A. Estimates for BRCA2 or pathogenic variants, or those from males or Other Labs, can be obtained by applying the corresponding relative rates in Table 2 to these plots. In both, the x-axis corresponds to the calendar year when the variant was initially classified. A) In this plot, the reclassification rate in 1997 was set to 1.00 for all ancestries, ignoring the substantial initial variation among ancestries in favor of illustrating ancestry-specific patterns of change in reclassification rate over time. B) In contrast, here each ancestry’s estimated annual growth in reclassification rate was applied to its own baseline rate from 1997, relative to the reference category of non-Hispanic European (NHE). From this second plot, we conclude that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the rate of reclassification eventually equaled or surpassed the NHE rate. Figure 3. View largeDownload slide Model-derived estimates of annual growth in rate of reclassification of BRCA1 variants, by ancestry. These panels illustrate two ways of plotting the ancestry-specific variation in reclassification of BRCA1/2 variants as reported in Table 2. For clarity, these plots use estimates for that model’s referent category, specifically nonpathogenic BRCA1 variants from females tested by Lab A. Estimates for BRCA2 or pathogenic variants, or those from males or Other Labs, can be obtained by applying the corresponding relative rates in Table 2 to these plots. In both, the x-axis corresponds to the calendar year when the variant was initially classified. A) In this plot, the reclassification rate in 1997 was set to 1.00 for all ancestries, ignoring the substantial initial variation among ancestries in favor of illustrating ancestry-specific patterns of change in reclassification rate over time. B) In contrast, here each ancestry’s estimated annual growth in reclassification rate was applied to its own baseline rate from 1997, relative to the reference category of non-Hispanic European (NHE). From this second plot, we conclude that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the rate of reclassification eventually equaled or surpassed the NHE rate. In contrast to BRCA1/2 variants, reclassification rates of non-BRCA1/2 variants did not vary over time (Table 2), a finding consistent with the fact that, unlike BRCA1/2 variants, non-BRCA1/2 variants were mostly accrued during a brief period near the end of the study (Figure 1). Moreover, overall reclassification rates were elevated for non-BRCA1/2 variants from most minority ancestries (African, Ashkenazi, Hispanic, and to lesser degree Middle Eastern and Chinese). The exceptions were non-BRCA1/2 variants from non-Chinese Asian and Native American ancestries, which had reclassification rates similar to variants from non-Hispanic European ancestry (Table 2). Among reclassified variants, few (26/268, 9.7%) underwent a net upgrade in pathogenicity. Excluding variant classifications (pathogenic, likely benign) never observed to be upgraded left 241 reclassified variants that could have undergone net upgrade. Per multivariable logistic regression (Table 3), reclassified variants were more likely to have been a net upgrade if they had been classified originally as likely pathogenic, belonged to a male participant, or had undergone initial testing earlier during the study period. Specifically, the chances that a reclassified variant represented a net upgrade decreased nonlinearly over time: following a sigmoid curve, that decrease in risk was initially minor (ie, approximately –8% at four years into the study) but accelerated over time (reaching –40% at 10 years, –70% at 15.5 years, and –90% at 18 years). Neither ancestry, type of mutation, nor BRCA1/2 status was associated with reclassification being a net upgrade. Table 3. Multivariable model: Associations with net upgrade among reclassified variants at risk* (n = 241) Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 * Variants observed to be at risk of net upgrade were those initially classified as likely pathogenic or variant of uncertain significance. † Because this model represents a secondary analysis, P values were unadjusted for multiple hypothesis testing. ‡ Covariates that did not improve the model’s fit were omitted from the final model. Table 3. Multivariable model: Associations with net upgrade among reclassified variants at risk* (n = 241) Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 * Variants observed to be at risk of net upgrade were those initially classified as likely pathogenic or variant of uncertain significance. † Because this model represents a secondary analysis, P values were unadjusted for multiple hypothesis testing. ‡ Covariates that did not improve the model’s fit were omitted from the final model. Incidentally, 17.1% of variants overall were obtained from persons who reported mixed ancestry. However, the proportion of mixed ancestry varied by group: It was absent by definition among participants with non-Hispanic European ancestry but common among persons with Ashkenazi (42.7% of whose variants were associated with mixed ancestry, chiefly non-Hispanic European), African (53.0% mixed ancestry, chiefly non-Hispanic European or Native American), or non-African Native American background (87.0% mixed ancestry, chiefly Hispanic or non-Hispanic European). Finally, 10.0% of Chinese, 12.4% of non-Chinese Asian, 15.6% of Middle Eastern, and 17.6% of Hispanic variants came from participants with mixed ancestry. Discussion This study of an ethnically diverse cohort of nonbenign variants followed for up to two decades reveals for the first time that, independent of covariates, the rate of variant reclassification varies by ancestry. Overall, current findings suggest a “catching up” in the reclassification of variants from minority ancestries after an excess in uncertain classifications (VUS) relative to non-Hispanic European tests. This pattern was likely facilitated by the availability of large population databases such as the National Heart, Lung, and Blood Institute Exome Sequencing Project and 1000 Genomes, followed by the more ethnically diverse Exome Aggregation Consortium (ExAC) database (4,12,13). Further, a “catching up” of reclassification would be consistent with a previous report that the frequency of VUS as a percentage of all variant classifications declined during the decades we studied (14), most steeply among African, Asian, Hispanic, and Middle Eastern ancestries. The current study also reports that the percentage of reclassifications that are net upgrades is low (less than half the 20% reported by an earlier study limited to BRCA1/2 variants) (15). Moreover, this percentage has declined steeply in recent years. Importantly for clinical care and genetic counseling, the latter finding argues against the view that VUS are commonly upgraded to pathogenic or likely pathogenic status, an assumption that may lead individuals who receive these results to undergo unnecessary procedures (ie, bilateral mastectomy) (16). We identify initial classification, sex, and calendar year of testing (but not ancestry, laboratory, or splice site mutation) (15) as risk factors for a reclassification being a net upgrade. Findings incidental to our primary hypothesis include two unanticipated associations with sex. Specifically, BRCA1/2 variants from male participants appear more likely to undergo reclassification. Also, regardless of gene, reclassified variants from male participants appear more likely to have undergone a net upgrade in pathogenicity. Neither of these associations with sex can be explained by any of the variant characteristics we studied. Possibly, these associations may be related to males’ greater reluctance to seek medical care generally (17). We speculate that, to present for cancer genetic risk assessment, males may tend to require a more compelling family history of cancer than females do. In turn, a more compelling family history may promote initiation and completion of the research necessary to issue a reclassification and may also be associated with genetic variants of greater pathogenicity. In any case, current incidental associations with sex should be regarded as preliminary until confirmed by separate studies. The current longitudinal analysis of time to reclassification suggests that, except for pathogenic variants, most nonbenign test results will ultimately be reclassified. For that reason, persons undergoing genetic testing should be encouraged to provide (and update) contact information to the provider ordering the test, so that they may be informed of reclassifications as they are issued. Current findings also indicate that the various classes of nonpathogenic variants (likely pathogenic, likely benign, VUS) are equally likely to undergo reclassification, while reclassifications of likely pathogenic variants are more likely than VUS reclassifications to be net upgrades. The latter finding supports the current clinical practice of treating likely pathogenic variants as similar to pathogenic variants (1). To our knowledge, only a limited description of time to variant reclassification has been published previously (3). That retrospective study (n = 107 women whose BRCA1/2 variants were exclusively VUS) did not provide median time to reclassification or rate per years of observation. In contrast, our prospective study has presented time to first reclassification by classification and modeled rate of reclassification within ancestry groups over time while controlling potential confounding factors. Moreover, our large sample was intended to be representative of contemporary clinical practice and thus accepted variants 1) from genes other than BRCA1/2, 2) with classifications not restricted to VUS, 3) from males as well as females, and 4) regardless of whether the participant carried a pathogenic BRCA1/2 variant. One methodological challenge inherent in research into reclassification of genetic variants is that, due to lack of specific regulations, internal proprietary data, and varying methodologies, commercial genetic laboratories do not follow a common protocol for issuing reclassifications. Some laboratories (ie, Lab A) have active, system-based approaches that generate reclassifications. Alternatively, laboratories may issue reclassifications only sporadically, for example, when new clinical or empirical evidence is brought forward by a health care provider or a laboratory director. Over time, laboratories may shift between sporadic and active reclassification programs without notice. Recognizing the complex heterogeneity in reclassification programs, we included all commercial laboratories used during the study period but took steps to prevent laboratory differences from confounding our investigation. Specifically, we took into account within-laboratory correlation, distinguished the most active laboratory from all others, and considered BRCA1/2 and non-BRCA1/2 variants separately. Another methodological challenge inherent in our study relates to the categorization of ancestry from self-report. To avoid oversimplifying ancestry, we recognized diversity within the “Asian” category, replacing it with categories of Chinese, Middle Eastern, and other Asian ancestry. On the other hand, clarity required us to identify systematically one ancestry per participant when several were reported. Mixed ancestry was reported by a minority of our participants, chiefly, as expected from studies of admixture, those with Ashkenazi, African, or Native American ancestry (18). In the future, studies of reclassification rates might benefit from analyzing ancestry using ethnic percentages or germline DNA-based categorization. Our study findings are specific to the era and genes that we studied. In the future, reference databases will become more ethnically diverse (4,6), routine genetic testing will include more genes, and new methodologies will become prevalent, such as polygenic risk scoring using single nucleotide polymorphisms, whole-exome and -genome sequencing, and secondary germline findings from tumor-based testing. As a result, the ancestry-based associations with reclassification that we have observed are likely to evolve. In conclusion, the current study investigates the role of ancestry in variant reclassification during two decades of clinical practice. Our findings demonstrate that independently of laboratory and initial classification, the rate at which a nonbenign cancer genetic variant is reclassified varies by ancestry in ways that depend on gene (BRCA1/2 vs others) and, when observation spans more than a few years, on the year when a genetic variant was classified. Funding The research reported in this publication was supported by the National Cancer Institute (NCI) of the National Institutes of Health (NIH) under award number P30CA33572 (Integrative Genomics and Biostatistics Cores). The City of Hope Clinical Cancer Genomics Community Research Network and the Hereditary Cancer Research Registry was supported in part by NCI NIH award number RC4CA153828 (PI: J. Weitzel). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Other sources of support include the 2015 STOP CANCER Research Career Development Award (PI: T. Slavin), the Oxnard Foundation (PI: T. Slavin), the American Cancer Society (PI: J. Weitzel), and the Avon Foundation (PI: J. Weitzel). Notes Affiliations of authors: Division of Clinical Cancer Genomics (TPS, LVT, IS, CR, BN, LK, MNS, KRB, KY, SS, DC, JH, SWG, JNW), Department of Information Sciences (CEB), and Integrative Genomics Core (ST), City of Hope, Duarte, CA; Cancer Genetics Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA (JOC). Present affiliations: Human Longevity Inc., San Diego, CA (CR); Dyson Center for Cancer Care, Poughkeepsie, NY (MNS). The funders had no role in the design of the study; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication. The authors thank the members of the the Clinical Cancer Genomics Community Research Network; research aides Rosa Mejia, Nancy Guerrero-Llamas, and Sophia Manjarrez; and all research participants. References 1 NCCN . NCCN clinical practice guidelines in oncology V.1.2018: Genetic/familial high-risk assessment: Breast and ovarian. NCCN Clinical Practice Guidelines. National Comprehensive Cancer Network, Inc; 2017 . https://www.nccn.org/professionals/physician_gls/default.aspx. Accessed February 20, 2018. 2 Richards S , Aziz N , Bale S , et al. . Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology . Genet Med. 2015 ; 17 5 : 405 – 424 . Google Scholar Crossref Search ADS PubMed 3 Murray ML , Cerrato F , Bennett RL , et al. . Follow-up of carriers of BRCA1 and BRCA2 variants of unknown significance: Variant reclassification and surgical decisions . Genet Med. 2011 ; 13 12 : 998 – 1005 . Google Scholar Crossref Search ADS PubMed 4 Lek M , Karczewski KJ , Minikel EV , et al. . Analysis of protein-coding genetic variation in 60,706 humans . Nature. 2016 ; 536 7616 : 285 – 291 . Google Scholar Crossref Search ADS PubMed 5 Manrai AK , Funke BH , Rehm HL , et al. . Genetic misdiagnoses and the potential for health disparities . N Engl J Med. 2016 ; 375 7 : 655 – 665 . Google Scholar Crossref Search ADS PubMed 6 Popejoy AB , Fullerton SM. Genomics is failing on diversity . Nature. 2016 ; 538 7624 : 161 – 164 . Google Scholar Crossref Search ADS PubMed 7 Ricker C , Culver JO , Lowstuter K , et al. . Increased yield of actionable mutations using multi-gene panels to assess hereditary cancer susceptibility in an ethnically diverse clinical cohort . Cancer Genet. 2016 ; 209 4 : 130 – 137 . Google Scholar Crossref Search ADS PubMed 8 Susswein LR , Marshall ML , Nusbaum R , et al. . Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing . Genet Med. 2016 ; 18 8 : 823 – 832 . Google Scholar Crossref Search ADS PubMed 9 Caswell-Jin JL , Gupta T , Hall E , et al. . Racial/ethnic differences in multiple-gene sequencing results for hereditary cancer risk . Genet Med. 2017; July 2017. Epub ahead of print. 10 Holm S. A simple sequentially rejective multiple test procedure . Scand J Stat. 1979 ; 6 2 : 65 – 70 . 11 Slavin T , Niell-Swiller M , Solomon I , et al. . Clinical application of multigene panels: Challenges of next-generation counseling and cancer risk management . Front Oncol. 2015 ; 5 . 12 National Heart L, and Blood Institute (NHLBI) . NHLBI Grand Opportunity Exome Sequencing Project (ESP). https://esp.gs.washington.edu/drupal/. Accessed February 20, 2018. 13 Pennisi E. 1000 Genomes Project gives new map of genetic diversity . Science. 2010 ; 330 6004 : 574 – 575 . Google Scholar Crossref Search ADS PubMed 14 Eggington JM , Bowles KR , Moyes K , et al. . A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes . Clin Genet. 2014 ; 86 3 : 229 – 237 . Google Scholar Crossref Search ADS PubMed 15 Easton DF , Deffenbaugh AM , Pruss D , et al. . A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes . Am J Hum Genet. 2007 ; 81 5 : 873 – 883 . Google Scholar Crossref Search ADS PubMed 16 Kurian AW , Li Y , Hamilton AS , et al. . Gaps in incorporating germline genetic testing into treatment decision-making for early-stage breast cancer . J Clin Oncol. 2017 ; 35 20 : 2232 – 2239 . Google Scholar Crossref Search ADS PubMed 17 Courtenay WH. Constructions of masculinity and their influence on men's well-being: A theory of gender and health . Soc Sci Med. 2000 ; 50 10 : 1385 – 1401 . Google Scholar Crossref Search ADS PubMed 18 Adhikari K , Chacon-Duque JC , Mendoza-Revilla J , et al. . The genetic diversity of the Americas . Annu Rev Genomics Hum Genet. 2017 ; 18 : 277 – 296 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI: Journal of the National Cancer Institute Oxford University Press

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Abstract

Abstract 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. Genetic testing for hereditary cancer susceptibility has dramatically improved delivery of precision cancer prevention or treatment to individuals who harbor pathogenic variants in cancer-associated genes. Increasingly, individuals undergoing a hereditary cancer risk assessment are tested with commercial multigene panels consisting of 25 genes or more (1). Laboratories classify genetic variants along a continuum of clinical significance (ie, pathogenic, likely pathogenic, variant of uncertain significance [VUS], likely benign, or benign) (2). As genomic knowledge evolves, however, variants can be reclassified from one category to another, often with clinical implications for the recipient. Because time to reclassification is highly variable and can exceed a decade or more (3), tested individuals often make health-related decisions based on initial rather than definitive interpretations of their genomic data. Despite the fact that variant reclassification may have profound implications for patient care and medical decision-making, little is known about the factors associated with variant reclassification. One factor that may be important in variant reclassification is ancestry. When classifying or reclassifying variants, labs rely heavily on reference databases of human genetic variation. However, because persons of European descent continue to be disproportionately overrepresented within reference genetic databases (4–6), individuals of non-European ancestry are more likely to receive uncertain test results (7–9). Given potential ethnic disparities in the initial classification of genetic variants, we hypothesized that the rate of reclassification of nonbenign variants likewise differs by ancestry, independent of potential confounding factors such as time, gene, laboratory, and initial classification. To address this hypothesis, we analyzed a prospective cohort of pathogenic, likely pathogenic, VUS, and likely benign variants identified during two decades of a tertiary care–based program for hereditary cancer risk assessment among an ethnically diverse population. Recognizing broad differences in the history and scale of efforts to research and reclassify variants of hereditary breast cancer genes (BRCA1/2) and other genes, we analyzed those two groups of variants separately. As secondary aims, we examined time to reclassification by initial classification and identified characteristics that distinguish reclassified variants that were upgraded. Methods Study Population The study was approved by the Institutional Review Board at City of Hope. Data were obtained from individuals who were referred for cancer genetic risk assessment and underwent genetic testing at one of two Southern California sites (City of Hope and Olive View Medical Center) from September 1996 through December 2016 and who gave informed consent to participate in the Clinical Cancer Genomics Community Research Network registry. Eligible for study were variants of actionable and commonly evaluated genes in hereditary cancer predisposition. In particular, because benign variants were not subject to reclassification, only nonbenign variants (pathogenic, likely pathogenic, VUS, likely benign) were studied. The number of genetic variants studied per person was unrestricted. Characteristics collected per variant were gene, mutation, laboratory, self-reported maternal and paternal ancestry, sex, and dates and results of initial classification and any subsequent reclassifications. Terminology for classifying variants was standardized across laboratories per Richards et al. (2). Variants listed as requiring special interpretation were grouped in the same category as VUS. Self-reported ancestry (defined as at least one grandparent) was assigned to mutually exclusive categories in the following sequence: any African, any Native American, any Chinese, including Taiwanese, any other Asian (Filipino, Japanese, Korean, Indian, Pakistani, Indonesian, Thai, Cambodian, Vietnamese, Nepali, Samoan), any Ashkenazi, any Hispanic, any Middle Eastern (Iranian, Armenian, Syrian, Lebanese, Egyptian, Turkish), non-Hispanic European. Within these categories, individuals were noted as having mixed ancestry if they reported ancestry from at least one additional category. A single laboratory (referred to as “Lab A”) issued most initial classifications and reclassifications under study. Corresponding data from the other 33 testing laboratories were sparse, necessitating their being grouped together (“Other Labs”) for analysis. Variants were reclassified by the commercial laboratory that had performed the original classification. Throughout the study period, variant reclassifications were recorded in the study database as they were received, assigned to the date on which the amended electronic or paper report was received from the laboratory. Prospective follow-up for reclassification was terminated either by study closure at the end of February 2017, by reclassification of the genetic variant to benign, or by receipt of a third reclassification (after which no further reclassification was observed). Follow-up was not terminated by the death of the individual who had been tested, because laboratories issue variant reclassifications regardless of the individual’s vital status. Statistical Analysis Duplicate variants were excluded from the study at both the family and the ancestry group levels. First, when members of a family were tested by the same laboratory for the same variant (“within-family duplicates”), only the first such variant from the family was retained for study. Then retained variants were put into random order, and any nonfamily duplicate variants (same ancestry, laboratory, and classification) were excluded. Rate of reclassification was defined as the number of reclassifications divided by total observation time. All statistical tests were two-sided. The primary hypothesis was that rate of reclassification differed between individual minority ancestries and the referent category, non-Hispanic European ancestry. This hypothesis was tested separately for variants from BRCA1/2 and non-BRCA1/2 genes using generalized linear modeling (log-linked, with Poisson distribution) of reclassification rate. Each model used a generalized estimating equation to take into account intralaboratory correlation and considered as potential confounding factors laboratory, individual genes with at least 50 observations apiece, classification of variant (handled as a time-dependent variable that changed at the time of each reclassification, initiating a new observation), year of current classification, and sex. A covariate or interaction term was retained in the model if it improved the fit to the observed data. When the model retained year of current classification, a quadratic term (year squared) and an interaction term for year by ancestry were considered also. The study’s overall type I error was limited to 5% by evaluating for statistical significance only those associations related to the primary hypothesis, namely the main effects of seven individual ancestries (relative to the referent ancestry, non-Hispanic European) and, when year of current classification was retained in the model, the seven potential interactions between individual nonreferent ancestries and year. Statistical testing was adjusted for multiple (n = 21) hypotheses using Holm-Bonferroni adjustment (10). For the first of two secondary aims, time to first reclassification was plotted graphically using the Kaplan-Meier method, by category of initial classification. For the other secondary aim, variants that underwent reclassification were categorized by net change (from original classification to most recent reclassification), with upgrade defined as any shift toward greater pathogenicity. Among variants at risk of upgrade (ie, not already pathogenic), multivariable logistic regression was used to test the following characteristics for independent association with net upgrade: original classification, type of mutation (splice site vs all others), gene (BRCA1/2 vs non-BRCA1/2), year of original testing, ancestry, and sex. For this secondary analysis, P values were unadjusted for multiple hypothesis testing. Results Not counting within-family duplicate variants, the study enrolled 1816 nonbenign variants. Those represented 96.6% of all eligible, nonbenign variants (n = 1880) identified at the participating hereditary cancer risk assessment sites during the study recruitment period. Further eliminating nonfamily duplicate variants yielded the study sample (n = 1483), comprised of 1282 unique variants in 42 actionable genes (Supplementary Table 1, available online) and another 201 variants that differed in ancestry, laboratory, or initial classification from otherwise similar variants. Discordant classification of variants between laboratories was rare, occurring in four (0.3%) variants (one each within BRCA1, BRCA2, CHEK2, and CDKN2A). Observation time per study variant ranged from 63 days to 20.2 years (median = 3.55 years). During follow-up, reclassification was observed for 268 (18.1%) variants, of which 40/268 (14.9%) were reclassified more than once (Supplementary Figure 1, available online). Overall, 693 (46.7%) variants involved BRCA1/2 (Figure 1). Nearly all BRCA1/2 variants (87.6%) were tested by Lab A, except in the final two years of the study, when Other Labs tested most (79.6%) BRCA1/2 variants. Overall, Other Labs also tested most (89.7%) variants of PTEN and all variants of another 18 genes; variants in the remaining 21 genes were tested approximately equally by Lab A and Other Labs (Supplementary Table 1, available online). Figure 1. View largeDownload slide Nonbenign variants (n = 1483) accrued per year, by class of gene. The volume of BRCA1/2 variants (hatched bars) fluctuated moderately from year to year. In contrast, the volume of non-BRCA1/2 variants (solid bars) went from negligible to very large in the final years of the study. We interpret the latter finding as consistent with the recent increase in use of multigene panel testing (1,11). Figure 1. View largeDownload slide Nonbenign variants (n = 1483) accrued per year, by class of gene. The volume of BRCA1/2 variants (hatched bars) fluctuated moderately from year to year. In contrast, the volume of non-BRCA1/2 variants (solid bars) went from negligible to very large in the final years of the study. We interpret the latter finding as consistent with the recent increase in use of multigene panel testing (1,11). Table 1 presents additional characteristics of BRCA1/2 and non-BRCA1/2 variants. In both groups, reclassification was more common among variants tested by Lab A and rare among pathogenic variants (Table 1). As illustrated in Figure 2, variants initially classified as pathogenic were almost never reclassified, but other classes of nonbenign variants were commonly reclassified and had similar times to first reclassification. Table 1. Variants (total and reclassified), by class of gene Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Table 1. Variants (total and reclassified), by class of gene Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Characteristic BRCA1/2 (n = 693) Reclassified No. (%) Non-BRCA1/2 (n = 790) Reclassified No. (%) Ancestry  African 42 18 (42.9) 41 10 (24.4)  Ashkenazi 41 10 (24.4) 55 12 (21.8)  Asian, Chinese 48 16 (33.3) 42 5 (11.9)  Asian, non-Chinese 64 15 (23.4) 81 3 (3.7)  Hispanic 176 32 (18.2) 205 26 (12.7)  Middle Eastern 32 11 (34.4) 58 8 (13.8)  Native American 39 10 (25.6) 30 3 (10.0)  Non-Hispanic European 251 70 (27.9) 278 20 (7.2) Initial classification  Likely benign 46 22 (47.8) 19 2 (10.5)  Of uncertain significance 288 154 (53.5) 501 74 (14.8)  Likely pathogenic 7 5 (71.4) 27 9 (33.3)  Pathogenic 352 1 (0.3) 243 2 (0.8) Laboratory  Lab A 607 181 (29.8) 322 64 (19.9)  Other Labs 86 1 (1.2) 468 23 (4.9) Sex  Male 20 2 (10.0) 133 12 (9.0)  Female 673 180 (26.8) 657 75 (11.4) Figure 2. View largeDownload slide Time to first reclassification of the variant, by initial classification. For ease of interpretation, this Kaplan-Meier plot illustrates follow-up of the 1483 variants through their first reclassification only. Variants are distinguished by their initial classification: likely benign (dotted line), likely pathogenic (dash-dot line), pathogenic (dashed line), variant of uncertain significance (solid line). Numbers of unreclassified variants remaining in follow-up are shown below the plot. As the plot indicates, variants initially classified as pathogenic were almost never reclassified. In contrast, the other three classes of variants were often reclassified, with a shared pattern of time to first reclassification that suggests that almost all nonpathogenic, nonbenign variants will be reclassified eventually. LB = likely benign; LP = likely pathogenic; P = pathogenic; VUS = variant of uncertain significance. Figure 2. View largeDownload slide Time to first reclassification of the variant, by initial classification. For ease of interpretation, this Kaplan-Meier plot illustrates follow-up of the 1483 variants through their first reclassification only. Variants are distinguished by their initial classification: likely benign (dotted line), likely pathogenic (dash-dot line), pathogenic (dashed line), variant of uncertain significance (solid line). Numbers of unreclassified variants remaining in follow-up are shown below the plot. As the plot indicates, variants initially classified as pathogenic were almost never reclassified. In contrast, the other three classes of variants were often reclassified, with a shared pattern of time to first reclassification that suggests that almost all nonpathogenic, nonbenign variants will be reclassified eventually. LB = likely benign; LP = likely pathogenic; P = pathogenic; VUS = variant of uncertain significance. Multivariable analyses of reclassification rate were conducted separately for BRCA1/2 and non-BRCA1/2 variants (Table 2). At the start of observation, BRCA1/2 variants from three ancestries (African, Ashkenazi, and Chinese) had reduced rates of reclassification; those from non-Chinese Asian, Hispanic, and Native American ancestries had rates no different than the non-Hispanic European referent category; and those from Middle Eastern ancestry had a higher rate. Over time, rate of reclassification increased in all ancestral categories of BRCA1/2, but it did so faster in each of the three ancestries initially associated with reduced rates (Table 2, Figure 3A). After a period of increase, each ancestry’s rate of BRCA1/2 reclassification began to decline toward its initial rate (Figure 3A), a pattern indicated by the quadratic term in the model (Table 2). For simplicity, Figure 3A ignores initial variation in reclassification rate among ancestries. Figure 3B, on the other hand, applies each ancestry’s initial rate to its rate over time, plotting relative rates of reclassification by year for each ancestry. In this way, it can be seen that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the annual rate of reclassification eventually equaled or surpassed the non-Hispanic European rate (Figure 3B). Table 2. Multivariable models: Association between ancestry and rate of variant reclassification Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* * To control error, statistical testing was limited to the primary hypothesis that the rates of BRCA1/2 and non-BRCA1/2 reclassification (initially and over time) varied for individual ancestries relative to the referent category, Non-Hispanic European. Covariates shown were retained in the models when they improved the fit to the observed data. † These covariates and the interaction of ancestry with time did not improve the non-BRCA1/2 model’s fit, so they were omitted. ‡ Terms for non-BRCA1/2 genes that had at least 50 observations (APC, ATM, CHEK2, MLH1, MSH2, MSH6) did not improve this model’s fit, so they were omitted. § The nonpathogenic category included the classifications of likely pathogenic, variant of uncertain significance, and likely benign. Their individual associations with reclassification were similar, so for efficiency, they were combined in the final model. Table 2. Multivariable models: Association between ancestry and rate of variant reclassification Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* Risk factor Rate ratio (95% confidence interval) BRCA1/2 variants (n = 693) Holm P* Non-BRCA1/2 variants (n = 790) Holm P* Ancestry  African 0.59 (0.45 to 0.78) .002 3.02 (2.38 to 3.81) .001  Ashkenazi 0.29 (0.20 to 0.44) .001 3.71 (2.27 to 6.04) .001  Asian, Chinese 0.28 (0.20 to 0.39) .001 1.38 (1.18 to 1.63) .001  Asian, non-Chinese 0.80 (0.63 to 1.02) .42 0.68 (0.47 to 0.97) .26  Hispanic 0.85 (0.64 to 1.11) 1.00 2.26 (1.85 to 2.75) .001  Middle Eastern 1.44 (1.21 to 1.72) .001 2.12 (1.32 to 3.40) .02  Native American 0.97 (0.84 to 1.12) 1.00 1.92 (0.35 to 10.53) 1.00  Non-Hispanic European 1.00 (reference) 1.00 (reference) Year of current classification, since 1997  Per additional year, by ancestry   African 1.46 (1.40 to 1.51) .001 —†   Ashkenazi 1.55 (1.46 to 1.64) .001 —†   Asian, Chinese 1.49 (1.42 to 1.56) .001 —†   Asian, non-Chinese 1.33 (1.29 to 1.37) 1.00 —†   Hispanic 1.29 (1.24 to 1.33) .16 —†   Middle Eastern 1.35 (1.31 to 1.39) .69 —†   Native American 1.37 (1.34 to 1.41) .42 —†   Non-Hispanic European 1.34 (1.29 to 1.40) (reference for year by ancestry interactions) —* —†  Per additional year, squared 0.983 (0.980 to 0.987) —* —† Gene  BRCA1 1.00 (reference) ‡  BRCA2 1.07 (1.06 to 1.08) —* —† Sex  Male 3.42 (3.21 to 3.63) —* —†  Female 1.00 (reference) Most recent nonbenign classification  Pathogenic 0.0055 (0.0055 to 0.0056) —* 0.03 (0.01 to 0.07) —*  Less than pathogenic§ 1.00 (reference) 1.00 (reference) Laboratory  Lab A 1.00 (reference) 1.00 (reference)  Other Labs 0.17 (0.02 to 1.54) —* 0.37 (0.25 to 0.55) —* * To control error, statistical testing was limited to the primary hypothesis that the rates of BRCA1/2 and non-BRCA1/2 reclassification (initially and over time) varied for individual ancestries relative to the referent category, Non-Hispanic European. Covariates shown were retained in the models when they improved the fit to the observed data. † These covariates and the interaction of ancestry with time did not improve the non-BRCA1/2 model’s fit, so they were omitted. ‡ Terms for non-BRCA1/2 genes that had at least 50 observations (APC, ATM, CHEK2, MLH1, MSH2, MSH6) did not improve this model’s fit, so they were omitted. § The nonpathogenic category included the classifications of likely pathogenic, variant of uncertain significance, and likely benign. Their individual associations with reclassification were similar, so for efficiency, they were combined in the final model. Figure 3. View largeDownload slide Model-derived estimates of annual growth in rate of reclassification of BRCA1 variants, by ancestry. These panels illustrate two ways of plotting the ancestry-specific variation in reclassification of BRCA1/2 variants as reported in Table 2. For clarity, these plots use estimates for that model’s referent category, specifically nonpathogenic BRCA1 variants from females tested by Lab A. Estimates for BRCA2 or pathogenic variants, or those from males or Other Labs, can be obtained by applying the corresponding relative rates in Table 2 to these plots. In both, the x-axis corresponds to the calendar year when the variant was initially classified. A) In this plot, the reclassification rate in 1997 was set to 1.00 for all ancestries, ignoring the substantial initial variation among ancestries in favor of illustrating ancestry-specific patterns of change in reclassification rate over time. B) In contrast, here each ancestry’s estimated annual growth in reclassification rate was applied to its own baseline rate from 1997, relative to the reference category of non-Hispanic European (NHE). From this second plot, we conclude that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the rate of reclassification eventually equaled or surpassed the NHE rate. Figure 3. View largeDownload slide Model-derived estimates of annual growth in rate of reclassification of BRCA1 variants, by ancestry. These panels illustrate two ways of plotting the ancestry-specific variation in reclassification of BRCA1/2 variants as reported in Table 2. For clarity, these plots use estimates for that model’s referent category, specifically nonpathogenic BRCA1 variants from females tested by Lab A. Estimates for BRCA2 or pathogenic variants, or those from males or Other Labs, can be obtained by applying the corresponding relative rates in Table 2 to these plots. In both, the x-axis corresponds to the calendar year when the variant was initially classified. A) In this plot, the reclassification rate in 1997 was set to 1.00 for all ancestries, ignoring the substantial initial variation among ancestries in favor of illustrating ancestry-specific patterns of change in reclassification rate over time. B) In contrast, here each ancestry’s estimated annual growth in reclassification rate was applied to its own baseline rate from 1997, relative to the reference category of non-Hispanic European (NHE). From this second plot, we conclude that, for variants from all minority ancestries except non-Chinese Asian and Hispanic, the rate of reclassification eventually equaled or surpassed the NHE rate. In contrast to BRCA1/2 variants, reclassification rates of non-BRCA1/2 variants did not vary over time (Table 2), a finding consistent with the fact that, unlike BRCA1/2 variants, non-BRCA1/2 variants were mostly accrued during a brief period near the end of the study (Figure 1). Moreover, overall reclassification rates were elevated for non-BRCA1/2 variants from most minority ancestries (African, Ashkenazi, Hispanic, and to lesser degree Middle Eastern and Chinese). The exceptions were non-BRCA1/2 variants from non-Chinese Asian and Native American ancestries, which had reclassification rates similar to variants from non-Hispanic European ancestry (Table 2). Among reclassified variants, few (26/268, 9.7%) underwent a net upgrade in pathogenicity. Excluding variant classifications (pathogenic, likely benign) never observed to be upgraded left 241 reclassified variants that could have undergone net upgrade. Per multivariable logistic regression (Table 3), reclassified variants were more likely to have been a net upgrade if they had been classified originally as likely pathogenic, belonged to a male participant, or had undergone initial testing earlier during the study period. Specifically, the chances that a reclassified variant represented a net upgrade decreased nonlinearly over time: following a sigmoid curve, that decrease in risk was initially minor (ie, approximately –8% at four years into the study) but accelerated over time (reaching –40% at 10 years, –70% at 15.5 years, and –90% at 18 years). Neither ancestry, type of mutation, nor BRCA1/2 status was associated with reclassification being a net upgrade. Table 3. Multivariable model: Associations with net upgrade among reclassified variants at risk* (n = 241) Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 * Variants observed to be at risk of net upgrade were those initially classified as likely pathogenic or variant of uncertain significance. † Because this model represents a secondary analysis, P values were unadjusted for multiple hypothesis testing. ‡ Covariates that did not improve the model’s fit were omitted from the final model. Table 3. Multivariable model: Associations with net upgrade among reclassified variants at risk* (n = 241) Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 Risk factor No. of reclassified variants No. of net upgrades (%) Odds ratio (95% confidence interval) P† Initial classification  Likely pathogenic 14 9 (64.3) 19.75 (5.38 to 72.48) <.001  Of uncertain significance 227 17 (7.5) 1.00 (reference) Gene  BRCA1/2 158 14 (8.9) —‡  Non-BRCA1/2 83 12 (14.5) — Type of mutation  Splice site 36 5 (13.9) —  All others 205 21 (10.2) — Laboratory  Lab A 218 21 (9.6) —  Other labs 23 5 (21.7) — Ancestry  African 26 1 (3.9) —  Ashkenazi 20 2 (10.0) —  Asian, Chinese 19 1 (5.3) —  Asian, non-Chinese 13 0 —  Hispanic 54 5 (9.3) —  Middle Eastern 18 1 (5.6) —  Native American 13 2 (15.4) —  Non-Hispanic European 78 14 (18.0) — Sex  Male 13 6 (46.2) 8.43 (2.06 to 34.53) .003  Female 228 20 (8.8) 1.00 (reference) Calendar year of initial testing per years since 1998, squared N/A N/A 0.995 (0.990 to 1.000) .05 * Variants observed to be at risk of net upgrade were those initially classified as likely pathogenic or variant of uncertain significance. † Because this model represents a secondary analysis, P values were unadjusted for multiple hypothesis testing. ‡ Covariates that did not improve the model’s fit were omitted from the final model. Incidentally, 17.1% of variants overall were obtained from persons who reported mixed ancestry. However, the proportion of mixed ancestry varied by group: It was absent by definition among participants with non-Hispanic European ancestry but common among persons with Ashkenazi (42.7% of whose variants were associated with mixed ancestry, chiefly non-Hispanic European), African (53.0% mixed ancestry, chiefly non-Hispanic European or Native American), or non-African Native American background (87.0% mixed ancestry, chiefly Hispanic or non-Hispanic European). Finally, 10.0% of Chinese, 12.4% of non-Chinese Asian, 15.6% of Middle Eastern, and 17.6% of Hispanic variants came from participants with mixed ancestry. Discussion This study of an ethnically diverse cohort of nonbenign variants followed for up to two decades reveals for the first time that, independent of covariates, the rate of variant reclassification varies by ancestry. Overall, current findings suggest a “catching up” in the reclassification of variants from minority ancestries after an excess in uncertain classifications (VUS) relative to non-Hispanic European tests. This pattern was likely facilitated by the availability of large population databases such as the National Heart, Lung, and Blood Institute Exome Sequencing Project and 1000 Genomes, followed by the more ethnically diverse Exome Aggregation Consortium (ExAC) database (4,12,13). Further, a “catching up” of reclassification would be consistent with a previous report that the frequency of VUS as a percentage of all variant classifications declined during the decades we studied (14), most steeply among African, Asian, Hispanic, and Middle Eastern ancestries. The current study also reports that the percentage of reclassifications that are net upgrades is low (less than half the 20% reported by an earlier study limited to BRCA1/2 variants) (15). Moreover, this percentage has declined steeply in recent years. Importantly for clinical care and genetic counseling, the latter finding argues against the view that VUS are commonly upgraded to pathogenic or likely pathogenic status, an assumption that may lead individuals who receive these results to undergo unnecessary procedures (ie, bilateral mastectomy) (16). We identify initial classification, sex, and calendar year of testing (but not ancestry, laboratory, or splice site mutation) (15) as risk factors for a reclassification being a net upgrade. Findings incidental to our primary hypothesis include two unanticipated associations with sex. Specifically, BRCA1/2 variants from male participants appear more likely to undergo reclassification. Also, regardless of gene, reclassified variants from male participants appear more likely to have undergone a net upgrade in pathogenicity. Neither of these associations with sex can be explained by any of the variant characteristics we studied. Possibly, these associations may be related to males’ greater reluctance to seek medical care generally (17). We speculate that, to present for cancer genetic risk assessment, males may tend to require a more compelling family history of cancer than females do. In turn, a more compelling family history may promote initiation and completion of the research necessary to issue a reclassification and may also be associated with genetic variants of greater pathogenicity. In any case, current incidental associations with sex should be regarded as preliminary until confirmed by separate studies. The current longitudinal analysis of time to reclassification suggests that, except for pathogenic variants, most nonbenign test results will ultimately be reclassified. For that reason, persons undergoing genetic testing should be encouraged to provide (and update) contact information to the provider ordering the test, so that they may be informed of reclassifications as they are issued. Current findings also indicate that the various classes of nonpathogenic variants (likely pathogenic, likely benign, VUS) are equally likely to undergo reclassification, while reclassifications of likely pathogenic variants are more likely than VUS reclassifications to be net upgrades. The latter finding supports the current clinical practice of treating likely pathogenic variants as similar to pathogenic variants (1). To our knowledge, only a limited description of time to variant reclassification has been published previously (3). That retrospective study (n = 107 women whose BRCA1/2 variants were exclusively VUS) did not provide median time to reclassification or rate per years of observation. In contrast, our prospective study has presented time to first reclassification by classification and modeled rate of reclassification within ancestry groups over time while controlling potential confounding factors. Moreover, our large sample was intended to be representative of contemporary clinical practice and thus accepted variants 1) from genes other than BRCA1/2, 2) with classifications not restricted to VUS, 3) from males as well as females, and 4) regardless of whether the participant carried a pathogenic BRCA1/2 variant. One methodological challenge inherent in research into reclassification of genetic variants is that, due to lack of specific regulations, internal proprietary data, and varying methodologies, commercial genetic laboratories do not follow a common protocol for issuing reclassifications. Some laboratories (ie, Lab A) have active, system-based approaches that generate reclassifications. Alternatively, laboratories may issue reclassifications only sporadically, for example, when new clinical or empirical evidence is brought forward by a health care provider or a laboratory director. Over time, laboratories may shift between sporadic and active reclassification programs without notice. Recognizing the complex heterogeneity in reclassification programs, we included all commercial laboratories used during the study period but took steps to prevent laboratory differences from confounding our investigation. Specifically, we took into account within-laboratory correlation, distinguished the most active laboratory from all others, and considered BRCA1/2 and non-BRCA1/2 variants separately. Another methodological challenge inherent in our study relates to the categorization of ancestry from self-report. To avoid oversimplifying ancestry, we recognized diversity within the “Asian” category, replacing it with categories of Chinese, Middle Eastern, and other Asian ancestry. On the other hand, clarity required us to identify systematically one ancestry per participant when several were reported. Mixed ancestry was reported by a minority of our participants, chiefly, as expected from studies of admixture, those with Ashkenazi, African, or Native American ancestry (18). In the future, studies of reclassification rates might benefit from analyzing ancestry using ethnic percentages or germline DNA-based categorization. Our study findings are specific to the era and genes that we studied. In the future, reference databases will become more ethnically diverse (4,6), routine genetic testing will include more genes, and new methodologies will become prevalent, such as polygenic risk scoring using single nucleotide polymorphisms, whole-exome and -genome sequencing, and secondary germline findings from tumor-based testing. As a result, the ancestry-based associations with reclassification that we have observed are likely to evolve. In conclusion, the current study investigates the role of ancestry in variant reclassification during two decades of clinical practice. Our findings demonstrate that independently of laboratory and initial classification, the rate at which a nonbenign cancer genetic variant is reclassified varies by ancestry in ways that depend on gene (BRCA1/2 vs others) and, when observation spans more than a few years, on the year when a genetic variant was classified. Funding The research reported in this publication was supported by the National Cancer Institute (NCI) of the National Institutes of Health (NIH) under award number P30CA33572 (Integrative Genomics and Biostatistics Cores). The City of Hope Clinical Cancer Genomics Community Research Network and the Hereditary Cancer Research Registry was supported in part by NCI NIH award number RC4CA153828 (PI: J. Weitzel). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Other sources of support include the 2015 STOP CANCER Research Career Development Award (PI: T. Slavin), the Oxnard Foundation (PI: T. Slavin), the American Cancer Society (PI: J. Weitzel), and the Avon Foundation (PI: J. Weitzel). Notes Affiliations of authors: Division of Clinical Cancer Genomics (TPS, LVT, IS, CR, BN, LK, MNS, KRB, KY, SS, DC, JH, SWG, JNW), Department of Information Sciences (CEB), and Integrative Genomics Core (ST), City of Hope, Duarte, CA; Cancer Genetics Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA (JOC). Present affiliations: Human Longevity Inc., San Diego, CA (CR); Dyson Center for Cancer Care, Poughkeepsie, NY (MNS). The funders had no role in the design of the study; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication. The authors thank the members of the the Clinical Cancer Genomics Community Research Network; research aides Rosa Mejia, Nancy Guerrero-Llamas, and Sophia Manjarrez; and all research participants. References 1 NCCN . NCCN clinical practice guidelines in oncology V.1.2018: Genetic/familial high-risk assessment: Breast and ovarian. NCCN Clinical Practice Guidelines. National Comprehensive Cancer Network, Inc; 2017 . https://www.nccn.org/professionals/physician_gls/default.aspx. Accessed February 20, 2018. 2 Richards S , Aziz N , Bale S , et al. . Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology . Genet Med. 2015 ; 17 5 : 405 – 424 . Google Scholar Crossref Search ADS PubMed 3 Murray ML , Cerrato F , Bennett RL , et al. . Follow-up of carriers of BRCA1 and BRCA2 variants of unknown significance: Variant reclassification and surgical decisions . Genet Med. 2011 ; 13 12 : 998 – 1005 . Google Scholar Crossref Search ADS PubMed 4 Lek M , Karczewski KJ , Minikel EV , et al. . Analysis of protein-coding genetic variation in 60,706 humans . Nature. 2016 ; 536 7616 : 285 – 291 . Google Scholar Crossref Search ADS PubMed 5 Manrai AK , Funke BH , Rehm HL , et al. . Genetic misdiagnoses and the potential for health disparities . N Engl J Med. 2016 ; 375 7 : 655 – 665 . Google Scholar Crossref Search ADS PubMed 6 Popejoy AB , Fullerton SM. Genomics is failing on diversity . Nature. 2016 ; 538 7624 : 161 – 164 . Google Scholar Crossref Search ADS PubMed 7 Ricker C , Culver JO , Lowstuter K , et al. . Increased yield of actionable mutations using multi-gene panels to assess hereditary cancer susceptibility in an ethnically diverse clinical cohort . Cancer Genet. 2016 ; 209 4 : 130 – 137 . Google Scholar Crossref Search ADS PubMed 8 Susswein LR , Marshall ML , Nusbaum R , et al. . Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing . Genet Med. 2016 ; 18 8 : 823 – 832 . Google Scholar Crossref Search ADS PubMed 9 Caswell-Jin JL , Gupta T , Hall E , et al. . Racial/ethnic differences in multiple-gene sequencing results for hereditary cancer risk . Genet Med. 2017; July 2017. Epub ahead of print. 10 Holm S. A simple sequentially rejective multiple test procedure . Scand J Stat. 1979 ; 6 2 : 65 – 70 . 11 Slavin T , Niell-Swiller M , Solomon I , et al. . Clinical application of multigene panels: Challenges of next-generation counseling and cancer risk management . Front Oncol. 2015 ; 5 . 12 National Heart L, and Blood Institute (NHLBI) . NHLBI Grand Opportunity Exome Sequencing Project (ESP). https://esp.gs.washington.edu/drupal/. Accessed February 20, 2018. 13 Pennisi E. 1000 Genomes Project gives new map of genetic diversity . Science. 2010 ; 330 6004 : 574 – 575 . Google Scholar Crossref Search ADS PubMed 14 Eggington JM , Bowles KR , Moyes K , et al. . A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes . Clin Genet. 2014 ; 86 3 : 229 – 237 . Google Scholar Crossref Search ADS PubMed 15 Easton DF , Deffenbaugh AM , Pruss D , et al. . A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes . Am J Hum Genet. 2007 ; 81 5 : 873 – 883 . Google Scholar Crossref Search ADS PubMed 16 Kurian AW , Li Y , Hamilton AS , et al. . Gaps in incorporating germline genetic testing into treatment decision-making for early-stage breast cancer . J Clin Oncol. 2017 ; 35 20 : 2232 – 2239 . Google Scholar Crossref Search ADS PubMed 17 Courtenay WH. Constructions of masculinity and their influence on men's well-being: A theory of gender and health . Soc Sci Med. 2000 ; 50 10 : 1385 – 1401 . Google Scholar Crossref Search ADS PubMed 18 Adhikari K , Chacon-Duque JC , Mendoza-Revilla J , et al. . The genetic diversity of the Americas . Annu Rev Genomics Hum Genet. 2017 ; 18 : 277 – 296 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

JNCI: Journal of the National Cancer InstituteOxford University Press

Published: Oct 1, 2018

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