J Genet Counsel (2015) 24:285–293 DOI 10.1007/s10897-014-9762-z
ORIGINAL RESEARCH
To Reflex or Not: Additional BRCA1/2 Testing in Ashkenazi Jewish Individuals Without Founder Mutations Nancie Petrucelli & Sarah Mange & Jennifer L. Fulbright & Lindsay Dohany & Dana Zakalik & Debra Duquette
Received: 20 December 2013 / Accepted: 19 August 2014 / Published online: 9 September 2014 # National Society of Genetic Counselors, Inc. 2014
Abstract This study determined the prevalence of nonAshkenazi Jewish BRCA1/2 mutations in the Ashkenazi Jewish population in the state of Michigan, current provider testing practices, and the use of mutation probability models in determining which Ashkenazi Jewish individuals should be offered further analysis following negative BRCA1/2 founder testing. Testing patterns, mutation probabilities, and testing results were assessed for 327 Ashkenazi Jewish individuals seen for BRCA1/2 counseling in the state of Michigan who underwent testing for the Ashkenazi Jewish founder mutations. Only one (0.6 %) Ashkenazi Jewish individual with sequencing after negative founder analysis was found to have a non-founder mutation; no rearrangements were identified. Testing patterns varied by clinic, with the proportion of Ashkenazi Jewish individuals undergoing additional sequencing ranging from 22.2 to 92.9 %. In Ashkenazi Jewish individuals with a pre-test BRCAPRO risk calculation, the mean risk was significantly higher in those with follow-up sequencing compared to those who did not pursue additional testing. The low prevalence of non-founder BRCA1/2 mutations in Ashkenazi Jewish individuals does not warrant automatically reflexing to full analysis after negative mutation testing. Increased use of mutation probability models may aid in determining which cases warrant additional testing.
N. Petrucelli (*) Cancer Genetic Counseling Service, Karmanos Cancer Institute, 4100 John R, HPO3GC, Detroit, MI 48201, USA e-mail: [email protected] S. Mange : D. Duquette Lifecourse Epidemiology and Genomics Division, Michigan Department of Community Health, Lansing, MI, USA J. L. Fulbright : L. Dohany : D. Zakalik Cancer Genetics Program, Beaumont Health System, Royal Oak, MI, USA
Keywords BRCA1/2 . Ashkenazi Jewish . Sequencing . Large rearrangement analysis
Introduction Individuals with suspected hereditary syndromes often undergo a diagnostic odyssey in their journey to discover a diagnosis. Although well-appreciated in reproductive and pediatric genetic settings, such diagnostic odysseys are arising more often in cancer genetics. With the increase in the number of clinically available genetic tests, individuals with a significant personal and/or family history of cancer can often undergo a series of cancer genetic tests in hopes of discovering a known mutation. The dilemma of whether to proceed with additional cancer genetic testing and when to cease the testing odyssey is complicated by multiple factors, including the ability to automatically reflex to additional testing, patient interest, insurance coverage, test cost, professional recommendations, likelihood of detecting a mutation, laboratory guidance, and institutional influences. Such a dilemma occurs when Ashkenazi Jewish (AJ) individuals with a personal and/or family history of breast and/or ovarian cancer test negative for BRCA1/2 founder mutations. It is well established that germline mutations within the BRCA1 and BRCA2 genes significantly increase the risk of various cancers such as male and female breast, ovarian, prostate, and pancreatic (Antoniou et al. 2003; Couch et al. 1997; Easton et al. 1997; Ford et al. 1998; Struewing et al. 1997). Since the discovery of the BRCA1/2 genes almost two decades ago, much research has been dedicated to the identification, prevalence, and penetrance of BRCA1/2 mutations in the AJ population. Three specific founder mutations account for greater than 90 % of mutations in AJ individuals: 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 (Kauff et al. 2002; Phelan et al. 2002). The prevalence of these
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three mutations is between 1.1 and 2.7 % in AJ populations unselected for personal or family cancer history (Metcalfe et al. 2010; Neuhausen et al. 1996; Roa et al. 1996; Struewing et al. 1997), and 10.2 % for AJ individuals with a family history of breast or ovarian cancer (Malone et al. 2006). Of those who do not carry one of the three founder mutations, approximately 0.6–4.0 % have been found to harbor a nonfounder BRCA1/2 mutation (Frank et al. 2002; Kauff et al. 2002; Phelan et al. 2002), and large genomic rearrangements in AJ individuals are reported to be rare (Palma et al. 2008; Stadler et al. 2010; Distelman-Menachem et al. 2009; Walsh et al. 2006). Since the three founder mutations comprise the majority of mutations in individuals of AJ ancestry, testing for these three mutations is more cost-effective and efficient than full sequencing, and is the preferred initial testing strategy. While recommendations emphasize genetic counseling and testing for founder mutations in the high risk AJ population based on a personal and/or family history of BRCA1/2-related cancers (United States Preventive Task Force [USPSTF] risk assessment, genetic counseling, and genetic testing for BRCArelated cancer in women: recommendation statement, 2013; National Comprehensive Cancer Network [NCCN] Clinical Practice Guidelines in Oncology: genetic/familial high-risk assessment: breast and ovarian, 2014), there is only one national guideline that addresses testing for non-founder mutations following negative founder testing. The NCCN guidelines indicate that testing for the three founder mutations should be performed first, and that sequencing should be considered if ancestry includes non-AJ relatives or if other criteria are met (NCCN Clinical Practice Guidelines in Oncology: genetic/familial high-risk assessment: breast and ovarian, 2014). There is no published literature on when to offer further testing after negative AJ founder analysis. For this reason, whether to proceed with costly sequencing with or without rearrangement analysis is not always clear. Many mutation probability models have been developed to calculate an individual’s risk of harboring a BRCA1/2 mutation including the BRCAPRO model, the Myriad II model, and the Tyrer-Cuzick model (Berry et al. 2002; Frank et al. 2002; Parmigiani et al. 1998). While the American Society of Clinical Oncology (ASCO) suggests that there is no numerical threshold for when to offer testing based on these models (American Society of Clinical Oncology policy statement update: genetic testing for cancer susceptibility 2003), the use of such models has been shown to help further discriminate which patients are more likely to have a BRCA1 or BRCA2 mutation, even among experienced providers (de la Hoya et al. 2003; Euhus et al. 2002) and thus, may be an effective way to determine which AJ individuals should be offered comprehensive BRCA1/2 sequencing and rearrangement analysis following negative testing for the three founder mutations.
Petrucelli et al.
In August 2011, we conducted an online survey of cancer genetic providers through the National Society of Genetic Counselors (NSGC) familial cancer special interest group (SIG) forum to determine provider practice following a negative result for the three founder mutations in AJ individuals with no known mutation in the family (unpublished data). This survey found that 56.6 % (94/166) of providers review the family history to determine if certain clinical criteria are met that would warrant further testing, while 10.8 % (18/166) perform a risk calculation using one or more mutation probability models for AJ patients without a BRCA1/2 founder mutation. Of the providers that review family history, 62.8 % (59/94) use NCCN criteria to determine if further testing is appropriate, while 2.1 % (2/94) use USPSTF. The remaining 33 providers either use institutional criteria, insurance criteria, or review on a case by case basis. More than one in five providers queried (22.9 %, 38/166) automatically reflex to sequencing with or without rearrangement analysis for all individuals of AJ ancestry with negative founder analysis. Given the lack of guidelines surrounding non-founder mutations in AJ individuals and an apparent discrepancy in practice by genetic providers nationally, we identified a need to investigate these issues in the State of Michigan using information on patients presenting for BRCA1/2 genetic counseling by board-certified/eligible genetic providers. The aims of this study were to 1) determine the prevalence of nonAJ mutations in the AJ population in the state of Michigan, 2) identify current provider testing practices in the State, and 3) assess the use of mutation probability models to aid in determining which AJ individuals should be offered sequencing and possible rearrangement analysis following negative testing for the three AJ founder mutations.
Methods Participants The Michigan Department of Community Health (MDCH) has developed a statewide surveillance network including 10 of the 11 clinical facilities providing BRCA1/2 counseling to Michigan patients by board-certified/eligible cancer genetic providers. These facilities are located in the state, with the exception of one which provides telephone counseling services. Participating facilities report all patients receiving BRCA1/2 counseling, including demographics, family and personal cancer history, and information derived during the counseling and testing process to MDCH. These data are consistent with public health surveillance and not research as defined by the code of federal regulations (CFR) 46.102 (D) and therefore were deemed exempt by the MDCH institutional review board (IRB).
Additional BRCA1/2 Testing in Ashkenazi Jews Without Founder Mutations
The database includes 5,960 individuals seen for BRCA1/2 counseling between October 1, 2007 and March 31, 2011. Any additional counseling visit information and testing outcomes for these patients were updated by the participating clinical facilities through September 30, 2013. All patients with AJ ancestry on at least one side of the family were included in the analysis. Because patients are sometimes referred for genetic counseling on the basis of prior genetic test results, those who had testing before counseling were omitted. We further excluded those individuals with a blood relative with a known BRCA1/2 mutation at the time of counseling since targeted testing would be performed in this situation and thus testing practices in these individuals would be different. Procedures Demographic and risk variables in our analyses included gender, race, family cancer history, personal cancer history, age of diagnosis, BRCAPRO and Myriad II mutation probabilities, BRCA1/2 test type ordered, and test results. Family cancer history was defined according to the 2005 USPSTF guidelines for counseling referral for AJ individuals (Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility; recommendation statement, 2005); those with a family history had a minimum of one first-degree relative or two second-degree relatives on the same side of the family with breast and/or ovarian cancer. Personal and family cancer histories and ages of diagnosis were based on self-report at the time of counseling, with possible verification through health system records. The definition of breast cancer included ductal carcinoma in situ (DCIS). If a provider generated a formal risk calculation for BRCA1/2 mutations using the Myriad II model and/or the BRCAPRO model, this calculation was rounded to the nearest whole number. The Myriad II model consists of two mutation prevalence tables, stratified by AJ ancestry and provides a combined mutation probability for both BRCA1 and BRCA2. BRCAPRO is a Bayesian probability model and is available as part of the CancerGene software. Both models use only first- and second-degree relatives. In some cases, mutation probability calculations with Myriad II and/or BRCAPRO were performed by the provider both prior to founder testing and following a negative founder test. Mutation probability calculations after an initial founder test can be performed using one of two methods to account for the negative test result. In one method, used for both the Myriad II and BRCAPRO models, AJ ancestry is discounted in an attempt to determine the likelihood that the individual is a carrier of a non-AJ founder mutation. An alternate method based on the assumption that founder mutations account for approximately 90 % of BRCA1/2 mutations in the AJ population involves inputting a negative BRCA1 and BRCA2 founder test result in the BRCAPRO model.
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BRCA1/2 testing and test results were continuously updated in the database until the time of analysis, including reclassifications of variants of unknown significance. All testing was performed by Myriad® Genetics, Inc. Subjects were grouped according to testing patterns, with either AJ founder testing alone, or founder testing and subsequent sequencing with or without large rearrangement analysis. Data Analysis Frequencies and percentages were calculated to describe testing patterns and test results for both the AJ patient population as a whole and at each clinical facility. A comparison of demographic and risk characteristics between those with founder testing only and those with additional testing was conducted using Chi-square testing for gender, race, and family history. Due to small sample sizes, the Fisher’s exact test was used to compare personal cancer history between the two testing groups. To account for unequal variances in both the first and second BRCAPRO calculations of those with and without additional testing after founder analysis, a Satterthwaite t-test was used to compare average risk calculation values. Pooled t-tests for equal variances were used to compare first and second Myriad II mutation probability calculation averages between the two testing groups. Using a Bonferroni correction for multiple statistical tests, findings were considered statistically significant at the level of 0.007. All analyses were conducted using the SAS 9.2 statistical package.
Results We identified 591 individuals of AJ ancestry who accessed BRCA1/2 counseling by a board-certified/eligible genetic provider from October 1, 2007 through March 31, 2011. Of these, 442 presented without previous BRCA1/2 testing and without a known familial BRCA1/2 mutation. Fifty-eight (13.1 %) of these patients did not pursue BRCA1/2 genetic testing after counseling. Of the remaining 384 patients, fifty-seven (14.8 %) had sequencing initially, while 327 (85.2 %) began the testing process with founder analysis. Patients who began testing with founder analysis were predominantly female (302, 92.4 %) and had a personal history of breast and/or ovarian cancer (188, 87.9 %; results not shown). None of AJ individuals were multi-racial, but it is unknown whether both their maternal and paternal ancestry was AJ. Thirty-two (9.8 %) were found to have one of the three AJ founder mutations, while one (0.3 %) had a variant of unknown significance after founder testing (Fig. 1). Of the 294 (89.9 %) without a founder mutation, 166 (56.5 %) proceeded to sequencing, with or without large
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Fig. 1 Testing patterns and results in Ashkenazi Jewish individuals without a known familial BRCA1/2 mutation
327 Ashkenazi patients who began testing with founder mutation testing
1 (0.3%) had a variant of unknown significance (VUS)
294 (89.9%) had no mutation
128 (43.5%) had no additional testing
166 (56.5%) had additional testing (sequencing with or without large rearrangement testinga)
163 (98.2%) had no mutation after sequencing
2 (1.2%) had a VUS after sequencing
25 (15.3%) had additional large rearrangement testing. None were found to have a deleterious mutation
1 had additional large rearrangement testing and had a VUS
32 (9.8%) had a deleterious mutation
1 (0.6%) had a deleterious mutation after sequencing
a
26 had large rearrangement testing
rearrangement analysis (Fig. 1). Compared to those who did not pursue further testing after negative founder analysis, those who went on for sequencing were more likely to be female and to have a family cancer history, defined as one first-degree relative or two second-degree relatives on the same side of the family with breast and/or ovarian cancer (Table 1). These patients were also significantly more likely to have been diagnosed with breast cancer at age 50 or younger, and less likely to be without a personal history of breast or ovarian cancer (Chi-square p-value 0.0013). Our primary finding was that only one (0.6 %) patient with sequencing after negative founder analysis was found to have a non-founder mutation (Fig. 1). This patient had a personal history of breast cancer diagnosed at age 56 and a family history of breast cancer in two paternal aunts and four paternal female first cousins. A paternal first cousin, diagnosed with breast cancer at age 54, was also later found to carry the known familial BRCA2 mutation. The patient’s maternal and paternal lineage is of AJ descent. One hundred sixty-three patients (98.2 %) with follow-up sequencing, including 25 with additional large rearrangement testing, were negative for non-founder mutations (Fig. 1). Just over half (173, 58.8 %) of patients negative for the AJ founder mutations had a BRCAPRO risk calculation prior to testing; the mean risk in those who had follow-up sequencing after founder testing was significantly higher than the mean risk in those who did not pursue follow-up sequencing (t-test p-value 0.001, Table 2). Only 20 (11.6 %) of the 173 patients with a pre-test mutation probability calculation had a second
BRCAPRO risk calculation post-founder analysis, and 6 (30.0 %) proceeded with sequencing. The grouped BRCAPRO risk calculation scores for the six with sequencing were not significantly higher than those who discontinued testing after negative founder analysis. Four of the 6 had BRCAPRO risk calculations under 10 %. There was no significant difference in the mean result of the Myriad risk calculation between those who did and did not pursue follow-up sequencing, either pre- or post- founder analysis. Only 27 (14.4 %) of the 188 with a Myriad pre-test risk calculation had a post-test risk calculation. Whether a formal post-test risk calculation was performed depended on the clinical facility; the majority of the ten reporting facilities with board-certified/eligible genetic providers did not record either BRCAPRO or Myriad risk calculations after a negative founder mutation test. Testing patterns among the reporting facilities were highly variable. At facilities with more than 25 AJ patients with negative founder analysis, the proportion going on for additional sequencing ranged from 22.2 to 92.9 % (data not shown).
Discussion Practice Implications In this study, we report a prevalence of 9.8 % for the three founder mutations in a sample of 327 AJ men and women presenting for genetic counseling. This population
Additional BRCA1/2 Testing in Ashkenazi Jews Without Founder Mutations
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Table 1 Comparison of Ashkenazi Jewish individuals with negative founder mutation testing who did and did not undergo additional BRCA1/2 analysis Demographics
Tests
Total
Founder mutation testing N=128
Founder mutation, comprehensive anda Large rearrangement N=166
113 (88.3) 15 (11.7)
158 (95.2) 8 (4.8)
Sex Female Male Race White Family historyb Yes No Personal cancer history Breast diagnosed ≤50 years of age Breast diagnosed >50 years of age Ovarian Both ovarian and breast at any age Neither ovarian or breast
Chi-square p-value
0.03
– 127
166
63 (50.8) 65 (49.2)
105 (63.3) 61 (36.8)
0.02
< 0.01c 11 (8.6) 41 (32.0) 6 (4.7) 0 70 (54.7)
39 (23.5) 57 (34.3) 8 (4.8) 3 (1.8) 59 (35.5)
a
Includes those with founder mutation analysis followed by sequencing only or both sequencing and large rearrangement testing
b
A first degree relative or two second degree relatives on the same side of the family with breast and/or ovarian cancer
c
Fisher’s exact test
predominantly had a personal history of breast and/or ovarian cancer, and the mutation prevalence is in general agreement with other reports involving selected breast cancer populations (John et al. 2007; Malone et al. 2006). John et al. found that among AJ women with breast cancer, 10.2 % carried a BRCA1 mutation and 1.1 % carried a BRCA2 mutation, while Malone et al. found a BRCA1 prevalence of 8.3 % among AJ individuals; the proportion with a mutation would be presumably higher had the study also estimated BRCA2 prevalence. The AJ population in Michigan is not known to be different from other AJ populations and therefore the mutation rate identified in this study is likely applicable to AJ populations outside the state of Michigan. In contrast, only 1 of 166 (0.6 %) individuals in our study had a BRCA1/2 non-founder mutation after full sequencing. This finding is lower than some previous reports (Frank et al. 2002; Kauff et al. 2002). In a series of 70 AJ kindreds seen by a clinical genetics service, Kauff et al. identified 3 (4.3 %) individuals with non-founder mutations, Frank et al. found 16 of 737 AJ individuals (2 %) who underwent BRCA1/2 sequencing had non-founder mutations. The lower prevalence in the current study may be the result of sample size and ascertainment differences. Similar to our study, Phelan et al. (Phelan et al. 2002) identified 1 (0.6 %) non-founder mutation in a group of 160 AJ families presenting for genetic evaluation and screened for BRCA1 mutations. The prevalence of a mutation may have been slightly higher had BRCA2 sequencing also been included as in the current study.
Regardless, our results and those of others in the literature indicate that the yield of sequencing in individuals of AJ ancestry is likely to be low. Large rearrangements were not identified in the BRCA1/2 genes (n=26) in our cohort of AJ individuals testing negative for founder and non-founder point mutations, which is consistent with previous reports (Palma et al. 2008; Stadler et al. 2010). These data are consistent with prior studies showing that founder mutations continue to account for the vast majority of all mutations in this population and suggest that routine screening for large rearrangements does not seem to be warranted in AJ individuals. Given this low prevalence of non-founder mutations in individuals of AJ ancestry, BRCA1/2 sequencing with or without rearrangement analysis for those who have tested negative for the three founder mutations invokes a significant cost for a low yield. The NCCN advises BRCA1/2 testing for individuals of AJ ancestry to include founder testing first with the consideration of sequencing if the ancestry also includes non-AJ relatives or if other BRCA1/2 testing criteria are met. Beyond this, the NCCN does not provide any further recommendations, leaving the decision of sequencing and rearrangement testing to the discretion of the provider ordering the test. Our online survey of cancer genetic providers from the NSGC Cancer SIG demonstrates that this ambiguity has resulted in some providers automatically reflexing to BRCA1/2 sequencing with or without rearrangement analysis after negative founder testing. Similarly, testing patterns among the
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Table 2 BRCA1/2 mutation probabilities for Ashkenazi Jewish individuals with negative founder mutation testing pre- and post-test Risk assessments
BRCAPRO assessment 1 N Mean (St. Dev) Median Range BRCAPRO assessment 2 N Mean (St. Dev) Median Range Myriad assessment 1 N Mean (St. Dev) Median Range Myriad assessment 2 N Mean (St. Dev) Median Range a
Tests
T-test
Founder mutation only N=128
Founder mutation, comprehensive anda Large rearrangement N=166
69 10.7 (15.3) 4.0 (0, 84)
104 20.0 (21.4) 12.0 (0, 96)
14 4.6 (6.9) 2.0 (1, 26)
6 20.2 (32.5) 6.0 (2, 85)
77 12.0 (8.8) 9.0 (3, 50)
111 13.3 (9.0) 12.0 (3, 64)
19 6.6 (4.2) 6.0 (3, 21)
8 9.8 (5.7) 9.0 (5, 22)
p-value
ORIGINAL RESEARCH
To Reflex or Not: Additional BRCA1/2 Testing in Ashkenazi Jewish Individuals Without Founder Mutations Nancie Petrucelli & Sarah Mange & Jennifer L. Fulbright & Lindsay Dohany & Dana Zakalik & Debra Duquette
Received: 20 December 2013 / Accepted: 19 August 2014 / Published online: 9 September 2014 # National Society of Genetic Counselors, Inc. 2014
Abstract This study determined the prevalence of nonAshkenazi Jewish BRCA1/2 mutations in the Ashkenazi Jewish population in the state of Michigan, current provider testing practices, and the use of mutation probability models in determining which Ashkenazi Jewish individuals should be offered further analysis following negative BRCA1/2 founder testing. Testing patterns, mutation probabilities, and testing results were assessed for 327 Ashkenazi Jewish individuals seen for BRCA1/2 counseling in the state of Michigan who underwent testing for the Ashkenazi Jewish founder mutations. Only one (0.6 %) Ashkenazi Jewish individual with sequencing after negative founder analysis was found to have a non-founder mutation; no rearrangements were identified. Testing patterns varied by clinic, with the proportion of Ashkenazi Jewish individuals undergoing additional sequencing ranging from 22.2 to 92.9 %. In Ashkenazi Jewish individuals with a pre-test BRCAPRO risk calculation, the mean risk was significantly higher in those with follow-up sequencing compared to those who did not pursue additional testing. The low prevalence of non-founder BRCA1/2 mutations in Ashkenazi Jewish individuals does not warrant automatically reflexing to full analysis after negative mutation testing. Increased use of mutation probability models may aid in determining which cases warrant additional testing.
N. Petrucelli (*) Cancer Genetic Counseling Service, Karmanos Cancer Institute, 4100 John R, HPO3GC, Detroit, MI 48201, USA e-mail: [email protected] S. Mange : D. Duquette Lifecourse Epidemiology and Genomics Division, Michigan Department of Community Health, Lansing, MI, USA J. L. Fulbright : L. Dohany : D. Zakalik Cancer Genetics Program, Beaumont Health System, Royal Oak, MI, USA
Keywords BRCA1/2 . Ashkenazi Jewish . Sequencing . Large rearrangement analysis
Introduction Individuals with suspected hereditary syndromes often undergo a diagnostic odyssey in their journey to discover a diagnosis. Although well-appreciated in reproductive and pediatric genetic settings, such diagnostic odysseys are arising more often in cancer genetics. With the increase in the number of clinically available genetic tests, individuals with a significant personal and/or family history of cancer can often undergo a series of cancer genetic tests in hopes of discovering a known mutation. The dilemma of whether to proceed with additional cancer genetic testing and when to cease the testing odyssey is complicated by multiple factors, including the ability to automatically reflex to additional testing, patient interest, insurance coverage, test cost, professional recommendations, likelihood of detecting a mutation, laboratory guidance, and institutional influences. Such a dilemma occurs when Ashkenazi Jewish (AJ) individuals with a personal and/or family history of breast and/or ovarian cancer test negative for BRCA1/2 founder mutations. It is well established that germline mutations within the BRCA1 and BRCA2 genes significantly increase the risk of various cancers such as male and female breast, ovarian, prostate, and pancreatic (Antoniou et al. 2003; Couch et al. 1997; Easton et al. 1997; Ford et al. 1998; Struewing et al. 1997). Since the discovery of the BRCA1/2 genes almost two decades ago, much research has been dedicated to the identification, prevalence, and penetrance of BRCA1/2 mutations in the AJ population. Three specific founder mutations account for greater than 90 % of mutations in AJ individuals: 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 (Kauff et al. 2002; Phelan et al. 2002). The prevalence of these
286
three mutations is between 1.1 and 2.7 % in AJ populations unselected for personal or family cancer history (Metcalfe et al. 2010; Neuhausen et al. 1996; Roa et al. 1996; Struewing et al. 1997), and 10.2 % for AJ individuals with a family history of breast or ovarian cancer (Malone et al. 2006). Of those who do not carry one of the three founder mutations, approximately 0.6–4.0 % have been found to harbor a nonfounder BRCA1/2 mutation (Frank et al. 2002; Kauff et al. 2002; Phelan et al. 2002), and large genomic rearrangements in AJ individuals are reported to be rare (Palma et al. 2008; Stadler et al. 2010; Distelman-Menachem et al. 2009; Walsh et al. 2006). Since the three founder mutations comprise the majority of mutations in individuals of AJ ancestry, testing for these three mutations is more cost-effective and efficient than full sequencing, and is the preferred initial testing strategy. While recommendations emphasize genetic counseling and testing for founder mutations in the high risk AJ population based on a personal and/or family history of BRCA1/2-related cancers (United States Preventive Task Force [USPSTF] risk assessment, genetic counseling, and genetic testing for BRCArelated cancer in women: recommendation statement, 2013; National Comprehensive Cancer Network [NCCN] Clinical Practice Guidelines in Oncology: genetic/familial high-risk assessment: breast and ovarian, 2014), there is only one national guideline that addresses testing for non-founder mutations following negative founder testing. The NCCN guidelines indicate that testing for the three founder mutations should be performed first, and that sequencing should be considered if ancestry includes non-AJ relatives or if other criteria are met (NCCN Clinical Practice Guidelines in Oncology: genetic/familial high-risk assessment: breast and ovarian, 2014). There is no published literature on when to offer further testing after negative AJ founder analysis. For this reason, whether to proceed with costly sequencing with or without rearrangement analysis is not always clear. Many mutation probability models have been developed to calculate an individual’s risk of harboring a BRCA1/2 mutation including the BRCAPRO model, the Myriad II model, and the Tyrer-Cuzick model (Berry et al. 2002; Frank et al. 2002; Parmigiani et al. 1998). While the American Society of Clinical Oncology (ASCO) suggests that there is no numerical threshold for when to offer testing based on these models (American Society of Clinical Oncology policy statement update: genetic testing for cancer susceptibility 2003), the use of such models has been shown to help further discriminate which patients are more likely to have a BRCA1 or BRCA2 mutation, even among experienced providers (de la Hoya et al. 2003; Euhus et al. 2002) and thus, may be an effective way to determine which AJ individuals should be offered comprehensive BRCA1/2 sequencing and rearrangement analysis following negative testing for the three founder mutations.
Petrucelli et al.
In August 2011, we conducted an online survey of cancer genetic providers through the National Society of Genetic Counselors (NSGC) familial cancer special interest group (SIG) forum to determine provider practice following a negative result for the three founder mutations in AJ individuals with no known mutation in the family (unpublished data). This survey found that 56.6 % (94/166) of providers review the family history to determine if certain clinical criteria are met that would warrant further testing, while 10.8 % (18/166) perform a risk calculation using one or more mutation probability models for AJ patients without a BRCA1/2 founder mutation. Of the providers that review family history, 62.8 % (59/94) use NCCN criteria to determine if further testing is appropriate, while 2.1 % (2/94) use USPSTF. The remaining 33 providers either use institutional criteria, insurance criteria, or review on a case by case basis. More than one in five providers queried (22.9 %, 38/166) automatically reflex to sequencing with or without rearrangement analysis for all individuals of AJ ancestry with negative founder analysis. Given the lack of guidelines surrounding non-founder mutations in AJ individuals and an apparent discrepancy in practice by genetic providers nationally, we identified a need to investigate these issues in the State of Michigan using information on patients presenting for BRCA1/2 genetic counseling by board-certified/eligible genetic providers. The aims of this study were to 1) determine the prevalence of nonAJ mutations in the AJ population in the state of Michigan, 2) identify current provider testing practices in the State, and 3) assess the use of mutation probability models to aid in determining which AJ individuals should be offered sequencing and possible rearrangement analysis following negative testing for the three AJ founder mutations.
Methods Participants The Michigan Department of Community Health (MDCH) has developed a statewide surveillance network including 10 of the 11 clinical facilities providing BRCA1/2 counseling to Michigan patients by board-certified/eligible cancer genetic providers. These facilities are located in the state, with the exception of one which provides telephone counseling services. Participating facilities report all patients receiving BRCA1/2 counseling, including demographics, family and personal cancer history, and information derived during the counseling and testing process to MDCH. These data are consistent with public health surveillance and not research as defined by the code of federal regulations (CFR) 46.102 (D) and therefore were deemed exempt by the MDCH institutional review board (IRB).
Additional BRCA1/2 Testing in Ashkenazi Jews Without Founder Mutations
The database includes 5,960 individuals seen for BRCA1/2 counseling between October 1, 2007 and March 31, 2011. Any additional counseling visit information and testing outcomes for these patients were updated by the participating clinical facilities through September 30, 2013. All patients with AJ ancestry on at least one side of the family were included in the analysis. Because patients are sometimes referred for genetic counseling on the basis of prior genetic test results, those who had testing before counseling were omitted. We further excluded those individuals with a blood relative with a known BRCA1/2 mutation at the time of counseling since targeted testing would be performed in this situation and thus testing practices in these individuals would be different. Procedures Demographic and risk variables in our analyses included gender, race, family cancer history, personal cancer history, age of diagnosis, BRCAPRO and Myriad II mutation probabilities, BRCA1/2 test type ordered, and test results. Family cancer history was defined according to the 2005 USPSTF guidelines for counseling referral for AJ individuals (Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility; recommendation statement, 2005); those with a family history had a minimum of one first-degree relative or two second-degree relatives on the same side of the family with breast and/or ovarian cancer. Personal and family cancer histories and ages of diagnosis were based on self-report at the time of counseling, with possible verification through health system records. The definition of breast cancer included ductal carcinoma in situ (DCIS). If a provider generated a formal risk calculation for BRCA1/2 mutations using the Myriad II model and/or the BRCAPRO model, this calculation was rounded to the nearest whole number. The Myriad II model consists of two mutation prevalence tables, stratified by AJ ancestry and provides a combined mutation probability for both BRCA1 and BRCA2. BRCAPRO is a Bayesian probability model and is available as part of the CancerGene software. Both models use only first- and second-degree relatives. In some cases, mutation probability calculations with Myriad II and/or BRCAPRO were performed by the provider both prior to founder testing and following a negative founder test. Mutation probability calculations after an initial founder test can be performed using one of two methods to account for the negative test result. In one method, used for both the Myriad II and BRCAPRO models, AJ ancestry is discounted in an attempt to determine the likelihood that the individual is a carrier of a non-AJ founder mutation. An alternate method based on the assumption that founder mutations account for approximately 90 % of BRCA1/2 mutations in the AJ population involves inputting a negative BRCA1 and BRCA2 founder test result in the BRCAPRO model.
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BRCA1/2 testing and test results were continuously updated in the database until the time of analysis, including reclassifications of variants of unknown significance. All testing was performed by Myriad® Genetics, Inc. Subjects were grouped according to testing patterns, with either AJ founder testing alone, or founder testing and subsequent sequencing with or without large rearrangement analysis. Data Analysis Frequencies and percentages were calculated to describe testing patterns and test results for both the AJ patient population as a whole and at each clinical facility. A comparison of demographic and risk characteristics between those with founder testing only and those with additional testing was conducted using Chi-square testing for gender, race, and family history. Due to small sample sizes, the Fisher’s exact test was used to compare personal cancer history between the two testing groups. To account for unequal variances in both the first and second BRCAPRO calculations of those with and without additional testing after founder analysis, a Satterthwaite t-test was used to compare average risk calculation values. Pooled t-tests for equal variances were used to compare first and second Myriad II mutation probability calculation averages between the two testing groups. Using a Bonferroni correction for multiple statistical tests, findings were considered statistically significant at the level of 0.007. All analyses were conducted using the SAS 9.2 statistical package.
Results We identified 591 individuals of AJ ancestry who accessed BRCA1/2 counseling by a board-certified/eligible genetic provider from October 1, 2007 through March 31, 2011. Of these, 442 presented without previous BRCA1/2 testing and without a known familial BRCA1/2 mutation. Fifty-eight (13.1 %) of these patients did not pursue BRCA1/2 genetic testing after counseling. Of the remaining 384 patients, fifty-seven (14.8 %) had sequencing initially, while 327 (85.2 %) began the testing process with founder analysis. Patients who began testing with founder analysis were predominantly female (302, 92.4 %) and had a personal history of breast and/or ovarian cancer (188, 87.9 %; results not shown). None of AJ individuals were multi-racial, but it is unknown whether both their maternal and paternal ancestry was AJ. Thirty-two (9.8 %) were found to have one of the three AJ founder mutations, while one (0.3 %) had a variant of unknown significance after founder testing (Fig. 1). Of the 294 (89.9 %) without a founder mutation, 166 (56.5 %) proceeded to sequencing, with or without large
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Fig. 1 Testing patterns and results in Ashkenazi Jewish individuals without a known familial BRCA1/2 mutation
327 Ashkenazi patients who began testing with founder mutation testing
1 (0.3%) had a variant of unknown significance (VUS)
294 (89.9%) had no mutation
128 (43.5%) had no additional testing
166 (56.5%) had additional testing (sequencing with or without large rearrangement testinga)
163 (98.2%) had no mutation after sequencing
2 (1.2%) had a VUS after sequencing
25 (15.3%) had additional large rearrangement testing. None were found to have a deleterious mutation
1 had additional large rearrangement testing and had a VUS
32 (9.8%) had a deleterious mutation
1 (0.6%) had a deleterious mutation after sequencing
a
26 had large rearrangement testing
rearrangement analysis (Fig. 1). Compared to those who did not pursue further testing after negative founder analysis, those who went on for sequencing were more likely to be female and to have a family cancer history, defined as one first-degree relative or two second-degree relatives on the same side of the family with breast and/or ovarian cancer (Table 1). These patients were also significantly more likely to have been diagnosed with breast cancer at age 50 or younger, and less likely to be without a personal history of breast or ovarian cancer (Chi-square p-value 0.0013). Our primary finding was that only one (0.6 %) patient with sequencing after negative founder analysis was found to have a non-founder mutation (Fig. 1). This patient had a personal history of breast cancer diagnosed at age 56 and a family history of breast cancer in two paternal aunts and four paternal female first cousins. A paternal first cousin, diagnosed with breast cancer at age 54, was also later found to carry the known familial BRCA2 mutation. The patient’s maternal and paternal lineage is of AJ descent. One hundred sixty-three patients (98.2 %) with follow-up sequencing, including 25 with additional large rearrangement testing, were negative for non-founder mutations (Fig. 1). Just over half (173, 58.8 %) of patients negative for the AJ founder mutations had a BRCAPRO risk calculation prior to testing; the mean risk in those who had follow-up sequencing after founder testing was significantly higher than the mean risk in those who did not pursue follow-up sequencing (t-test p-value 0.001, Table 2). Only 20 (11.6 %) of the 173 patients with a pre-test mutation probability calculation had a second
BRCAPRO risk calculation post-founder analysis, and 6 (30.0 %) proceeded with sequencing. The grouped BRCAPRO risk calculation scores for the six with sequencing were not significantly higher than those who discontinued testing after negative founder analysis. Four of the 6 had BRCAPRO risk calculations under 10 %. There was no significant difference in the mean result of the Myriad risk calculation between those who did and did not pursue follow-up sequencing, either pre- or post- founder analysis. Only 27 (14.4 %) of the 188 with a Myriad pre-test risk calculation had a post-test risk calculation. Whether a formal post-test risk calculation was performed depended on the clinical facility; the majority of the ten reporting facilities with board-certified/eligible genetic providers did not record either BRCAPRO or Myriad risk calculations after a negative founder mutation test. Testing patterns among the reporting facilities were highly variable. At facilities with more than 25 AJ patients with negative founder analysis, the proportion going on for additional sequencing ranged from 22.2 to 92.9 % (data not shown).
Discussion Practice Implications In this study, we report a prevalence of 9.8 % for the three founder mutations in a sample of 327 AJ men and women presenting for genetic counseling. This population
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Table 1 Comparison of Ashkenazi Jewish individuals with negative founder mutation testing who did and did not undergo additional BRCA1/2 analysis Demographics
Tests
Total
Founder mutation testing N=128
Founder mutation, comprehensive anda Large rearrangement N=166
113 (88.3) 15 (11.7)
158 (95.2) 8 (4.8)
Sex Female Male Race White Family historyb Yes No Personal cancer history Breast diagnosed ≤50 years of age Breast diagnosed >50 years of age Ovarian Both ovarian and breast at any age Neither ovarian or breast
Chi-square p-value
0.03
– 127
166
63 (50.8) 65 (49.2)
105 (63.3) 61 (36.8)
0.02
< 0.01c 11 (8.6) 41 (32.0) 6 (4.7) 0 70 (54.7)
39 (23.5) 57 (34.3) 8 (4.8) 3 (1.8) 59 (35.5)
a
Includes those with founder mutation analysis followed by sequencing only or both sequencing and large rearrangement testing
b
A first degree relative or two second degree relatives on the same side of the family with breast and/or ovarian cancer
c
Fisher’s exact test
predominantly had a personal history of breast and/or ovarian cancer, and the mutation prevalence is in general agreement with other reports involving selected breast cancer populations (John et al. 2007; Malone et al. 2006). John et al. found that among AJ women with breast cancer, 10.2 % carried a BRCA1 mutation and 1.1 % carried a BRCA2 mutation, while Malone et al. found a BRCA1 prevalence of 8.3 % among AJ individuals; the proportion with a mutation would be presumably higher had the study also estimated BRCA2 prevalence. The AJ population in Michigan is not known to be different from other AJ populations and therefore the mutation rate identified in this study is likely applicable to AJ populations outside the state of Michigan. In contrast, only 1 of 166 (0.6 %) individuals in our study had a BRCA1/2 non-founder mutation after full sequencing. This finding is lower than some previous reports (Frank et al. 2002; Kauff et al. 2002). In a series of 70 AJ kindreds seen by a clinical genetics service, Kauff et al. identified 3 (4.3 %) individuals with non-founder mutations, Frank et al. found 16 of 737 AJ individuals (2 %) who underwent BRCA1/2 sequencing had non-founder mutations. The lower prevalence in the current study may be the result of sample size and ascertainment differences. Similar to our study, Phelan et al. (Phelan et al. 2002) identified 1 (0.6 %) non-founder mutation in a group of 160 AJ families presenting for genetic evaluation and screened for BRCA1 mutations. The prevalence of a mutation may have been slightly higher had BRCA2 sequencing also been included as in the current study.
Regardless, our results and those of others in the literature indicate that the yield of sequencing in individuals of AJ ancestry is likely to be low. Large rearrangements were not identified in the BRCA1/2 genes (n=26) in our cohort of AJ individuals testing negative for founder and non-founder point mutations, which is consistent with previous reports (Palma et al. 2008; Stadler et al. 2010). These data are consistent with prior studies showing that founder mutations continue to account for the vast majority of all mutations in this population and suggest that routine screening for large rearrangements does not seem to be warranted in AJ individuals. Given this low prevalence of non-founder mutations in individuals of AJ ancestry, BRCA1/2 sequencing with or without rearrangement analysis for those who have tested negative for the three founder mutations invokes a significant cost for a low yield. The NCCN advises BRCA1/2 testing for individuals of AJ ancestry to include founder testing first with the consideration of sequencing if the ancestry also includes non-AJ relatives or if other BRCA1/2 testing criteria are met. Beyond this, the NCCN does not provide any further recommendations, leaving the decision of sequencing and rearrangement testing to the discretion of the provider ordering the test. Our online survey of cancer genetic providers from the NSGC Cancer SIG demonstrates that this ambiguity has resulted in some providers automatically reflexing to BRCA1/2 sequencing with or without rearrangement analysis after negative founder testing. Similarly, testing patterns among the
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Table 2 BRCA1/2 mutation probabilities for Ashkenazi Jewish individuals with negative founder mutation testing pre- and post-test Risk assessments
BRCAPRO assessment 1 N Mean (St. Dev) Median Range BRCAPRO assessment 2 N Mean (St. Dev) Median Range Myriad assessment 1 N Mean (St. Dev) Median Range Myriad assessment 2 N Mean (St. Dev) Median Range a
Tests
T-test
Founder mutation only N=128
Founder mutation, comprehensive anda Large rearrangement N=166
69 10.7 (15.3) 4.0 (0, 84)
104 20.0 (21.4) 12.0 (0, 96)
14 4.6 (6.9) 2.0 (1, 26)
6 20.2 (32.5) 6.0 (2, 85)
77 12.0 (8.8) 9.0 (3, 50)
111 13.3 (9.0) 12.0 (3, 64)
19 6.6 (4.2) 6.0 (3, 21)
8 9.8 (5.7) 9.0 (5, 22)
p-value
