Nicolette M. Chun

Performance of BRCA1/2 Mutation Prediction Models in Asian Americans

PURPOSE There are established differences in breast cancer epidemiology between Asian and white individuals, but little is known about hereditary breast cancer in Asian populations. Although increasing numbers of Asian individuals are clinically tested for BRCA1/2 mutations, it is not known whether computer models that predict mutations work accurately in Asian individuals. We compared the performance in Asian and white individuals of two widely used BRCA1/2 mutation prediction models, BRCAPRO and Myriad II. PATIENTS AND METHODS We evaluated BRCAPRO and Myriad II in 200 Asian individuals and a matched control group of 200 white individuals who were tested for BRCA1/2 mutations at four cancer genetics clinics, by comparing numbers of observed versus predicted mutation carriers and by evaluating area under the receiver operating characteristic curve (AUC) for each model. RESULTS BRCAPRO and Myriad II accurately predicted the number of white BRCA1/2 mutation carriers (25 observed v 24 predicted by BRCAPRO; 25 predicted by Myriad II, P > or = .69), but underpredicted Asian carriers by two-fold (49 observed v 25 predicted by BRCAPRO; 26 predicted by Myriad II; P < or = 3 x 10(-7)). For BRCAPRO, this racial difference reflects substantial underprediction of Asian BRCA2 mutation carriers (26 observed v 4 predicted; P = 1 x 10(-30)); for Myriad II, separate mutation predictions were not available. For both models, AUCs were nonsignificantly lower in Asian than white individuals, suggesting less accurate discrimination between Asian carriers and noncarriers. CONCLUSION Both BRCAPRO and Myriad II underestimated the proportion of BRCA1/2 mutation carriers, and discriminated carriers from noncarriers less well, in Asian compared with white individuals.

The MLH1 c.-27C>A and c.85G>T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype.

Germline mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2, and deletions affecting the EPCAM gene adjacent to MSH2, underlie Lynch syndrome by predisposing to early-onset colorectal, endometrial and other cancers. An alternative but rare cause of Lynch syndrome is constitutional epimutation of MLH1, whereby promoter methylation and transcriptional silencing of one allele occurs throughout normal tissues. A dominantly transmitted constitutional MLH1 epimutation has been linked to an MLH1 haplotype bearing two single-nucleotide variants, NM_000249.2: c.−27C>A and c.85G>T, in a Caucasian family with Lynch syndrome from Western Australia. Subsequently, a second seemingly unrelated Caucasian Australian case with the same MLH1 haplotype and concomitant epimutation was reported. We now describe three additional, ostensibly unrelated, cancer-affected families of European heritage with this MLH1 haplotype in association with constitutional epimutation, bringing the number of index cases reported to five. Array-based genotyping in four of these families revealed shared haplotypes between individual families that extended across ≤2.6–≤6.4 megabase regions of chromosome 3p, indicating common ancestry. A minimal ≤2.6 megabase founder haplotype common to all four families was identified, which encompassed MLH1 and additional flanking genes and segregated with the MLH1 epimutation in each family. Our findings indicate that the MLH1 c.−27C>A and c.85G>T variants are borne on a European ancestral haplotype and provide conclusive evidence for its pathogenicity via a mechanism of epigenetic silencing of MLH1 within normal tissues. Additional descendants bearing this founder haplotype may exist who are also at high risk of developing Lynch syndrome-related cancers.

Ductal Lavage of Fluid-Yielding and Non–Fluid-Yielding Ducts in BRCA1 and BRCA2 Mutation Carriers and Other Women at High Inherited Breast Cancer Risk

Objective: Nipple fluid production and atypical breast duct cells in women at high risk of breast cancer have been associated with further increased risk. Most publications on ductal lavage for cell collection report cannulating fluid-yielding ducts only. We report lavage of fluid-yielding and non–fluid-yielding ducts in women at high inherited breast cancer risk. Methods: A pilot breast cancer screening study including ductal lavage was conducted in 75 women at high inherited risk, 56 (74.7%) of whom had BRCA1/2 mutations. Ductal lavage was attempted in any duct identifiable with a catheter. Results: Ducts were successfully catheterized in 60 of 75 patients (80%). Successfully catheterized patients were younger (median age 41 versus 53 years, P = 0.0003) and more often premenopausal (51.7% versus 20%, P = 0.041). Thirty-one successfully catheterized patients [51.6%, 95% confidence interval (39.4-63.9%)] had non–fluid-yielding ducts only. Seventeen patients [28.3% (18.5-40.9%)] had atypical cells. Twelve of seventeen [70.6% (46.8-87.2%)] samples with atypia were from non–fluid-yielding ducts. Patients with non–fluid-yielding ducts (versus fluid-yielding ducts) were more likely to have had prior cancer (48.4% versus 17.2%, P = 0.014) or chemotherapy (45.2% versus 17.2%, P = 0.027); this was also true in patients with atypia from non–fluid-yielding ducts. Conclusion: Successfully lavaged women were younger and more often premenopausal. Atypical cells can be found in non–fluid-yielding ducts in patients at high inherited breast cancer risk. Non–fluid-yielding ducts, and atypia from non–fluid-yielding ducts, are more common in patients with prior cancer and chemotherapy. Larger studies are needed to identify risk factors and prognostic significance associated with atypia and non–fluid-yielding ducts in high-risk populations, and define their role as biomarkers.

Identification of an intronic single nucleotide polymorphism leading to allele dropout during validation of a CDH1 sequencing assay: implications for designing polymerase chain reaction-based assays

Purpose: The CDH1 gene encodes the cell adhesion protein E-cadherin, and CDH1 germline mutations are associated with hereditary diffuse gastric cancer. Identification of individuals at high risk of developing diffuse gastric cancer affords the opportunity for endoscopic screening or elective prophylactic gastrectomy. We set out to develop a CDH1 sequencing assay for clinical use.Methods: All exons of the CDH1 gene were amplified and sequenced with published and modified primers.Results: While validating the assay, we encountered a case in which a single nucleotide polymorphism located in intron 15 led to allele dropout and therefore to a false-negative result. The polymorphism leading to allele dropout was located within a primer-binding sequence, five bases away from the 3′ end of the primer. A frameshift mutation in exon 15 was detected by an alternative primer that binds away from the polymorphic site. A search of the University of California Santa Cruz single nucleotide polymorphism database revealed other polymorphisms located within primer-binding sites. A total of 12 primers in nine primer sets were modified to minimize allele dropout risk.Conclusion: The approach of designing primers to avoid known single nucleotide polymorphisms can be generalized to the design of any polymerase chain reaction-based assay and should be employed whenever possible.

Parent decision-making around the genetic testing of children for germline TP53 mutations

Li‐Fraumeni syndrome is a rare genetic cancer predisposition syndrome caused by germline TP53 mutations. Up to 20% of mutation carriers develop cancer during childhood. The benefits of TP53 mutation testing of children are a matter of debate and knowledge of parent decision‐making around such testing is limited. The current study examined how parents make decisions regarding TP53 testing for their children.

Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation

Our patient was a 52-year-old man who was diagnosed with signet ring cell gastric adenocarcinoma. An extensive family history of gastric cancer raised suspicion for hereditary diffuse gastric cancer. Sequencing of the patients CDH1 gene revealed a novel point mutation in a strictly conserved splice site within intron 6, c.833-2 A > G. This mutation was predicted to result in loss of function due to defective RNA splicing. To characterize the pathogenic mechanism of this mutation, we amplified the patients CDH1 gene products by reverse transcriptase polymerase chain reaction. Primers flanking the region of the mutation detected 3 distinct transcripts. In addition to the wild-type product, a larger product consistent with activation of a cryptic splice site within intron 6 and a smaller product shown to result from exon 7 skipping were detected. In summary, we have identified a novel CDH1 mutation in a large hereditary diffuse gastric cancer kindred and identified its pathogenic mechanism.

Identification of a novel p53 in-frame deletion in a Li–Fraumeni-like family

We describe a 2‐year‐old female with a completely resected cerebral pilocytic astrocytoma who subsequently developed B‐progenitor acute lymphoblastic leukemia (ALL). Her father and paternal uncle were previously diagnosed with glioblastoma multiforme. Sequence analysis of the patients p53 gene revealed a novel germline three base‐pair deletion (339_341delCTT) in exon 4, resulting in removal of an evolutionarily conserved phenylalanine amino acid residue at codon 113. The same mutation was found in the patients two clinically unaffected siblings. The in‐frame deletion we describe has not previously been reported and adds to our understanding of the biologic effects of p53 gene mutation in Li–Fraumeni syndrome (LFS). Pediatr Blood Cancer 2008;50:914–916.

Racial/ethnic differences in multiple-gene sequencing results for hereditary cancer risk

PurposeWe examined racial/ethnic differences in the usage and results of germ-line multiple-gene sequencing (MGS) panels to evaluate hereditary cancer risk.MethodsWe collected genetic testing results and clinical information from 1,483 patients who underwent MGS at Stanford University between 1 January 2013 and 31 December 2015.ResultsAsians and Hispanics presented for MGS at younger ages than whites (48 and 47 vs. 55; P = 5E-16 and 5E-14). Across all panels, the rate of pathogenic variants (15%) did not differ significantly between racial groups. Rates by gene did differ: in particular, a higher percentage of whites than nonwhites carried pathogenic CHEK2 variants (3.8% vs. 1.0%; P = 0.002). The rate of a variant of uncertain significance (VUS) result was higher in nonwhites than whites (36% vs. 27%; P = 2E-4). The probability of a VUS increased with increasing number of genes tested; this effect was more pronounced for nonwhites than for whites (1.1% absolute difference in VUS rates testing BRCA1/2 vs. 8% testing 13 genes vs. 14% testing 28 genes), worsening the disparity.ConclusionIn this diverse cohort undergoing MGS testing, pathogenic variant rates were similar between racial/ethnic groups. By contrast, VUS results were more frequent among nonwhites, with potential significance for the impact of MGS testing by race/ethnicity.

Patient communication of cancer genetic test results in a diverse population

Research on the communication of genetic test results has focused predominately on non-Hispanic White (NHW) mutation-positive families with high-risk hereditary cancer conditions. Little is known about this process for racially and ethnically diverse individuals or for those with mutations in moderate risk genes. The communication behaviors of study participants who carry a gene mutation were analyzed 3 months after disclosure of genetic test results. Participants were queried about communication of their results, as part of a prospective study of multi-gene panel genetic testing. The responses of particpants who tested positive were analyzed by race/ethnicity and by level of cancer risk (high vs. moderate). Of the 216 mutation-positive study participants, 136 (63%) responded. Self-reported race/ethnicity was 46% NHW, 41% Hispanic, 10% Asian, and 2% Black. The majority (99.0%, n = 135) had shared their results with someone and 96% had told a family member (n = 130). Hispanic respondents were less likely to have told a healthcare provider about their results than NHW (29% vs. 68%, p < .0001). Asian respondents were less likely than NHW to encourage family members to undergo testing (OR = 0.1, p = .03); but Asian family members were more likely to undergo testing (OR = 8.0, p = .03). There were no differences in communication between those with a mutation in a high- or moderate-risk gene. Three months post genetic testing, communication of results was very high; 30% reported a family member underwent genetic testing. Further studies are needed to better understand the communication process in individuals from diverse racial/ethnic backgrounds.

Ductal lavage of non-fluid yielding ducts in BRCA1 and BRCA2 mutation carriers and other women at high genetic risk for breast cancer

9535 Background: Atypical breast duct cells in patients at increased risk of breast cancer have been predictive of subsequent breast cancer. It has been reported that women whose breasts yield no fluid on nipple suction aspiration have lower risk for breast cancer than women whose breasts yield fluid; publications on ductal lavage (DL) have reported attempt of DL only on patients with fluid-yielding ducts (FY) on initial suction aspiration. We report preliminary results of a study of DL including evaluation of non-fluid yielding ducts (NFY) in women at increased genetic risk of breast cancer. METHODS A pilot study of screening with breast MRI, mammogram, and ductal lavage was conducted in 41 women at increased genetic risk of breast cancer; 26 (63%) had BRCA mutations, and 15 (37%) were included because family history conveyed greater than 10% breast cancer risk at 10 years via the Claus model. Whether or not fluid was obtained on suction aspiration, ducts which could be identified with a dilator underwent attempted lavage. Cytologic diagnostic categories were benign, atypical, or malignant. Fishers exact test, 2 tailed, was used for statistical analysis. RESULTS DL was successful in 34 of 41 patients (82.9%); 17 (50%) had NFY. Ten patients (29.4%) had atypical cells; none had malignant cells. Seven of 17 patients (41.2%, 95% confidence interval 18.4-67.1%) with NFY had atypical cells. Patients with atypical cells from NFY were more likely than those with atypical cells from FY to have a BRCA mutation (57.1 versus 0%, p 0.20). Patients with NFY were more likely than those with FY to have had breast or ovarian cancer (58.8% versus 17.6%, p 0.03), and to have received chemotherapy (47.1% versus 17.6%, p 0.14). CONCLUSIONS Atypical cells can be found in NFY patients at high risk for breast cancer, with trends toward higher incidence of atypical cells from NFY in BRCA mutation carriers, and of NFY in cancer survivors. Larger studies are needed to confirm this finding, and to identify risk factors and outcomes associated with atypical cells and NFY. No significant financial relationships to disclose.