Non-founder Mutations Research Articles

Abstract PS8-18: Describing the cancer spectrum in families with CHEK2 pathogenic and likely pathogenic variants by mutation type

Abstract Background: Deleterious CHEK2 variants have well-defined risks for breast and colorectal cancers. Some studies have also suggested association with a variety of other cancers, including leukemia, kidney, prostate, thyroid and gastric cancers. However, most CHEK2 studies are based on European founder mutations, and there is a lack of data on non-founder mutations. Here, we describe the cancer family histories of patients with both founder and non-founder CHEK2 mutations. Methods: This is a retrospective review of patients who underwent germline testing of 31 cancer risk genes. All pathogenic (P) and likely pathogenic (LP) variants in CHEK2 were selected from our database and categorized as founder or non-founder mutations. Personal and family histories of cancer reported by the ordering provider were recorded for each case. Cancer rates were calculated as the number of patients with a personal and/or family history of each cancer divided by the total number of patients. Ethnicities in our CHEK2+ cohort and our overall database were calculated as a percentage. Results: A total of 132 patients were found to have P/LP variants in CHEK2. This CHEK2+ cohort was largely Caucasian (79.5%), despite our overall database being ethnically diverse (56% Caucasian). Nineteen different mutations were identified, 75% of which were founder and 25% were non-founder mutations. Personal histories of cancer were identified in 7% of patients. Twenty-eight different cancers were reported in patients’ personal/family histories. The rates of select cancers in the total cohort, founder mutation group and non-founder mutation group are reported in Table 1. Discussion: These findings support previous reports that prostate, gastric and kidney cancers may be part of the CHEK2 cancer spectrum, which has typically only included breast and colorectal cancer. We also identify ovarian and pancreatic cancer as potential CHEK2-associated cancers. Cancer rates are similar between CHEK2 patients with founder and non-founder mutations. Differences in cancer rates were observed between these two groups, but this cohort was too small to determine significance. Additional studies are needed to understand the overlap of cancer risk with non-founder mutations and those well-described for founder mutations. Table 1: Select cancer rates by CHEK2 mutation typeBreast FemaleOvarianProstateColorectalPancreaticUterineGastricKidneyAll80.3%39.4%25%23.5%13.6%8.3%8.3%6.1%Founder81.8%40.4%30.3%23.2%18.2%9.1%9.1%8.1%Non-founder75.8%36.3%9.1%24.2%0%6%6%0% Citation Format: Julia G Moroney, Michele S Basiliere. Describing the cancer spectrum in families with CHEK2 pathogenic and likely pathogenic variants by mutation type [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-18.

Analysis of mutational and proteomic heterogeneity of gastric cancer suggests an effective pipeline to monitor post-treatment tumor burden using circulating tumor DNA

Circulating tumor DNA (ctDNA) is released from tumor cells into blood in advanced cancer patients. Although gene mutations in individual tumors can be diverse and heterogenous, ctDNA has the potential to provide comprehensive biomarker information. Here, we performed multi-region sampling (three sites) per resected specimen from 10 gastric cancer patients followed by targeted sequencing and proteomic profiling using reverse-phase protein arrays. A total of 126 non-synonymous mutations were identified from 30 samples from 10 tumors. Of these, 16 (12.7%) were present in all three regions and were designated as founder mutations. Variant allele frequencies (VAFs) of founder mutations were significantly higher than those of non-founder mutations. Phylogenetic analysis also demonstrated a good concordance between founder and truncal mutations, defined as mutations shared by all simulated clones at the trunk of the tumor phylogenetic tree. These findings led us to prioritize founder mutations for quantitative ctDNA monitoring by digital PCR with individually-designed primer/probe sets. In preoperative plasma, the average ctDNA VAF of founder mutations was significantly higher than that of non-founder mutations (p = 0.039). Proteomic heterogeneity was present across the tumor regions both within and between patients independent of mutational status. Our results suggest that, in practice, mutations having high VAF identified without multi-regional sequencing may be immediately useful for quantitative ctDNA monitoring but do not provide sufficient information to predict the proteomic composition of tumors.

Abstract 2229: Molecular profile of histological and mutational heterogeneity of adenocarcinoma of the stomach in tumor burden monitoring using circulating tumor DNA

Abstract PURPOSE: This study aimed to perform molecular profiling of the heterogeneous histological and mutational background of primary adenocarcinoma of the stomach for pre- and post-operational tumor burden monitoring using circulating tumor DNA (ctDNA). METHODS: Ten patients with gastric adenocarcinoma who underwent surgical resection for clinical Stage IB or higher were included. Multi-region samples (three sites) per resected specimen were used for cancer cellulality estimation, a 151-gene ClearSeq panel sequencing on Next Generation Sequencer (NGS), and proteomic profiling with 294 proteins using reverse-phase protein arrays (RPPAs). The ctDNA level was evaluated by digital PCR (dPCR) using 25 originally-designed sets with the Hypercool Primer & ProbeTM technology based on mutations of individual tumors. With the median follow-up of 808 days, a total of 187 serial plasma samples were examined for ctDNA. RESULTS: A total of 103 mutations were detected in 30 regions from 10 tumors. Twenty founder mutations (i.e., mutations found in all three regions) were observed in eight tumors whereas two tumors had only non-founder mutations. Twenty-three and 60 non-founder mutations were detected in two regions and one region per tumor, respectively. Variant allele frequencies (VAFs) of founder mutations were higher than those of non-founder mutations (31.2% vs 14.5%; p < 0.01) in primary tumors. With sample regions in which mutations were not detected in trio by NGS, non-founder mutations could be detected by dPCR at low VAFs (ranging from 0.02 to 2.2%) in 95% (19/20) regions. In preoperative patient plasma, ctDNA was detected in 30% (3/10) patients (with a mean VAF of 0.59%). The rate of preoperative ctDNA detection was lower than that of esophageal squamous cell cancer (24/26, 92.3%) and colorectal cancer (10/12, 83.3%) patients. With respect to the tumor stromal effect to ctDNA, no ctDNA was detected in three cases of scirrhous type cancers (0/3, 0%), whereas three of six cases of the other stromal types (3/6, 50%) showed detectable ctDNA (VAF>0.03%). In two relapsed cases with peritoneal dissemination, the elevation of ctDNA was not apparent even at the time of diagnosis of the relapse by CT scan. Among 42 gene-protein matched pairs, the level of proteins did not seem to predict the coding gene mutation. However, tumors with TP53 mutations had significantly higher levels of p53 than those with the wild-type, which was likely due to protein stabilization (p = 0.0004). CONCLUSIONS: Detecting ctDNA in gastric cancer patients may not be as feasible as in other gastrointestinal cancer patients likely due to the heterogeneous histological and mutational background. If applicable, founder mutations are the most suitable marker for detection and monitoring ctDNA. Mutations in the primary tumor itself did not appear to predict protein levels except for TP53. Citation Format: Noriyuki Sasaki, Takeshi Iwaya, Takehiro Chiba, Masashi Fujita, Fumitaka Endo, Mizunori Yaegashi, Ryo Sugimoto, Tamotsu Sugai, Doris Siwak, Lance Liotta, Yilling Lu, Gordon Mills, Hidewaki Nakagawa, Satashi S. Nishizuka. Molecular profile of histological and mutational heterogeneity of adenocarcinoma of the stomach in tumor burden monitoring using circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2229.

Abstract 1353: The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis

Abstract Background : Circulating tumor DNA (ctDNA) is considered to be a new class of tumor-specific personalized biomarkers. Since the variant allele frequency (VAF) of ctDNA is very low (0.01-1%), it is more important to quantify tumor-specific mutations using specific probes than to perform exploratory analyses. A molecular targeting drug is often selected by mutations, and it has not been verified whether the mutations reflect the level of proteins as the drug target molecule. Purpose: To clarify: (a) the utility of ctDNA from tumors with genetic heterogeneity; (b) the ability of early detection for relapse; and (c) the validity of protein level prediction by gene mutation. Methods: Tumor DNA was extracted from three regions of 14 Stage III/IV surgically-resected colorectal tumors. Plasma DNA was also extracted from pre-/post-operative as well as follow-up blood samples every three months. The multi-regional sequencing analysis was conducted using a next generation sequencer (NGS) with a panel of 151 genes and phylogenetic trees were subsequently generated. Of the detected non-synonymous mutations, ctDNA was monitored using digital PCR (dPCR) for which primers and probes were designed by the Hypercool Primer & Probe™ technology to facilitate a specific PCR with short (~70bp) DNA fragments. A reverse phase protein array (RPPA) was used to quantitate 294 proteins in cancer tissues for three regions. Results: The average number of mutations per tumor and per region was nine and six, respectively. The average number of founder mutations (i.e., mutations found in all three regions) and non-founder mutations was three and seven, respectively. Phylogenetic analysis revealed that mutations with high VAF tended to emerge at the trunk of the evolutional tree. Interestingly, when non-founder mutations in the regions that had not exhibited the mutation by NGS were detected using dPCR in 5 of 13 regions. mutations were still detected in 5 of 13 regions (38.5%). The VAFs of founder mutations were significantly higher than those of non-founder mutations. In pre-operative plasma, ctDNA was detected in 10 of 12 patients (83.3%) with low VAF (range; 0.028-13.3%). Founder mutations in ctDNA were detected in 8 of 10 patients with low VAF (range; 0.04-13.3%). In each patient, founder mutations appeared to exhibit higher VAFs in ctDNA than those of non-founder mutations. Importantly, the VAF of ctDNA started to increase 6 months before the relapse was diagnosed by CT scan. Among 42 gene-protein matched pairs, the level of phosphorylated proteins that could be molecular targets did not seem to be predicted by the coding gene mutation. Conclusions: To select mutations for ctDNA monitoring, founder mutations with high VAF in a tumor are mandatory. Since mutations in colorectal cancer do not seem to predict the protein level, the proper clinical utility of ctDNA is a tumor marker over the entire course of colorectal cancer therapy. Citation Format: Mizunori Yaegashi, Takeshi Iwaya, Masashi Fujita, Zhenlin Ju, Doris Siwak, Kei Sato, Fumitaka Endo, Ryo Sugimoto, Tamotsu Sugai, Lance Liotta, Yiling Lu, Gordon Mills, Hidewaki Nakagawa, Satoshi Nishizuka. The clinical utility of ctDNA in colorectal cancer validated by multiregional sequencing and protein analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1353.

APPLICATION OF THE TARGETED MULTIGENE SEQUENCING FOR THE SEARCH OF HEREDITARY BREAST CANCER MUTATIONS IN RUSSIAN PATIENTS

Understanding of the molecular-genetic pathogenesis of hereditary cancer syndrome is extremely important for developing of personal therapeutic approaches and for improving the effectiveness of preventive measures. Today, the optimal solution for the search of causative germ-line mutations in hereditary breast cancer (BC) patients is the next-generation sequencing-based multigene mutational screening. The authors have assembled a targeted panel of 31 genes, based on their potential involvement in the cancer susceptibility and taking into account the frequency of pathogenic alleles in the Russian population. It includes the “canonical” genes of hereditary breast cancer (BRCA1, BRCA2, BRIP1, PALB2, TP53, ATM, NBN), the recently identified “novel” genes (BLM, FANCD2, POLE, FANCM, RAD51C, MRE11A, RECQL, as well as some other genes involved in DNA repair, apoptosis and genome stability maintenance. 94 patients with hereditary BC of unknown genetic etiology were subjected to targeted sequencing. As a result, causative germ-line mutations were identified in 21/94 (22.3%) patients. Importantly, 19 patients harbored rare non-founder BRCA1 and BRCA2 mutations. In the remaining two cases, the functions of the ATM (p.Glu73fs) and POLE (p.Leu1171fs) genes were disrupted. The obtained data are of evident clinical importance; they argue for the expanding of diagnostic panels for monitoring at-risk individuals and for moving the standards of routine clinical diagnostics towards the targeted next-generation sequencing of multigene panels.

Abstract P5-09-16: Multiple mutations in a single lineage: is it time for a shift in predictive testing?

Abstract Germline cancer genetic testing is a critical part of patient care influencing cancer risk reduction, prevention and now treatment decision-making. Single syndrome and mutation testing has been available for twenty years, however massively parallel sequencing technology has made multigene panel tests more readily available and at lower costs. Currently, it remains unclear if there is benefit related to panel testing in cancer families and patients already harboring a pathogenic mutation. We report 5 families found to harbor additional pathogenic mutations identified with multigene panel testing. One Ashkenazi family was found to harbor both a founder and non-founder BRCA mutation in a single lineage. Four additional families were found to harbor a second mutation in either a novel breast/ovary cancer gene or in a gene related to an established cancer syndrome. All patients were either initially BRCA2 or PALB2 positive and were subsequently found to have a second mutation in either CHEK2, RAD51D, ATM, MUTYH or PMS2. In 2 cases, the secondary mutation either increased frequency and/or decreased start age of colonoscopy screening. Interestingly, 2 of the additional mutations were first reported as variants of uncertain significance later reclassified as likely pathogenic. In all cases the traditional approach of single site/syndrome analysis for predictive testing would have failed to identify the second pathogenic mutation resulting in an underestimate of cancer risk in negative family members. Notably, the majority of secondary mutations were present within the same lineage as the known familial mutation. In 3 of 5 cases, the finding of a second mutation in already identified mutation carriers led to additional changes in medical management. As multigene panel testing becomes more accessible in both cost and provider acceptance, this case series illustrates the need to strongly consider use of multigene panel testing for relatives undergoing testing for known familial mutations regardless of family history, and ultimately indicating a role for re-testing established mutation carriers. Citation Format: Scalia Wilbur J, Laprise J, Beaston M, Legare RD. Multiple mutations in a single lineage: is it time for a shift in predictive testing? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-09-16.

Reflex Testing for Germline BRCA1, BRCA2, PALB2, and ATM Mutations in Pancreatic Cancer: Mutation Prevalence and Clinical Outcomes From Two Canadian Research Registries.

We investigated the translational value of reflex testing for germline mutations in four homology-directed DNA repair predisposition genes (BRCA1, BRCA2, PALB2, and ATM) in consecutive patients with pancreatic adenocarcinoma. One hundred fifty patients with French-Canadian (FC) ancestry were evaluated for founder mutations, and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation-dependent probe amplification for nonfounder mutations. Two hundred thirty-six patients unselected for ancestry were also assessed for mutations by full gene sequencing. The FC founder mutation prevalence among the 150 patients was 5.3% (95% CI, 2.6% to 10.3%), and the nonfounder mutation prevalence across the four genes among the 114 patients tested was 2.6% (95% CI, 0.6% to 7.8%). In the case series unselected for ancestry, 10.0% (95% CI, 2.7% to 26.4%) of patients reporting Ashkenazi Jewish (AJ) ancestry carried an AJ founder mutation, with no nonfounder mutations identified. The mutation prevalence among patients without FC/AJ ancestry was 4.9% (95% CI, 2.6% to 8.8%). Mutations were more frequent in patients diagnosed at ≤ 50 years of age (P = .03) and in patients with either two or more first- or second-degree relatives with pancreas, breast, ovarian or prostate cancer, or one such relative and a second primary of one of these cancer types (P < .001). BRCA1, BRCA2, and PALB2 carriers with late-stage (III or IV) disease had an overall survival advantage (P = .049), particularly if treated with platinum-based chemotherapies (P = .030). Considering these results, we recommend reflex founder mutation testing of patients with FC/AJ ancestry and full gene sequencing of patients who are ≤ 50 years or meet the identified family history criteria. Reflex testing of all incident patients for these four genes may become justified as full gene sequencing costs decline.

High frequency of pathogenic non-founder germline mutations in BRCA1 and BRCA2 in families with breast and ovarian cancer in a founder population

BackgroundPathogenic BRCA1 founder mutations (c.4035delA, c.5266dupC) contribute to 3.77% of all consecutive primary breast cancers and 9.9% of all consecutive primary ovarian cancers. Identifying germline pathogenic gene variants in patients with primary breast and ovarian cancer could significantly impact the medical management of patients. The aim of the study was to evaluate the rate of pathogenic mutations in the 26 breast and ovarian cancer susceptibility genes in patients who meet the criteria for BRCA1/2 testing and to compare the accuracy of different selection criteria for second-line testing in a founder population.MethodsFifteen female probands and 1 male proband that met National Comprehensive Cancer Network (NCCN) criteria for BRCA1/2 testing were included in the study and underwent 26-gene panel testing. Fourteen probands had breast cancer, one proband had ovarian cancer, and one proband had both breast and ovarian cancer. In a 26-gene panel, the following breast and/or ovarian cancer susceptibility genes were included: ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MEN1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. All patients previously tested negative for BRCA1 founder mutations.ResultsIn 44% (7 out of 16) of tested probands, pathogenic mutations were identified. Six probands carried pathogenic mutations in BRCA1, and one proband carried pathogenic mutations in BRCA2. In patients, a variant of uncertain significance was found in BRCA2, RAD50, MRE11A and CDH1. The Manchester scoring system showed a high accuracy (87.5%), high sensitivity (85.7%) and high specificity (88.9%) for the prediction of pathogenic non-founder BRCA1/2 mutations.ConclusionA relatively high incidence of pathogenic non-founder BRCA1/2 mutations was observed in a founder population. The Manchester scoring system predicted the probability of non-founder pathogenic mutations with high accuracy.

Breast cancer risk is similar for CHEK2 founder and non-founder mutation carriers

CHEK2 mutations are associated with increased cancer risks, including breast; however, published risk estimates are limited to those conferred by CHEK2 founder mutations, presenting uncertainty in risk assessment for carriers of other CHEK2 mutations. This study aimed to assess phenotypes and molecular characteristics of CHEK2 mutation carriers (CHEK2 + s) from a multi-gene panel testing (MGPT) cohort, focusing on comparing phenotypes of founder and non-founder CHEK2 + s. Clinical histories and molecular results were reviewed from 45,879 patients who underwent MGPT including CHEK2 at a commercial laboratory. Of individuals tested, 2.4% (n = 1085) were CHEK2 + s. Sixteen individuals harbored biallelic CHEK2 mutations, bringing the total number of CHEK2 mutations detected in this cohort to 1101. Personal/family cancer histories were compared between founder (n = 576; included c.1100delC, p.S428F, c.444 + 1G > A, and EX8_9del) and non-founder (n = 259) CHEK2 + s using Fisher's exact test and multivariate logistic regression analysis. Individuals carrying the p.I157T moderate risk founder mutation (n = 231), additional mutations in non-CHEK2 genes (n = 83), or biallelic mutations (n = 16) were excluded from phenotype analysis, as were cases with no clinical information provided. No significant phenotypic differences were observed between founder and non-founder CHEK2 + s. These data suggest that cancer risks reported for founder mutations may be generalizable to all CHEK2 + s, particularly for breast cancer.

Breast cancer in high-risk Afrikaner families: Is BRCA founder mutation testing sufficient?

Germline pathogenic mutations in cancer susceptibility genes result in inherited cancer syndromes. In the Afrikaner population of South Africa (SA), three founder mutations in the BRCA genes that lead to hereditary breast and ovarian cancer syndrome (HBOCS) have been identified. To investigate the uptake and type of molecular testing performed on patients for HBOCS, to determine the prevalence of the three Afrikaner founder BRCA mutations as well as non-founder BRCA mutations in the study population, and to analyse the utility of two mutation prediction models (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) and Manchester scoring method) in assisting with the decision for the most cost-effective testing option. A retrospective file review was performed on counsellees of self-reported Afrikaner ancestry from Johannesburg, SA (2001 - 2014), with a personal or family history of breast and/or ovarian cancer. Demographic and family history information was recorded and Manchester and BOADICEA scores were calculated for each patient. Of 86 unrelated counsellees whose files were reviewed, 54 (62.8%) underwent BRCA genetic testing; 18 (33.3%) tested positive for a mutation, and 14 of these (77.8%) for an Afrikaner founder mutation. Twelve counsellees had the BRCA2 c.7934delG mutation. Four non-founder mutations were identified. BOADICEA scores were significantly higher in counsellees who tested positive for a mutation than in those who tested negative. Founder mutation testing should be performed as a first-line option. BOADICEA is very useful in identifying counsellees at high risk for a BRCA mutation and also assists with the decision to pursue further testing following a negative founder mutation result. These findings assist in guiding an informed genetic counselling service for at-risk individuals with an Afrikaner background.

Screening for germline mutations in breast/ovarian cancer susceptibility genes in high-risk families in Israel.

We evaluated the clinical utility of screening for mutations in 34 breast/ovarian cancer susceptibility genes in high-risk families in Israel. Participants were recruited from 12, 2012 to 6, 2015 from 8 medical centers. All participants had high breast/ovarian cancer risk based on personal and family history. Genotyping was performed with the InVitae™ platform. The study was approved by the ethics committees of the participating centers; all participants gave a written informed consent before entering the study. Overall, 282 individuals participated in the study: 149 (53%) of Ashkenazi descent, 80 (28%) Jewish non-Ashkenazi descent, 22 (8%) of mixed Ashkenazi/non-Ashkenazi origin, 21 (7%) were non-Jewish Caucasians, and the remaining patients (n=10-3.5%) were of Christian Arabs/Druze/unknown ethnicity. For breast cancer patients (n=165), the median (range) age at diagnosis was 46 (22-90) years and for ovarian cancer (n=15) 54 (38-69) years. Overall, 30 cases (10.6%) were found to carry a pathogenic actionable mutation in the tested genes: 10 BRCA1 (3 non-founder mutations), 9 BRCA2 (8 non-founder mutations), and one each in the RAD51C and CHEK2 genes. Furthermore, actionable mutations were detected in 9 more cases in 4 additional genes (MSH2, RET, MSH6, and APC). No pathogenic mutations were detected in the other genotyped genes. In this high-risk population, 10.6% harbored an actionable pathogenic mutation, including non-founder mutations in BRCA1/2 and in additional cancer susceptibility genes, suggesting that high-risk families should be genotyped and be assigned a genotype-based cancer risk.

Candidate gene analysis of BRCA1/2 mutation-negative high-risk Russian breast cancer patients

Twenty one DNA repair genes were analyzed in a group of 95 BC patients, who displayed clinical features of hereditary disease predisposition but turned out to be negative for mutations in BRCA1 and BRCA2 entire coding region as well as for founder disease-predisposing alleles in CHEK2, NBN/NBS1 and ATM genes. Full-length sequencing of CHEK2 and NBN/NBS1 failed to identify non-founder mutations. The analysis of TP53 revealed a woman carrying the R282W allele; further testing of additional 108 BC patients characterized by a very young age at onset (35 years or earlier) detected one more carrier of the TP53 germ-line defect. In addition, this study confirmed non-random occurrence of PALB2 truncating mutations in Russian hereditary BC patients. None of the studied cases carried germ-line defects in recently discovered hereditary BC genes, BRIP1, FANCC, MRE11A and RAD51C. The analysis of genes with yet unproven BC-predisposing significance (BARD1, BRD7, CHEK1, DDB2, ERCC1, EXO1, FANCG, PARP1, PARP2, RAD51, RNF8, WRN) identified single women carrying a protein-truncating allele, WRN R1406X. DNA sequencing of another set of 95 hereditary BC cases failed to reveal additional WRN heterozygous genotypes. Since WRN is functionally similar to the known BC-predisposing gene, BLM, it deserves to be analyzed in future hereditary BC studies. Furthermore, this investigation revealed a number of rare missense germ-line variants, which are classified as probably protein-damaging by online in silico tools and therefore may require further consideration.

Incidence of BRCA1 and BRCA2 non-founder mutations in patients of Ashkenazi Jewish ancestry.

An estimated 1:40 individuals of Ashkenazi Jewish (AJ) ancestry carry one of three common founder mutations in BRCA1 or BRCA2, resulting in the inherited cancer condition, Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Targeted testing for these three mutations (BRCA1 187delAG, BRCA1 5385insC, and BRCA2 6174delT) is therefore recommended for all AJ breast and ovarian cancer patients, regardless of age of diagnosis or family history. Comprehensive analysis of both genes is recommended for a subset of AJ patients in whom founder mutations are not identified, but estimates of the yield from comprehensive analysis in this population vary widely. We sought to determine the proportion of non-founder mutations as a percentage of all mutations in BRCA1 and BRCA2 among AJ patients to inform decisions about HBOC testing strategies in this population. We analyzed the genetic testing results for 37,952 AJ patients for whom clinical testing of BRCA1 and BRCA2 was performed at Myriad Genetic Laboratories from January 2006 through August 2013. Analysis was limited to AJ-only patients for whom the initial test order was either (1) comprehensive testing, or (2) founder mutation testing with instructions to automatically "reflex" to comprehensive analysis if negative. Cases were excluded if a separate follow-up order was placed to reflex to comprehensive analysis only after the founder mutation testing was reported out as negative. Among all BRCA1 and BRCA2 mutations detected in these groups, the percentage of non-founder mutations was 13 % (104/802) and 7.2 % (198/2,769). One-hundred and eighty-nine unique non-founder mutations were detected, 76 in BRCA1 and 113 in BRCA2. Non-founder mutations make up between 7.2 and 13.0 % of all BRCA1 and BRCA2 mutations in Ashkenazi Jews. A wide range of mutations are present, most of which are also seen in non-AJ individuals.

To reflex or not: additional BRCA1/2 testing in Ashkenazi Jewish individuals without founder mutations.

This study determined the prevalence of non-Ashkenazi 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.

Proffered Papers and Posters Submitted to the Fifth International Symposium on Hereditary Breast and Ovarian Cancer, BRCA: Twenty Years of Advances

Objectives: It is estimated that 1–2% of individuals of Ashkenazi Jewish (AJ) ancestry carry one of three pathogenic founder mutations in BRCA1 and BRCA2. Targeted testing for these mutations (BRCA1 187delAG and 5385insC, and BRCA2 6174delT) is therefore recommended for all AJ breast and ovarian cancer patients, regardless of age of diagnosis or family history. Comprehensive analysis of both genes is recommended for a subset of AJ patients in whom founder mutations are not identified, but estimates of the yield from comprehensive analysis in this population vary widely. Methods: We sought to establish the proportion of non-founder mutations in AJ patients undergoing clinical testing in our laboratory from January 2006 through August 2013. Analysis included AJ patients for whom: (1) comprehensive testing was ordered as the initial test, or (2) founder mutation testing was ordered with instructions to “reflex” to comprehensive analysis if negative. The latter group was limited to cases where the reflex testing was ordered on the original test request form, and not cancelled for any reason other than the detection of a founder mutation. Results: The percentage of non-founder mutations detected in these groups was 13% (104/802) and 7.2% (198/2769) respectively. We detected 189 unique non-founder mutations, 76 in BRCA1 and 113 in BRCA2. BRCA2 4075delGT was detected in 15 patients. The next most common mutations, found in 7 patients each, were BRCA1 5055delG, BRCA2 1982delA, and BRCA2 R3128X. Conclusions: Non-founder mutations make up between 13% and 7.2% of BRCA1 and BRCA2 mutations in patients reporting AJ ancestry. These numbers may represent underestimates if some patients were ascertained for testing based on the identification of a founder mutation in a relative. These numbers suggest that the prevalence of non-founder mutations in AJ individuals may be comparable to the prevalence of BRCA1/2 mutations in non-AJ individuals.

Abstract P4-04-10: Clinically relevant tumor mutation profiles in patients with triple negative breast cancer (TNBC)

Abstract TNBC account for ∼15% of breast cancers and are most difficult to treat. However, TNBC patient outcome is very heterogeneous. In an effort to characterize the biological characteristics of TNBC, we examined 190 routinely diagnosed tumor tissues from early high-risk breast cancer patients treated with adjuvant chemotherapy (anthracyclines and/or taxanes). Highly multiplexed PCR primer pools were designed for 43 genes previously implicated in TNBC with Ion Ampliseq optimized for FFPE samples. Upon library construction and clonal amplification, amplicons were massively sequenced on Ion Proton PI chips and base-called (Torrent Suite 3.6). Variants were called and annotated (Ion Reporter 1.6), and accepted for analysis upon stringent read quality filtering at p&amp;lt;0.001. Informative results were obtained in 183 cases (96.3%). Deleterious and hot-spot mutations (Ingenuity and Oncomine databases) were observed in 39/43 genes, and were more frequent in TP53 (77%), CDH1 (29%), PIK3CA (16.4%), ARID1B (10.4%). TP53 was affected as a single gene in 55/183 (30%) of the cases. Most TNBC (103/183, 56.3%) were mutant in more than one genes; double deleterious/damaging TP53 mutations were observed in 59 (32.2%) tumors at relatively high incidence; and, 29 (15.8%) tumors had extremely variable mutation profiles with &amp;gt;4 mutations per sample, most of them at low incidence but still indicative of dynamic clonal expansion. CDH1 mutations seldom occurred alone (2% of all tumors) and were probably non-founders, while mutations in some genes, e.g., AKT1 (9/12) and ARID1B (13/19) preferentially occurred in CDH1mutant tumors (p&amp;lt;0.001). In comparison to the 12 patients with tumors free of mutations in any of the genes tested (no events up to 120 mo after treatment start), patients with tumors bearing any number of mutations (n = 171) had significantly shorter disease-free survival (DFS, median: 56 mo, log-rank p = 0.031). Mutations in single genes were not significantly associated with disease outcome. The 29 patients with tumors bearing &amp;gt;4 mutations had longer median DFS (91 mo) as compared to those with 1 mutation (n = 68, median DFS 46.5 mo) or with 4 mutations (n = 21, median DFS 39 mo) (HR: 0.4; 95%CI: 0.2-0.8; Wald's p = 0.010). In comparison to patients with TP53&amp;CDH1 non-mutant tumors (n = 26, median DFS 62 mo), patients with TP53mutant/CDH1non-mutant tumors relapsed significantly earlier (n = 103, median DFS 48 mo, HR 3.9, 95%CI 1.2-12.9, Wald's p = 0.023) and tended to have shorter overall survival. CDH1mutant/TP53non-mutant and tumors mutated in both genes did not show such associations (interaction Wald's p = 0.044). No interactive effects on patient outcome were observed between mutation markers and treatment with taxanes, adjuvant radiotherapy, or with standard clinicopathologic parameters. In conclusion, TNBC may be assigned as a TP53 disease. However, the present data underline the need for a broad assessment of tumor mutational profiles, including non-founder mutations, since they may interfere with patient outcome upon standard treatments. The application of targeted parallel sequencing on routinely processed FFPE tissue samples seems feasible and may help in assessing clinically relevant genomic variant profiles of these highly intra- and inter-heterogeneous tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-10.

Common MUTYH mutations and colorectal cancer risk in multiethnic populations

MUTYH is associated with colorectal cancer (CRC) risk. We studied the frequency of MUTYH and risk of CRC in Arabs, North African and European Jews. Participants were all 593 Sephardi Moroccan Jews (232 cases, 361 controls) and all 631 Arabs (327 cases, 304 controls) recruited into a population-based study of colorectal cancer in Israel, as well as a random sample of 189 Ashkenazi Jewish cases. Two MUTYH mutations, G396D and Y179C, were studied in 1,413 individuals, with MUTYH sequence analysis in 46 cases with CRC in a sibling or adenoma. No carriers of mutations in MUTYH were identified in Ashkenazi Jews and only one in Arabs. In Sephardi Jews, 28 carriers of G396D, 25 (4.2%) heterozygotes and 3 (0.5%) homozygotes were identified. Four (0.7%) were heterozygote carriers of the Y179C mutation. Two compound heterozygous carriers of Y179C and G396D were identified. Homozygote carriers of G396D had nonsignificantly elevated risk of CRC (OR=11.0, 95% CI: 0.91-213.9, p=0.06), and combined bi-allelic carriers of G396D and Y179C had increased risk, OR=17.4, 95% CI=(1.9-316.7, p=0.009). Four of five bi-allelic carriers reported a family history of CRC. Sequencing of 46 colorectal cancer cases with family history and additional adenomas, did not identify any other non-founder mutations. MUTYH carriers of the two common founder mutations are profoundly under-represented among both Ashkenazi Jews and Arabs. The prevalence of MUTYH carriers of the common mutations is much higher in Sephardi Jews. Bi-allelic carriers of mutations in MUTYH, are associated with highly risk of colorectal cancer.

Non-founder BRCA1 mutations in Russian breast cancer patients

A few founder BRCA1 mutations (5382insC, 4154delA, 185delAG) account for up to 15% of high-risk (young-onset or familial or bilateral) breast cancer (BC) cases in Russia. The impact of non-founder BRCA1 mutations in this country is less studied; in particular, there are no reports analyzing gross rearrangements of this gene in the Russian patient series. We selected for the study 95 founder mutation negative high-risk BC cases. Combination of high-resolution melting (HRM) and sequencing revealed six presumably BC-associated alleles (2080delA, 4808C > G, 5214C > T, 5236G > A, 5460G > T, 5622C > T) and one variant of an unknown significance (4885G > A). The pathogenic role of the 5236G > A mutation leading to G1706E substitution was further confirmed by the loss of heterozygosity analysis of the corresponding tumor tissue. Multiplex ligation-dependent probe amplification (MLPA) revealed two additional BRCA1 heterozygotes, which carried BRCA1 deletions involving exons 1–2 and 3–7, respectively. Based on the results of this investigation and the review of prior Russian studies, three BRCA1 mutations (2080delA, 3819del5, 3875del4) were considered with respect to their possible founder effect and tested in the additional series of 210 high-risk BC patients; two BRCA heterozygotes (2080delA and 3819del5) were revealed. We conclude that the non-founder mutations constitute the minority of BRCA1 defects in Russia.

Absence of genomic BRCA1 and BRCA2 rearrangements in Ashkenazi breast and ovarian cancer families

A substantial proportion of Ashkenazi Jewish (AJ) breast and ovarian cancer families carry one of three founder mutations in BRCA1 (185delAG, 5382InsC) and BRCA2 (6174delT). Non-founder mutations are identified in another 2-4% of such families. The extent to which major genomic rearrangements in BRCA contribute to breast and ovarian cancer in the Ashkenazim is not well understood. We identified AJ individuals with breast and/or ovarian cancer undergoing hereditary breast/ovarian cancer risk assessment since 2006 without evidence of a deleterious mutation on BRCA gene sequencing who were screened for major gene rearrangements in BRCA1 and BRCA2. For each proband, the pre-test probability of identifying a deleterious BRCA mutation was estimated using the Myriad II model. We identified 108 affected individuals who underwent large rearrangement testing (80 breast cancer, 19 ovarian cancer, nine both breast and ovarian cancer). The mean estimated AJ specific pre-test probability of a deleterious mutation in BRCA1 and BRCA2 was 24.7% (range: 4.4-88.9%). No genomic rearrangements were identified in either the entire group or in the 26 subjects with pre-test mutation prevalence estimates exceeding 30%. Major gene rearrangements involving the BRCA1 and BRCA2 genes appear to contribute little to the burden of inherited predisposition to breast and ovarian cancer in the Ashkenazim.

Phenotypic spectrum of Fukutinopathy: Most severe phenotype of Fukutinopathy

Fukuyama-type congenital muscular dystrophy (FCMD), Walker–Warburg syndrome (WWS), and muscle–eye–brain (MEB) disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, cobblestone lissencephaly, and eye anomalies. Among them, WWS is the most severe syndrome. Causative genes for FCMD ( Fukutin), WWS ( POMT1), and MEB ( POMGnT1) have been identified. The vast majority of Japanese FCMD patients carry at least one copy of an ancestral founder insertion mutation. Patients homozygous for this insertion show a milder phenotype than do compound heterozygotes, carrying the insertion in combination with a missense or nonsense mutation on the other allele. No Japanese FCMD patients have been identified with nonfounder mutations on both alleles. A Turkish boy with characteristics of WWS was detected to have a homozygous nonsense mutation in exon 5 of Fukutin. This is the first case worldwide in which a Fukutin mutation has been found outside the Japanese population. Later, another Turkish boy with WWS phenotype was found to have a homozygous nonsense mutation in exon 4 of Fukutin. These two Turkish boys represent the most severe end of the phenotypic spectrum of Fukutin mutations. The Japanese FCMD patients carrying at least one copy of a founder mutation in the noncoding region may produce a lower level of mature Fukutin than normal and generate a relatively mild FCMD phenotype. The homozygous nonsense mutations within the coding region identified in Turkish patients are predicted to cause a total loss of fukutin activity and are likely to produce a more severe phenotype which closely resembles WWS.