Brian A. Allen

BRAF Mutation Is Frequently Present in Sporadic Colorectal Cancer with Methylated hMLH1, But Not in Hereditary Nonpolyposis Colorectal Cancer

Purpose: The BRAF gene encodes a serine/threonine kinase and plays an important role in the mitogen-activated protein kinase signaling pathway. BRAF mutations in sporadic colorectal cancer with microsatellite instability (MSI) are more frequently detected than those in microsatellite stable cancer. In this study, we sought to compare the frequencies of BRAF mutations in sporadic colorectal cancer with MSI with those in hereditary nonpolyposis colorectal cancer (HNPCC). Experimental Design: We analyzed BRAF mutations in 26 colorectal cancer cell lines, 80 sporadic colorectal cancers, and 20 tumors from HNPCC patients by DNA sequencing and sequence-specific PCR. The methylation status of the hMLH1 gene was measured by either sequencing or restriction enzyme digestion after NaHSO3 treatment. Results: We observed a strong correlation of BRAF mutation with hMLH1 promoter methylation. BRAF mutations were present in 13 of 15 (87%) of the colorectal cell lines and cancers with methylated hMLH1, whereas only 4 of 91 (4%) of the cell lines and cancers with unmethylated hMLH1 carried the mutations (P < 0.00001). Sixteen of 17 mutations were at residue 599 (V599E). A BRAF mutation was also identified at residue 463 (G463V) in one cell line. In addition, BRAF mutations were not found in any cancers or cell lines with K-ras mutations. In 20 MSI+ cancers from HNPCC patients, however, BRAF mutations were not detectable, including a subset of 9 tumors with negative hMLH1 immunostaining and methylated hMLH1. Conclusions: BRAF mutations are frequently present in sporadic colorectal cancer with methylated hMLH1, but not in HNPCC-related cancers. This discrepancy of BRAF mutations between sporadic MSI+ cancer and HNPCC might be used in a strategy for the detection of HNPCC families.

Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next‐generation sequencing with a 25‐gene panel

Next‐generation sequencing (NGS) allows for simultaneous sequencing of multiple cancer susceptibility genes and, for an individual, may be more efficient and less expensive than sequential testing. The authors assessed the frequency of deleterious germline mutations among individuals with breast cancer who were referred for BRCA1 and BRCA2 (BRCA1/2) gene testing using a panel of 25 genes associated with inherited cancer predisposition.

Frequency of Germline Mutations in 25 Cancer Susceptibility Genes in a Sequential Series of Patients With Breast Cancer

PURPOSE Testing for germline mutations in BRCA1/2 is standard for select patients with breast cancer to guide clinical management. Next-generation sequencing (NGS) allows testing for mutations in additional breast cancer predisposition genes. The frequency of germline mutations detected by using NGS has been reported in patients with breast cancer who were referred for BRCA1/2 testing or with triple-negative breast cancer. We assessed the frequency and predictors of mutations in 25 cancer predisposition genes, including BRCA1/2, in a sequential series of patients with breast cancer at an academic institution to examine the utility of genetic testing in this population. METHODS Patients with stages I to III breast cancer who were seen at a single cancer center between 2010 and 2012, and who agreed to participate in research DNA banking, were included (N = 488). Personal and family cancer histories were collected and germline DNA was sequenced with NGS to identify mutations. RESULTS Deleterious mutations were identified in 10.7% of women, including 6.1% in BRCA1/2 (5.1% in non-Ashkenazi Jewish patients) and 4.6% in other breast/ovarian cancer predisposition genes including CHEK2 (n = 10), ATM (n = 4), BRIP1 (n = 4), and one each in PALB2, PTEN, NBN, RAD51C, RAD51D, MSH6, and PMS2. Whereas young age (P < .01), Ashkenazi Jewish ancestry (P < .01), triple-negative breast cancer (P = .01), and family history of breast/ovarian cancer (P = .01) predicted for BRCA1/2 mutations, no factors predicted for mutations in other breast cancer predisposition genes. CONCLUSION Among sequential patients with breast cancer, 10.7% were found to have a germline mutation in a gene that predisposes women to breast or ovarian cancer, using a panel of 25 predisposition genes. Factors that predict for BRCA1/2 mutations do not predict for mutations in other breast/ovarian cancer susceptibility genes when these genes are analyzed as a single group. Additional cohorts will be helpful to define individuals at higher risk of carrying mutations in genes other than BRCA1/2.

Genotype and phenotype of patients with both familial adenomatous polyposis and thyroid carcinoma.

The incidence of thyroid carcinoma in familial adenomatous polyposis (FAP) is thought to be 1%–2%, with the majority of cases being female. We have investigated the phenotype and genotype of 16 patients with FAP associated thyroid carcinoma. Among 1194 FAP patients studied in two high risk registries in North America (Familial Gastrointestinal Cancer Registry, Toronto and University California, San Francisco), 16 (1.3%) unrelated patients with FAP associated thyroid cancers were identified. Adenomatous polyposis coli (APC) gene testing was performed in 14 of the 16 cases. The average age of diagnosis for FAP and thyroid carcinoma was 29 years (range 17–52 years) and 33 years (range 17–55 years), respectively. All FAP patients except 1 had more than 100 colonic adenomas. Extracolonic manifestations, beside thyroid cancer, were presented in 81% (n = 13) of the patients, including gastric and duodenal polyps, desmoid tumor, osteoma, epidermoid cyst, sebaceous cyst and lipoma. Colorectal cancer was diagnosed in 38% (n = 6) of the patients. The pathology of the FAP associated thyroid cancer was predominantly papillary carcinoma. Germline mutations were identified in 12 of 14 patients tested. Mutations proximal to the mutation cluster region (1286–1513) were detected in 9 cases. Thyroid cancer in our FAP population was rare, predominantly in females and showed papillary carcinoma histology. Additionally, thyroid cancer in our patients occurred in the setting of classic FAP phenotype. Germline mutations were located predominantly outside the APC mutation cluster region.

Hereditary polyposis syndromes and hereditary non-polyposis colorectal cancer

Colorectal cancer due to hereditary syndromes comprises approximately 5% of the overall colorectal cancer burden. Conditions fall into two distinct categories, the polyposis syndromes and hereditary non-polyposis colorectal cancer. It is important for the clinician to have a working knowledge of both as screening and surveillance recommendations differ significantly from those applicable to the general population. The polyposis syndromes include familial adenomatous polyposis, Peutz-Jeghers syndrome, juvenile polyposis, and Cowden syndrome. For each condition, a review of both the intestinal and extra-intestinal clinical findings is presented as well as the genetic basis, genetic testing, screening, surveillance and treatment options. As genetic testing for several of these conditions has recently become both commercially available and standard practice, special attention is given to indications and strategies for genetic testing in hereditary colorectal cancer syndromes.

Cancer Susceptibility Gene Mutations in Individuals With Colorectal Cancer

Purpose Hereditary factors play an important role in colorectal cancer (CRC) risk, yet the prevalence of germline cancer susceptibility gene mutations in patients with CRC unselected for high-risk features (eg, early age at diagnosis, personal/family history of cancer or polyps, tumor microsatellite instability [MSI], mismatch repair [MMR] deficiency) is unknown. Patients and Methods We recruited 1,058 participants who received CRC care in a clinic-based setting without preselection for age at diagnosis, personal/family history, or MSI/MMR results. All participants underwent germline testing for mutations in 25 genes associated with inherited cancer risk. Each gene was categorized as high penetrance or moderate penetrance on the basis of published estimates of the lifetime cancer risks conferred by pathogenic germline mutations in that gene. Results One hundred five (9.9%; 95% CI, 8.2% to 11.9%) of 1,058 participants carried one or more pathogenic mutations, including 33 (3.1%) with Lynch syndrome (LS). Twenty-eight (96.6%) of 29 available LS CRCs demonstrated abnormal MSI/MMR results. Seventy-four (7.0%) of 1,058 participants carried non-LS gene mutations, including 23 (2.2%) with mutations in high-penetrance genes (five APC, three biallelic MUTYH, 11 BRCA1/2, two PALB2, one CDKN2A, and one TP53), 15 of whom lacked clinical histories suggestive of their underlying mutation. Thirty-eight (3.6%) participants had moderate-penetrance CRC risk gene mutations (19 monoallelic MUTYH, 17 APC*I1307K, two CHEK2). Neither proband age at CRC diagnosis, family history of CRC, nor personal history of other cancers significantly predicted the presence of pathogenic mutations in non-LS genes. Conclusion Germline cancer susceptibility gene mutations are carried by 9.9% of patients with CRC. MSI/MMR testing reliably identifies LS probands, although 7.0% of patients with CRC carry non-LS mutations, including 1.0% with BRCA1/2 mutations.

Low rate of microsatellite instability in young patients with adenomas: reassessing the Bethesda guidelines.

BACKGROUND AND AIM:Screening adenomas for microsatellite instability (MSI) in patients younger than 40 yr of age has been recommended by the Bethesda Guidelines as a means of identifying patients at risk for hereditary nonpolyposis colorectal cancer (HNPCC). We sought to determine the rate of MSI in adenomas removed from individuals under 40 yr of age over a 5-yr period in a university general gastroenterology practice.METHODS:We identified patients between 18 and 39 yr of age with endoscopically removed adenomatous colorectal polyps. Patients with polyposis syndromes, inflammatory bowel disease, or colorectal carcinoma were excluded. A three-generation family history was obtained via telephone interview. Endoscopic and histology reports were reviewed. Adenomas were tested for MSI using the BAT26 and BAT40 microsatellite markers, and expression of the MSH2 and MLH1 proteins was assessed by immunostaining.RESULTS:A total of 34 patients had 46 adenomas removed endoscopically. Out of 34 patients, 14 (41%) had a family history of colorectal cancer and 3 were from Amsterdam criteria positive families. A total of 28 of 46 adenomas (61%) were distal to the splenic flexure. Polyps ranged in size from 2 to 20 mm and averaged 6.6 mm. Five polyps (11%) were tubulovillous adenomas, and the remainder were tubular adenomas. None of the polyps were serrated adenomas and none demonstrated high-grade dysplasia. Among the 40 adenomas available for testing, none demonstrated MSI using either BAT26 (0/40) or BAT40 (0/21), nor did any of the polyps tested demonstrate loss of either MSH2 or MLH1 expression (0/16).CONCLUSION:Screening adenomas from patients younger than 40 yr of age for MSI was ineffective in identifying potentially new cases of HNPCC. New strategies that improve on the current clinical and molecular screening methods should be developed so that at-risk individuals can be identified and referred for germline testing before developing their first cancer.

Awareness of gynecologic surveillance in women from hereditary non-polyposis colorectal cancer families

ObjectiveTo determine knowledge of gynecologic cancer risk and screening in women with HNPCC.Study designForty-three women with HNPCC were counseled through a gastrointestinal cancer risk program, and later sent a questionnaire regarding their screening practices for gynecologic neoplasms.ResultsTwenty-seven (63%) of 43 responded. Fifteen (55%) of 27 had previously been diagnosed with cancer. Among 16 women with a uterus, 11 (69%) reported surveillance by ultrasound or endometrial sampling. Among 21 respondents with ovaries, 13 (62%) reported screening by ultrasound or CA125. Twenty-two (81%) of 27 had seen a gynecologist after receiving their HNPCC diagnosis, but only 12% recalled hearing about risks from their gynecologist, and␣8% from their gynecologic oncologist. Genetic counselors were cited as the most common source (48%) of gynecologic cancer risk information.ConclusionsWhile the effectiveness of surveillance remains in question, gynecologists can be a source of information regarding gynecologic cancer risk for women from HNPCC families.

Homologous Recombination Deficiency in Patients With Pancreatic Ductal Adenocarcinoma and Response to Chemotherapy

PurposeMutations or copy number abnormalities of genes involved in homologous recombination (HR) occur in pancreatic ductal adenocarcinoma (PDAC). DNA-based measures of HR deficiency (HRD) have been developed and may help identify tumors with better response to DNA-damaging agents. This study aimed to describe the HR pathway mutations and HRD status and determine their association with treatment response and outcome in patients with PDAC.Patients and MethodsWe performed a retrospective analysis of tumor samples from patients treated at Indiana University for locally advanced or metastatic PDAC. Patients were included if they received gemcitabine plus nanoparticle albumin-bound paclitaxel (control) or fluorouracil, oxaliplatin, leucovorin, and irinotecan (FOLFIRINOX) and had adequate follow-up to assess survival and response to therapy. Tumor analysis generated a three-biomarker HRD score and mutation data for 44 genes.ResultsNinety-one samples met inclusion criteria, and 78 samples (formalin-fixed paraffi...