Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions.

Cholangiocarcinoma is a highly lethal cancer of the biliary tract. The intrahepatic subtype of cholangiocarcinoma is increasing in incidence globally. Despite technologic advancements over the past decade, little is known about the somatic changes that occur in these tumors. The goal of this study was to determine the frequency of common oncogenes in resected cholangiocarcinoma specimens that could provide potential therapeutic targets for patients diagnosed with cholangiocarcinoma. Formalin-fixed, paraffin-embedded tissue blocks from 94 resected cholangiocarcinomas were used to extract DNA from areas comprising more than 20% tumor. Specimens were evaluated using the Sequenom MassARRAY OncoCarta Mutation Profiler Panel (San Diego, CA). This matrix-assisted laser desorption/ionization-time of flight mass spectrometry single genotyping panel evaluates 19 oncogenes for 238 somatic mutations. Twenty-five mutations were identified in 23 of the 94 cholangiocarcinomas within the following oncogenes: KRAS (n = 12), PIK3CA (n = 5), MET (n = 4), EGFR (n = 1), BRAF (n = 2), and NRAS (n = 1). Mutations were identified in 7 (26%) of 27 extrahepatic cholangiocarcinomas and 16 (24%) of 67 intrahepatic cholangiocarcinomas. When combined with IDH1/2 testing, 40 (43%) of the 94 cholangiocarcinomas had a detectable mutation. MassARRAY technology can be used to detect mutations in a wide variety of oncogenes using paraffin-embedded tissue. Clinical testing for somatic mutations may drive personalized therapy selection for cholangiocarcinomas in the future. The variety of mutations detected suggests that a multiplexed mutation detection approach may be necessary for managing patients with biliary tract malignancy.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and risk for pancreatic adenocarcinoma.

BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are common in white persons and are associated with pancreatic disease. The purpose of this case-control study was to determine whether CFTR mutations confer a higher risk of pancreatic cancer. METHODS: In a case-control study, the authors compared the rates of 39 common cystic fibrosis-associated CFTR mutations between 949 white patients with pancreatic adenocarcinoma and 13,340 white controls from a clinical laboratory database for prenatal testing for CFTR mutations. The main outcome measure was the CFTR mutation frequency in patients and controls. RESULTS: Overall, 50 (5.3%) of 949 patients with pancreatic cancer carried a common CFTR mutation versus 510 (3.8%) of 13,340 controls (odds ratio [OR], 1.40; 95% confidence interval [CI], 1.04-1.89; P = .027). Among patients who were younger when their disease was diagnosed (<60 years), the carrier frequency was higher than in controls (OR, 1.82; 95% CI, 1.14-2.94; P = .011). In patient-only analyses, the presence of a mutation was associated with younger age (median 62 vs 67 years; P = .034). In subgroups, the difference was seen only among ever-smokers (60 vs 65 years, P = .028). Subsequent sequencing analysis of the CFTR gene detected 8 (16%) compound heterozygotes among the 50 patients initially detected to have 1 mutation. CONCLUSIONS: Carrying a disease-associated mutation in CFTR is associated with a modest increase in risk for pancreatic cancer. Those affected appear to be diagnosed at a younger age, especially among smokers. Clinical evidence of antecedent pancreatitis was uncommon among both carriers and noncarriers of CFTR mutations.

Development of genomic DNA reference materials for genetic testing of disorders common in people of ashkenazi jewish descent.

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research.

Development of genomic reference materials for cystic fibrosis genetic testing.

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812-1G>A (c.1680-1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Comparison of three methods for genotyping the UGT1A1 (TA)n repeat polymorphism.

OBJECTIVES: The UGT1A1 promoter contains a (TA)n repeat polymorphism. The 7 repeat allele is associated with decreased enzyme activity and patients homozygous for this allele treated with irinotecan may experience life-threatening toxicity. Here, we have compared three methods [DNA sequencing, fragment analysis, and the Invader assay (Third Wave Technologies)] for genotyping this polymorphism. RESULTS: All of the DNA samples (n=119) had concordant genotype calls between the sequencing and size-based methods. The Invader method was also concordant if the genotypes were 6/6, 6/7, or 7/7. Both the size-based method and the Invader method had straightforward data analysis, while interpretation of the sequencing results was occasionally more challenging. The Invader method required more concentrated DNA for analysis, was more expensive, and had a limited genotyping spectrum. CONCLUSION: All three methods were valuable for genotyping the UGT1A1 (TA)n repeat, with the sequencing and size-based assays having the fewest drawbacks.

RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.

Single bp mutations in the RET proto-oncogene can cause multiple endocrine neoplasia type 2 syndromes. The conventional approach for genotyping RET mutations is sequencing the exons. A closed-tube RET genotyping assay using a saturating DNA dye, unlabeled probes, and amplicon high-resolution melting analysis was developed. The method required two sequential polymerase chain reaction stages, a primary and secondary assay. The primary assay analyzed RET exons 10, 11, 13, 14, and 16 with a total of seven reactions using eight unlabeled probes. The primary assay genotyped wild-type exons, a common exon 13 polymorphism, and an exon 16 mutation, whereas other RET sequence variation was detected. The primary unlabeled probe data limited the possible genotypes for the detected RET sequence variation, which permitted genotyping in a secondary assay with only two to five reactions. Six probes were designed with the masking technique and masked selected sequence variations to allow unambiguous analysis of other mutations elsewhere under the probe. After this two-stage RET genotyping assay, less than 0.2% of exons tested would require sequencing for genotype. A blinded study generated from five wild type and 29 available RET sequence variation samples was 100% concordant with sequencing. Amplicon high-resolution melting analysis with unlabeled probes and the masking technique is a fast, accurate method for genotyping the >50 RET sequence variations.

Mutation scanning of the RET protooncogene using high-resolution melting analysis.

BACKGROUND: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. METHODS: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen, and the high-resolution melter, HR-1, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. RESULTS: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. CONCLUSION: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.