Increased sensitivity of KRAS mutation detection by high‐resolution melting analysis of COLD‐PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low‐level tumor specific KRAS mutations. Coamplification at lower denaturation temperature‐PCR (COLD‐PCR) is a new form of PCR that selectively amplifies mutation‐containing templates based on the lower melting temperature of mutant homoduplexes versus wild‐type homoduplexes. We have developed a fast COLD‐PCR and high‐resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD‐PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen^® KRAS mutation kit. The sensitivity was increased by 5‐ to 100‐fold for melting temperature decreasing mutations when using COLD‐PCR compared to standard PCR. Mutations, undetectable by the TheraScreen^® kit in clinical samples, were detected by COLD‐PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD‐PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Major improvement in the detection of microsatellite instability in colorectal cancer using HSP110 T17 E‐ice‐COLD‐PCR

Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI) to screen for Lynch syndrome. Evaluation of MSI status involves screening tumor DNA for the presence of somatic deletions in DNA repeats using PCR followed by fragment analysis. While this method may lack sensitivity due to the presence of a high level of germline DNA, which frequently contaminates the core of primary colon tumors, no other method developed to date is capable of modifying the standard PCR protocol to achieve improvement of MSI detection. Here, we describe a new approach developed for the ultra‐sensitive detection of MSI in CRC based on E‐ice‐COLD‐PCR, using HSP110 T17, a mononucleotide DNA repeat previously proposed as an optimal marker to detect MSI in tumor DNA, and an oligo(dT)₁₆ LNA blocker probe complementary to wild‐type genotypes. The HT17 E‐ice‐COLD‐PCR assay improved MSI detection by 20–200‐fold compared with standard PCR using HT17 alone. It presents an analytical sensitivity of 0.1%–0.05% of mutant alleles in wild‐type background, thus greatly improving MSI detection in CRC samples highly contaminated with normal DNA. HT17 E‐ice‐COLD‐PCR is a rapid, cost‐effective, easy‐to‐implement, and highly sensitive method, which could significantly improve the detection of MSI in routine clinical testing.     

KRAS (but not BRAF) mutations in ovarian serous borderline tumour are associated with recurrent low‐grade serous carcinoma

BRAF and KRAS mutations in ovarian serous borderline tumours (OSBTs) and ovarian low‐grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or whether those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumour samples from 23 recurrent LGSC patients with a known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for five patients, and either OSBTs or LGSCs were available for another 18 patients. Tumour cells from paraffin‐embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumours that appeared to have wild‐type KRAS by conventional PCR–Sanger sequencing were further analysed by full COLD (co‐amplification at lower denaturation temperature)‐PCR and deep sequencing. Full COLD‐PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in ten patients. Full COLD‐PCR deep sequencing detected low‐abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in seven OSBT samples and six LGSC samples. Surprisingly, patients with the KRAS G12V mutation have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumour cells with or without detectable KRAS mutations. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay

Detection of low‐abundance mutations in cell‐free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR‐LDR‐qPCR assay capable of detecting point mutations at a single‐molecule resolution in the presence of an excess of wild‐type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer‐binding regions as well as wild‐type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single‐well version of the assay detected 2–5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild‐type human genomic DNA (hgDNA) from buffy coat. A 12‐well (pixel) version of the assay was capable of single‐molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild‐type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.     

Comparison of Targeted Next‐Generation Sequencing (NGS) and Real‐Time PCR in the Detection of EGFR,KRAS, and BRAF Mutations on Formalin‐Fixed,Paraffin‐Embedded Tumor Material of Non‐Small Cell Lung Carcinoma—Superiority of NGS

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next‐generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real‐time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue specimens from 81 non‐small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real‐time PCR. Moreover, targeted NGS revealed seven nonsynonymous single‐nucleotide variations and one insertion‐deletion variation in EGFR not detectable by the real‐time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material. © 2012 Wiley Periodicals, Inc.     

Genetic analyses of isolated high‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) reveal paucity of alterations in TP53 and SMAD4

High‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) is the major precursor of pancreatic ductal adenocarcinoma (PDAC) and is an ideal target for early detection. To characterize pure HG‐PanIN, we analysed 23 isolated HG‐PanIN lesions occurring in the absence of PDAC. Whole‐exome sequencing of five of these HG‐PanIN lesions revealed a median of 33 somatic mutations per lesion, with a total of 318 mutated genes. Targeted next‐generation sequencing of 17 HG‐PanIN lesions identified KRAS mutations in 94% of the lesions. CDKN2A alterations occurred in six HG‐PanIN lesions, and RNF43 alterations in five. Mutations in TP53, GNAS, ARID1A, PIK3CA, and TGFBR2 were limited to one or two HG‐PanINs. No non‐synonymous mutations in SMAD4 were detected. Immunohistochemistry for p53 and SMAD4 proteins in 18 HG‐PanINs confirmed the paucity of alterations in these genes, with aberrant p53 labelling noted only in three lesions, two of which were found to be wild type in sequencing analyses. Sixteen adjacent LG‐PanIN lesions from ten patients were also sequenced using targeted sequencing. LG‐PanIN harboured KRAS mutations in 94% of the lesions; mutations in CDKN2A, TP53, and SMAD4 were not identified. These results suggest that inactivation of TP53 and SMAD4 are late genetic alterations, predominantly occurring in invasive PDAC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genotyping by Induced Förster Resonance Energy Transfer (iFRET) Mechanism and Simultaneous Mutation Scanning

Multiple genotyping techniques were developed on the basis of real‐time PCR. In this article, we present a genotyping technique extending the induced Förster resonance energy transfer (iFRET) mechanism in conjunction with simultaneous mutation scanning. Rapid, asymmetric PCR was performed with SYTO9, polymerase lacking 5′ → 3′ exonuclease activity, two primers, and a probe labeled with 6‐Carboxy‐X‐rhodamine. Six primers and probe sets were designed to detect germline mutations in BRCA1, a singular polymorphism in CCND1 and somatic mutations in KRAS and BRAF genes. The validation set consisted of 140 archival DNA samples from patients with previously confirmed BRCA1 mutation and 42 archival formalin‐fixed and paraffin‐embedded tissues from patients with colorectal cancer or malignant melanoma. BRCA1 and CCND1 genotyping by iFRET probe showed 100% agreement with Sanger sequencing and other validated methods. A combination of iFRET and high‐resolution melting analysis (HRMA) detected a spectrum of six different mutations in the KRAS gene and three different mutations in the BRAF gene. Due to an allele enrichment effect, the sensitivity of mutation detection of iFRET–HRMA genotyping and sequencing of iFRET–HRMA PCR products was significant, increasing from 1.5% to 6.2%, respectively. The technique presented in this article is a useful and cost‐effective method for the detection of both germline and somatic mutations.     

Novel intra‐genic large deletions of CTNNB1 gene identified in WT desmoid‐type fibromatosis

A wait and see approach for desmoid tumors (DT) has become part of the routine treatment strategy. However, predictive factors to select the risk of progressive disease are still lacking. A translational project was run in order to identify genomic signatures in patients enrolled within an Italian prospective observational study. Among 12 DT patients (10 CTNNB1‐mutated and 2 wild type) enrolled from our institution only two patients (17%) showed a progressive disease. Tumor biopsies were collected for whole exome sequencing. Overall, DT exhibited low somatic sequence mutation rate and no additional recurrent mutation was found. In the two wild type (WT) cases, two novel alterations were detected: a complex deletion of APC and a pathogenic mutation of LAMTOR2. Focusing on WT DT subtype, deep sequencing of CTNNB1, APC and LAMTOR2 was conducted on a retrospective series of 11 WT DT using a targeted approach. No other mutation of LAMTOR2 was detected, while APC was mutated in two cases. Low‐frequency (mean reads of 16%) CTNNB1 mutations were discovered in five samples (45%) and two novel intra‐genic deletions in CTNNB1 were detected in two cases. Both deletions and low frequency mutations of CTNNB1 were highly expressed. In conclusion, a minority of DT is WT for either CTNNB1, APC or any other gene involved in the WNT pathway. In this subgroup novel and hard to be detected molecular alterations in APC and CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases.     

Prognostic significance of alterations in KRAS isoforms KRAS‐4A/4B and KRAS mutations in colorectal carcinoma

Somatic KRAS mutation is an early well‐known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico‐pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real‐time PCR assay, to study the protein expression of KRAS4A and ‐4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0 . 0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5‐year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0 . 0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0 . 0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0 . 0234) and inversely correlated with p27kip1 protein (p = 0 . 0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Targeted next‐generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas

Intraductal neoplasms are important precursors to invasive pancreatic cancer and provide an opportunity to detect and treat pancreatic neoplasia before an invasive carcinoma develops. The diagnostic evaluation of these lesions is challenging, as diagnostic imaging and cytological sampling do not provide accurate information on lesion classification, the grade of dysplasia or the presence of invasion. Moreover, the molecular driver gene mutations of these precursor lesions have yet to be fully characterized. Fifty‐two intraductal papillary neoplasms, including 48 intraductal papillary mucinous neoplasms (IPMNs) and four intraductal tubulopapillary neoplasms (ITPNs), were subjected to the mutation assessment in 51 cancer‐associated genes, using ion torrent semiconductor‐based next‐generation sequencing. P16 and Smad4 immunohistochemistry was performed on 34 IPMNs and 17 IPMN‐associated carcinomas. At least one somatic mutation was observed in 46/48 (96%) IPMNs; 29 (60%) had multiple gene alterations. GNAS and/or KRAS mutations were found in 44/48 (92%) of IPMNs. GNAS was mutated in 38/48 (79%) IPMNs, KRAS in 24/48 (50%) and these mutations coexisted in 18/48 (37.5%) of IPMNs. RNF43 was the third most commonly mutated gene and was always associated with GNAS and/or KRAS mutations, as were virtually all the low‐frequency mutations found in other genes. Mutations in TP53 and BRAF genes (10% and 6%) were only observed in high‐grade IPMNs. P16 was lost in 7/34 IPMNs and 9/17 IPMN‐associated carcinomas; Smad4 was lost in 1/34 IPMNs and 5/17 IPMN‐associated carcinomas. In contrast to IPMNs, only one of four ITPNs had detectable driver gene (GNAS and NRAS) mutations. Deep sequencing DNA from seven cyst fluid aspirates identified 10 of the 13 mutations detected in their associated IPMN. Using next‐generation sequencing to detect cyst fluid mutations has the potential to improve the diagnostic and prognostic stratification of pancreatic cystic neoplasms. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Diagnostic approach to pancreatic tumors with the specimens of endoscopic ultrasound‐guided fine needle aspiration

Endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) has enabled clinicians to histologically diagnose pancreatic tumors. However, EUS‐FNA specimens often result in tiny fragmented tissues, so auxiliary utilities are necessary. Using immunostaining of CK7, CDX2, neuroendocrine markers and KRAS mutation analysis, we examined 57 FNA cell block sections and 61 surgically‐resected specimens (25 invasive ductal carcinomas, 25 endocrine tumors, and 11 acinar cell tumors). In the majority of the matched pairs, the diagnoses between EUS‐FNA and surgical specimens were concordant using the following criteria: neuroendocrine markers negative, CK7 positive, and mutated KRAS gene for invasive ductal carcinomas; neuroendocrine markers diffusely positive, CK7 and CDX2 negative, and wild‐type KRAS gene for well‐differentiated endocrine tumors; and neuroendocrine markers no more than focal positive, CK7 and CDX2 with various staining patterns, and wild‐type KRAS gene for acinar cell carcinomas. Expression of CK7 and/or CDX2 in addition to KRAS mutations were occasionally seen in endocrine carcinomas, but not in well‐differentiated endocrine tumors, suggesting that ductal differentiation in an endocrine tumor may be a predictor of aggressive disease. The usefulness of these markers was confirmed using 13 additional pancreatic tumors, prospectively. Although minimal in selection, these markers are helpful in making diagnosis from EUS‐FNA specimens of the major pancreatic tumors.     

Meier‐Gorlin syndrome

Infantile‐ and juvenile‐onset spinal cerebellar ataxia (SCA) is associated with expansion of 130 to more than 200 CAG‐repeats in the SCA2 and SCA7 genes. Routine clinical assays for SCA2 and SCA7, which use polymerase chain reaction (PCR) and denaturing PAGE (polyacrylamide gel electrophoresis), will not reliably detect such large expansions. An assay based on separation of PCR products on an agarose gel, blotting, and hybridization with a (CAG)6 oligonucleotide probe was used to test DNA from individuals more than 10 years of age who had a possible diagnosis of SCA. Among 25 cases, the PCR‐blot assay confirmed the presence of SCA2 expansions between 230 and 500 repeats in four unrelated individuals, but did not detect any cases of extreme expansion in the SCA7 gene. The PCR‐blot assay provides reliable detection of extreme expansion mutations. Routine incorporation of this assay in clinical laboratories may reveal that infantile–juvenile forms of SCA2 and SCA7 are more prevalent than previously recognized. © 2002 Wiley‐Liss, Inc.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue

KRAS, BRAF, and PIK3CA mutation testing before administration of anti-epidermal growth factor receptor therapy of metastatic colorectal cancer (CRC) has become important. However, considerable uncertainty exists regarding which detection method can be applied in a reproducible, sensitive, and simple manner in the routine diagnostic setting. We compared the detection rates of KRAS, BRAF, and PIK3CA mutations in 92 routine formalin-fixed, paraffin-embedded CRC specimens by 2 discrete methods: direct sequencing and peptide nucleic acid (PNA)-mediated PCR. The detection rates for KRAS, BRAF, and PIK3CA mutations by direct sequencing were 20.7%, 3.3%, and 1.1%, respectively. PNA-mediated PCR clamping significantly increased the percentages of KRAS, BRAF, and PIK3CA mutations by up to 7.6%, 1.2%, and 5.4%, respectively, compared to the detection rate of regular PCR followed by direct sequencing (p = 0.039, p = 0.250, and p = 0.031, respectively). The tumor volume of discordant cases was not significantly different from concordant cases (56.2 ± 28.7% vs. 67.6 ± 17.9%, p = 0.41), which implies that there is a minor population of mutant alleles in the heterogeneous tumor population. The PNA-mediated PCR clamping method is highly sensitive and is efficiently applicable to the detection of KRAS, BRAF, and PIK3CA mutations in a clinical setting.     

MCM3AP and POMP Mutations Cause a DNA‐Repair and DNA‐Damage‐Signaling Defect in an Immunodeficient Child

Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center‐associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole‐exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B‐cell maturation by nuclear targeting of activation‐induced cytidine deaminase (AID) and to control AID‐dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF‐κB signaling. Patient‐derived cells were characterized by impaired homologous recombination, moderate radio‐ and cross‐linker sensitivity associated with accumulation of damage, impaired DNA damage‐induced NF‐κB signaling, and reduced nuclear AID levels. Complementation by wild‐type (WT)‐GANP normalized DNA repair and WT‐POMP rescued defective NF‐κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.     

The extended spectrum of RAS‐MAPK pathway mutations in colorectal cancer

Current clinical guidelines recommend mutation analysis for select codons in KRAS and NRAS exons 2, 3, and 4 and BRAF V600E to guide therapy selection and prognostic stratification in advanced colorectal cancer. This study evaluates the impact of extended molecular testing on the detection of RAS‐MAPK pathway mutations. Panel next‐generation sequencing results of colorectal cancer specimens from 5795 individuals from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) were included. Mutations in RAS‐MAPK pathway genes were analyzed and functionally annotated. Colorectal cancers had recurrent pathogenic pathway activating mutations in KRAS (44%), NRAS (4%), HRAS (<1%), BRAF (10%), MAP2K1 (1%), RAF1 (<1%), and PTPN11 (<1%). The proportion of colorectal cancers with pathogenic RAS pathway mutations was 37% when only KRAS codon 12 and 13 mutations were considered, 46% when also including select KRAS and NRAS exons 2, 3, and 4 mutations, 53% when including BRAF V600E mutations, and 56% when including all pathogenic mutations. Panel next‐generation sequencing testing identifies additional RAS‐MAPK pathway driver mutations beyond current guideline recommendations. These mutations have potential implications in treatment selection for patients with advanced colorectal cancer.     

A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA

Pasanen T, Korkeila M, Mero S, Tarkka E, Piiparinen H, Vuopio‐Varkila J, Vaara M, Tissari P. A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA. APMIS 2010; 118: 74–80. We analyzed the performance of a selective enrichment broth combined with Taqman‐based real‐time duplex nuc‐mecA‐PCR to expedite the screening of methicillin‐resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4–256 μg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc‐mecA‐PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24–48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.     

Frequent lack of GNAS mutations in colorectal adenocarcinoma associated with GNAS‐mutated villous adenoma

Colorectal villous adenoma is thought to be associated with a high risk of progression to adenocarcinoma. To better characterize the genetic alterations involved in colorectal carcinogenesis related to villous adenoma, we analyzed mutations in APC, BRAF, KRAS, TP53, and GNAS in 12 colorectal adenocarcinomas associated with villous adenomas. APC, KRAS, and BRAF mutations were identified in five, 11, and one lesion, respectively, and most of these mutations were shared between the villous adenoma and the adenocarcinoma components in the respective lesions, except in one lesion with APC mutations and in two lesions with KRAS mutations. TP53 mutations were observed exclusively in four adenocarcinoma components, consistent with their role in the progression from adenoma to adenocarcinoma. Activating GNAS mutations were found in nine villous adenomas; however, unexpectedly, these mutations were shared only in three associated adenocarcinomas. Notably, all six adenocarcinomas with discordant GNAS mutation statuses were nonmucinous type, whereas all the other adenocarcinomas, including three adenocarcinomas associated with GNAS wild‐type villous adenomas, were mucinous type. The current study suggests that GNAS‐mutated villous adenomas may not necessarily be direct precursors of associated adenocarcinomas. At the same time, our observations support the role of activating GNAS mutations in increased mucin production in colorectal neoplasms. © 2014 Wiley Periodicals, Inc.