Authors’ Reply to Uguen: “Comparison of Five Different Assays for the Detection of BRAF Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Metastatic Melanoma”

e14147 Background: In patients with metastatic colorectal cancer (mCRC), activating mutations within KRAS, which result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC have been significantly associated with lack of response to cetuximab or panitumumab therapy. Although current guidelines recommend testing for frequent KRAS codons 12/13 mutations, emerging data indicate that additional KRAS and BRAF mutations are also predictive of non-responsiveness to anti-EGFR antibodies in mCRC. This study is aimed to analyze the prevalence of low-penetrance KRAS and BRAF V600 mutations in caucasian mCRC population. Methods: A two-institution retrospective cohort of 1,238 consecutive KRAS wild type mCRC patients previously studied for 7 mutations in codons 12/13 (G12D, G12A, G12V, G12S, G12R, G12C and G13D) by the CE-IVD marked ARMS-scorpion real-time polymerase chain reaction PCR (Therascreen, Qiagen) was assayed by the diagnostic TaqMelt PCR assay cobas KRAS mutation and cobas BRAF V600 mutation tests (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61 (including G12F, G13C, G13R, G13S, G13A, G13V, G13I, Q61H, Q61K, Q61R, Q61L, Q61E and Q61P) and BRAF V600 (V600E, V600K and V600D) mutations. An additional cohort of 146 KRAS mutated patients by ARMS-scorpion PCR was studied. DNA was obtained by cobas DNA preparation kit from one single 5µm formalin-fixed paraffin-embedded tissue section. Results: In all samples, sufficient DNA was obtained for KRAS and BRAF mutational studies. Among 1238 KRAS codons 12/13 wild-type patients by ARMS-scorpion PCR,166 (13.4%) showed KRAS mutations, 117 (9.5%) in codons 12/13, and 49 (4%) in codon 61. BRAF V600 mutations were detected in 9% cases. In ARMS-scorpion PCR KRAS mutated patients, mutations were confirmed by cobas in all cases. Conclusions: The cobas mutation tests are robust and reproducible assays that, 1) detects a higher incidence (13.4%) of mutations in codons 12, 13, and 61 of the KRAS gene in wild-type mCRC population, 2) a relevant rate of BRAF mutations is present in the same population, and 3) requires a very small amount of tissue.

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Identification of differentially expressed genes in matched formalin-fixed paraffin-embedded primary and metastatic melanoma tumor pairs

Pigment Cell & Melanoma Research ◽

10.1111/j.1755-148x.2011.00965.x ◽

2012 ◽

Vol 25 (2) ◽

pp. 284-286 ◽

Cited By ~ 4

Author(s):

Rosalyn Jewell ◽

Angana Mitra ◽

Caroline Conway ◽

James Iremonger ◽

Christy Walker ◽

...

Keyword(s):

Metastatic Melanoma ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Melanoma Tumor ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Integrated routine workflow using next-generation sequencing and a fully-automated platform for the detection of KRAS, NRAS and BRAF mutations in formalin-fixed paraffin embedded samples with poor DNA quality in patients with colorectal carcinoma

PLoS ONE ◽

10.1371/journal.pone.0212801 ◽

2019 ◽

Vol 14 (2) ◽

pp. e0212801 ◽

Cited By ~ 8

Author(s):

Claire Franczak ◽

Ludovic Dubouis ◽

Pauline Gilson ◽

Marie Husson ◽

Marie Rouyer ◽

...

Keyword(s):

Next Generation Sequencing ◽

Colorectal Carcinoma ◽

Next Generation ◽

Braf Mutations ◽

Dna Quality ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Automated Platform ◽

Generation Sequencing ◽

Formalin Fixed

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Automated PCR detection of BRAF mutations in colorectal adenocarcinoma: a diagnostic test accuracy study

Journal of Clinical Pathology ◽

10.1136/jclinpath-2015-203345 ◽

2015 ◽

Vol 69 (5) ◽

pp. 398-402 ◽

Cited By ~ 22

Author(s):

Richard Colling ◽

Lai Mun Wang ◽

Elizabeth Soilleux

Keyword(s):

Digital Pcr ◽

Turnaround Time ◽

Test Accuracy ◽

Braf Mutations ◽

System A ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Accuracy Study ◽

Automated Platform ◽

Formalin Fixed

BackgroundTesting for BRAF mutations in colorectal carcinoma (CRC) is important in the screening pathway for Lynch syndrome and is of prognostic value to guide management. This is a diagnostic accuracy study of the Idylla system, a novel and automated alternative PCR system.Methods100 consecutive formalin-fixed, paraffin-embedded CRC resection cases were tested for BRAF mutations using the Idylla automated platform and compared with standard (Cobas) PCR.ResultsThe sensitivity of the Idylla BRAF test was 100% and the specificity was 96%. Only one discordant Idylla positive/standard PCR negative result occurred and on Droplet Digital PCR demonstrated a mutation not identified by traditional PCR in this case.ConclusionThis study has validated the Idylla system for BRAF testing in CRC and demonstrated a possibly greater sensitivity, in addition to cost effectiveness and shorter turnaround time, when compared with standard PCR.

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Splenic B-Cell Lymphoma/Leukemia, Unclassifiable in Japan

Blood ◽

10.1182/blood-2018-99-113239 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5301-5301

Author(s):

Takaharu Suzuki ◽

Hiroaki Miyoshi ◽

Keisuke Kawamoto ◽

Joji Shimono ◽

Jun Takizawa ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cyclin D3 ◽

Hairy Cell ◽

Cell Leukemia ◽

Braf Mutations ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Abstract Introduction Splenic B-cell lymphoma/leukemia, unclassifiable is defined as being unable to be classified as any other B-cell neoplasm infiltrating the spleen (World Health Organization Classification of Tumors of Haematopoietic and Lymphoid Tissue 2017). In splenic B-cell lymphoma/leukemia, unclassifiable, splenic diffuse red pulp small cell lymphoma (SDRPL) and hairy cell leukemia variant (HCL-v) are provisionally defined. The incidence of these diseases is rare. It has been reported that they account for 9% of splenic B-cell lymphomas (Haematologica 2010;95:1122-1129). There are no reports of histopathological examinations or BRAF mutations (V600E) in Japan. It has been shown that BRAF mutations (V600E) are found in all patients with Hairy cell leukemia-classical (HCL-c) (N Engl J Med. 2011 364: 2305-2315). This study aimed to clarify the pathological features of splenic B-cell lymphoma/leukemia, unclassifiable. Methods We analyzed 5 cases of suspected splenic B-cell lymphoma/leukemia, unclassifiable in terms of splenectomy. Five cases include 0 cases of SDRPL, 2 cases of HCL-v, and 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable not applicable to either case. We analyzed patients undergoing splenectomy who were pathologically diagnosed at the Kurume University Pathology Course between July 2012 and May 2017. Splenic B-cell lymphoma/leukemia, unclassifiable was suspected in 5 cases. From the findings of peripheral blood, bone marrow, and BRAF mutation (V600E) analyses, 2 patients were diagnosed with HCL-v. The other 3 patients were diagnosed with splenic B-cell lymphoma/leukemia, unclassifiable. Pathological examinations were performed in these cases. Marker expression analysis of the B-cell line was performed using immunohistochemical (IHC) staining and flow cytometry (FCM). IHC staining was performed on formalin-fixed paraffin-embedded samples. For FCM analysis, we confirmed the clonality of B cells and analyzed their expression. A positive judgment was made at 30% or greater. For BRAF mutation (V600E) analysis, deoxyribonucleic acid was extracted from formalin-fixed paraffin-embedded samples and performed using the Sanger sequencing method. Results The results of the analysis are shown in Table 1. HCL-v cases included 1 man and 1 woman, and the age was 62-71 years old. Splenic B-cell lymphoma/leukemia, unclassifiable cases included 2 men and 1 woman, and the age was 56-66 years. Splenic B-cell lymphoma/leukemia, unclassifiable cases involved bone marrow infiltration of B-cell lymphoma in 2 cases. In 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable, there were no cases in which hairy cells were recognized in the peripheral blood. In all cases of splenic B-cell lymphoma/leukemia, unclassifiable, BRAF mutations (V600E) were not observed. All cases of splenic B-cell lymphoma/leukemia, unclassifiable and HCL-v tested positive for cluster of differentiation (CD)20, CD19, and B-cell lymphoma (Bcl-2) and tested negative for CD103 and CD10. All HCL-v cases and 2 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for cyclin D3. One case of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for CD5, but negative for lymphoid enhancer-binding factor 1 (LEF1), cyclin D1, and Sox11. All other cases tested negative for CD5, LEF1, cyclin D1, and Sox11. One of two cases of HCL-v and 2 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for CD11c. All HCL-v cases tested negative and 1 of 3 cases of splenic B-cell lymphoma/leukemia, unclassifiable tested positive for Bcl-6. IHC staining was performed because FCM was not performed in 1 case of HCL-v. There was no obvious bias in κ and λ. One patient with HCL-v who underwent FCM was κ-positive. Splenic B-cell lymphoma/leukemia, unclassifiable was κ-positive in all cases. Conclusions In this analysis, splenic B-cell lymphoma/leukemia, unclassifiable did not involve BRAF mutations (V600E) in all cases. It has been reported that cyclin D3 is highly expressed in SDRPL. Splenic B-cell lymphoma/leukemia, unclassifiable and HCL-v confirmed the same tendency (Blood. 2017; 129:1042-1045). Further molecular biological analysis for splenic B-cell lymphoma/leukemia, unclassifiable is desired. Disclosures No relevant conflicts of interest to declare.

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