Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues

Dufort, Sandrine; Richard, Marie‐Jeanne; Fraipont, Florence de

doi:10.1016/j.ab.2009.05.027

Cited by 65 publications

(48 citation statements)

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“…This recommendation was based on a number of clinical studies, performed following the initial identification of specific mutations in the EGFR tyrosine kinase domain in 2004 (15,16), including a 2009 IPASS report (1,2,18,19). Previously, EGFR mutation testing was recommended prior to systemic chemotherapy for all patients with advanced NSCLC excluding SCC (20).…”

Section: Discussionmentioning

confidence: 99%

“…The technique is a simple, robust, fast and sensitive method that is also cost-effective. There are a number of studies that have applied the pyrosequencing technique to analyze genetic variations, including k-ras and BRAF (15)(16)(17). However, there are currently no studies on the application of pyrosequencing for the detection of EGFR mutations in clinical practice.…”

Section: Introductionmentioning

confidence: 99%

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Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

Kim

et al. 2012

Oncology Letters

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Abstract. Direct sequencing is the standard method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer, however, its relatively low sensitivity limits its clinical use. Pyrosequencing is a bioluminometric, real-time non-electrophoretic DNA sequencing technique with a number of advantages compared with direct sequencing, including higher sensitivity, speed, automation and costeffectiveness. Clinical specimens from 202 lung cancer patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21 using the pyrosequencing method following genomic DNA extraction from paraffin-embedded tissue specimens. All clinical data and tumor specimens were obtained from the Konkuk University Hospital (Korea) between July 2006 and December 2008. The results and clinical responses to EGFR-tyrosine kinase inhibitors (TKIs) were compared. Overall, EGFR mutation-positive rate was 26.7% (54/202). Activating EGFR mutations were observed more frequently in female (52.1 vs. 13.0%), non-smoking (47.8 vs. 15.8%) and adenocarcinoma (35.2 vs. 5.2%) patients. However, significant numbers of EGFR mutation-positive patients were identified as male, former or current smokers and non-adenocarcinoma patients. The combinations of favorable clinicopathological factors, including female, non-smoking and adenocarcinoma, were not identified to significantly increase the positive EGFR mutation rate (female, 52.1%; female and non-smoker, 52.6%; female, non-smoker and adenocarcinoma, 51.9%). The present findings indicate that EGFR mutation analysis is a highly useful method for the prediction of response to EGFR-TKI and the use of favorable clinicopathological factors to perform this analysis is not suitable. Exon 19 deletion was the most common mutation (63.6%) and exon 21 L858R substitution was measured at 32.7%. The exon 20 T790M mutation was identified in 1 patient prior to EGFR-TKI treatment. EGFR mutation status is associated with response to EGFR-TKI and the overall response rate in patients who have the activating EGFR mutation was 82.4 vs. 5.9% in patients with a wild-type EGFR. The present study demonstrates that EGFR mutations analyzed by the pyrosequencing method are well correlated with clinicopathological parameters and that this method may be useful in the clinical practice.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

Kim

et al. 2012

Oncology Letters

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“…The limit for mutant allele detection ranges between 2.5 and 5% with the pyrosequencing technique, compared with the 10-20% obtained with direct sequencing. [27][28][29] As a positive control, we used a colon tumor harboring the V600E mutation, which was detected by both direct sequencing and pyrosequencing ( Figure 2). Direct sequencing was performed in all prostate tumors and in 17 of the bladder tumors from G2, and we did not detect any mutation.…”

Section: Braf Mutational Analysis By Direct Sequencing and Pyrosequenmentioning

confidence: 99%

PI3K signaling pathway is activated by PIK3CA mRNA overexpression and copy gain in prostate tumors, but PIK3CA, BRAF, KRAS and AKT1 mutations are infrequent events

et al. 2011

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The phosphatidylinositol 3-kinase (PI3K)-AKT and RAS-MAPK pathways are deregulated in a wide range of human cancers by gain or loss of function in several of their components. Our purpose has been to identify genetic alterations in members of these pathways in prostate cancer. A total of 102 prostate tumors, 79 from prostate cancer alone (group G1) and 23 from bladder and prostate cancer patients (G2), are the subject of this study. In 20 of these 23, the bladder tumors were also analyzed. PIK3CA, KRAS, BRAF and AKT1 mutations were analyzed by direct sequencing, and BRAF also by pyrosequencing. PIK3CA quantitative mRNA expression and fluorescence in situ hybridization (FISH) gains were tested in 25 and 32 prostate tumors from both groups (G1 and G2), respectively. Immunohistochemistry for pAKT was performed in 55 prostate tumors. Of 25 prostate tumors, 10 (40%) had PIK3CA mRNA overexpression that was statistically associated with Gleason score Z7 (P ¼ 0.018). PIK3CA copy gain was detected in 9 of 32 (28%) prostate tumors. Of 20 bladder tumors, 3 (15%) displayed mutations in PIK3CA, KRAS and AKT1, the corresponding prostate tumors being wt. We also detected a previously not reported PIK3CA polymorphism (IVS9 þ 91) in two prostate tumors. In all, 56% of prostate tumors overexpressed pAKT. There is a statistical association (Po0.0001) of strong pAKT immunostaining with high Gleason score, and with PIK3CA alterations (mRNA overexpression and/or FISH gains). PIK3CA gene is deregulated by mRNA overexpression and DNA gain in B40 and 28% of prostate tumors, respectively. High-grade prostate tumors are associated with PIK3CA mRNA overexpression, but not with FISH status. PIK3CA, BRAF, KRAS and AKT1 mutations are very infrequent events in prostate tumors. However, PI3K signaling pathway is activated by PIK3CA FISH gain and/or mRNA overexpression, leading to an increased pAKT protein expression.

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“…Most studies have demonstrated that the detection limit of pyrosequencing is approximately 1.25% to 6% mutant DNA. 26,30,32,[34][35][36] The increased sensitivity of pyrosequencing is especially useful where KRAS mutant DNA is likely to be heavily diluted in wild-type DNA, such as CRC with microsatellite instability and significant inflammatory infiltrates or desmoplastic pancreatic tumors with few malignant cells and abundant stromal cells. 35 Additionally, because the detection of each pyrosequencing light emission is more uniform than the detection of different fluorophores in Sanger sequencing, interpreting low-level signals is less subjective in pyrosequencing than in Sanger sequencing.…”

Section: Sequencing Methods In Kras Testingmentioning

confidence: 99%

KRAS Testing: A Tool for the Implementation of Personalized Medicine

et al. 2012

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Activating point mutations in codons 12, 13, and 61 of the KRAS proto-oncogene are common in colorectal, non-small cell lung, pancreatic, and thyroid cancers. Constitutively activated KRAS mutations are strongly associated with a resistance to anti-epidermal growth factor receptor (EGFR) therapies, such as panitumumab and cetuximab used for treating metastatic colorectal carcinoma and EGFR tyrosine inhibitors used for advanced non-small cell lung cancers. Since anti-EGFR therapies are costly and may exert deleterious effects on individuals without activating mutations, KRAS mutation testing is recommended prior to the initiation of anti-EGFR therapy for these malignancies. The goal of this review is to summarize the KRAS mutation testing methods. Testing is now routinely requested in the clinical practice to provide data to assign the most appropriate anticancer chemotherapy for each given patient. Review of the most relevant literature was performed. Several areas were considered: ordering of the test, selection of the sample to be tested, and review of the testing methodologies. We found that several different methods are used for clinical KRAS mutation testing. Each of the methodologies is described, and information is provided about their performance, cost, turnaround times, detection limits, sensitivities, and specificities. We also provided "tips" for the appropriate selection and preparation of the sample to be tested. This is an important aspect of KRAS testing for clinical use, as the results of the test will affect clinical decisions with consequences for the patient.

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Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues

Cited by 65 publications

References 10 publications

Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

PI3K signaling pathway is activated by PIK3CA mRNA overexpression and copy gain in prostate tumors, but PIK3CA, BRAF, KRAS and AKT1 mutations are infrequent events

KRAS Testing: A Tool for the Implementation of Personalized Medicine

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