High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

Do, Hongdo; Krypuy, Michael; Mitchell, Paul; Fox, Stephen B.; Dobrovic, Alexander

doi:10.1186/1471-2407-8-142

Cited by 185 publications

(187 citation statements)

References 52 publications

(64 reference statements)

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“…32 Performed before DNA sequencing, high-resolution melting is very attractive because the analysis of the real-time amplification curves allows (i) the calculation of the Ct value that is directly related to the amount of amplifiable DNA and (ii) the identification of heterozygous genetic changes even in samples containing only 10% of mutated cells. 33 In our series of DNA extracted from the manually dissected specimens, all Ct values were o30, whereas higher values were obtained for the paired laser capture microdissected samples (data not shown), suggesting a negative impact of laser capture microdissection on DNA. Concerning the allele-specific PCR assay, the first kit approved for diagnostic purposes was the TheraScreen kit that can detect as few as 1% of mutated alleles in a wild-type background and is a simple 'mix-andmeasure' test.…”

Section: Discussionmentioning

confidence: 69%

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with antiepidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allelespecific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

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Section: Discussionmentioning

confidence: 69%

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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“…The sensitivity of our assays is 10 and 1% of mutant DNA for hybridisation-probe assay and for clamped-probe assay, respectively, and might correspond to a more sensitive test, applicable for routine purposes without macro or micro dissection. Another new rapid method for mutated EGFR and K-Ras detection by high resolution melting analysis (HRM) has been recently reported (Do et al, 2008). Even HRM is a promising method, it can be compromised by a low proportion of tumour cells in the analysed sample and by the difficulty to detect homozygous mutations, as the sensitivity of K-Ras mutations detection was only 5 -10%.…”

Section: Discussionmentioning

confidence: 99%

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

et al. 2009

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Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P <0.005), among which five in samples with low tumour cellularity. In adenocarcinoma, K-Ras mutation frequency increased from 7 out of 55 (13%) by direct sequencing to 15 out of 55 (27%) by clamped-PCR ( P <0.005). K-Ras mutations detected by these sensitive techniques lost its prognostic value. In conclusion, a rapid and sensitive PCR-clamping test avoiding macro or micro dissection could be proposed in routine analysis especially for NSCLC samples with low percentage of tumour cells such as bronchial biopsies or after neoadjuvant chemotherapy.

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“…An example of this is the development of KRAS mutation status testing in patients' tumors in order to decide about the appropriate treatment [6,[12][13][14][15][16][17]. The optimal use of EGFR-targeted therapies requires accurate KRAS mutation testing with specific guideline recommendations and a European quality assurance program [18].…”

Section: Resultsmentioning

confidence: 99%

“…The relatively low sensitivity of sequencing analysis in somatic mutation detection [5] is a particular problem because tumor material often contains only a small proportion of neoplastic cells. Therefore is it necessary to use other approaches for detection of KRAS mutation status in tumors [6].…”

Section: Introductionmentioning

confidence: 99%

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients

Zavodna

Konečný²,

Krivulcik³

et al. 2009

neo

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colorectal carcinoma (crc) represents a serious problem worldwide: in the slovak republic are diagnosed about 2600 new crc cases annually and its incidence is increasing. colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of crc or changing the existing therapeutic procedures. recent progresses have been made in understanding of eGFr pathway involved in crc carcinogenesis, especially the role of ras protein. mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (eGFr) inhibitor therapies, such as panitumumab and cetuximab. consequently, screening for KRAS mutations status may be used as a prognostic marker, because the crc patients with KRAS positive tumors have a worse prognosis.The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFpe) tissues dNa. We applied real Time pcr analysis (Therascreen Kras mutation Test Kit) and sequencing analysis (optimised for the analysis of FFpe tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of dNa isolated from 25 colorectal FFpe tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). our results demonstrate that the real Time pcr analysis can be used for detection of somatic KRAS mutations in FFpe clinical samples. however, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for KRAS mutations status screening, but with care of method sensitivity.

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High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

Cited by 185 publications

References 52 publications

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Detection of K-Ras mutations in tumour samples of patients with non-small cell lung cancer using PNA-mediated PCR clamping

Genetic analysis of KRAS mutation status in metastatic colorectal cancer patients

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