Comparison of<i>KRAS</i>Mutation Tests in Colorectal Cancer Patients

Hancer, Veysel Sabri; Buyukdogan, Murat; Türkmen, İlknur; Başsüllü, Nuray; Altuğ, Tuncay; Diz‐Küçükkaya, Reyhan; Bulbul-Dogusoy, Gulen; Demir, Gökhan

doi:10.1089/gtmb.2011.0027

Cited by 12 publications

(9 citation statements)

References 16 publications

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“…EntroGen provides a commercially available real‐time PCR kit for KRAS and BRAF mutation detection, which uses allele‐specific primers without employing a pre‐amplification step. For the EntroGen assay, 50 ng gDNA from tissues was amplified with gene specific primers and the KRAS codons 12, 13 and 61 and BRAF V600E mutations were detected using allele‐specific probes (EntroGen), as described previously . For CTC samples, a nested PCR was first performed on 10 ng gDNA extracted from CellSearch‐enriched CTCs, followed by a second PCR with the allele‐specific probes as described before …”

Section: Methodsmentioning

confidence: 99%

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Mostert

Jiang

Sieuwerts

et al. 2013

Intl Journal of Cancer

130

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Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch TM . DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic TM ), real-time PCR (EntroGen TM ) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior.

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Section: Methodsmentioning

confidence: 99%

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

Mostert

Jiang

Sieuwerts

et al. 2013

Intl Journal of Cancer

130

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“…In fact, in a recent review of 578 cases referred to our laboratory, we found that 528 (91.3%) specimens contained more than 30% of neoplastic cells 61. Besides the percentage of neoplastic cells, the limit of detection (LOD) of this technique partly depends on the specific mutation and on the experience of the person interpreting the data 62. When a KRAS mutation is identified by direct sequencing, both mutant and wild-type alleles are seen on the sequencing electropherograms.…”

Section: The Methodsmentioning

confidence: 99%

KRAS testing in metastatic colorectal carcinoma: challenges, controversies, breakthroughs and beyond

et al. 2013

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Metastatic colorectal cancer harbouring a mutation in codon 12 or 13 of the KRAS gene does not benefit from therapy with antibodies targeting the epidermal growth factor receptor (EGFR). The implementation of community KRAS testing is generating a rapid flow of new data that have implications for the pathologist and testing guidelines besides the physician. Therefore, it seems timely to draw together the threads of this large body of information in order that pathologists can be knowledgeable partners in the multidisciplinary process of targeted cancer therapy and to help refine current testing guidelines. This review addresses (1) the most relevant methodological and technical aspects of KRAS testing in terms of sample site (primary/metastatic), test specimens (resection/biopsy/cytology) and the diverse molecular methods available; (2) the issues related to daily practice, namely, the timing of the test, its turnaround time and the quality control procedures; and (3) the evidence related to the relationship between KRAS genetic intratumoural heterogeneity, clinical sensitivity of mutational detection tools and anti-EGFR treatment outcome. Hopefully, in the near future, elucidation of the potential of biomarker panels and of the mechanisms underlying primary and acquired resistance to anti-EGFR therapy will refine even further personalised treatment regimens for patients with metastatic colorectal cancer.

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“…However, this assay requires a minimum of 30-50% tumor cells to successfully detect a mutation. 2, 10, 15, 19 Other methods in use, such as pyrosequencing or high resolution melt analysis, have higher sensitivities and require lower percentages of malignant DNA to successfully detect a mutation. 4, 9, 20 Nonetheless, all methods, by definition, have a limit of analytic sensitivity which represents the minimum percentage of tumor cells in a tissue sample required to correctly identify a mutation.…”

Section: Introductionmentioning

confidence: 99%

“…4, 9, 20 Nonetheless, all methods, by definition, have a limit of analytic sensitivity which represents the minimum percentage of tumor cells in a tissue sample required to correctly identify a mutation. 10 …”

Section: Introductionmentioning

confidence: 99%

Automated Objective Determination of Percentage of Malignant Nuclei for Mutation Testing

Viray

Coulter

et al. 2014

Applied Immunohistochemistry &Amp; Molecular Morphology

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Detection of DNA mutations in tumor tissue can be a critical companion diagnostic test prior to prescription of a targeted therapy. Each method for detection of these mutations is associated with an analytic sensitivity that is a function of the percentage of tumor cells present in the specimen. Currently, tumor cell percentage is visually estimated resulting in an ordinal and highly variant result for a biologically continuous variable. We proposed that this aspect of DNA mutation testing could be standardized by developing a computer algorithm capable of accurately determining the percentage of malignant nuclei in an image of a hematoxylin and eosin (H&E) stained tissue. Using INform software (Caliper Life Sciences/Perkin Elmer), we developed an algorithm, to calculate the percentage of malignant cells in histologic specimens of colon adenocarcinoma. A criterion standard was established by manually counting malignant and benign nuclei. Three pathologists also estimated the percentage of malignant nuclei in each image. Algorithm #9 had a median deviation from the criterion standard of 5.4% on the training set and 6.2% on the validation set. Compared to pathologist estimation, Algorithm #9 showed a similar ability to determine percentage of malignant nuclei. This method represents a potential future tool to assist in determining the percent of malignant nuclei present in a tissue section. Further validation of this algorithm or an improved algorithm may have value to more accurately assess percentage of malignant cells for companion diagnostic mutation testing.

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Comparison ofKRASMutation Tests in Colorectal Cancer Patients

Cited by 12 publications

References 16 publications

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue

KRAS testing in metastatic colorectal carcinoma: challenges, controversies, breakthroughs and beyond

Automated Objective Determination of Percentage of Malignant Nuclei for Mutation Testing

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