Limits...
KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis.

Deschoolmeester V, Boeckx C, Baay M, Weyler J, Wuyts W, Van Marck E, Peeters M, Lardon F, Vermorken JB - Br. J. Cancer (2010)

Bottom Line: Direct sequencing was used to confirm and characterise HRMA results.Kaplan-Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation.HRMA was found to be a valid screening method for KRAS mutation detection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp/Antwerp University Hospital, Wilrijk 2610, Belgium. vanessa.deschoolmeester@ua.ac.be

ABSTRACT

Background: The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.

Methods: After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.

Results: HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan-Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.

Conclusions: HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients.

Show MeSH

Related in: MedlinePlus

Inter- and intra-variation analysis in four different cell lines (three wild type: ECV304, CAL27 and SQD9; one mutant: A549) on 3 consecutive days (A: day 1, B: day 2 and C: day 3). Within each replicate of the same experiment, the mutant cell line (A549) is clearly discriminated from the three wild-type cell lines.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2990591&req=5

fig4: Inter- and intra-variation analysis in four different cell lines (three wild type: ECV304, CAL27 and SQD9; one mutant: A549) on 3 consecutive days (A: day 1, B: day 2 and C: day 3). Within each replicate of the same experiment, the mutant cell line (A549) is clearly discriminated from the three wild-type cell lines.

Mentions: Cancer cell lines with or without a known KRAS mutation (Table 1) were first used to test the HRMA methodology. The HRMA was able to discriminate between the wt DNA and the different mutations present in the mutant cell line DNA. As seen in the melting (Figure 2A) and derivative plot (Figure 2B), HCT116, MDA-MB-231 and NCI-H292 showed typical heteroduplex melting patterns and were readily distinguishable from the wt cell lines CAL27 and ECV304. The lung cancer cell line A549 has a homozygous mutation (Table 1) and, as expected, it showed a similar shape to the wt pattern but with earlier melting of the amplified product, which is consistent with the lower thermal stability of AT base pairs relative to GC base pairs. Furthermore, dideoxy sequencing analysis confirmed the HRMA results in all cases (data not shown). In addition, an inter- and intra-variation analysis was performed on three different days in four different cell lines (A549, CAL27, ECV304 and SQD9). The results showed that inter- and intra-variation was present and could hamper the interpretation of the results (Figure 3). However, within one experiment, it was still possible to discriminate between DNA of the mutant cell line and that of the three wt cell lines (Figure 4). These results indicate that it is necessary to include positive (mutant) and negative (wt) controls in each experiment. In addition, owing to the inter-variation, it is inadvisable to compare plots generated on different days.


KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis.

Deschoolmeester V, Boeckx C, Baay M, Weyler J, Wuyts W, Van Marck E, Peeters M, Lardon F, Vermorken JB - Br. J. Cancer (2010)

Inter- and intra-variation analysis in four different cell lines (three wild type: ECV304, CAL27 and SQD9; one mutant: A549) on 3 consecutive days (A: day 1, B: day 2 and C: day 3). Within each replicate of the same experiment, the mutant cell line (A549) is clearly discriminated from the three wild-type cell lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990591&req=5

fig4: Inter- and intra-variation analysis in four different cell lines (three wild type: ECV304, CAL27 and SQD9; one mutant: A549) on 3 consecutive days (A: day 1, B: day 2 and C: day 3). Within each replicate of the same experiment, the mutant cell line (A549) is clearly discriminated from the three wild-type cell lines.
Mentions: Cancer cell lines with or without a known KRAS mutation (Table 1) were first used to test the HRMA methodology. The HRMA was able to discriminate between the wt DNA and the different mutations present in the mutant cell line DNA. As seen in the melting (Figure 2A) and derivative plot (Figure 2B), HCT116, MDA-MB-231 and NCI-H292 showed typical heteroduplex melting patterns and were readily distinguishable from the wt cell lines CAL27 and ECV304. The lung cancer cell line A549 has a homozygous mutation (Table 1) and, as expected, it showed a similar shape to the wt pattern but with earlier melting of the amplified product, which is consistent with the lower thermal stability of AT base pairs relative to GC base pairs. Furthermore, dideoxy sequencing analysis confirmed the HRMA results in all cases (data not shown). In addition, an inter- and intra-variation analysis was performed on three different days in four different cell lines (A549, CAL27, ECV304 and SQD9). The results showed that inter- and intra-variation was present and could hamper the interpretation of the results (Figure 3). However, within one experiment, it was still possible to discriminate between DNA of the mutant cell line and that of the three wt cell lines (Figure 4). These results indicate that it is necessary to include positive (mutant) and negative (wt) controls in each experiment. In addition, owing to the inter-variation, it is inadvisable to compare plots generated on different days.

Bottom Line: Direct sequencing was used to confirm and characterise HRMA results.Kaplan-Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation.HRMA was found to be a valid screening method for KRAS mutation detection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp/Antwerp University Hospital, Wilrijk 2610, Belgium. vanessa.deschoolmeester@ua.ac.be

ABSTRACT

Background: The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.

Methods: After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.

Results: HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan-Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.

Conclusions: HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients.

Show MeSH
Related in: MedlinePlus