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KRAS mutation detection in paired frozen and Formalin-Fixed Paraffin-Embedded (FFPE) colorectal cancer tissues.

Solassol J, Ramos J, Crapez E, Saifi M, Mangé A, Vianès E, Lamy PJ, Costes V, Maudelonde T - Int J Mol Sci (2011)

Bottom Line: KRAS mutations were found in 47/131 (35.8%) using both approaches.Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations.Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, Center Hospital University, Montpellier 34000, France; E-Mails: a-mange@chu-montpellier.fr (A.M.); e-vianes@chu-montpellier.fr (E.V.); t-maudelonde@chu-montpellier.fr (T.M.).

ABSTRACT
KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.

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Related in: MedlinePlus

DNA (A) and KRAS 164-bp PCR products (B) run on 2% agarose gel for samples 8, 29, and 127 frozen of fixed in formaldehyde. Non-degraded DNA exhibited bands of high molecular weight. DNA extracted from blood samples were used as a positive control. MW: molecular weight. F: frozen samples. P: FFPE samples.
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f4-ijms-12-03191: DNA (A) and KRAS 164-bp PCR products (B) run on 2% agarose gel for samples 8, 29, and 127 frozen of fixed in formaldehyde. Non-degraded DNA exhibited bands of high molecular weight. DNA extracted from blood samples were used as a positive control. MW: molecular weight. F: frozen samples. P: FFPE samples.

Mentions: Evaluation of the degree of DNA degradation (preservation) is of major importance when handling FFPE samples; otherwise, real-time PCR and sequencing results may not be interpreted appropriately. In our study, we performed a 2% agarose gel electrophoresis to check the DNA degradation level in each sample. As expected, the frozen samples were not degraded, whereas the FFPE samples were partially fragmented. However, when we checked for PCR product amplifications, we observed a correct amplification in both the FFPE and frozen tissues, demonstrating that our PCR conditions were adapted to the FFPE samples. We showed examples of DNA fragmentation (Figure 4A) and KRAS 164-bp PCR products (Figure 4B) in 3 FFPE and matched frozen samples with discrepant nucleotide changes.


KRAS mutation detection in paired frozen and Formalin-Fixed Paraffin-Embedded (FFPE) colorectal cancer tissues.

Solassol J, Ramos J, Crapez E, Saifi M, Mangé A, Vianès E, Lamy PJ, Costes V, Maudelonde T - Int J Mol Sci (2011)

DNA (A) and KRAS 164-bp PCR products (B) run on 2% agarose gel for samples 8, 29, and 127 frozen of fixed in formaldehyde. Non-degraded DNA exhibited bands of high molecular weight. DNA extracted from blood samples were used as a positive control. MW: molecular weight. F: frozen samples. P: FFPE samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116185&req=5

f4-ijms-12-03191: DNA (A) and KRAS 164-bp PCR products (B) run on 2% agarose gel for samples 8, 29, and 127 frozen of fixed in formaldehyde. Non-degraded DNA exhibited bands of high molecular weight. DNA extracted from blood samples were used as a positive control. MW: molecular weight. F: frozen samples. P: FFPE samples.
Mentions: Evaluation of the degree of DNA degradation (preservation) is of major importance when handling FFPE samples; otherwise, real-time PCR and sequencing results may not be interpreted appropriately. In our study, we performed a 2% agarose gel electrophoresis to check the DNA degradation level in each sample. As expected, the frozen samples were not degraded, whereas the FFPE samples were partially fragmented. However, when we checked for PCR product amplifications, we observed a correct amplification in both the FFPE and frozen tissues, demonstrating that our PCR conditions were adapted to the FFPE samples. We showed examples of DNA fragmentation (Figure 4A) and KRAS 164-bp PCR products (Figure 4B) in 3 FFPE and matched frozen samples with discrepant nucleotide changes.

Bottom Line: KRAS mutations were found in 47/131 (35.8%) using both approaches.Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations.Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, Center Hospital University, Montpellier 34000, France; E-Mails: a-mange@chu-montpellier.fr (A.M.); e-vianes@chu-montpellier.fr (E.V.); t-maudelonde@chu-montpellier.fr (T.M.).

ABSTRACT
KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.

Show MeSH
Related in: MedlinePlus