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Technical Advance |
From the Departments of Investigation,
* Pathology,
Gastroenterology,
and Oncology,
** Hospital General Universitario de Alicante, Alicante; Department of Gastroenterology,
Hospital del Mar, Barcelona; Department of Gastroenterology,
|| Hospital Universitario Germans Trias i Pujol, Badalona; and Department of Gastroenterology,
¶ Institut de Malalties Digestives, Hospital Clinic, Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain
Abstract
Mutation V600E of BRAF, a kinase-encoding gene from the RAS/RAF/MAPK pathway, in colorectal carcinoma (CRC) suggests a sporadic origin of the disease, providing an exclusion criterion for hereditary nonpolyposis colorectal cancer. Here we describe detection of this mutation by real-time chemistry TaqMan MGB probes, confirmed by direct DNA sequencing as the gold standard. DNA was extracted from paraffin-embedded tissue from 112 tumors obtained from the EPICOLON study. Seventy-two tumors were CRC with defective DNA mismatch repair (MMR; microsatellite instability and/or loss of protein expression by immunohistochemical analysis), and 40 were proficient MMR controls. BRAF mutation was detected in 20/72 (27.8%) CRC with defective MMR and in 3/40 (7.5%) proficient MMR controls (P = 0.011). BRAF mutation was detected in 19/51 (37.3%) tumors with loss of MLH1 expression and in none of the tumors with loss of MSH2 expression (0/13). BRAF mutation was not found in cases with germline mutation of MLH1 (4/112) or MSH2 (3/112) genes. The sensitivity and specificity of our real-time chemistry were both 100% for detecting the V600E mutation. Because real-time chemistry methodology has advantages in cost, time, and labor, we consider it a valuable alternative to automatic direct sequencing, particularly for serial measurements.
Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary colon cancer syndrome, accounting for 0.9 to 2.5% of all colorectal cancer (CRC).1 HNPCC is due to a germline mutation of DNA mismatch repair (MMR) genes, mainly MLH1 or MSH2, and cancers occurring in this condition show high-level DNA microsatellite instability (MSI-H). According to the international criteria for HNPCC diagnosis, cancer patients with Amsterdam clinical criteria or Bethesda criteria showing MSI-H should receive genetic counseling and be offered testing for germline mutations of MMR genes.2 Furthermore, 5 to 10% of unselected CRC show MSI-H phenotype that is primarily due to hypermethylation of the MLH1 gene promoter, not germline mutation.3 Thus, it would be desirable to exclude this group from MMR gene testing.
Recently, an oncogenic V600E hotspot mutation in BRAF, a kinase-encoding gene from the RAS/RAF/MAPK pathway, has been found in diverse human cancers,4 especially in CRC with MMR deficiency.5 Interestingly, BRAF mutations have not been detected in those cases with a germline mutation in MLH1, MSH2, or MSH6; thus, detection of a positive BRAF-V600E mutation in CRC suggests a sporadic origin of the disease.6 These findings have potential impact for the genetic testing for HNPCC diagnostics and suggest a potential use of BRAF as an exclusion criterion for HNPCC.7, 8
So far, detection of this mutation has been achieved by restriction fragment length polymorphism analysis,9 allele-specific polymerase chain reaction (PCR),10 single-strand conformation analysis,11 and direct automatic sequencing.8 The last method is widely accepted as the standard, but this technique is very time consuming. For this reason, and to perform routine detection of V600E mutation, we assessed the performance of alternative real-time chemistry TaqMan MGB probes in a 7500 sequence detection system designed for allelic discrimination of single nucleotide polymorphisms. The aim of this research was to validate and determine the cost effectiveness of real-time chemistry methodology.
Materials and Methods
Colorectal Cancer Tissue Samples and DNA Extraction
CRC specimens were obtained from researchers participating in the EPICOLON study12, 13
at Hospital del Mar (M.A., X.B.). The study was approved by the institutional ethics committee, and informed written consent for molecular studies was obtained from all patients. Specimens were previously sequenced for the detection of BRAF V600E mutation (Figure 1A
and 1B)
. DNA was extracted from paraffin-embedded tissue after deparaffination by the standard procedure involving digestion with proteinase K and phenol-chloroform extraction. Then, extracted DNA was checked and quantified in a Biophotometer (Eppendorf AG, Hamburg, Germany) and stored at –20°C until later use.
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Real-Time PCR
Once we had chosen our primers and TaqMan MGB probes, real-time PCR was performed with DNA extracted from CRC tissues. Real-time PCR was performed in a final reaction volume of 20 µl containing 10 µl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nmol/L of each primer, 250 nmol/L of each probe, and 5 µl of DNA solution. PCR was performed in MicroAmp optical 96-well plates with optical adhesive covers (Applied Biosystems). Amplification and detection were performed with an ABI prism 7500 sequence detection system (Applied Biosystems). The amplification conditions were 2 minutes at 50°C for AmpErase uracil-N-glycosylase activity and 10 minutes at 95°C for AmpliTaq Gold activation, followed by 50 cycles of 15 seconds at 92°C for denaturation and 1.5 minutes at 60°C for annealing and extension. After amplification, end point detection of fluorescence was performed at 60°C. The fluorescence data were analyzed with the allelic discrimination software of the ABI Prism 7500 instrument, and amplification plots produced in the PCR reaction were also checked. A time, cost, and labor analysis was also performed to establish the most efficient technique per sample comparing real-time PCR in an ABI Prism 7500 versus automatic sequencing in ABI Prism 310. For real-time PCR versus automatic sequencing, time was established at 4 minutes (6 hours/96 samples) versus 10 hours, cost at 2 
($2.60) versus 8 ($10.40), and labor at 1.25 minutes (2 hours/96 samples) versus 16 minutes, respectively.
Results and Discussion
We tested DNA from 112 tumors obtained from the EPICOLON study, a clinical epidemiology survey aimed at establishing the incidence of HNPCC in Spain.12, 13 Seventy-two tumors were CRC with defective MMR (MSI and/or loss of protein expression in immunohistochemical analysis), and 40 were proficient MMR controls. Mutation analysis of MLH1/MSH2 was performed in all cases with defective MMR.
BRAF mutation was detected in 20/72 (27.8%) CRC with defective MMR and in 3/40 (7.5%) proficient MMR controls (P = 0.011) (Figure 1C
and 1D)
. In tumors with loss of MLH1 expression, BRAF mutation was detected in 19/51 (37.3%) tumors and in none of the tumors with loss of MSH2 expression (0/13). BRAF mutation was not found in those cases with germline mutation of the MLH1 (4/112) or MSH2 (3/112) genes. These findings are along the same lines as those of other authors7, 8
.
Considering as gold standard the direct sequencing methodology, the sensitivity and specificity of the real-time PCR procedure were both 100%. Therefore, this real-time PCR method with TaqMan MGB probes is a robust and convenient way to determine the presence of V600E mutation. The use of TaqMan MGB probes gives clear discrimination of genotypes independent of the amount of input DNA.
In conclusion, real-time PCR is a highly sensitive and specific methodology for detecting V600E mutation. Because real-time chemistry methodology has advantages in cost, time, and labor, we consider that it may be an alternative to automatic direct sequencing, particularly for serial measurements. The combination of TaqMan MGB probes with universal assay conditions allows an efficient workflow when the number of different single-nucleotide polymorphisms that are detected in the clinical laboratory increases.
Acknowledgments
We thank Dolores Durá, Juan Pulido, and Estefania Rojas for technical assistance.
Footnotes
Address reprint requests to Susana Benlloch, Unidad de Investigación, Hospital General Universitario de Alicante, C/ Maestro Alonso, 109, 03010 Alicante, Spain. E-mail: benlloch_sus{at}gva.es
This work was supported by grant from the Sociedad Española de Oncología Médica (SEOM) and Instituto de Salud Carlos III (CO3/02).
C.A. and S.B. are recipients of a research contract of Spanish Ministry of Health, Instituto de Salud Carlos III (CM04/00035 and 01/3080).
Accepted for publication June 21, 2006.
References
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