Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.
BACKGROUND
Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series.


METHODS
In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up.


RESULTS
Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively).


CONCLUSIONS
Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.
Department of Pathology, GROW - School for Oncology & Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Muriel X G
MX
Draht
Department of Radiation Oncology (MAASTRO), GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
Kim M
KM
Smits
Registre Bourguignon des cancers digestifs, INSERM U866, Universite de Bourgogne, Centre Hospitalier Universitaire de Dijon, Dijon, France.
Valérie
V
Jooste
Service de Pathologie, Centre Hospitalier Universitaire de Dijon, Dijon, France.
Benjamin
B
Tournier
Zuyd University of Applied Sciences, Heerlen, The Netherlands ; Chemelot Innovation and Learning Labs, Geleen, The Netherlands.
Martijn
M
Vervoort
Zuyd University of Applied Sciences, Heerlen, The Netherlands.
Chantal
C
Ramaekers
Service de Pathologie, Centre Hospitalier Universitaire de Dijon, Dijon, France.
Caroline
C
Chapusot
Department of Epidemiology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
Matty P
MP
Weijenberg
Department of Pathology, GROW - School for Oncology & Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Manon
M
van Engeland
Department of Pathology, GROW - School for Oncology & Developmental Biology, Maastricht University Medical Center, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Veerle
V
Melotte
D000368:Aged; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology*; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011401:Promoter Regions, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D016019:Survival Analysis
D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human
Analytic sensitivity; Clinical sensitivity; DNA methylation; MS-HRM; MSP; RET; pyrosequencing