Evaluation of microsatellite instability, hMLH1 expression and hMLH1 promoter hypermethylation in defining the MSI phenotype of colorectal cancer.
BACKGROUND
About 15% of all colorectal cancers (CRCs) demonstrate high levels of microsatellite instability (MSI-H) and are currently best identified by molecular analysis of microsatellite markers. Most sporadic CRCs with MSI-H are known to be associated with the methylation of the hMLH1 promoter. Promoter methylation coincided with lack of hMLH1 expression. We aimed to investigate the association between MSI status, hMLH1 protein expression and methylation status of the hMLH1 promoter, and to determine the usefulness of each method in defining the MSI phenotype in sporadic CRCs.


METHODS
CRCs from 173 patients from the Cancer and Leukemia Group B (CALGB) were assessed for their MSI status. An additional cohort of 18 MSI-H tumors from the University of California San Diego (UCSD) was included in the analysis of the MSI-H subgroup. MSI testing was performed by PCR using five standard MSI markers. hMLH1 promoter analysis was investigated by methylation specific PCR (MSP), and expression of the MMR genes hMLH1 and hMSH2 was examined by immunohistochemistry (IHC).


RESULTS
Of the 173 CALGB tumors, 111 (64%) were MSS, 35 (20%) were MSI-L and 27 (16%) MSI-H, respectively. Data on hMLH1 protein expression, hMSH2 protein expression and hMLH1 methylation are available on 128, 173 and 81 of these tumors, respectively. Presence of hMLH1 and hMSH2 protein expression was significantly associated with MSI status. Four of 45 (8.9%) MSI-H tumors and 0 of 146 (0%) MSS/MSI-L tumors did not express hMSH2 (p = 0.0028). hMLH1 protein expression was present in 107 of 108 (99%) MSS and MSI-L tumors versus 11 of 20 (55%) MSI-H tumors (p < 0.0001). Of 61 MSS and MSI-L cancers studied for methylation, 11 (18%) were methylated at the hMLH1 promoter whereas 14 of 20 (70%) MSI-H cancers were methylated (p = 0.0001). In 27 MSI-H tumors studied for hMLH1 protein expression and methylation, 93% of tumors with loss of expression (93%) were also methylated while 42% (5/12) with positive immunostaining for hMLH1 were methylated at the hMLH1 promoter (p = 0.009).


CONCLUSIONS
Promoter methylation and hMLH1 expression are significantly associated with the MSI-H phenotype in CRC. Promoter methylation analysis provides a useful means to screen for MSI-H tumors. Our data further suggests that hMLH1 promoter methylation analysis alone cannot replace MSI testing, as a significant number of MSI-H tumors could be potentially overseen by such an approach. We suggest that phenotypic evaluation of CRC is performed most reliably with MSI testing, although expression analysis and investigation of the promoter methylation status may complement the screening process.
Arnold
Christian N
CN
Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California, USA.
Goel
Ajay
A
Compton
Carolyn
C
Marcus
Victoria
V
Niedzwiecki
Donna
D
Dowell
Jeannette M
JM
Wasserman
Linda
L
Inoue
Toru
T
Mayer
Robert J
RJ
Bertagnolli
Monica M
MM
Boland
C Richard
CR
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D002352:Carrier Proteins; D015179:Colorectal Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics*; D005819:Genetic Markers; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D004273:DNA, Neoplasm; D005819:Genetic Markers; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1