MGMT methylation in diffuse large B-cell lymphoma: validation of quantitative methylation-specific PCR and comparison with MGMT protein expression.
OBJECTIVE
(1) To validate a quantitative real time methylation specific PCR assay (MethyLight) for the detection of O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status (MS) in diffuse large B-cell lymphoma (DLBCL). (2) To determine the immunohistochemical (IHC) expression of the MGMT protein and correlate it with MS. Both IHC and MethyLight results were compared with patient's outcome.


METHODS
71 patients with primary nodal DLBCL were studied. MGMT immunoreactivity was detected using a specific monoclonal antibody. The MS of MGMT gene was analysed in 52/71 DLBCL using MethyLight. A selected subset of 40 DLBCL was also analysed using qualitative methylation-specific PCR (MSP). Statistical analysis of overall survival (OS), lymphoma-specific survival (LSS) and progression free survival (PFS) was performed according to IHC and MS results.


RESULTS
19/71 DLBCLs (27%) were MGMT-negative at IHC; all were analysed, together with 33/52 MGMT-positive DLBCLs. MethyLight showed a better performance than MSP. There was a good correlation between the presence of MGMT expression and the unmethylated status; the absence of IHC expression was poorly correlated with the presence of methylation. Better OS, LSS and PFS was found in DLBCLs with MGMT gene methylation. DLBCLs not expressing MGMT at IHC showed a longer PFS.


CONCLUSIONS
The quantitative real-time methylation-specific PCR assay for the detection of MGMT gene hypermethylation has been validated for the first time in DLBCL. Immunohistochemistry seems to represent an useful preliminary test to identify unmethylated cases; MS analysis may be performed in non-immunoreactive cases to identify truly methylated DLBCLs, which bear a better prognosis.
Uccella
S
S
Dept of Human Morphology, Unit of Pathology, University of Insubria-Ospedale di Circolo, Via O. Rossi, 9, Varese 21100, Italy. silvia.uccella@uninsubria.it
Cerutti
R
R
Placidi
C
C
Marchet
S
S
Carnevali
I
I
Bernasconi
B
B
Proserpio
I
I
Pinotti
G
G
Tibiletti
M G
MG
Furlan
D
D
Capella
C
C
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D002879:Chromosomes, Human, Pair 10 / Q000235:genetics; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction / Q000379:methods; D016019:Survival Analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes