Abstract MGMT is a tumor suppressor gene, which becomes methylated in many different cancers including diffuse large B-cell lymphoma (DLBCL). Methylation of MGMT has been associated with the T-allele of the rs16906252 SNP found in the MGMT promoter. However, allelic MGMT methylation patterns and a possible association with the T-allele of this SNP have not been investigated in DLBCL. Today, standard treatment for DLBCL patients is rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and methylation of MGMT has been shown to predict chemosensitivity to the alkylating agent cyclophosphamide in the era before rituximab. We developed a novel method for the analysis of allelic MGMT methylation patterns based on pyrosequencing of antisense methylation specific PCR (MSP) products including the rs16906252 SNP. Samples from 148 DLBCL patients, of which 75 received R-CHOP, were studied using this assay. Standard bisulfite pyrosequencing and bisulfite sequencing of single clones were used to confirm data obtained by allelic MSP pyrosequencing. The allelic MSP pyrosequencing assay could reliably analyze allelic methylation patterns in standards of known allelic methylation status even when diluted in unmethylated DNA down to 1.25% methylation. MGMT methylation was observed for 28 patients of which seven were heterozygous. Thus, an association between the T-allele and MGMT methylation was observed (p-value =0.04). Among the seven methylated and heterozygous patients, two were methylated at both alleles, four were methylated only at the T-allele, and one only at the C-allele. Finally, we found no statistical significant associations between MGMT methylation or SNP genotypes with clinical parameters among the R-CHOP treated patients. In conclusion, allelic MSP pyrosequencing is a fast and cost-efficient method for evaluation of allelic methylation patterns, which may have wide implications for locus specific methylation analysis of tumor suppressor genes in future cancer research. Citation Format: Lasse Sommer Kristensen, Marianne Bach Treppendahl, Mia Seremet Girkov, Fazila Asmar, Helene Myrtue Nielsen, Tina Kjeldsen, Elisabeth Ralfkiaer, Lise Lotte Hansen, Kirsten Grønbæk. Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B28.