Bone marrow gene methylation detection for prognostic evaluation in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) features significant clinical and molecular heterogeneity, and current molecular biomarkers are insufficient for precise prognostic stratification. DNA methylation is well-validated in solid tumor diagnosis but its value in DLBCL prognosis remains unclear. The purpose of this study was to evaluate the predictive value of bone marrow RASSF1A, P16, and PAX5 methylation detection for prognosis in patients with diffuse large B-cell lymphoma (DLBCL).

A total of 60 newly diagnosed DLBCL patients and 61 controls were retrospectively enrolled. Methylation-specific real-time fluorescent PCR (MS-PCR) was used to detect the methylation status of RASSF1A, P16, and PAX5 in pre-treatment bone marrow samples. The optimal threshold and AUC values were determined by ROC curve analysis. The correlations between methylation results and complete blood count, endocrine-related metabolic indicators, treatment regimens, and immunohistochemical markers were analyzed. Factors affecting patient prognosis were screened through LASSO and Cox regression.

The methylation levels of RASSF1A, P16, and PAX5 showed varying degrees of correlation with pre-treatment complete blood count and endocrine-related metabolic indicators. In the CART treatment group, the positive rates of P16 and PAX5 methylation were significantly lower than those in the untreated group (P < 0.05), while in the transplantation treatment group, only the positive rate of P16 methylation was significantly reduced (P = 0.011). In terms of immunohistochemistry, the positive rates of RASSF1A and P16 methylation were significantly elevated in the Ki67-positive group (P < 0.05), the positive rates of all three methylation markers were significantly elevated in the CD34-positive group (all P < 0.05), the positive rates of P16 and PAX5 methylation were significantly elevated in the CD56-positive group (P < 0.01), and only the positive rate of P16 methylation was significantly elevated in the P53-positive group (P = 0.047). LASSO regression and multivariate Cox regression analysis showed that RASSF1A methylation (HR=0.921, P = 0.022), P16 methylation (HR=0.933, P = 0.042), and Ki67 proliferation index (HR=1.042, P < 0.001) were independent predictors of PFS, while CD56 expression showed a positive correlation trend (HR=2.866, P = 0.062).

Bone-marrow-based gene methylation markers may act as candidate epigenetic biomarkers for risk stratification in DLBCL. Given limitations of this single-center retrospective study with limited outcome events, our results cannot yet support direct clinical judgement for hematopoietic stem cell transplantation. Further multi-center external validation is required before clinical translation. Bone marrow gene methylation detection shows promising research value for prognostic evaluation of DLBCL.