Azacitidine Priming Prior to R-CHOP Is Feasible and Results in Global Demethylation, Restoration of TGF-Beta Pathway, and Improved Chemotherapy Sensitivity in Patients with Newly Diagnosed DLBCL

Diffuse large B cell lymphoma (DLBCL) is potentially curable with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but many patients either do not respond to treatment or relapse after achieving remission. Since outcomes after relapse are poor, the optimal strategy to improve survival would be to improve initial therapy. Aberrant DNA methylation contributes to the phenotype of many tumors, including DLBCL. DNA methyltransferase inhibitor (MTI) drugs such as azacitidine can reactivate epigenetically silenced genes resulting in direct anti-tumor effects and/or chemosensitization. Preclinical studies show that combination therapy with MTIs and chemotherapy is synergistic in killing DLBCL xenografts while non-toxic to the host. This chemosensitization effect is mediated, in part, by TGF-beta signaling restoration due to SMAD1 demethylation and re-expression. We therefore hypothesized that the combination of the MTI azacitidine with R-CHOP would be feasible and improve outcomes in patients with DLBCL.

We performed a phase I study of epigenetic priming with azacitidine in patients with newly diagnosed DLBCL receiving R-CHOP. Patients were eligible if they had not received previous therapy, had preserved organ function, and did not have active viral hepatitis. Patients were treated with subcutaneous azacitidine daily for 5 days at escalating doses, followed by standard R-CHOP administered on day 8. Cycles were repeated every 21 days for 6 cycles. Hematopoietic growth factor was administered according to ASCO guidelines. Azacitidine dose escalation was undertaken according to the continuous reassessment model using the following dose levels: dose level 1: 25 mg/m² daily; dose level 2: 50 mg/m² daily; dose level 3: 75 mg/m²daily. Dose limiting toxicity (DLT) was defined as grade 3 or greater non-hematologic toxicity or grade 4 hematologic toxicity lasting more than 7 days. Neutropenic fever was considered DLT only if grade 4. Tumor and blood samples for correlative studies were obtained prior to initiation of treatment and after cycle one of azacitidine, prior to initiation of R-CHOP.

The study has completed accrual, with 12 patients enrolled and treated: one at dose level 1, one at dose level 2, and ten at dose level 3. Eleven patients were of high or high intermediate risk by International Prognostic Index (IPI), and one was of low intermediate risk. Eight patients have completed or discontinued therapy, and 4 remain on treatment. One patient on dose level 3 experienced DLT consisting of hepatitis C reactivation after 2 cycles of study therapy; that patient completed an additional 4 cycles of treatment with CHOP alone. All patients experienced grade 3 or 4 neutropenia, none lasting more than 7 days. Four patients experienced grade 3 neutropenic fever. One patient discontinued study therapy after cycle 1 due to complications of a GI bleed presumed secondary to tumor response; he completed therapy with R-CHOP. All 7 patients who have completed post-treatment restaging have achieved complete remission and are eligible for secondary efficacy analysis. At median follow up of 10 months (range 1.5 to 25 months), one patient (who experienced DLT and completed therapy with CHOP alone) has relapsed. Correlative analysis of tumor and blood samples obtained prior to and after the first cycle of azacitidine show global hypomethylation, decreased SMAD1 promoter methylation and increased SMAD1 expression following azacitidine treatment. Paired patient tumor samples showed improved sensitivity to chemotherapy after treatment with azacitidine ex vivo.

Epigenetic priming with azacitidine prior to standard R-CHOP is feasible and shows promising clinical outcomes in patients with previously untreated DLBCL. Correlative studies support a role for TGF-beta signaling pathway in mediating chemosensitization in these patients.

Off Label Use: Azacitidine is approved for use in MDS. Discussion here is off label. Martin:Millennium Pharmaceuticals, Inc.: Speakers Bureau. Leonard:Celgene: Consultancy, Honoraria.