Epigenetic mechanisms may drive CD20 negative transformation in B-cell lymphoma

Resistance to rituximab (Rituxan) has emerged as a considerable problem as the drug has become widely used to treat B-cell lymphomas, such as non-Hodgkin’s lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. A recent study in Blood suggested that resistance to rituximab is related to downregulation of CD20 expression via epigenetic mechanisms.

“Combination chemotherapies with rituximab have achieved significantly favorable results for CD20 positive B-cell lymphoma, but acquired resistance occurs in about 30% of patients. Not all patients with relapse or progression develop resistance to rituximab. Some patients are sensitive to re-treatment with rituximab-containing salvage chemotherapies,” wrote Akihiro Tomita, MD, and colleagues at the Nagoya University Graduate School of Medicine in Japan (Blood 113:4885-4893, 2009).

Over the past five years, several groups have reported a CD20 negative phenotypic change in CD20 positive lymphomas after rituximab treatment that appears to account for resistance. “Now it appears that this phenomenon is not rare,” Dr. Tomita said.

The current study followed 124 patients in whom CD20 positive B-cell lymphomas were diagnosed and who initially were treated with combination chemotherapy with rituximab between September 2001 and November 2006. Median age was 58 years at the time rituximab treatment was initiated.

Results
Relapse/disease progression was observed in 36 patients as of July 31, 2007. Tumors from 19 of these 36 patients were re- sampled, and CD20 expression was assessed by immunohistochemistry (IHC) or flow cytometry (FCM) in tumors harvested from lymph nodes, bone marrow, peripheral blood, or spinal fluid. Total RNA and protein were extracted from tumor cells and were analyzed with RT-PCR and immunoblotting.

CD20 protein expression was not detected or was significantly decreased in five patients. Thus, a CD20 negative phenotype transformation after rituximab treatment was found in 26.3% of the 19 patients whose tumor cells were re-sampled at the time of relapse or progression.Although all five patients were treated with salvage chemotherapy without rituximab, they all died of disease progression within 11 months of confirmed CD20 negative transformation. Patients with CD20 negative relapsing/progressive disease tend to have a shorter survival time than patients with CD20 positive relapsing/progressive disease. A larger study is needed to confirm these observations and the prognostic significance of CD negative transformation, according to the authors.

In vitro treatment with 5-Aza
Cells cultures from some of the tumors were treated with and without 5-aza-2´ deoxycytidine (5-Aza). Then the cells were harvested and assessed for total RNA and protein extraction. In vitro assays of CD20 negative transformed lymphoma cells showed that treatment with 5-Aza was able to partially restore rituximab sensitivity compared with cells not treated with 5-Aza. Further experiments are warranted to explore this strategy of re-sensitization.

Dr. Tomita said that the restoration of CD20 mRNA and protein expression within three days after cells were treated with 5-Aza may support the idea that expression is regulated by epigenetic mechanisms rather than by alteration of several tumor clones. In this study, diffuse large B-cell lymphoma (DLBCL) was diagnosed in all five patients in whom a CD20 negative phenotypic change developed; in two, follicular lymphoma was initially diagnosed, but it transformed to DLBCL when a CD20 negative phenotype was observed.

“These findings may suggest that the clinical entity and progression pattern is partly related to CD20 negative phenotypic transformation. Further study is needed to confirm this,” Dr. Tomita said.

In addition, the in vitro findings suggest that a combination of rituximab and a DNA methyltransferase inhibitor such as 5-Aza may be a unique salvage strategy for some individuals in whom CD20 negative transformed B-cell malignancies develop, he told Oncology News International.