Epigenetic Modulation of CD30 Gene Expression in Alemtuzumab Resistant T Cell Malignancy:Rationale and Efficacy of Brentuximab Vedotin.

T-Cell prolymphocytic leukemia (T-PLL) is an aggressive hematological disease mainly affecting adults. It is characterized by skin involvement and high lymphocyte counts. It affects mature T-cells mainly of the CD4+/CD8- phenotype. Although treatment with alemtuzumab, an anti-CD52 monoclonal antibody (mAb), induces complete remission (CR) in >50% of patients, it is short lived (median: 6 mos) and resistance is common. Epigenetic therapy with the histone deacetylase inhibitors (HDACi) vorinostat (SAHA) or romidepsin and the hypomethylating agent cladribine (2-CdA), can circumvent lack of response to alemtuzumab. Brentuximab Vedotin (SGN-35) is an antibody drug conjugate ADC) targeted against CD30 (TNFRSF8) and we have show here this ADC has activity in combination with epigenetic agents in T-PLL.

Five T-PLL patients were studied. Diagnosis was made by hematopathology, flow cytometry and TCR rearrangement studies. Alemtuzumab (30 mg SQ d1,3,5) and 2-CdA (5mg/m2 IVd1–5), followed by alemtuzumab, 2-CdA and SAHA (400mg po daily X 5 days) or romidepsin (14mg/m²IV d 1,8,15) was given. After, epigenetic therapy with 2-CdA, SAHA, and romidepsin expression of CD30 in skin and blood was assayed where applicable.SGN-35 was administered at 1.8 mg/kg IV q 3 weeks. Peripheral blood was obtained and assayed under an IRB approved protocol. Genes of interest were analyzed by qRT-PCR. Chromatin Immunoprecipitation (ChIP) assays were used to study epigenetic changes at the CD30 promoter.

Of the 5 patients studied, three were alemtuzumab refractory with disease in blood and/or skin. With the addition of 2-CdA and SAHA or romidepsin, all three patients' peripheral blood counts normalized and flow cytometry and PCR assays were negative for residual disease. One patient at the time of relapse developed skin lesions that were unresponsive to treatment with alemtuzumab, romedpsin, 2-CdA, or palatrexate. Lack of response to alemtuzumab in this patient was circumvented through induction of the cell surface receptor CD30, the target for the potent ADC, brentuximab vedotin. qRT-PCR assays on leukemic blood demonstrated that 4/5 of the T-PLL patients demonstrated increased CD30 mRNA following epigenetic therapy (mean: 14; range: 1.3–69 fold). These increases in CD30 mRNA expression correlated with increases in CD30 protein levels by Western blot analysis. CD30 and CD52 expression in the skin was confirmed by immunohistochemical analysis. In two patients, ChIP analysis of the CD30 promoter showed decreases in histone methylation markers and increases in Pol II binding at the coding sequence after treatment with epigenetic agents, consistent with increased CD30 expression. The patient with skin lesions had a dramatic response of his skin and normalization of his WBC count, with flow and PCR studies of skin and blood negative for residual disease after two cycles of SGN35.

In contrast, a second patient with T-PLL who was refractory to up front alemtuzumab (IV) responded and achieved remission with the addition of 2-CdA, Her leukemic cells 2 days post treatment expressed CD30 mRNA in. At relapse 16 months later, her leukemic cells did not express CD30 and treatment with 2-CdA and romedepsin was unable to derepress CD30 gene expression. Correlative studies with ChiP assays differed from the previous patient. Further elaboration and follow up will be presented with both patients.

Therapy with HDACi and 2-CdA epigenetically augments clinical activity of alemtuzumab and SGN-35, as evidenced by de-repression of TNFSFR8 (CD30) and increased response to treatment in one patient. While CD30 is a gene that can be epigenetically regulated in T cell malignancies, further complexities in epigenetic regulation of CD30 require additional investigation. Correlative studies are underway to elucidate the regulation of CD30 by epigenetic mechanisms. We present a rationale for the use of epigenetic drugs in combination with mAbs such as alemtuzumab and ADCs in T-PLL. A prospective multicenter clinical trial is warranted.

Off Label Use: Brentuximab vedotin for PLL. Epner:Seattle Genetics: Speakers Bureau.