• Hirdesh Uppal, Estelle Doudement, Kaushiki Mahapatra, Walter C Darbonne, Daniela Bumbaca, Ben-Quan Shen, Xiaoyan Du, Ola Saad, Kristin Bowles, Steve Olsen, Lewis PhillipsGail DGDDepartment of Molecular Oncology, Genentech, Inc, South San Francisco, California., Dylan Hartley, Mark X Sliwkowski, Sandhya Girish, Donna Dambach, and Vanitha Ramakrishnan.
• Department of Safety Assessment, Genentech, Inc, South San Francisco, California.
• Clin Cancer Res. 2015 Jan 1; 21 (1): 123-33.

PurposeTrastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia.Experimental DesignThe effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR.ResultsT-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure.ConclusionsThese data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.©2014 American Association for Cancer Research.

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