Abstract A135: T-DM1-induced thrombocytopenia results from impaired platelet production in a HER2-independent manner.
Authors: Mahapatra Kaushiki , Darbonne Walter , Bumbaca Daniela , Shen Ben , Du Xiaoyan , Tibbitts Jack , Olsen Steve , Sliwkowski Mark , Girish Sandhya , Hartley Dylan , Dambach Donna , Ramakrishnan Vanitha , Uppal Hirdesh 
Published: June 2012

Abstract
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising trastuzumab, a stable thioether linker, and the maytansine derivative DM1, a microtubule inhibitor. T-DM1 has been evaluated for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer in several phase 2 trials. Unexpectedly, the dose-limiting toxicity in patients treated with T-DM1 was dose- and duration-dependent thrombocytopenia, even though HER2 is not detected on platelets. To understand the mechanism of T-DM1-induced thrombocytopenia, we evaluated the effect of T-DM1 on platelet (PLT) function and formation from hematopoietic stem cells (HSCs).
Using PLT-rich plasma (PRP) or washed PLT (WP) from healthy human donors, PLT function was assessed by aggregometry and flow cytometry to evaluate activation markers (PAC1; CD62P) in the presence of T-DM1, trastuzumab, control ADC (anti-CD22 conjugated to DM1), free DM1, or vehicle. Human HSCs (CD133+/CD34+) were purified from healthy donor bone marrow, expanded in hematopoietic expansion media, and transferred to megakaryocyte (Mk) differentiation media for 14 days to obtain mature Mks, in the presence of T-DM1, trastuzumab, ADC control (5B6, anti-gD conjugated to DM1), and vehicle. T-DM1 uptake and catabolism in Mks were evaluated by incubating the cells with T-[3H]DM1.
Incubation with T-DM1, free DM1, or any controls at clinically relevant concentrations had no observable effect on platelet aggregation in PRP or WP and no direct impact on PLT activation (PAC1 binding or CD62P expression). Neither T-DM1 nor free DM1 inhibited PLT aggregation and activation induced by the PLT agonists collagen and TRAP6. Treatment of mature Mks with T-DM1 and control ADC resulted in a decrease in Mk number and a significant decrease (∼3-fold) in Mk production from HSC precursors, as measured by a reduction in CD41+/CD61+ cells. Incubation of Alexa488-conjugated trastuzumab and T-DM1 with Mks resulted in surface binding and internalization as assessed by immunofluorescence and flow cytometry. Preincubation with anti-CD32 decreased Alexa488-conjugated T-DM1 antibody binding and uptake (∼2-fold), suggesting that FcR IIb contributes to this process. To determine the fate of the internalized antibody, we quantified the uptake and catabolism of T-DM1 in Mks using T-[3H]DM1. Total cellular T-[3H]DM1 increased over the 3-day incubation with peak concentrations occurring during the first 8 hours of incubation, and a peak total uptake in the Mks of 0.4% of the total dose. The metabolized intracellular⌝-free [3H]DM1 levels were 0.1% of the total dose. By quantification of the HSCs and various ploidy populations, T-DM1 was found to primarily inhibit Mk production from HSCs rather than directly impact mature Mks. Furthermore, the similarity of effects between T-DM1 and 5B6-DM1 suggests that the DM1 moiety is responsible for the observed platelet reduction.
Our experiments show that T-DM1 did not have a direct effect on PLT function but did impair Mk and PLT production. DM1-conjugated antibodies were internalized in a target-independent, partially Fc-dependent manner into Mks, and intracellular release of DM1 resulted in a reduction of the stem cell population. These data support the hypothesis that the thrombocytopenia observed in clinical trials may be a consequence of impaired PLT production from Mks in the bone marrow.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A135.