Differential expression of microRNAs induced by trastuzumab emtansine (T-DM1) during megakaryocytopoiesis.

Abstract

645 Background: Treatment with the HER2-targeted antibody–drug conjugate T-DM1 resulted in significantly longer PFS and OS vs lapatinib + capecitabine in patients previously treated with trastuzumab and a taxane in the phase 3 study EMILIA. Thrombocytopenia (TCP) was the dose-limiting toxicity for patients treated with T-DM1, although platelets do not express HER2. In EMILIA, grade 3/4 TCP was observed in 12.9% of T-DM1-treated patients. We have previously shown that T-DM1 inhibits megakaryocyte (Mk) production and differentiation. Here, we investigated the effect of T-DM1 on microRNAs (miRNAs) associated with megakaryocytopoiesis. Methods: Human stem cells (HSCs; CD133+/CD34+) from 8 donors were differentiated into Mks in the presence of T-DM1, trastuzumab, or vehicle. Total RNA was extracted using the miRNeasy MiniKit. cDNA was prepared using the Taqman miRNA RT Kit, FAM-MGB probes, and stem-loop RT primer pool set. miRNA expression was measured using the 96.96 Dynamic Array Chip on the Biomark HD Reader. Data were analyzed using Fluidigm real-time analysis software Spotfire 5, and SAS 9.2. hsa−let−7g and hsa−miR−671−3p were chosen as reference miRNAs due to their low variation between treatments and time points. Median normalization was also applied. Results: A total of 526 miRNA RT-qPCR assays were used to map miRNA expression during differentiation of HSCs from 8 separate donors to Mks in vitro over 30 days. Several miRNAs demonstrated temporal changes in their expression profiles during maturation, suggesting these miRNAs are potential drivers of Mk differentiation. T-DM1 treatment inhibited Mk production and differentiation. Concomitant modifications in the expression of specific miRNAs were observed. These modifications were not present in trastuzumab- or vehicle-treated cells, suggesting these miRNAs may be involved in the development of T-DM1–induced TCP. Conclusions: These results suggest that the miRNAs have the potential to be used as biomarkers for TCP in patients treated with T-DM1 and possibly other DM1 conjugates. Specific miRNA alterations related to T-DM1 treatment will be discussed following investigations using clinical samples to validate these preliminary data.