title: Potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1).
abstract: OBJECTIVE
  Trastuzumab-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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  T-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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Department of Safety Assessment, Genentech, Inc, South San Francisco, California.
authors.forename: Hirdesh
authors.initials: H
authors.lastname: Uppal
authors.affiliation: Department of Safety Assessment, Genentech, Inc, South San Francisco, California.
authors.forename: Estelle
authors.initials: E
authors.lastname: Doudement
authors.affiliation: Department of Safety Assessment, Genentech, Inc, South San Francisco, California.
authors.forename: Kaushiki
authors.initials: K
authors.lastname: Mahapatra
authors.affiliation: Department of Oncology Biomarker Development, Development Sciences, gRED, Genentech, Inc, South San Francisco, California.
authors.forename: Walter C
authors.initials: WC
authors.lastname: Darbonne
authors.affiliation: Department of Preclinical and Translational Pharmacokinetics and Pharmodynamics, Genentech, Inc, South San Francisco, California.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bumbaca
authors.affiliation: Department of Preclinical and Translational Pharmacokinetics and Pharmodynamics, Genentech, Inc, South San Francisco, California.
authors.forename: Ben-Quan
authors.initials: BQ
authors.lastname: Shen
authors.affiliation: Department of Oncology Biomarker Development, Development Sciences, gRED, Genentech, Inc, South San Francisco, California.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Du
authors.affiliation: Department of Bioanalytical Sciences, Genentech, Inc, South San Francisco, California.
authors.forename: Ola
authors.initials: O
authors.lastname: Saad
authors.affiliation: Department of Protein Chemistry, Genentech, Inc, South San Francisco, California.
authors.forename: Kristin
authors.initials: K
authors.lastname: Bowles
authors.affiliation: Department of Product Development, Genentech, Inc, South San Francisco, California.
authors.forename: Steve
authors.initials: S
authors.lastname: Olsen
authors.affiliation: Department of Molecular Oncology, Genentech, Inc, South San Francisco, California.
authors.forename: Gail D
authors.initials: GD
authors.lastname: Lewis Phillips
authors.affiliation: Department of Safety Assessment, Genentech, Inc, South San Francisco, California.
authors.forename: Dylan
authors.initials: D
authors.lastname: Hartley
authors.affiliation: Department of Molecular Oncology, Genentech, Inc, South San Francisco, California.
authors.forename: Mark X
authors.initials: MX
authors.lastname: Sliwkowski
authors.affiliation: Department of Development Sciences, Genentech, Inc, South San Francisco, California.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Girish
authors.affiliation: Department of Small Molecule and Investigative Toxicology, Genentech, Inc, South San Francisco, California.
authors.forename: Donna
authors.initials: D
authors.lastname: Dambach
authors.affiliation: Department of Project Management and Operations, Genentech, Inc, South San Francisco, California. vanithar@gene.com.
authors.forename: Vanitha
authors.initials: V
authors.lastname: Ramakrishnan
mesh_terms: D000080044:Ado-Trastuzumab Emtansine; D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage / Q000009:adverse effects*; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000009:adverse effects*; D001943:Breast Neoplasms / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects; D005260:Female; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000009:adverse effects; D008453:Maytansine / Q000008:administration & dosage / Q000009:adverse effects / Q000031:analogs & derivatives*; D008533:Megakaryocytes / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D053657:Platelet-Rich Plasma / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D013921:Thrombocytopenia / Q000139:chemically induced / Q000209:etiology / Q000473:pathology*; D000068878:Trastuzumab
chemical_list: D000080044:Ado-Trastuzumab Emtansine; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D018796:Immunoconjugates; D008453:Maytansine; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab