title: Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents.
abstract: Very-late-antigen-4 (VLA-4, α4β1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker.
  
  
  METHODS
  Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in α4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5.
  
  
  RESULTS
  (64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe.
  
  
  CONCLUSIONS
  (64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.
authors.affiliation: Division of Oncology, Department of Medicine, Washington University Medical School, St. Louis, Missouri.
authors.forename: Deepti
authors.initials: D
authors.lastname: Soodgupta
authors.affiliation: Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri.
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania; and.
authors.forename: Wissam
authors.initials: W
authors.lastname: Beaino
authors.affiliation: Division of Oncology, Department of Medicine, Washington University Medical School, St. Louis, Missouri.
authors.forename: Lan
authors.initials: L
authors.lastname: Lu
authors.affiliation: Division of Oncology, Department of Medicine, Washington University Medical School, St. Louis, Missouri.
authors.forename: Michael
authors.initials: M
authors.lastname: Rettig
authors.affiliation: Department of Genetics, Washington University Medical School, St. Louis, Missouri.
authors.forename: Mark
authors.initials: M
authors.lastname: Snee
authors.affiliation: Department of Genetics, Washington University Medical School, St. Louis, Missouri.
authors.forename: James
authors.initials: J
authors.lastname: Skeath
authors.affiliation: Division of Oncology, Department of Medicine, Washington University Medical School, St. Louis, Missouri.
authors.forename: John F
authors.initials: JF
authors.lastname: DiPersio
authors.affiliation: Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Akers
authors.affiliation: Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri.
authors.forename: Richard
authors.initials: R
authors.lastname: Laforest
authors.affiliation: Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania; and.
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Anderson
authors.affiliation: Division of Oncology, Department of Medicine, Washington University Medical School, St. Louis, Missouri mshokeen@wustl.edu tomasson@dom.wustl.edu.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Tomasson
authors.affiliation: Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri mshokeen@wustl.edu tomasson@dom.wustl.edu.
authors.forename: Monica
authors.initials: M
authors.lastname: Shokeen
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D003301:Copper Radioisotopes; D004151:Dipeptides / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009101:Multiple Myeloma / Q000000981:diagnostic imaging* / Q000378:metabolism*; D010671:Phenylurea Compounds / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D049268:Positron-Emission Tomography; D011487:Protein Conformation; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D014018:Tissue Distribution; D051598:Whole Body Imaging
chemical_list: D014408:Biomarkers, Tumor; D003301:Copper Radioisotopes; D004151:Dipeptides; D039041:Integrin alpha4beta1; C512274:LLP2A compound; D010671:Phenylurea Compounds; D019275:Radiopharmaceuticals
keywords: LLP2A; VLA-4 specificity; animal imaging; dosimetry; molecular imaging; multiple myeloma; radiobiology/dosimetry