A SENSITIVE MULTILAYER IMMUNOALKALINE PHOSPHATASE METHOD FOR DETECTION OF P-GLYCOPROTEIN IN LEUKEMIC AND TUMOR-CELLS IN THE BONE-MARROW

BACKGROUND: Relatively low levels of the multidrug resistance P-glycop rotein have correlated with poor prognosis in rhabdomyosarcoma, neurob lastoma, acute myelogenous leukemia, lymphoma, myeloma and breast carc inoma. A sensitive, nonradioactive method, less costly and time-consum ing than the present molecular biologic techniques, is desirable for d irect measurement of P-glycoprotein in tumor cells versus normal cells . EXPERIMENTAL DESIGN: We have devised an immunoalkaline phosphatase m ethod using four antibody layers to amplify the primary signal conside rably and refined staining conditions to optimize the 'signal-to-noise ' ratio. Immunoalkaline phosphatase is preferred to immunoperoxidase f or testing leukemic and tumor cells in bone marrow, because it avoids myeloperoxidase staining in myeloblasts and myeloid progenitors that i nterferes with P-glycoprotein interpretation. RESULTS: Multilayer immu noalkaline phosphatase detected low levels of P-glycoprotein overexpre ssion, that could not be identified by conventional immunoperoxidase o r immunoblot, in a low-resistance (8-fold) cell line with a barely det ectable transcript. Our technique also detected increased P-glycoprote in in malignant cells in bone marrow of relapsed acute lymphoblastic l eukemia (10/11), acute myelogenous leukemia (2/2), lymphoma (1/1), neu roblastoma (7/7), and rhabdomyosarcoma (2/2). Increased P-glycoprotein was not identified at diagnosis in 6 patients with acute lymphoblasti c leukemia, and two with stage IV and three with stage IVS neuroblasto ma that remained relapse-free in the long-term, but was detected in 4 patients with stage IV neuroblastoma and three with rhabdomyosarcoma w ho ultimately relapsed. CONCLUSIONS: Our new technique is more sensiti ve than conventional immunoperoxidase and immunoblot for assaying P-gl ycoprotein in low-resistance cell lines. It may be potentially applica ble for detecting low levels of P-glycoprotein overexpression in leuke mic and tumor cells in bone marrow. Early identification of low levels of multidrug resistance may be clinically relevant by allowing poor-p rognostic patients to receive alternative therapy,