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-glycoprotein have correlated with poor prognosis in rhabdomyosarcoma, neuroblastoma, acute myelogenous leukemia, lymphoma, myeloma and breast carcinoma. A sensitive, nonradioactive method, less costly and time-consuming than the present molecular biologic techniques, is desirable for direct measurement of P-glycoprotein in tumor cells versus normal cells.


METHODS
We have devised an immunoalkaline phosphatase method using four antibody layers to amplify the primary signal considerably and refined staining conditions to optimize the 'signal-to-noise' ratio. Immunoalkaline phosphatase is preferred to immunoperoxidase for testing leukemic and tumor cells in bone marrow, because it avoids myeloperoxidase staining in myeloblasts and myeloid progenitors that interferes with P-glycoprotein interpretation.


RESULTS
Multilayer immunoalkaline phosphatase detected low levels of P-glycoprotein overexpression, that could not be identified by conventional immunoperoxidase or immunoblot, in a low-resistance (8-fold) cell line with a barely detectable transcript. Our technique also detected increased P-glycoprotein in malignant cells in bone marrow of relapsed acute lymphoblastic leukemia (10/11), acute myelogenous leukemia (2/2), lymphoma (1/1), neuroblastoma (7/7), and rhabdomyosarcoma (2/2). Increased P-glycoprotein was not identified at diagnosis in 6 patients with acute lymphoblastic leukemia, and two with stage IV and three with stage IVS neuroblastoma that remained relapse-free in the long-term, but was detected in 4 patients with stage IV neuroblastoma and three with rhabdomyosarcoma who ultimately relapsed.


CONCLUSIONS
Our new technique is more sensitive than conventional immunoperoxidase and immunoblot for assaying P-glycoprotein in low-resistance cell lines. It may be potentially applicable for detecting low levels of P-glycoprotein overexpression in leukemic and tumor cells in bone marrow. Early identification of low levels of multidrug resistance may be clinically relevant by allowing poor-prognostic patients to receive alternative therapy.
Department of Pediatrics, Hospital for Sick Children.
G
G
Haddad
P S
PS
Thorner
G
G
Bradley
W S
WS
Dalton
V
V
Ling
H S
HS
Chan
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000378:metabolism; D000469:Alkaline Phosphatase; D015153:Blotting, Western; D001853:Bone Marrow / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D018432:Drug Resistance, Multiple; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007938:Leukemia / Q000378:metabolism / Q000473:pathology*; D008223:Lymphoma / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology*; D009101:Multiple Myeloma / Q000175:diagnosis / Q000473:pathology; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000473:pathology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000473:pathology; D012208:Rhabdomyosarcoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000469:Alkaline Phosphatase