1. A Strong Expression of CD44-6v Correlates With Shorter Survival of Patients With Acute Myeloid Leukemia
by S. Legras, U. Günthert, R. Stauder, F. Curt, S. Oliferenko, H.C. Kluin-Nelemans, J.P. Marie, S. Proctor, C. Jasmin, and F. Smadja-Joffe
Blood
Volume 91(9):
May 1, 1998
©1998 by American Society of Hematology

2. Specific gating of AML blasts.
Specific gating of AML blasts. Cell suspensions containing more than 95% of AML blasts were prepared by removing CD2+ CD19+ cells (lymphocytes) using specific immunoadsorption on Dynabeads coated with specific MoAbs as described in Materials and Methods. In the example shown here, which is from an M1 AML patient, CD14+ monocytes have also been removed. AML blasts, which are characterized by large forward and side scattering values37 (1), are gated in the CD2neg, CD19neg (2) and CD14neg (3) windows.
S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

3. Scatter plot of total CD44 expression on normal myeloid cells and on AML leukemic cells.
Scatter plot of total CD44 expression on normal myeloid cells and on AML leukemic cells. Normal Myeloid Cells: CD34+ cells (very immature hematopoietic cells); GP, granulocytic precursors (CD34−, CD15+ BM cells); PMN, polymorphonuclear cells; MP, monocytic precursors (CD34−, CD14+ BM cells); Mono, mature monocytes (circulating CD14+ cells). AML Leukemic Cells: from AML patients with the following FAB types M1/M2, myeloblastic AML; M3, promyelocytic AML; M4, myelomonocytic AML; M5, monoblastic AML. Mature monocytes and PMN from PB were identified according to their forward angle and side scatter.37CD34+ cells and MP were double-stained with (1) an FITC-conjugated MoAb directed to either CD34, which is specific for very immature hematopoietic cells, or CD14, a monocyte-specific antigen and (2) a PE-conjugated anti-CD44 MoAb F , which binds to an epitope located in the constant part of the CD44 molecule. Negative controls were cells labeled with FITC and PE-conjugated isotype-matched control antibodies. The MFI was determined by flow cytometry, relative to cells labeled with PE-conjugated IgG2a (negative controls), as described in Materials and Methods. Each symbol refers to the MFI value per patient, and the horizontal bars indicate the mean MFI value in each category of patients. The normal BM cells and the leukemic cell populations were isolated as described in Materials and Methods. The gating of leukemic cells is shown in Fig 1.
S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

4. S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

5. S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

6. Scatter plot of CD44v protein expression on AML leukemic cells and normal myeloid cells.
Scatter plot of CD44v protein expression on AML leukemic cells and normal myeloid cells. Normal myeloid cells: CD34+ cells (very immature BM hematopoietic cells). GP, granulocytic precursors (CD34−, CD15+ BM cells); PMN, polymorphonuclear cells; MP, monocytic precursors (CD34neg, CD14+ BM cells); Monocytes: mature monocytes (circulating CD14+ cells). AML leukemic cells from AML patients with the following FAB types M1/M2, myeloblastic AML; M3, promyelocytic AML; M4, myelomonocytic AML; M5, monoblastic AML. CD34+ cells, GP, and MP were identified by double-staining with (1) an FITC-conjugated MoAb directed to CD34, CD15 for GP and CD14 for MP, respectively, (2) unconjugated MoAbs directed to variant epitopes 3v, 6v, and 9v plus PE-goat anti-mouse (PE-GAM). Leukemic cells were identified by specific electronic gating.37 Flow cytometric analysis was performed as described in Materials and Methods. The staining intensity of variant CD44 isoforms was evaluated by measuring the percentage of labeled cells over the background (cells incubated with isotype-matched control antibodies). Each symbol (▪) refers to the percent CD44v cells per patient. Expression of CD44v was considered as negative (−) when less than 5% of the cells were labeled, low (+) when 5% to less than 20% of cells were labeled, and high (++) when more than 20% of the cells were CD44v+.
S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

7. Correlation of CD44-6v isoform expression with survival of AML patients.
Correlation of CD44-6v isoform expression with survival of AML patients. Survival curves of AML patients according to the expression of CD44-6v on leukemic cells at diagnosis, have been computed using the Kaplan-Meier method.38 Statistical comparisons between curves were based on log-rank tests. (A, B, and C) Overall survival curves of AML patients treated by conventional chemotherapy36 (anthracyclin plus cytosin arabinosine). (D and E) Disease-free survival curves of AML patients induced in complete remission by conventional chemotherapy. M1/M2, myeloblastic AML; M4, myelomonocytic AML; M5, monoblastic AML. Significant numbers have been obtained by grouping M4 and M5 patients for overall survival, but not for disease-free survival analysis.
S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

8. S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology

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©1998 by American Society of Hematology

10. S. Legras et al. Blood 1998;91:
©1998 by American Society of Hematology