Treatment of myelodysplastic syndromes with 5q deletion before the lenalidomide era; the GFM experience with EPO and thalidomide.

Anemia in MDS with 5q deletion was generally considered, until the advent of lenalidomide, unresponsive to available treatments. We analyzed erythroid response to erythropoetin (EPO) or darbepoetin (DAR) and thalidomide in MDS with 5q deletion treated by French centers (GFM) and in whom karyotype was successfully performed. Of 345 patients treated with EPO or DAR+/-G-CSF, 48 had 5q deletion. The response rate was 46% (31% major, 15% minor) according to International Working Group (IWG) 2000 criteria versus 64% in patients without 5q deletion (p=0.03). According to IWG 2006 criteria, the response rate in patients with 5q deletion was 39% versus 52% in patients without 5q deletion (p=0.10). Mean duration of response was 14 months versus 25 months (IWG 2000) and 13 months versus 27 months (IWG 2006) in 5q deletion and non-5q deletion patients (p=0.019 and 0.003, respectively). Of 120 MDS treated with thalidomide, all of whom had successful cytogenetic analysis, 37% of the 24 patients with 5q deletion responded (IWG 2000 criteria, 20% major, 17% minor) with a mean duration of 9.5 months, versus 32% (18% major, 14% minor) in MDS without 5q deletion and a mean response duration of 9 months (p=NS). Our results confirm that response rates to EPO or DAR and thalidomide are clearly inferior to those obtained with lenalidomide.

Detection of serum anti p53 antibodies and their correlation with p53 mutations in myelodysplastic syndromes and acute myeloid leukemia.

In solid tumors, p53 antibodies are found in 30% of the patients with p53 mutations, and their analysis is an interesting method for the detection of p53 mutations. We looked for circulating p53 antibodies in 83 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), by an ELISA technique. Detection of p53 mutations was made by single stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene and confirmed by direct sequencing. Circulating antibodies to p53 were seen in three of the 83 (3.5%) patients analyzed, and a p53 point mutation was found in ten cases. Two of the three patients with p53 antibodies had a p53 mutation, but the remaining case had no detectable mutation. The other eight mutated cases had no detectable p53 antibodies. Our findings show that serological analysis of p53 antibodies is rarely positive in MDS and AML. This could be due to the relatively low incidence of p53 mutations seen in those disorders, but also to the immune depression to which they are often associated.

Absence of amplification of MDM2 gene, a regulator of p53 function, in myelodysplastic syndromes.

The MDM2 gene is a gene whose product binds to p53 and regulates its function. Amplification of MDM2 has been found in human sarcomas, where it leads to inactivation of p53. In 64 cases of generally advanced myelodysplastic syndromes, we found no amplification or rearrangement of MDM2 gene by Southern analysis. MDM2 RNA was also normal in the 15 cases where Northern analysis was made. Thus, amplification of MDM2 is not seen or must be very rare in myelodysplastic syndrome (MDS). Because P53 gene mutations are not frequent in MDS, inactivation of p53 seems to be, overall, a rare pathogenetic event in MDS.

Detection of p53 mutations in hematological malignancies: comparison between immunocytochemistry and DNA analysis.

The wild type p53 protein has a short half-life and cannot be detected by immunohistochemistry on tissue sections. Mutated p53, on the other hand, has a prolonged half-life and becomes detectable by this method, so that its detection by immunohistochemistry in solid tumors is almost synonymous with mutation. We assessed the value of immunocytochemical analysis of p53 protein on blood or bone marrow slides in the detection of p53 mutation in hematological malignancies, by comparison with single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene. One hundred and twenty eight patients with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic syndromes (MDS), or chronic lymphocytic leukemia (CLL) were studied by both methods. Immunocytochemistry showed detectable levels of intracellular p53 in 19 cases (including 2/19 AML, 2/21 ALL, 11/48 MDS, 4/40 CLL). Staining by p53 antibodies was restricted to the nucleus of blasts in AML, ALL, and MDS, and of lymphocytes in CLL. In 16 of the 19 cases, SSCP analysis, followed by direct sequencing, showed a p53 missense mutation in exons 4 to 8 of the gene. In the remaining three cases, where the number of cells stained by p53 antibodies was small, no p53 mutation could be detected. On the other hand, SSCP and sequence analysis identified a p53 mutation in two patients who had negative immunocytochemical findings. Both cases had a nonsense mutation, presumably leading to reduced levels of truncated p53. Thus, overall, immunocytochemistry and SSCP gave concordant results in 123 of the 128 (96%) patients analyzed. Our findings show that immunocytochemistry on blood and bone marrow smears is a sensitive method of p53 mutation detection in hematological malignancies, except in the rare patients with chain-terminating mutations. Positive immunocytochemistry is found in some patients with normal SSCP findings, and could correspond to overexpression of a non-mutated p53, but also to p53 mutation in a minor proportion of the malignant cells, undetectable by SSCP.

Relationship between p53 gene mutations and multidrug resistance (mdr1) gene expression in myelodysplastic syndromes.

P glycoprotein, the product of multidrug resistance (mdr1) gene, is frequently expressed in advanced myelodysplastic syndromes (MDS) with an excess of bone marrow blasts and could explain their frequent resistance to chemotherapy. P53 gene mutations are also found in 10 to 15% of advanced MDS. Because it has recently been suggested that normal p53 suppressed, but that mutated p53 activated, the mdr1 gene promoter, we tried to correlate p53 mutations and P glycoprotein expression in 34 patients with MDS and an excess of bone marrow blasts (> 5%). P glycoprotein expression was assessed by immunocytochemistry using JSB1 monoclonal antibody and was found positive in 13 out of the 34 patients. p53 mutations were detected both by immunocytochemistry using three different monoclonal antibodies and by single stranded conformation polymorphism (SSCP) analysis of exons 5 to 8 of the P53 gene. Both methods detected a point mutation in 5 out of the 34 patients. Only one out of the 5 patients with a p53 mutation expressed P glycoprotein, as compared to 12 out of the 29 patients without p53 mutations. This suggested the mutant and normal p53 are not major determinants of the regulation of mdr1 expression in vivo, at least in MDS.

Expression of the multidrug resistance P-glycoprotein and its relationship to hematological characteristics and response to treatment in myelodysplastic syndromes.

Expression of P-glycoprotein (PGP), the product of the multi-drug resistance mdr1 gene was studied by immunocytochemistry on bone marrow slides using JSB1 monoclonal antibody and the alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin-peroxidase (ABC) techniques in 82 cases of untreated myelodysplastic syndromes (MDS), of whom ten had evolved to AML (MDS-AML). The relationship between PGP expression, myeloperoxidase activity and immunophenotype of blast cells, karyotype and outcome was also analyzed. PGP expression was found in the blasts of 34 of the 82 patients (41%), the majority of blasts being stained in positive cases. PGP positivity was rare in 'low risk' MDS (RA and RARS: 2/12 cases) as opposed to 'high risk' MDS (RAEB, RAEB-T, CMML: 25/60 cases) and MDS-AML (7/10 cases) (p = 0.04). PGP expression was positively correlated to the presence of myeloperoxidase activity in less than 3% of blasts (p = 0.025), and CD34 antigen expression (p = 0.04), whereas CD33 antigen expression had borderline significance (p = 0.07), demonstrating that PGP expression predominated in blasts with an immature phenotype. An abnormal karyotype, and especially the presence of monosomy 7, was not correlated to a higher incidence of PGP expression, however. There was a trend for more frequent progression to AML and for shorter survival in PGP-positive cases, but differences with PGP-negative cases were not significant. Twenty patients received intensive anthracycline-Ara-C chemotherapy and ten (50%) achieved complete response, including 9/13 (69%) PGP-negative cases and 1/7 (14%) PGP-positive cases (p = 0.03). Twenty other patients were treated with low-dose Ara-C and ten (50%) responded (complete or partial response). PGP-positivity did not negatively affect response to low-dose Ara-C: 4/11 responses in PGP-negative, and 6/9 responses in PGP-positive patients (p = 0.18). Because the treatment choice in advanced MDS (especially between anthracycline-Ara-C or low-dose Ara-C, chemotherapy) is difficult, our preliminary therapeutic results suggest that the analysis of PGP expression could have practical importance in MDS. These findings however, will have to be confirmed on larger numbers of patients. Clinical trials using drugs potentially reverting mdr, activity could also be warranted in MDS.

Expression of the multidrug resistance P glycoprotein in newly diagnosed adult acute lymphoblastic leukemia: absence of correlation with response to treatment.

We analyzed P glycoprotein (PGP) expression and its correlation with hematological parameters and outcome in 50 cases of newly diagnosed adult acute lymphoblastic leukemia (ALL). PGP expression was evaluated by flow cytometry using MRK16 monoclonal antibody (MoAb) and/or immunocytochemistry on marrow slides, using JSB1 MoAb. Thirty-two of the 50 patients (64%) were PGP positive by at least one of the two methods, which gave concordant results in 15 of the 18 cases in which they were both used. No correlation between PGP expression and clinical and hematological parameters including WBC counts, immunophenotype and karyotype was seen, although there was a trend for more frequent CD34 expression in PGP-positive cases. All patients were treated with intensive chemotherapy. We found no difference in complete remission (CR) rate, actuarial disease-free survival and survival in PGP-positive and PGP-negative cases. Our findings suggest that the clinical significance of PGP expression is less clear in ALL than in AML. Wider use of functional techniques of evaluation of mdr1 gene expression, which assess the 'pumping' activity of PGP, and their correlation with quantitative analysis of mdr1 mRNA and protein, would probably improve knowledge of the role of PGP in ALL. Analysis of other mechanisms of drug resistance, especially multidrug resistance-associated protein (MRP) expression, would also be useful.

bcl-2 expression in myelodysplastic syndromes and its correlation with hematological features, p53 mutations and prognosis.

We looked for bcl-2 protein expression by immunocytochemistry on bone marrow slides from 51 cases of myelodysplastic syndrome (MDS), of whom 25 received some form of chemotherapy. Forty-six of them had at least 20% bcl-2 positive blasts and the median percentage of positive blasts was 80%, whereas myeloid cells beyond blasts were always negative. No correlation was found between bcl-2 expression and the FAB type of MDS, CD34 expression and P-glycoprotein expression. A strong correlation between weak bcl-2 expression and the presence of a p53 mutation detected by SSCP analysis and direct sequencing was found. Response to chemotherapy (intensive chemotherapy or low-dose Ara-C) and survival were not significantly influenced by the intensity of bcl-2 expression in blasts, although there was a trend for better response to chemotherapy and longer survival in patients with strong bcl-2 expression. This trend was no longer found, however, if patients with a p53 mutation were excluded. Our findings show that blasts from a majority of MDS cases have bcl-2 expression and that strong bcl-2 expression is not associated with a poor prognosis. The correlation between weak bcl-2 expression and p53 mutation suggests a possible downregulation of bcl-2 gene expression by mutated p53, the mechanism of which remains to be established.

Philadelphia negative, BCR-ABL positive adult acute lymphoblastic leukemia (ALL) in 2 of 39 patients with combined cytogenetic and molecular analysis.

We performed cytogenetic and molecular analysis of the BCR-ABL rearrangement by polymerase chain reaction (PCR) in 39 consecutive cases of adult acute lymphoblastic leukemia (ALL). Eleven patients had a Philadelphia (Ph) chromosome. Thirteen patients had a BCR-ABL rearrangement, involving minor breakpoint cluster region (m-bcr, situated in intron 1 of the BCR gene) in 11 cases, and major breakpoint cluster region (M-bcr, 'specific' of chronic myeloid leukemia) in the remaining two cases. All of the 12 BCR-ABL cases studied immunologically were of early B, CALLA-positive immunophenotype. The 13 BCR-ABL positive cases included the 11 Ph-positive cases, and two patients with normal karyotype at diagnosis. In the two Ph-negative BCR-positive cases, seven (patient 1) and 18 (patient 2) mitoses had been examined at diagnosis. In patient 1, Ph negativity at diagnosis could certainly be explained by the small number of mitoses analyzed, as a Ph chromosome was found at relapse. This was less probable in patient 2, who raised the issue of whether authentic Ph-negative BCR-ABL-positive ALL exists (as in the chronic myeloid leukemia model) or not. Whatever the explanation, our results suggest that molecular detection of BCR-ABL should be more widely used in B-lineage ALL.

Prognostic significance of p16INK4a immunocytochemistry in adult ALL with standard risk karyotype.

The p16INK4a gene is frequently inactivated in acute lymphoblastic leukemia (ALL), by homozygous deletion. However, p16INK4a protein expression also varies widely in ALL blasts. We investigated the p16INK4a protein expression by immunocytochemistry (ICC) analysis in 76 cases adult ALL. We observed a great variation of the percentage of ICC-positive leukemic cells between samples even in which FISH analysis did not find p16INK4a gene deletion. All patients carrying a p16INK4a gene homozygous deletion were also negative by ICC. ALL with negative p16INK4a ICC were more frequently of T lineage, but no significant differences for white blood cell count, presence of bulky disease, karyotype, hemoglobin level, complete remission rate, overall and event-free survival (EFS) were found. However overall survival and EFS were significantly lower in patients negative by ICC, when analysis was performed in ALL with standard risk karyotype. We also analyzed sequentially at diagnosis and relapse nine cases and observed that one case lost p16INK4a expression between diagnosis and relapse, but that on the contrary three other samples showed increased expression at relapse. These findings suggest that p16INK4a ICC and deletion analysis provide distinct information about ALL cells and that the simple ICC method may be of prognostic value in standard risk adult ALL.

Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoïesis and a high incidence of P53 mutations.

We looked for correlations between cytogenetic rearrangements leading to 17p deletion and presence of dysgranulopoïesis and p53 mutations in MDS and AML. Forty-nine (4.3%) of the MDS and AML studied cytogenetically at our institution over a period of 11 years had detectable 17p deletion, through monosomy 17 (14 cases) or rearrangements of chromosome 17 (generally unbalanced translocations between 17p and another chromosome) (35 cases). Most of the patients had additional complex cytogenetic findings, and 10 cases were therapy related. In 70% of the patients with 17p deletion, a particular type of dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils was seen in > 5% marrow neutrophils, whereas 69% of the patients had a p53 mutation, generally in a missense mutation involving exons 5 to 8 of the p53 gene. FISH analysis, performed in eight cases, confirmed loss of one P53 allele in all of them. No DNA fragmentation suggesting increased apoptosis was found in marrow samples. Response to chemotherapy was almost uniformly poor and median survival was only 3 months. Analysis of dysgranulopoïesis and p53 mutations were also made in 'control' groups of MDS and AML without 17p deletion. 'Typical' dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils in > 5% marrow neutrophils, was not seen in any of the 47 MDS and AML without 17p deletion analyzed and without p53 mutation (P = 10(-4) with patients having 17p deletion), and was seen in one of five patients without 17p deletion but with a p53 mutation. Only 3.1% of 256 MDS and AML without 17p deletion had a p53 mutation (P = 10(-4) with patients having 17p deletion). These findings suggest that 17p deletion, in MDS and AML, is strongly correlated to the presence of a particular type of dysgranulopoïesis and to a high incidence of p53 mutations, and that MDS and AML with 17p deletion could constitute a new morphological-cytogenetic-molecular entity in myeloid disorders.

Myelodysplasia during the course of myeloma. Restriction of 17p deletion and p53 overexpression to myeloid cells.

We report a case of myelodysplastic syndrome (MDS) occurring during the course of multiple myeloma (MM) treated by alkylating agents. Karyotype showed unbalanced t(5;17), resulting in 17p deletion. Dysgranulopoïesis typical of the '17p-syndrome' and p53 mutation and overexpression were present. A combination of FISH and immunophenotype analysis (FICTION, analysis) showed that 17p deletion was restricted to myeloid cells, and that p53 overexpression was also restricted to myeloid cells. These findings strongly argue against a common clonal origin of MM and MDS, and support the hypothesis that MM and MDS were clonally unrelated, and that MDS was indeed secondary to treatment with alkylating agents.

A prospective study of autologous bone marrow or peripheral blood stem cell transplantation after intensive chemotherapy in myelodysplastic syndromes. Groupe Français des Myélodysplasies. Group Ouest-Est d'étude des Leucémies aiguës myéloïdes.

We prospectively assessed autologous stem cell transplantation for consolidation treatment in a trial of intensive chemotherapy in high risk myelodysplastic syndromes (MDS). In this trial, patients aged 55 years or less with no HLA-identical sibling and achieving CR were scheduled to receive unmanipulated autologous bone marrow transplantation (ABMT) preceded by a consolidation chemotherapy course. Forty-two of the 83 patients aged 55 years or less included in the trial (51%) achieved CR. Three were allografted in CR. Twenty-four of the remaining 39 patients who achieved CR (62%) received ABMT (16 patients) or autologous peripheral blood stem cell transplantation (APSCT) (eight patients). Indeed, as bone marrow harvest was often insufficient, APSCT was subsequently proposed after mobilization by consolidation chemotherapy followed by G-CSF. The conditioning regimen combined cyclophosphamide and busulfan. ABMT and APSCT were performed 1-7 months (median 3) after CR achievement. Hematological reconstitution occurred in all patients and tended to be faster after APSCT than ABMT although not significantly. Three patients died from the procedure, nine relapsed after 2-26 months and 12 (50%) were still in CR after 8-55 months. In autografted patients, median Kaplan-Meier disease-free survival and survival were 29 and 33 months from the autograft, respectively. Thus, ABMT or APSCT can be performed in almost two-thirds of MDS patients who achieve CR with intensive chemotherapy. PBSC collection may yield higher numbers of stem cells than marrow collection in some cases, and could improve the percentage of MDS patients autografted in CR. Longer follow-up is required to determine if autograft will prolong CR duration in at least some patients.

Translocation t(10;17)(p13;q12) in two cases of acute nonlymphocytic leukemia with phagocytic activity of blasts. A new cytogenetic entity?

We report two cases of translocation t(10;17)(p13;q12) found in a series of 278 cytogenetically studied acute nonlymphocytic leukemia cases. Blast cells, in both cases, were undifferentiated and had phagocytic properties. These patients might represent cases of a new cytogenetic entity.

Translocation t(1;19)(q23;p13) in acute lymphoblastic leukemia. A report on six new cases and an unusual t(17;19)(q11;q13), with special reference to prognostic factors.

We report clinical, immunologic, and cytogenetic characteristics of six patients with a t(1;19)(q23;p13) that was balanced in one case and of the unbalanced type [-19,der(19)t(1;19)(q23;p13)] in the remaining five cases. Intracytoplasmic immunoglobulins (cIg) were positive in the three cases where they were found. We also report on another patient, with a t(17;19) involving 17q11 and probably 19q13 regions, although involvement of 19p13 could not be excluded. In this patient, cIg were also present, thus raising the issue of whether such a rearrangement could be a variant of t(1;19). Clinically, five patients belonged to the high-risk acute lymphoblastic leukemia (ALL) group, because of high leukocytosis, central nervous system (CNS) disease at presentation, or massive organomegaly. Cytologically, all cases were FAB type L1. Except for the two cases allografted in the first complete remission (CR) all patients relapsed, three of them within 13 months. Two CNS relapses were seen in spite of adequate CNS prophylaxis. ALL with t(1;19) appears to be a poor-risk ALL subgroup and probably requires a reinforcement of therapeutic modalities that might include, when possible, allografting at first CR.

Fas/APO-1 (CD95) expression in myelodysplastic syndromes.

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.

Expression of lung resistance protein and correlation with other drug resistance proteins and outcome in myelodysplastic syndromes.

The major vault lung resistance protein LRP is a cytoplasmic protein involved in drug resistance, especially in acute myeloid leukemia. We looked for LRP overexpression, using immunocytochemistry with LRP 56 monoclonal antibody, on marrow slides from 41 cases of myelodysplastic syndromes (MDS). LRP overexpression (LRP+) was defined by expression of LRP 56 in at least 20% of marrow blasts. LRP overexpression was seen in 19 (46%) cases. Concordant results between LRP overexpression and P-glycoprotein (PGP) expression were seen in 66% of the cases (p = 0.03), and discordant results (LRP+ and PGP-, or LRP- and PGP+) in 33% of the cases. No correlation was seen between LRP overexpression and FAB type, karyotype, CD34, p53 expression and bcl2 overexpression in blasts. Furthermore, in the 18 cases treated with anthracycline-AraC intensive chemotherapy and the 7 cases treated with low dose AraC, the response rate was not significantly different in LRP+ and LRP- patients. Survival was also similar in LRP+ and LRP- patients. In conclusion, LRP overexpression is probably more frequent in MDS than in de novo AML and, as in AML, is only partially correlated with PGP expression. In our experience, however, LRP was not a prognostic factor for response to chemotherapy and survival in MDS.

Detection of monocyte tissue factor after endotoxin stimulation: comparison of one functional and three immunological methods.

Monocyte procoagulant activity is mainly due to tissue factor (TF) expression, but functional assays may not be sufficiently accurate in clinical use, making useful a determination of TF antigen level. The aim of this study was to compare the results of one functional and three immunological TF assays (ELISA, immunocytochemical staining on slides and flow immuno cytometric analysis), in normal monocytes, after standardized stimulation by endotoxin. TF expression was determined in blood mononuclear cells isolated by gradient centrifugation and cultured, with or without various concentrations of endotoxin. On lysed cells, TF activity was determined by amidolytic assay and TF antigen level was determined, after triton extraction, by ELISA (Imubind, American Diagnostica). Mouse monoclonal antibody against TF (4508, American Diagnostica) was used for 1) immunocytochemical (ICC) staining on cytocentrifuge slides (Avidine-Biotine-peroxidase-Complex revelation) and 2) flow cytometric analysis using indirect labeling (Fab'2 Fluoresceine Isothyocyanate revelation). The determination of TF activity and TF antigen by ELISA method were equally sensitive to low concentration of endotoxin (0.005 EU/ml) and well correlated in the presence of higher concentrations of endotoxin. ICC led to a qualitative detection with a similar sensitivity to endotoxin stimulation. Flow cytometric analysis was poorly sensitive to increasing stimulation of monocytes. Of note, the functional, ELISA and immunocytochemical assays for monocyte TF expression were sensitive to endotoxin concentrations as low as 0.005 EU/ml.

Assessment of P-glycoprotein expression by immunocytochemistry and flow cytometry using two different monoclonal antibodies coupled with functional efflux analysis in 34 patients with acute myeloid leukemia.

Drug resistance often results in failure of anticancer chemotherapy in leukemias. A large number of studies have been published on the effect of P-glycoprotein (Pgp) expression on prognosis in AML. However, a consensus has been difficult to reach, due to the variable results obtained by different laboratories. Pgp expression was investigated here in bone marrow samples from 34 patients with AML including 19 newly diagnosed cases and 15 relapsing patients. Pgp expression was performed by immunocytochemistry (ICC) using the aviding-biotin-peroxydase technique with JSB1 and UIC2 MoAbs. Flow cytometry (FCM) analysis of Pgp expression was performed using UCI2 MoAbs in an indirect immunofluorescent assay without cell permeabilization. Rhodamine 123 (Rh 123) uptake was measured in the presence or absence of verapamil. Result was discordant in only 1/20 samples studied with both JSB1 and UIC2 by ICC. Results of Pgp expression were consistent on FCM and ICC in 23 of the 28 (82%) samples tested. Overall, Pgp expression was observed by ICC or FCM in 23 (67%) patients, including 11 (58%) newly diagnosed patients and 12 (80%) patients in relapse. Functional Rh123 efflux (Rh123+) was observed in 20 cases (59%): 10 de novo AML (53%) vs 10 AML in relapse (67%). The functional efflux was correlated with Pgp expression in 25 of the 34 cases analyzed (p = 0.013). 3 (9%) and 6 (18%) samples were Pgp-/Rh123+ and Pgp+/Rh123- respectively. Nine of the 14 pts (64%) treated with intensive anthracyclin-Ara C chemotherapy achieved complete remission, including 5/5 (100%) Pgp- cases vs 4/9 (44%) Pgp+ cases (p = 0.04) and 4/6 (67%) Rh 123- vs 4/7 (57%) Rh123+ cases (p = 0.5). In conclusion, assessment of Pgp expression by ICC and FMC using 2 different MoAbs coupled with functional efflux analysis confirms that Pgp expression is correlated with disease stage and response to treatment in AML. Discordant Pgp/Rh123 cases suggest a non functional Pgp or another alteration of drug transport.

[Assessment of P glycoprotein expression by immunocytochemistry and flow cytometry coupled with functional efflux analysis: application to acute myeloid leukemia]. / Evaluation de l'expression de la glycoprotéine P en immunocytochimie et en cytométrie de flux associée au test fonctionnel: application aux leucémies aiguës myéloïdes.

UNLABELLED: P glycoprotein (Pgp) expression is associated with failure of anticancer chemotherapy in acute myeloid leukemia (AML). However, a consensus has been difficult to reach, due to the variable results obtained by different methods. Samples of 27 patients with AML were studied here according to international recommendations (Beck, et al. , Cancer Research 1996; 56: 3010-20). Pgp expression was performed by immunocytochemistry (ICC) using the avidin-biotin peroxidase technique with JSB1 and UIC2 monoclonal antibodies. Flow cytometry (FCM) analysis of Pgp was investigated using UIC2 in an indirect immunofluorescent assay. UIC2 staining was measured by the Kolmogorov-Smirnov statistical test and fluorescence intensity ratio. Finally, the rhodamine 123 test (Rh 123) with or without verapamil was performed to detect functional activity. RESULTS: by ICC, results of JSB1 and UIC2 were consistent in 94% of the cases. In 74% of the cases, concordant conclusions were observed by ICC and FCM. Overall, Pgp expression was detected in 67% of the cases (ICC/JSB1+ and ICC/UIC2+ or FCM/UIC2+). Functional activity of Pgp was shown in 59% of the patients. Rh 123 efflux was correlated with Pgp expression in 70% of the 27 studied cases but 3 cases were Pgp-/Rh 123+ and 5 Pgp+/Rh 123-. In conclusion, assessment of Pgp expression by ICC and FCM using two different monoclonal antibodies coupled with functional efflux test is required to identify discordant expression/function cases suggesting a non functional Pgp or another alteration of drug transport.