[Waldenström's macroglobulinemia]. / Macroglobulinémie de Waldenström.

Waldenström's macroglobulinemia (WM) is a B-cell disorder characterized primarily by bone marrow infiltration with lymphoplasmacytic cells, along with the presence of an IgM monoclonal gammopathy in the blood. WM remains incurable with a median of 8-year of overall survival for patients with symptomatic WM. Treatment is postponed for asymptomatic patients and progressive anemia is the most common indication for initiation of treatment. The main therapeutic options include alkylating agents, nucleoside analogues, and rituximab, either alone or in combination. Studies involving new combination chemotherapy are ongoing and preliminary results are encouraging. However, there are several limitations to these approaches. The complete response rate is low and the treatment free survival is short in many patients, no specific agent or regimen has been shown to be superior to another, and no treatment has been specifically approved for WM. As such, new therapeutic agents are needed for the treatment of WM. In ongoing efforts, we and others have sought to exploit advances made in the understanding of the biology of WM so as to better target therapeutics for this malignancy. These efforts have led to the development of proteasome inhibitors as bortezomib, several Akt/mTor inhibitors, such as perifosine and Rad001. Many other agents and monoclonal antibodies are currently being tested in clinical trials and seem promising. This article provides an update of the current preclinical studies and clinical efforts for the development of novel agents in the treatment of WM.

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.

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.

Expression of the multidrug resistance-associated protein in myelodysplastic syndromes.

In the myelodysplastic syndromes (MDS), P-glycoprotein (P-gp) expression is clinically associated with drug resistance, whereas the clinical significance of multidrug resistance-associated protein (MRP1) is uncertain. Bone marrow from 56 patients with MDS, including six with refractory anaemia (RA)/RA with ringed sideroblasts (RARS), 23 cases of RA with excess blasts/in transformation (RAEB/T), four patients with chronic myelomonocytic leukaemia (CMML) and 23 cases of MDS having progressed to acute myeloid leukaemia (MDS-AML), were studied. MRP1 expression was investigated by immunocytochemistry (ICC) and by flow cytometry using MRPm6 monoclonal antibody. The efflux test using calcein-AM (CAM) +/- probenecid to evaluate MRP1 activity was performed in ten of the 56 patients. Twenty-eight of the 56 cases (50%) expressed MRP1. MRP1 expression was more frequent in MDS-AML than in MDS (70% vs. 36%). The efflux test using CAM was positive in three out of the ten patients tested. The results were in agreement with expression of MRP1 in six cases, and were discordant in four cases (1 MRP-/CAM+, 3 MRP+/CAM-). No correlation was observed between MRP1 expression and P-gp, lung resistance-associated protein (LRP) or CD34 expression, although there was a trend for more frequent MRP1 expression in P-gp-positive cases in MDS-AML (P = 0.08). Ten of the 26 patients treated with intensive chemotherapy achieved complete remission including six out of 16 MRP1+ and four out of ten MRP1- cases (P = NS). In conclusion, MRP1 expression was correlated with disease stage in MDS in our study. As for P-gp, discordant expression/function of MRP1 could be found in some cases, suggesting the existence of non-functional transport proteins in MDS. MRP1 expression did not seem to be a prognostic factor in MDS in our experience.

[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.

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.

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.

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.

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.

Very low incidence of p53 antibodies in adult non-Hodgkin's lymphoma and multiple myeloma.

In several types of solid tumours, circulating antibodies to p53 are seen in about a third of cases with a p53 mutation, but are absent in cases without p53 mutation. Therefore detection of those antibodies has relatively low sensitivity but high specificity in the detection of p53 mutations. We looked for circulating p53 antibodies by ELISA in 56 adult non-Hodgkin's lymphoma (NHL) and 80 multiple myeloma cases. A certain or highly probable p53 mutation was found by SSCP analysis, immunocyto- or immunohistochemistry in 8/35 (23%) NHL cases and 2/19 (10%) MM cases analysed by these techniques. None of the 80 MM cases and only one of the 56 cases of NHL had circulating p53 antibodies. The positive case had Burkitt's lymphoma and a p53 missense mutation at codon 273. Thus, very few MM and NHL patients with a p53 mutation develop p53 antibodies and this test does not appear to be useful in haematological malignancies.

Long-term follow-up of de novo myelodysplastic syndromes treated with intensive chemotherapy: incidence of long-term survivors and outcome of partial responders.

The percentage of long-term survivors after intensive chemotherapy and the outcome of MDS patients who achieve partial remission (PR) with intensive chemotherapy (IC) are not known. Between 1981 and 1996 we treated 99 patients with de novo MDS who had high-risk MDS or progression to AML, with IC. 41 (41%) achieved CR, 16 (16%) achieved partial remission (PR), 26 (26%) had failure, and 16 (16%) died in aplasia. Eight of the patients who achieved CR were autografted, three were allografted and the remaining cases received moderate consolidation chemotherapy. After IC, the 16 PR patients fulfilled the criteria for RA in 15 cases and CMML in one case. Median PR duration was 17 months, and three PR were > 3 years (39, 50+, 82+ months). Median actuarial survival of patients who achieved PR and CR was 18 months and 20 months from the onset of IC, respectively (difference not significant). Of the 71 patients treated before 1993, with sufficient follow-up, 10 (14%) had survived > 4 years (long-term survivors). Four of them were alive in first CR after 49+ to 110+ months and probably cured, two were alive in PR after 50+ and 82+ months and four had died after 49-78 months. Long-term survivors were characterized by a significantly higher incidence of RAEB-T at diagnosis, and with normal or favourable cytogenetic findings. In patients with RAEB-T at diagnosis included before 1993, 8/23 (35%) cases who had no unfavourable karyotype had survived > 4 years. Our findings suggest that MDS patients who achieve PR with IC, and not only those who achieve CR, can benefit from this type of treatment. The percentage of long-term survivors remains low, however, and is almost restricted to patients with RAEB-T at diagnosis and no unfavourable karyotype.

[Multi-drug resistance and myelodysplastic syndromes: a possible role for remission inducing agents?]. / Résistance multidrogue et syndromes myélodysplasiques: un rôle possible des révertants?

Expression of P-glycoprotein (PGP), the product of the multidrug resistance gene (mdr1), is common in myelodysplastic syndromes (MDSs) and explains in part the low rate of complete remissions (CRs) obtained after aggressive chemotherapy. Reversion of the mdr phenotype to restore chemosensitivity has been the focus of many studies over the last ten years. Two phase III studies evaluated quinine for obtaining reversion of mdr gene expression in MDSs treated by aggressive chemotherapy. Results suggested better response rates and longer survival times in quinine-treated MDR-positive patients. However, the toxicity of quinine warrants further work aimed at developing other mdr phenotype reversion-inducing agents. Some such agents have proved superior over quinine in in vitro studies. Reversion of other mechanisms underlying chemoresistance in MDSs is a promising avenue of research.

Transfer of p16inka/CDKN2 gene in leukaemic cell lines inhibits cell proliferation.

The gene encoding for p16ink4a, a negative regulator of transition between G1 and S phase, is homozygously deleted in a large proportion of acute lymphoblastic leukaemias (ALL). Transfer of p16ink4a gene in several solid tumour cell lines with functional pRb and lacking both p16ink4a alleles has resulted in a dramatic reduction of cell proliferation, and the aim of this work was to confirm this effect in leukaemic (especially ALL) cell lines. We tested the proliferation in liquid medium and in soft agar after transfer of p16ink4a gene by a retroviral vector in leukaemic cell lines with homozygous p16ink4a gene deletion (K562, CEM, Jurkat cell lines) or with p16ink4a gene hemizygous deletion and a point mutation inactivating the remaining allele (HL60 cell line). The viral titre obtained after transfection of PA317 amphotropic packaging cell line, which has a p16ink4a gene homozygous deletion, was low, suggesting that p16ink4a gene expression could impair viral production of retroviral packaging cell lines derived from the NIH3T3 cell line. After retroviral transfer of p16ink4a in cell lines and G418 selection in liquid medium, a strong cell proliferation inhibition was observed for K562, CEM and Jurkat, but no inhibition was seen for HL60. A strong growth reduction in soft agar was also observed with p16ink4a-transduced CEM, Jurkat and K562 cells, with a moderate growth reduction in the HL60 cell line. The growth inhibition in liquid culture, of K562 and Jurkat cell lines, was confirmed by electroporation transfer of the p16ink4a gene. Our findings show that p16ink4a gene transfer has a growth-inhibitory effect in leukaemic cell lines with p16ink4a gene homozygous deletion. These data suggest that p16 could be a suitable gene for gene therapy in ALL.

Is apoptosis a massive process in myelodysplastic syndromes?

We looked for increased apoptosis in fresh bone marrow aspirates in 40 cases of myelodysplastic syndrome (MDS), by detection of DNA fragmentation using TdT incorporation of nucleotides on 3' ends of DNA (TUNEL technique). No DNA laddering was seen. In six cases (15%) the TUNEL technique showed a moderate increase in the percentage of apoptotic cells (2.5-5% in comparison with < 2% in controls). In seven of the 34 patients with normal findings by TUNEL analysis, apoptosis was reanalysed after short-term (18 h) bone marrow culture without inducers of apoptosis. Increased apoptosis was shown in four of the seven cases by morphological analysis and/or the TUNEL technique. Increased apoptosis predominated on erythroblasts in three of them. The percentage of apoptotic cells, however, was < 40% in all samples. Our findings suggest that increased apoptosis can be detected in one half of MDS cases after cell culture. Furthermore, the precise relationship between increased apoptosis of myeloid precursors and cytopenias will have to be more precisely explored 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.

Combined immunophenotyping and in situ hybridization (FICTION): a rapid method to study cell lineage involvement in myelodysplastic syndromes.

We present a study in which we used a recently described method combining fluorescence in situ hybridization (FISH) and immunophenotyping, i.e. FICTION, to assess the involvement of different cell lineages in myelodysplastic syndrome (MDS) with monosomy 7 (-7), trisomy 8 (+8) or loss of Y chromosome (-Y). Blood or marrow smears or cytocentrifuge preparations were stained both by antibodies to granulocytes (CD15), monocytes (CD14), T lymphocytes (CD3), B lymphocytes (CD20) and by probes specific for chromosomes 7, 8 or Y. Of nine cases of MDS with -7, four with +8 and two with -Y studied, none showed lymphocytic involvement by the chromosome abnormality. In contrast, -7, +8 and -Y were found in granulocytes and monocytes in all patients studied, but they involved a variable proportion of those cells. The partial involvement by -7 and +8 seen in some cases suggests that myelopoïesis was only partially clonal in those cases, or that the chromosome abnormality was a secondary event in the MDS process. FICTION therefore appears to be a simple and easily reproducible method that can be used for the assessment of lineage involvement in MDS and other haematological malignancies.