[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.

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.

FISH analysis with a YAC probe improves detection of LAZ3/BCL6 rearrangement in non-Hodgkin's lymphoma.

INTRODUCTION: Chromosomal translocations involving the chromosome 3q27 region are common in B-cell non-Hodgkin's lymphoma (NHL), mainly diffuse large cell lymphoma (DLCL) and less often in follicular lymphoma. Most of these rearrangements involve the same major translocation cluster (MTC) on the 3q27 region, disrupting the LAZ3/BCL6 gene. Some of those translocations are difficult to detect by cytogenetic analysis and/or Southern-blot analysis. In the present report we used a FISH assay to improve the detection of LAZ3/BCL6 rearrangements. METHODS: We isolated a YAC clone (803g3), containing the BCL6 gene, in order to analyze by FISH 19 cases of B-cell non-Hodgkin's lymphoma with cytogenetically detectable 3q27 rearrangement, including reciprocal translocation in 11 cases, deletion in two cases, and addition of undefined chromosomal material on 3q27 in six cases. RESULTS: In the 11 cases with reciprocal translocation, FISH results confirmed cytogenetic data and showed disruption of the LAZ3 region: four t(3;4)(q27;p13), two t(3;11)(q27;q23.1), four t(3;14)(q27;q32) and one t(2;3)(p12;q27). In two of the cases, reciprocal t(3;14) was associated with other cytogenetically detectable abnormalities of 3q27, but FISH showed that they did not affect the LAZ3 gene region. FISH demonstrated a reciprocal translocation with LAZ3 gene rearrangement in two of the six patients with add 3q27: one t(3;11) and one t(3;14). In the two patients with del(3q27), one had two 3q27 FISH signals and one had only one 3q27 FISH signal, but no LAZ3 gene rearrangement was observed. CONCLUSION: We have identified a YAC containing the LAZ3/BCL6 gene. This YAC probe could be useful in clinical practice to demonstrate LAZ3 rearrangements by FISH analysis on tumor samples in NHL.

p190 bcr-abl rearrangement: a secondary cytogenetic event in some chronic myeloid disorders?

BACKGROUND AND OBJECTIVE: A small number of chronic myeloproliferative disorders with hematologic features of chronic myelomonocytic leukemia (CMML) or atypical chronic myeloid leukemia and Ph1 chromosome with m-BCR rearrangement have been reported (p190 CMPD). We report here 3 new cases of p190 CMPD that had unusual features. In 2 of the cases the m-BCR rearrangement appeared to be a secondary event. DESIGN AND METHODS: Patients were studied by cytogenetic, FISH, and molecular biology analyses and followed-up clinically. RESULTS: The first patient initially had typical 5q- syndrome, without m-BCR rearrangement. Five years later, she developed hematologic features of CMML, with t(9;22) translocation, m-BCR rearrangement and high levels of p190 BCR-ABL transcript. The second patient initially had hematologic characteristics of chronic myeloid leukemia (CML) with t(9;22) translocation and m-BCR rearrangement but also other complex cytogenetic findings including 17p rearrangement. Monocytosis developed during the course of the disease. The third patient initially had agnogenic myeloid metaplasia (AMM). Five years later, while the hematologic characteristics were still those of AMM, a first karyotype showed a t(9;22) translocation and molecular analysis showed a very low level of p190 BCR-ABL transcript. Four years later, the patient developed hematologic features of atypical CML with blood monocytosis, t(9;22) and much greater (100 fold) p190 BCR-ABL transcript levels. INTERPRETATION AND CONCLUSIONS: Our 3 cases and review of the previously published cases show the variability of clinical features of p190 positive CMPD. Our results also suggest that, at least in some cases, p190 BCR-ABL rearrangement could be a secondary event in the course of a myeloid disorder.

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.

17p Deletion in acute myeloid leukemia and myelodysplastic syndrome. Analysis of breakpoints and deleted segments by fluorescence in situ.

Recently, we and other groups reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) a strong correlation between cytogenetic rearrangements leading to 17p deletion, a typical form of dysgranulopoiesis combining pseudo-Pelger-Huët hypolobulation and small vacuoles in neutrophils, and p53 mutation. To gain further insight into this "17p-syndrome," we studied 17 cases of AML and MDS with 17p deletion by whole chromosome painting (WCP) and fluorescence in situ hybridization (FISH) with probes spanning the 17p arm, including a p53 gene probe. Cytogenetically, 15 patients had unbalanced translocation between chromosome 17 and another chromosome (chromosome 5 in nine cases and unidentified chromosome -add 17p- in three cases), one patient had monosomy 17, and one had i(17q). All rearrangements appeared to result in 17p deletion. Sixteen patients had additional cytogenetic rearrangements. WCP analysis confirmed the cytogenetic interpretation in all cases and identified one of the cases of add 17p as a t(17;22). WCP also identified chromosome 17 material on a marker or ring chromosome in two cases of t(5;17). FISH analysis with 17p markers made in 16 cases showed no deletion of the 17p markers studied in the last two patients, who had no typical dysgranulopoiesis; p53 mutation analysis in one of them was negative. In the 14 other cases, FISH showed a 17p deletion of variable extent but that always included deletion of the p53 gene. All 14 patients had typical dysgranulopoiesis, and all but one had p53 mutation and/or overexpression. These findings reinforce the morphologic, cytogenetic, and molecular correlation found in the 17p-syndrome and suggest a pathogenetic role for inactivation of tumor suppressor gene(s) located in 17p, especially the p53 gene.

Identification of a YAC spanning the translocation breakpoint t(8;22) associated with acute monocytic leukemia.

Using a series of yeast artificial chromosomes (YAC) from the Bp11-12 chromosome region, we have analyzed a t(8;22) translocation present in two patients suffering from acute leukemia by using fluorescence in situ hybridization (FISH). We have identified a YAC that spans the breakpoint in both cases.

Monoclonal gammopathy of undetermined significance: chromosome changes are a common finding within bone marrow plasma cells.

We used two indirect approaches [image analysis (Feulgen staining) and fluorescence in situ hybridization (FISH)] to study bone marrow plasma cells (BMPC) in 28 patients fulfilling criteria for MGUS. 61% of patients were found to be aneuploid after image analysis: three were hypodiploid and 14 were hyperdiploid. 12/14 hyperdiploid patients also revealed abnormalities after FISH: 12-72% of BMPC exhibited trisomy for at least one of chromosomes 3, 7, 9 and 11. These latter chromosomes are the four chromosomes most frequently implicated (in the shape of trisomy) in MM, confirming the tight relationship between both conditions. After a median follow-up of 19 months (12-41 months) no patient developed overt MM. Also, we failed to find any relationship between currently available biological parameters and DNA findings. As literature data give a transformation rate of 20-30% after a follow-up of 20-35 years, it is worth presuming that some aneuploid patients will evolve to MM, whereas others (also with aneuploid bone marrow plasma cells) will never develop cancer. Our findings indicate that numeric abnormalities, as they are shared both by MGUS and MM patients, are certainly an additional or a prerequisite event, but are not related to an overt disease. They also emphasize the importance of cytogenetic study in the pathophysiology of MGUS.

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.

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.

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.

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.

[Decrease of fecal beta-galactosidase activity in Crohn disease]. / L'activité bêta-galactosidasique fécale est diminuée au cours de la maladie de Crohn.

An increased degradation of colonic mucus by bacterial enzymes might participate in the development of mucosal lesions in inflammatory bowel disease. The biodisponibility of drugs used in the treatment of such disease relies upon the metabolic activity of colonic bacterial flora. This activity can be indirectly assessed by measuring fecal enzymatic activities. The aim of this study was to compare fecal beta-galactosidase (beta-gal) activity in controls, in patients suffering from extradigestive inflammatory disease and in patients with Crohn's disease (CD). Three groups were studied including 11 healthy volunteers (6 F, 5 M) mean age 29 years (21-37), 20 patients with rheumatoid arthritis (RA) (17 F, 3 M), mean age 61.5 years, and 34 patients with non operated CD (21 F, 13 M) mean age: 27 years (13-50). The Crohn disease activity index (CDAI) was > 150 in 24 and < 150 in 10. beta-gal activity was measured in fecal extracts by its ability to hydrolyze paranitrophenyl beta-D-galactopyranoside and expressed as units of enzymatic activity/gram of fecal proteins. beta-gal activity was significantly decreased in patients with CD (16 +/- 4.5 U/g) (m +/- sem) as compared with patients with RA (353 +/- 64 U/g) (P < 0.0001) and to controls (263 +/- 40 U/g) (P = 0.002). beta-gal activity was not significantly different in controls and in patients with RA. Patients with active CD had a significantly lower beta-gal activity than patients with quiescent CD (9.5 +/- 3.7 U/g vs 31.4 +/- 11.5 U/g) (P = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)