Deletion cartography around the D13S25 locus in B cell chronic lymphocytic leukemia and accurate mapping of the involved tumor suppressor gene.

The presence of an unidentified tumor suppressor gene on the long arm of chromosome 13 which could be involved in the development of B cell chronic lymphocytic leukemia has been suspected because of frequent deletions of the locus D13S25 which lies 1.6 cM telomeric to the retinoblastoma gene. In order to accurately map this gene, cells from 25 B cell chronic lymphocytic leukemia tumors have been analyzed for allelic loss using a panel of microsatellite markers located in this region. These markers, which stretch from the retinoblastoma gene to the Wilson disease gene, have been ordered for their rank from centromere to telomere. In addition to the data obtained from deletion pattern of these markers, results from preliminary pulse-field electrophoresis studies enable us to redefine the minimal deleted area from more than 1 cM to 280 kilobase around D13S25.

CD28, a marker associated with tumoral expansion in multiple myeloma.

CD28 expression was thoroughly investigated on plasma cells of monoclonal gammopathy of undetermined significance, multiple myeloma (MM), and human myeloma cell lines. CD28+ plasma cells were detected in 19% of 31 monoclonal gammopathy of undetermined significance, 41% of 116 MM, and 100% of 13 human myeloma cell lines. CD28+ myeloma cells were detected in 21 of 79 (26%) MM cases at diagnosis, 13 of 22 (59%) at medullary relapse (P < 0.009), and 14 of 15 (93%) at extramedullary relapse (P = 0.05), including 10 of 10 (100%) secondary plasma cell leukemias (P = 0.05). Serial studies in individual patients confirmed the emergence of CD28+ myeloma cells with tumoral expansion and treatment failure. This was significantly correlated with the expression of CD28 ligand, i.e., CD86 (but not CD80), and with an increase in the proliferative activity (labeling index) of myeloma cells in bone marrow. Whereas the expression of CD56 defines a particular subset of myeloma patients, CD28 is the only antigen for which expression correlates with tumor progression. Our data show that an aggressive compartment of CD28+ and CD86+ myeloma cells emerges during the course of MM in vivo, indicating that CD28 could be aberrantly expressed on highly malignant (possibly mutated) myeloma cells. Conversely, a subset of proliferative plasmablasts coexpressing CD28 and CD86 could be the normal counterpart of the clonogenic myeloma stem cell because a subset of CD28+ plasma cells was observed in 6 of 6 cases of reactive plasmocytosis.

High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis.

Using allele-specific amplification method (ARMS), a highly sensitive one-stage allele-specific PCR, we have evaluated the incidence of NRAS and KRAS2 activating mutations (codons 12, 13, and 61) in 62 patients with either monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM), primary plasma-cell leukemia (P-PCL), and also in human myeloma cell lines (HMCL). NRAS and/or KRAS2 mutations were found in 54.5% of MM at diagnosis (but in 81% at the time of relapse), in 50% of P-PCL, and in 50% of 16 HMCL. In contrast, the occurrence of such mutations was very low in MGUS and indolent MM (12.50%). Of note, KRAS2 mutations were always more frequent than NRAS. The validity of the technique was assessed by direct sequencing of cell lines and of some patients. Multiple mutations found in two patients were confirmed by subcloning exon PCR amplification products, testing clones with our method, and sequencing them. Thus, these early mutations could play a major role in the oncogenesis of MM and P-PCL.

Cyclin D1 expression in patients with multiple myeloma.

INTRODUCTION: Chromosomal abnormalities are detected in 50 to 70% of patients with multiple myeloma (MM). By conventional cytogenetic analysis, a t(11;14)(q13;q32) is observed at a frequency of 3 to 14%. MATERIALS AND METHODS: To demonstrate a cyclin D1 expression in MM patients or MM cell lines, 14 patients with multiple myeloma (MM) and nine human multiple myeloma cell lines (HMCL) were screened by a competitive RT-PCR and/or Northern blot analysis for cyclin D1 expression. Furthermore, we screened 10 MM patients with FISH to demonstrate a relationship between the cyclin D1 expression and the presence of the t(11;14). RESULTS: Five HMCL had a cyclin D1 overexpression: three of them had a t(11;14)(q13;q32) and two had extra copies of chromosome 11. A cyclin D1 expression was found at diagnosis in seven out of 14 untreated MM patients (50%). Out of 14 MM patients, FISH studies were performed in 10 patients. A t(11;14) was detected in three out of 10 patients and extra copies of chromosome 11 were found in two additional patients. CONCLUSION: Cyclin D1 expression is a common event in MM patients (50%) and is associated either with a t(11;14)(q13;q32) or extra copies of chromosome 11. The prognostic role of the cyclin D1 expression and the level of this expression, as compared to other B-cell chronic lymphoproliferative disorders such as mantle cell lymphoma or hairy cell leukemia, remains to be determined in the pathogenesis of multiple myeloma.

PCR detection of residual Bcl-2/IgH-positive cells after high-dose therapy with autologous stem cell transplantation is a prognostic factor for event-free survival in patients with low-grade follicular non-Hodgkin's lymphoma.

This study was designed to evaluate the results of high-dose therapy followed by purged autologous stem cell transplantation (ASCT) for patients with low-grade follicular non Hodgkin's lymphoma (LGFL), and the prognostic significance of PCR detection of residual Bcl-2/IgH-positive cells after ASCT. Between 1992 and 1998, 49 patients with LGFL received total body irradiation and high-dose cyclophosphamide followed by purged ASCT. PCR amplification of the Bcl-2/IgH rearrangement was performed at diagnosis, on stem cell collections before and after purging and on bone marrow and blood samples after ASCT. With a median follow-up of 76 months (37-103) 34 patients remain alive and event-free. A total of 20 patients had disease recurrence, three patients developed secondary myelodysplastic syndrome (MDS). In all, 11 patients died; 10 deaths were because of recurrent disease, one because of MDS. Kaplan-Meier estimates of event-free survival (EFS) and overall survival (OS) at 5 years were 65% (+/-7%) and 77% (+/-6%), respectively. Patients who achieved a sustained molecular complete response (CR) had a lower risk of disease recurrence and experienced significantly longer EFS (93% (+/-6%) vs 11% (+/-7%) P=0.0008) and OS (100 vs 55% (+/-12%) P=0.0057). In conclusion, myeloablative therapy followed by purged ASCT may induce long EFS in patients with LGFL. The achievement of sustained molecular CR after ASCT improves EFS and OS.

Chromosome mapping and expression of the human interleukin-13 receptor.

Interleukin-13 (IL-13) is a cytokine secreted by activated T cells and shares most but not all biological activities with interleukin-4 (IL-4). Both cytokines play an important role as a switch factor directing synthesis of IgE; they act on monocytes and endothelial cells, but unlike IL-4, IL-13 does not act on T cells. These cytokines have both common and distinct components in their respective receptors. Based on sequence similarity shared by cytokine receptor family members, we have identified a cDNA encoding the human IL-13 receptor (IL-13R). This cDNA was used to examine the pattern of IL-13R mRNA expression by Northern blot analyses of poly(A)+ RNA purified from different human tissues and cell lines. Among several myeloma cell lines analyzed, the U266 cell line was the only one found to express IL-13R transcripts. This cell line is also the only one described as producing IgE. The IL-13R gene was mapped to chromosome Xq24 by in situ hybridization. Interestingly, this locus is near that of the CD40 ligand gene, the product of which is also involved, like IL-13, in proliferation and IgE isotype switching of human B cells. The human IL-13R gene maps between two cytokine receptor genes located on the chromosome arm Xq region: the interleukin-2 receptor gamma chain gene (Xq13.1) and the interleukin-9 receptor gene (Xq28). The lack of nucleotide sequence similarity suggests unrelated evolutionary pathways between these receptor genes.

IL-6 upregulates its own receptor on some human myeloma cell lines.

Interleukin 6 (IL-6) is the major survival factor of myeloma cells. In this study, we demonstrate that IL-6, oncostatin M (OSM) and leukemia inhibitory factor (LIF) upregulate membrane IL-6 receptor alpha (IL-6Ralpha) on OPM-2 myeloma cell line at transcriptional level. In OPM-2 cells, IL-6, OSM and LIF induce both signal transducers and activators of transcription (STAT), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-K) activation. We show that the cytokine-induced upregulation of IL-6Ralpha can be abolished by a janus kinase (JAK)-2 specific inhibitor, i.e. AG490, suggesting an involvement of the JAK/STAT pathway in this process. Finally, IL-6Ralpha upregulation was also inhibited by wortmannin, an inhibitor of the PI 3-kinase pathway. In conclusion, IL-6 can upregulate its own receptor on OPM-2 cells probably through the JAK/STAT and PI 3-kinase pathways.

High incidence of deletions but infrequent inactivation of the retinoblastoma gene in human myeloma cells.

We have studied the retinoblastoma (RB-1) susceptibility gene status and pRB expression in 22 human myeloma cell lines (HMCL) and in 10 patients with advanced multiple myeloma (MM). Deletions of the RB-1 gene were observed in 81% (17/21) of the informative HMCL, regardless of their paracrine or autocrine interleukin-6 (IL-6) status. Among the deleted HMCL, only one (U266) had a biallelic deletion and lacked pRB expression. Monoallelic deletions had no consequence on the RB-1 gene activation and pRB expression. One patient of 10 presented the same biallclic deletion as U266 and six of 10 had monoallelic deletions. We conclude that monoallelic deletions of the RB-1 gene are frequent in HMCL and MM patients but have no consequence on gene activation and pRB expression.

Molecular cytogenetic abnormalities in multiple myeloma and plasma cell leukemia measured using comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to identify recurrent regions of DNA sequence loss and gain in 21 multiple myeloma (MM) and plasma cell leukemia (PCL) primary tumor specimens and cell lines. Multiple regions of non-random sequence loss and gain were observed in 8/8 primary advanced stage tumors and 13/13 cell lines. Identification of sequence copy number changes was facilitated by statistical analyses that reduce subjectivity associated with identification of copy number changes and by requiring that sequence changes are visible using both red- and green-labeled tumor DNA. Loss of sequence on 13q and 14q and gain of sequence on 1q and chromosome 7 occurred in 50-60% of the population. In general, cell lines carry more and larger regions of sequence gain and loss than primary tumors. Regions of sequence copy number change that recur among MM cell lines and primary tumors include, in order of prevalence, enh(1q12qter), dim(13), enh(7), enh(3q22q29), enh(11q13.3qter), dim(14q11.2q31), enh(8q21qter), enh(3p25pter), dim(17p11.2p13), and dim(6q22.1q23). Population distributions of genome-wide changes in primary tumors reveal "hot-spots" of sequence loss from 13q12.1-q21, 13q32-q34, 14q11.2-q13, and 14q23-q31. Genomic changes detected using CGH are consistent with those identified using banding analyses, although recurrent involvement of additional regions of the genome are also evident. A higher prevalence of genomic changes is visible using CGH compared to banding. Identification of recurrent regions of sequence gain and loss provides opportunities to identify regions of the genome that may be involved in the malignant phenotype and/or disease progression.

Metalloproteinases in multiple myeloma: production of matrix metalloproteinase-9 (MMP-9), activation of proMMP-2, and induction of MMP-1 by myeloma cells.

Multiple myeloma is a very devastating cancer with a high capacity to destroy bone matrix. Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor invasion. In this study, we have investigated the involvement of interstitial collagenase (MMP-1) and gelatinases (MMP-2 and MMP-9) in the biology of multiple myeloma. We show (1) that myeloma cells express MMP-9 and (2) that this expression is not subjected to regulation either by interleukin-6 (IL-6), the major myeloma cell growth factor, or by other cytokines involved in the multiple myeloma cytokine network. In the tumoral environment, we show that bone marrow stromal cells express MMP-1 and MMP-2. Whereas MMP-1 is positively regulated by IL-1beta, tumor necrosis factor-alpha, and Oncostatin M, MMP-2 is not modulated by any of these cytokines. To evaluate whether myeloma cells can modify the bone marrow stromal environment, we have examined these MMP activities in coculture. Interestingly, we have observed an upregulation of MMP-1 and a partial conversion of the proMMP-2 into its activated form. We conclude that the increase of MMP activity produced or induced by myeloma cells in these cocultures could favor bone resorption and tumor invasion. Inhibition of such activities could represent a new therapeutical approach in multiple myeloma.

Comparative genomic hybridization detects genomic abnormalities in 80% of follicular lymphomas.

Comparative genomic hybridization (CGH) was used to analyse 34 follicular lymphoma (FL) samples. 27 samples showed DNA sequence copy number changes of at least one genomic region (26 samples with at least one gain and nine with at least one loss). Some chromosomes or chromosomal regions were preferentially involved. The most frequently gained regions were chromosome 18q (29% of samples), chromosome X (21%), chromosome 7 (18%), chromosomes 2, 6p and 8q (12%). Two regions were preferentially lost: 6q (12%) and 17p (9%). All these gained and lost regions have been previously reported in cytogenetic studies, confirming the accuracy of CGH in detecting genetic abnormalities in FL. 21% of samples displayed normal profiles, probably reflecting the absence of unbalanced abnormality, which is also in agreement with the cytogenetic data. In conclusion, we showed that CGH is an accurate, reliable and rapid method and we propose the inclusion of CGH in the evaluation of FL at diagnosis.

Myeloma cell growth arrest, apoptosis, and interleukin-6 receptor modulation induced by EB1089, a vitamin D3 derivative, alone or in association with dexamethasone.

We have previously shown that malignant plasma cells expressed the specific receptor for 1,25-dihydroxyvitamin D3 and that this derivative could significantly inhibit the proliferation of such malignant cells. More recently, new vitamin D3 derivatives have been generated with extraordinarily potent inhibitory effects on leukemic cell growth in vitro. These new data prompted us to (re)investigate the capacity of such new vitamin D3 derivatives to inhibit myeloma cell growth in comparison with that of dexamethasone, a potent antitumoral agent in multiple myeloma. In the current study, we show that EB1089, a new vitamin D3 derivative, (1) induces G1 growth arrest of human myeloma cells, which is only partially reversed by interleukin-6 (IL-6); (2) induces apoptosis in synergy with dexamethasone, IL-6, leukemia-inhibitory factor, and Oncostatin M, with an agonistic anti-gp130 monoclonal antibody being unable to prevent this apoptosis; (3) downregulates both the gp80 (ie, the alpha chain of the IL-6 receptor [IL-6Ralpha]) expression on malignant plasma cells and the production of soluble IL-6Ralpha, and finally (4) inhibits the deleterious upregulation of gp80 expression induced by dexamethasone while limiting the dexamethasone-induced upregulation of gp130 expression. Considering that these in vitro effects of EB1089 have been observed at doses obtainable in vivo (without hypercalcemic effects), our present data strongly suggest that EB1089 could have a true interest in the treatment of multiple myeloma, especially in association with dexamethasone.

The cancer germ-line genes MAGE-1, MAGE-3 and PRAME are commonly expressed by human myeloma cells.

In this study, we have investigated the mRNA expression of the cancer germ-line genes MAGE, BAGE, GAGE, RAGE and the tumor-overexpressed gene PRAME by human myeloma cell lines and malignant plasma cells from patients with multiple myeloma (MM). By reverse transcription-PCR, we show that all myeloma cell lines (n = 16) express at least one of these genes, except RAGE-1 that was never expressed. We show that malignant plasma cells from the majority of MM patients (n = 21) expressed MAGE-1, MAGE-3 and PRAME. On the contrary, polyclonal reactive plasma cells did not express any of these genes. By flow cytometry, we show that mage-1 protein is expressed within myeloma cells and cell lines and that anti-mage-1.HLA-A1 cytotoxic T lymphocytes efficiently killed MAGE-1+HLA-A1+ MDN myeloma cells. Taken together, our data show that mage-1 and mage-3 could constitute specific targets for tumor immunotherapy of MM patients.

Amplification of the 11q23 region in acute myeloid leukemia.

Cytogenetic abnormalities involving the 11q23 region are found in both acute lymphoblastic leukemia (ALL) and myeloid leukemia (AML). Molecular consequences of 11q23 translocations are the formation of chimeric genes, all of them involving the MLL (mixed-lineage leukemia) gene. To evaluate the usefulness of fluorescence in situ hybridization (FISH) in detecting MLL rearrangements in AML, we analyzed 181 patients with an MLL-specific probe. Among them, we detected three patients with multiple FISH signals, reflecting genomic amplification of this chromosomal region. Extra copies of MLL have been reported previously in four patients, but did not correspond to a true gene amplification. For the first time, we describe genomic amplification of the 11q23 region (up to more than 50 copies) in AML patients. This genomic amplification could affect MLL, but other genes in the vicinity could also be the primary target. Genes Chromosomes Cancer 26:166-170, 1999.

Detection of t(11;14) using interphase molecular cytogenetics in mantle cell lymphoma and atypical chronic lymphocytic leukemia.

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty-eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL-type immunophenotype, i.e., CD19-, CD5+, CD23-/low, CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual-color interphase FISH. We selected an IGH-specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH-CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH-CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)-positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities.