NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype.

Cervical cancer is associated with human papilloma virus infection. However, this infection is insufficient to induce transformation and progression. Loss of heterozygosity analyses suggest the presence of a tumor suppressor gene (TSG) on chromosome 6p21.3-p25. Here we report the cloning NOL7, its mapping to chromosome band 6p23, and localization of the protein to the nucleolus. Fluorescence in situ hybridization analysis demonstrated an allelic loss of an NOL7 in cultured tumor cells and human tumor samples. Transfection of NOL7 into cervical carcinoma cells inhibited their growth in mouse xenografts, confirming its in vivo tumor suppressor activity. The induction of tumor dormancy correlated with an angiogenic switch caused by a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These data suggest that NOL7 may function as a TSG in part by modulating the expression of the angiogenic phenotype.

High incidence of translocations t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with plasma cell malignancies.

Abnormalities involving the 14q32 region are recurrent chromosomal changes in plasma cell malignancies. Recent preliminary molecular analyses found IGH rearrangements in almost 100% of human myeloma cell lines and in 75% of patients. However, no systematic study analyzing the nature of the partner chromosomal regions have been reported thus far. To define the exact incidence of illegitimate IGH rearrangements and the respective incidence of partner genes cloned to date, we analyzed 141 patients with either multiple myeloma (MM, n = 127) or primary plasma cell leukemia (PCL, n = 14) using fluorescence in situ hybridization. The overall incidence of illegitimate recombinations was 57% (80 of 141 patients). Analysis of this incidence according to Durie and Salmon stage, patients' status, i.e., MM versus primary PCL and diagnosis versus relapse, immunoglobulin type and subtype, and beta2-microglobulin value, did not show any correlation. To analyze the nature of the partner chromosomal region, we selected probes specific for the following genes: FGFR3 (4p16), MYC (8q24), CCND1 (11q13), MAF (16q23), and BCL2 (18q21). These probes, combined with differentially labeled 14q32 probes, were used for dual-color fluorescence in situ hybridization on interphase plasma cells. Among the 80 patients with illegitimate IGH rearrangement, we identified 23 IGH-CCND1 fusion cases [i.e., t(11;14)], 17 IGH-FGFR3 fusion cases [i.e., t(4;14)], 3 IGH-MYC fusion cases [i.e., t(8;14)], and only one IGH-MAF fusion case. No IGH-BCL2 fusion case was detected. In 37 of 80 patients, none of these partner genes was involved. Analysis of cases with specific translocations according to their bioclinical features at diagnosis did not show any correlation. This study demonstrated that CCND1 and FGFR3 genes are involved together in about 50% of MM and primary PCL patients with illegitimate IGH rearrangements.

Deletion of chromosome 20q associated with hypereosinophilic syndrome. A report of two cases.

Deletion of the long arm of chromosome 20--del(20q)--is commonly associated with myeloid malignancies, in particular with myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), and acute myelogenous leukemia (AML). Its association with the hypereosinophilic syndrome (HES) had never been reported. In the present study we describe two patients with long-standing hypereosinophilia and features of atypical MPD or MDS carrying a del(20q) as a constant and isolated chromosomal abnormality. One patient, with an aggressive clinical course, died within 2 years, despite several lines of treatment. The other patient had a more indolent course consistent with that of an atypical MDS with eosinophilia.

A "miniaturized" method for the karyotypic analysis of bone marrow or blood samples in hematological malignancies.

Standard techniques of karyotypic analysis of bone marrow or peripheral blood cells generally use large numbers of cells. Thus, the quantity of cells harvested from one bone marrow or blood puncture frequently represents a limiting factor for other assays such as molecular biology or flow cytometry, which are often essential for the diagnosis of hematological diseases. To resolve this problem, we developed a "miniaturized technique" of cytogenetic analysis that we tested on bone marrow (BM) cells from 20 patients with multiple myeloma (MM), 5 patients with acute leukemia (AL), as well as on CD34+ cells purified from the blood of 8 patients with agnogenic myeloid metaplasia (AMM). We used 3 x 10(6) BM cells from MM patients (for testing 6 different culture conditions), 10(6) BM cells from AL (2 culture conditions) and 4 x 10(3) CD34+ cells from AMM patients. In most patients, 20 good quality metaphases per slide were easily analyzed, showing that a 10-40 times reduction of the number of cells used for cytogenetics allows a reliable karyotypic analysis in hematological malignancies.

Hypodiploidy is a major prognostic factor in multiple myeloma.

Conventional karyotypes performed before any treatment in 208 patients with multiple myeloma were reviewed by the Groupe Français de Cytogénétique Hématologique. A total of 138 patients displayed complex chromosomal abnormalities (CCAs). According to the chromosome number pattern, a first group of 75 patients had a hyperdiploid karyotype. A second group of 63 patients referred to as the hypodiploid group had either pseudodiploid, hypodiploid, or near-tetraploid karyotypes. Of 159 treated patients available for survival analysis, 116 had an abnormal karyotype. The comparison of overall survival (OS) between hyperdiploid and hypodiploid patients showed a highly significant difference (median OS 33.8 vs 12.6 months, respectively, P <.001). The presence of 14q32 rearrangements (36 of 116 patients) worsened the prognosis (median OS 17.6 vs 29.9 months, P <.02). The presence of chromosome 13q abnormalities (13qA, 63 patients) did not modify OS in CCA patients (median OS 20.6 vs 27.8 months, P <.59). However, taking into account the whole series including normal karyotypes, 13qA (63 of 159 patients) had a significant impact on OS (median 20.6 vs 37.1 months, P <.04). In the same way, the presence of a hypodiploid karyotype (52 of 159 patients) had a strong prognostic value (OS 12.8 vs 44.5 months, P <.000 01). A multivariate analysis including stage, beta2-microglobulin, bone marrow plasmocytosis, treatment type, 13qA, and hyperdiploidy and hypodiploidy showed that a hypodiploid karyotype was the first independent factor for OS (P <.001), followed by treatment approach. These results confirm that the chromosome number pattern of malignant plasma cells is a very powerful prognostic factor in newly diagnosed multiple myeloma patients.

Cytogenetic study of 30 patients with multiple myeloma: comparison of 3 and 6 day bone marrow cultures stimulated or not with cytokines by using a miniaturized karyotypic method.

Cytogenetics in multiple myeloma (MM) cases are generally difficult to perform due to the low proliferation index of malignant plasma cells (PC) in most cases. Although IL-6 and GM-CSF stimulate the in vitro proliferation of malignant plasma cells, their usefulness for improving cytogenetic results in multiple myeloma patients remains questionable, because results which compare various culture conditions in a sufficient number of patients are not available. By using a miniaturized karyotypic method, we compared in 30 multiple myeloma patients the number and percentage of clonal abnormal mitoses obtained from 3 and 6 d bone marrow cultures performed without or with two combinations of cytokines: IL-6 + GM-CSF or IL-6 + GM-CSF + IL-2 + IL-4 + TNFalpha. The percentage of patients with an abnormal karyotype, which varied with the Durie and Salmon stage of the disease, as well as the type of numerical and structural karyotypic abnormalities that we detected, were in agreement with published results. The detection of clonal karyotypic abnormalities was better after 3d of culture without cytokine than in all other culture conditions. The higher percentage of patients at all stages of MM with an abnormal karyotype in our study (76.6%) than in previous ones (20% to 60%) is largely explained by the large number of mitoses analysed in six different culture conditions due to the use of a miniaturized karyotypic method.

Is treatment with hydroxyurea leukemogenic in patients with essential thrombocythemia? An analysis of three new cases of leukaemic transformation and review of the literature.

From 1981 to 1995, we diagnosed, followed and treated at our institution fifty-eight cases of essential thrombocythemia (ET), using hydroxyurea (HU) as first-line therapy in these patients. Three patients who were continuously receiving HU had a leukemic transformation after a chronic phase of respectively 47, 81 and 90 months. One patient developed an acute leukemia with minimal myeloid differentiation (AML MO) and soon died of refractory disease; the second developed a refractory anemia with excess blasts in transformation (t-RAEB) and survived one year; the third patient developed a chronic myelomonocytic leukemia (CMML) and is alive at 21 months. The two former patients had complex nonrandom bone marrow karyotypic abnormalities, suggestive of therapy-related leukemia, whereas the latter one had a normal karyotype throughout the chronic and leukemic phase. These findings, together with recently published results on myeloproliferative disorders (MPD) treated with HU, suggest that this drug might be as leukemogenic as other myelosuppressive therapies in patients with ET. Longterm HU therapy should be reserved for patients in whom the treatment benefits obviously outweigh the risk of inducing leukemia.

Glial fibrillary acidic protein expression in a new human glioma cell line in culture before and after xenogenic transplantation into nude mice.

A human glioma cell line, SA146, was initiated on precoated extracellular matrix from a stereotactic biopsy of a glioblastoma. We report modulation in the expression of glial fibrillary acidic protein (GFAP) by SA146 passed in vitro before or after xenogenic transplantation into nude mice. Immunofluorescence data show a decrease in the percentage of GFAP-expressing cells with increasing in vitro passages but a full reexpression (100% of GFAP-positive cells among vimentin-positive cells) was observed in cultures just derived from the xenotransplanted tumor. These changes are correlated with the mRNA content (Northern blot probed with a cDNA for GFAP) and with the protein level (cytoskeletal fraction analyzed by two-dimensional gel electrophoresis and Western blots probed with a monoclonal antibody). At the optimal level of GFAP expression, a large range of micro-heterogeneity in GFAP isoforms is reached for which post-translational events are clearly involved since mRNA translation in cell free system would provide at best three isomers. We suggest that SA146 would be an appropriate model to study the regulation of GFAP expression in the context of human glial tumor biology.

Cytogenetic, interphase, and multicolor fluorescence in situ hybridization analyses in primary plasma cell leukemia: a study of 40 patients at diagnosis, on behalf of the Intergroupe Francophone du Myélome and the Groupe Français de Cytogénétique Hématologique.

Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM-a higher incidence of t(11;14) (33% vs 16%; P <.025) and of t(14;16) (13% vs 1%; P <.002) though incidences of t(4;14) were identical and a higher incidence of monosomy 13 (68% vs 42%; P =.005). Hypodiploid karyotypes and monosomy 13 may explain, at least in part, the poorer prognosis of primary PCL. In contrast, significantly longer survival was observed in patients displaying t(11;14) in comparison with those lacking this translocation (P =.001).

Monosomy 13 is associated with the transition of monoclonal gammopathy of undetermined significance to multiple myeloma. Intergroupe Francophone du Myélome.

Chromosomal abnormalities are present in most (if not all) patients with multiple myeloma (MM) and primary plasma cell leukemia (PCL). Furthermore, recent data have shown that numerical chromosomal changes are present in most individuals with monoclonal gammopathy of undetermined significance (MGUS). Epidemiological studies have shown that up to one third of MM may emerge from pre-existing MGUS. To clarify further possible stepwise chromosomal aberrations on a pathway between MGUS and MM, we have analyzed 158 patients with either MM or primary PCL and 19 individuals with MGUS using fluorescence in situ hybridization (FISH). Our FISH analyses were designed to detect illegitimate IGH rearrangements at 14q32 or monosomy 13. Whereas translocations involving the 14q32 region were observed with a similar incidence (60%) in both conditions, a significant difference was found in the incidence of monosomy 13 in MGUS versus MM or primary PCL. It was present in 40% of MM/PCL patients, but in only 4 of 19 MGUS individuals. Moreover, whereas monosomy 13 was found in the majority of plasma cells in MM, it was observed only in cell subpopulations in MGUS. It is noteworthy that, in a group of 20 patients with MM and a previous MGUS history, incidence of monosomy 13 was 70% versus 31% in MM patients without a known history of MGUS (P =.002). Thus, this study highlights monosomy 13 as correlated with the transformation of MGUS to overt MM and may define 2 groups of MM with possible different natural history and outcome, ie, post-MGUS MM with a very high incidence of monosomy 13 and de novo MM in which other genetic events might be involved. Serial analyses of individuals with MGUS will be needed to validate this model.

High incidence of cryptic translocations involving the Ig heavy chain gene in multiple myeloma, as shown by fluorescence in situ hybridization.

Cytogenetic studies have shown rearrangements of the Ig heavy chain (IGH) gene at 14q32 in 10-60% of patients with multiple myeloma (MM) or primary plasma cell leukemia (PCL). Analysis of MM patients and human myeloma cell lines (HMCL) using interphase fluorescence in situ hybridization (FISH) and molecular techniques has shown IGH rearrangements in 75% of MM cases and in up to 100% of HMCL. A review of the literature revealed at least 18 different partner chromosomal regions. To investigate whether some of these translocations were recurrent and possibly to identify new partner regions, we developed a set of FISH probes to detect every IGH recombination. We analyzed 28 MM and 4 primary PCL patients with abnormal karyotypes and 12 HMCL. Whereas conventional cytogenetics detected a 14q32 abnormality in only 15% of the patients, FISH detected it in 47% of patients and in 75% of HMCL. The partner chromosome was identified in 10 of 15 patients with a 14q32 rearrangement. Interestingly, the same t(4; 14)(p16;q32) was detected in five patients and three HMCL, i.e., 33% of patients and HMCL with an IGH rearrangement. New partner chromosomal regions have also been identified, i.e., 9p13, 12p11, 12p13, and Xq28, besides the previously reported 8q24, 11q13, 12q24, and 16q24 rearrangements. The genes involved in these new translocations are not known, except for 9p13, where PAX5 was shown to be the partner gene. We conclude that: I) IGH recombinations are frequent but not constant in MM, 2) these rearrangements often occur through cryptic translocations, and 3) the t(4;14)(p16;q32) is one of the most frequent translocations, but many other chromosomal regions may be involved.