RNA for reverse PCR analysis can be obtained from bone marrow cells prepared for cytogenetic analysis and stored at -20 degrees C for several years.
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Base Sequence↗
Biomedical subjects
C Preudhomme
Publications and source records attributed to C Preudhomme.
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.
Gene Expression↗
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.
Acute Disease↗
Transformation of follicular lymphoma with both t(14;18) and t(8;22).
The majority of low grade non-Hodgkin's follicular lymphoma undergo clinical progression to intermediate and high grade lymphoma, but the molecular mechanisms involved in this transformation are not yet well understood. In this article, we describe the case of a 66 year old man with follicular non-Hodgkin's lymphoma (NHL), in whom a centroblastic leukaemic transformation led to death in six months, despite a transient period of remission. At the time of transformation, cytogenetic analysis revealed the original coexistence of t(14;18)(q32;q21) and t(8;22)(q24;q11). These results were confirmed by fluorescent in situ hybridization, while molecular analysis showed a BCL2-JH rearrangement but failed to detect a c-myc rearrangement or any additional p53 mutation. Our observations would therefore suggest other mechanisms to be involved in the transformation of follicular NHL.
Aged↗
p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies.
We analyzed the prognostic value of p53 mutations for response to chemotherapy and survival in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL). Mutations were detected by single-stranded conformation polymorphism (SSCP) analysis of exons 4 to 10 of the P53 gene, and confirmed by direct sequencing. A p53 mutation was found in 16 of 107 (15%) AML, 20 of 182 (11%) MDS, and 9 of 81 (11%) CLL tested. In AML, three of nine (33%) mutated cases and 66 of 81 (81%) nonmutated cases treated with intensive chemotherapy achieved complete remission (CR) (P = .005) and none of five mutated cases and three of six nonmutated cases treated by low-dose Ara C achieved CR or partial remission (PR) (P = .06). Median actuarial survival was 2.5 months in mutated cases, and 15 months in nonmutated cases (P < 10(-5)). In the MDS patients who received chemotherapy (intensive chemotherapy or low-dose Ara C), 1 of 13 (8%) mutated cases and 23 of 38 (60%) nonmutated cases achieved CR or PR (P = .004), and median actuarial survival was 2.5 and 13.5 months, respectively (P < 10(-5)). In all MDS cases (treated and untreated), the survival difference between mutated cases and nonmutated cases was also highly significant. In CLL, 1 of 8 (12.5%) mutated cases treated by chemotherapy (chlorambucil and/or CHOP and/or fludarabine) responded, as compared with 29 of 36 (80%) nonmutated cases (P = .02). In all CLL cases, survival from p53 analysis was significantly shorter in mutated cases (median 7 months) than in nonmutated cases (median not reached) (P < 10(-5)). In 35 of the 45 mutated cases of AML, MDS, and CLL, cytogenetic analysis or SSCP and sequence findings showed loss of the nonmutated P53 allele. Our findings show that p53 mutations are a strong prognostic indicator of response to chemotherapy and survival in AML, MDS, and CLL. The usual association of p53 mutations to loss of the nonmutated P53 allele, in those disorders, ie, to absence of normal p53 in tumor cells, suggests that p53 mutations could induce drug resistance, at least in part, by interfering with normal apoptotic pathways in tumor cells.
Adult↗
Immunoglobulin and T-cell receptor delta gene rearrangements are rarely found in myelodysplastic syndromes in chronic phase.
Clonality, in MDS, can only be assessed in patients with chromosomal rearrangements or in females heterozygote for X chromosome restricted polymorphisms. "Illegitimate" rearrangements of the immunoglobulin heavy chain (IgH) gene and incomplete rearrangements involving V delta 2 and D delta 3 segments of the T-cell receptor delta (TcR delta) gene are seen in some cases of AML, and AML post-SMD, and can be detected by a sensitive PCR method. In order to analyse clonality in additional cases in MDS, we looked for Ig H and TcR delta gene rearrangement by PCR in 95 cases of MDS. A rearrangement of the Ig H gene was seen in 2 of the 95 patients: in the circulating blood of 2 of the 36 cases of chronic myelomonocytic leukaemia (CMML) and in none of the marrow samples of the other 59 MDS. A rearrangement of the TcR delta gene (involving V delta 2 and D delta 3 segments) was seen in three cases (in the circulating blood of two other CMLL patients, and in the bone marrow of another MDS patient). Twenty-five of the 90 cases of MDS with negative PCR findings, in addition to the five cases with positive PCR findings underwent Southern blot analysis of Ig H and TcR delta genes, and PCR analysis of V delta 1 and J delta 1 segments of the TcR delta gene. Those examinations were normal in all the cases tested. In patients with positive PCR findings for Ig H or V delta 2 D delta 3 rearrangements, the proportion of rearranged cells was evaluated at 1-5% in four cases, and 5-10% in the remaining patient. Because the analysis was performed on total circulating leukocytes or total nucleated marrow cells, the nature of the clonal population in positive cases (lymphoid cells? myeloid cells? blasts?) could not be determined. From a practical point of view, Ig H and TcR delta gene rearrangements seem to very rare in MDS, and cannot be used as clonality markers in most cases.
Anemia, Refractory↗
MDM2 gene amplification in human breast cancer.
The MDM2 gene is a gene whose product binds to p53 and regulates its functions. The amplification of the MDM2 gene has been found in one third of human sarcomas, and a differential expression of MDM2 gene in relation with oestrogen receptor status was recently found in human breast cancer cell lines. We analysed 60 breast cancers for MDM2 gene amplification by Southern blot. This event was observed in 1 case with high levels of oestrogen receptor (ER). Thus, MDM2 gene amplification seems to be a rare event in breast cancer. Further studies are needed to define precisely the relationship between MDM2 amplification and ER status.
Blotting, Southern↗
Inactivation of the retinoblastoma gene appears to be very uncommon in myelodysplastic syndromes.
Rearrangements of the retinoblastoma (RB) gene have been reported in a few cases of myelodysplastic syndromes (MDS). In addition, low or absent expression of the RB protein is found in 20-30% of cases of acute myeloid leukaemias (AML), particularly in AML with a monocytic component (M4 or M5). We performed Southern blot analysis of the RB gene in 90 cases of MDS, including 37 cases of chronic myelomonocytic leukaemia (CMML). None of them had progressed to AML at the time of study. In 37/90 patients (including 20 CMML) Northern blot analysis, study of RB protein by immunocytochemistry on bone marrow slides, and detection of point mutations in exons 20-24 of the RB gene was also made, using single strand conformation polymorphism analysis (SSCP). No abnormal Southern profile was found in any of the 90 patients. Northern blot and immunocytochemical study of RB protein were normal in the 37 cases studied. SSCP analysis detected a point mutation in 2/37 patients tested. Direct sequencing confirmed the mutation in each case, which involved intron 21 and intron 23, respectively, and was located outside splicing sites of the neighbouring exons. These findings suggest that abnormalities of the RB gene and its expression must be very rare in MDS, and play a minor role, if any, in the pathophysiology of those disorders, at least before progression to AML.
Base Sequence↗
Over-expression of the MDM2 gene is found in some cases of haematological malignancies.
We looked for MDM2 gene amplification and over-expression by Southern and Northern blot analysis in 135 and 66 cases of haematological malignancies, including ALL, AML, CML in chronic phase, CLL, MDS, PLL, non-Hodgkin's lymphoma (NHL) and myeloma. No amplification of the gene was found. An over-expression of MDM2 RNA was seen in 9/66 (14%) patients tested, including 3/9 ALL, 3/24 AML, 2/4 myelomas, 1/1 PLL, but 0/2 CML, 0/2 NHL and 0/21 MDS. None of the patients over-expressing MDM2 had modifications of P53 gene transcript or p53 mutations. Most of the patients over-expressing MDM2 gene had poor prognostic features (including 'unfavourable' cytogenetic abnormalities), poor response to chemotherapy and short survival. Our findings suggest that over-expression of MDM2 is seen in a relatively small number of haematological malignancies, and is associated with poor prognosis.
Aged↗
Significance of circulating plasma cells in multiple myeloma.
The number of circulating plasma cells (CPC) was determined on mononuclear cell preparations after Giemsa (morphology) and light chain staining (immunocytochemistry). Both methods gave reproducible and identical results when CPC were 1% or more. Using this limit, no CPC were observed in MGUS (0/11) and primary amyloidosis (0/2), whereas 45/98 (45.9%) multiple myeloma (MM) pts had > or = 1% CPC. 3/14 pts (21.4%) in stage I, 5/13 pts (38.5%) in stage II, 20/28 pts (71.4%) in stage III, 4/25 pts (16%) at plateau phase, and 13/18 pts (72.2%) at relapse had > or = 1% CPC (p < 0.001). Mean beta-2 microglobulin was 3.77 mg/l and 6.08 mg/l for pts without or with > or = 1% CPC, respectively p = 0.0001). Presence of CPC was also correlated with an higher percentage of bone marrow PC, but not with the number of Ki-67 positive BM-PC, and not with CRP or LDH levels. K/L and Gamma/Alpha CPC isotype ratio showed these cells as monotypic in nearly all pts. The prognostic value could not really be assessed in this study, as only the initial response to therapy was investigated, and the latter failed to give any difference between pts with and without CPC. So, presence of CPC is not an infrequent finding, but is highly related to tumor mass and active disease; in most if not all patients they are monotypic and certainly belong to the malignant clone. Their prognostic value is unclear but under current investigation; CPC are correlated with B-2M level.
Cell Count↗
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.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Inactivation of the p53 gene in leukemias and myelodysplastic syndrome (MDS) with 17p monosomy.
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Chromosomes, Human, Pair 17↗
Absence of BCR-ABL rearrangement (with m-bcr breakpoint) in chronic myelomonocytic leukemia.
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Aged↗
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.
Anemia↗
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.
Antibodies, Neoplasm↗
Absence of rearrangement of the neurofibromatosis 1 (NF1) gene in myelodysplastic syndromes and acute myeloid leukemia.
Neurofibromatosis 1 (NF1) disease is associated with an increased incidence of leukemias and the NF1 gene product acts as a negative regulator of the product of RAS genes which are often activated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) through point mutations. Thus, we looked for abnormalities of the NF1 gene by Southern analysis in 35 cases of MDS and eight cases of AML, using cDNA probes covering the whole coding sequence. Fourteen of the patients had monosomy 17 (i.e. had lost one allele of the NF1 gene, situated in 17q11-2). Neither rearrangement nor deletion was found in any patient, suggesting that gross abnormalities of the NF1 gene must be very rare in MDS and AML. This does not exclude the possibility of more subtle abnormalities, such as point mutations, in some cases.
Adult↗
Fluorescence in situ hybridization improves the detection of monosomy 7 in myelodysplastic syndromes.
We performed conventional cytogenetic (CC) and interphase fluorescence in situ hybridization (FISH) analysis with an alpha satellite chromosome 7 specific DNA centromeric probe (p alpha 7t1) on bone marrow material prepared for CC in 11 controls and 80 cases of myelodysplastic syndromes (MDS). In controls, a mean of 4.3 +/- 1% of the 700 cells examined showed only one FISH signal for chromosome 7, and the finding of > 6.3% (mean +2 standard deviations) of cells with one FISH signal was considered to indicate the presence of a clone with -7. By CC, clonal -7 was found in 11 patients, whereas two patients had -7 in only one mitose (non-clonal -7). In eight of the 11 cases of clonal -7 by CC, interphase FISH confirmed -7. In the remaining three patients, 5.1%, 6.3% and 18.4% respectively of the cells had one signal. Those three patients had, in addition to -7 by CC, a marker chromosome which was shown to be constituted of chromosome 7 pericentromeric material by FISH analysis on metaphase spreads (metaphase FISH). Of the two patients with non-clonal -7 by CC, one had a -7 clone by interphase FISH whereas the other patient had normal FISH results. Five of the 67 patients with no -7 mitose by CC had clonal -7 by interphase FISH, with one chromosome 7 signal in 14.4 to 39% of the cells examined. At least three mitoses with -7 were found in two of them by metaphase FISH. Three of the five patients were reexamined 12 to 17 months later: CC and metaphase FISH found no -7, whereas interphase FISH still showed a -7 clone. Three of the patients with clonal -7 by CC and by FISH were reexamined in complete hematological remission after intensive therapy. CC found no -7 and interphase FISH was normal in all three patients. Our findings suggest that interphase FISH may improve the detection of -7 in MDS. Conventional cytogenetics should still be performed in parallel to FISH, however, because of possible false negative FISH results when a pericentromeric chromosome 7 marker is present in patients with -7. Larger numbers of cases with minor -7 clones, detectable by FISH only, and longer follow-up in those cases will be necessary to determine the significance of this finding, the evolution of this minor clone, and the outcome of the patients.
Bone Marrow↗
Analysis of p53 antibodies in patients with various cancers define B-cell epitopes of human p53: distribution on primary structure and exposure on protein surface.
p53 antibodies have been found in sera of patients with breast and lung carcinomas and in children with B-lymphomas. We report here the presence of p53 antibodies in sera of patients with 11 different types of cancer. The frequency of seropositives for p53 varied among the different types of cancer, but a correlation with the frequency of p53 gene alteration was established. Using a powerful peptide enzyme-linked immunosorbent assay, we demonstrated that the immune response of patients with p53 antibodies was restricted to a small subset of peptides localized in the amino and carboxy termini of p53, whatever the type of cancer. Given the similarities of the patterns of immune responses in patients with p53 antibodies and animals hyperimmunized with human p53, we propose that the p53 humoral response is the result of a self-immunization process which is itself the consequence of p53 protein accumulation in tumor cells.
Amino Acid Sequence↗