Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

Methylation of the p15(INK4b) gene in myelodysplastic syndromes is frequent and acquired during disease progression.

p15(INK4b) gene is an inhibitor of cyclin-dependent kinase (CDK) 4 and CDK6 whose expression is induced by transforming growth factor (TGF)beta. Recent reports suggest frequent methylation of the p15(INK4b) gene promoter in leukemias, and it has been proposed that this methylation could be necessary for leukemic cells to escape TGF beta regulation. We investigated the methylation status of p15(INK4b) gene in 53 myelodysplastic syndromes (MDS) cases, including nine that had progressed to acute myeloid leukemia (AML), using a recently described sensitive method where polymerase chain reaction (PCR) is preceded by bisulfite modification of DNA (methylation specific PCR). p15(INK4b) methylation was observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients with greater than 10% bone marrow blasts had p15(INK4b) methylation (including all nine patients who had progressed to AML) as compared with none of MDS patients with <10% bone marrow blasts. No correlation between karyotypic abnormalities and methylation status was found. Patients with p15(INK4b) methylation had a worse prognosis, but the prognostic significance of p15(INK4b) methylation was no more found by multivariate analysis, due to its strong correlation to the percentage of marrow blasts. In 10 MDS cases, sequential DNA samples were available. In five of them, methylation of the p15(INK4b) gene was detected at leukemic transformation, but not at diagnosis. Our results showed that methylation of the p15(INK4b) gene in MDS is correlated with blastic bone marrow involvement and increases with disease evolution toward AML. It suggests that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle, and possibly of TGF beta inhibitory effect.

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.

Glutathione S transferase theta 1 gene defects in myelodysplastic syndromes and their correlation with karyotype and exposure to potential carcinogens.

Glutathione S transferase theta 1 (GSTT1) is implicated in the detoxification of different substances, including carcinogens. Recently, an increased incidence of GSTT1 null genotype was found in myelodysplastic syndromes (MDS) by comparison with a control population. We analyzed GSTT1 gene by PCR in 174 MDS cases and 100 controls. The incidence of GSTT1 null genotype was 22% in MDS in 19% in controls (P = 0.53). The incidence of GSTT1 null genotype in MDS did not differ according to gender, FAB classification, karyotype and whether MDS were therapy related or 'de novo'. In 86 of the de novo cases, data on previous occupational and environmental exposure to a list of 170 substances were available. In those MDS patients, a significantly lower frequency of GSTT1 null genotype was seen in cases with previous jobs exposed to chemicals, and with previous exposure to mineral dusts and exhaust gases. A lower frequency (but with only borderline significance) was seen in MDS patients who had been coal miners and those who had been exposed to any of the 70 substances analyzed. Overall, GSTT1 null genotype occurred at a similar incidence (19%) in controls and in MDS cases previously exposed to any substance, but tended to be higher in unexposed MDS patients (40%, P = 0.07). Our results do not confirm the higher incidence of GSTT1 null genotype observed in MDS. The lower incidence of GSTT1 null genotype in MDS cases exposed to some compounds previously found associated with MDS is apparently unexpected. However, it could be explained by the fact that GSTT1 enzyme, which has a detoxification role for some compounds, could also have an activating role for other substances, including solvents.

Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL).

We sequentially performed cytogenetic analysis and RT-PCR analysis of BCR-ABL transcripts in 17 cases of Ph1-positive ALL who had achieved hematological complete remission (CR) with intensive chemotherapy (CT). Sixteen cases were studied prospectively. All but one of the patients had reached cytogenetic CR, but cytogenetic has low sensitivity in predicting relapse. Twelve patients relapsed, three died in first CR and two were alive in first CR. Two of five, two of four, and five of nine patients who were allografted (in first or second CR), autografted and received consolidation CT, respectively, achieved negative two-round PCR in the bone marrow (BM): three died in CR, three remained in CR with negative two-step PCR in the BM and three relapsed after 22 to 28 months. In all cases, relapse was preceded by switch to PCR positivity in the BM by 4 to 6 months. The remaining nine patients remained PCR-positive in the BM and relapsed after 2 to 16 months. In the four autografted cases, PCR was positive at the time of bone marrow harvest. The two patients who received a purged transplant achieved negative PCR and prolonged CR, whereas the two patients who received an unpurged transplant remained PCR positive and relapsed. In 34% of the samples where analysis was concomitant, sensitivity of PCR proved lower in the blood than in the BM. These findings show that RT-PCR is a useful tool in the monitoring of MRD in Ph1 positive ALL.

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.

Differential efficacy of adenoviral mediated gene transfer into cells from hematological cell lines and fresh hematological malignancies.

As a first step to evaluate the possibility of gene therapy using adenoviral vectors in hematological malignancies in vivo, we tested the efficacy of gene transfer by a recombinant adenovirus in cell lines and fresh cells from various hematological neoplasms. Thirteen cell lines and samples from 27 patients were studied. Cells were infected by a recombinant adenovirus expressing beta galactosidase gene (Ad RSV betagal) and efficacy of transduction assessed by evaluating betagal expression in cells with a histochemical method. After infection of the cells at a multiplicity of infection (MOI) of 200 p.f.u./cell, the percentage of beta gal-positive cells after 48h was high in two cell lines. K562 (64%) and RPMI 8226 (a myeloma cell line, 65%), relatively large in the two myeloma cell lines tested (41% and 20%, respectively) and in MT4 (an adult T cell leukemia cell line, 38%) and low or absent in other cell lines. In fresh samples from AML, ALL, CLL, NHL, myeloma and MDS, no betagal positive cells were seen 48h and 72h after infection, except in one case of myeloma and one case of CLL (where 10% and 2% of betagal positive cells were seen after infection, respectively). Exposure of fresh malignant cells to GM-CSF before and during adenoviral infection, in three cases, did not increase the number of transfected cells. This suggests that adenoviral vectors, at least in their present form, cannot efficiently be used for direct gene transfer in hematological malignant cells.

The retinoblastoma gene (RB-1) status in multiple myeloma: a report on 35 cases.

We looked for abnormalities of the retinoblastoma (RB-1) gene and of RB protein expression in 35 patients with multiple myeloma (MM). Mutations in exons 20 to 24 of the RB-1 gene (exons where mutations predominate in retinoblastoma and other solid tumors) were analyzed by single stranded conformation polymorphism (SSCP). RB-1 protein was studied in bone marrow plasma cells by immunocytochemistry (ABC peroxidase technique) with a specific monoclonal antibody. Southern blot analysis of RB-1 gene was also performed in 20 of the patients. Twenty two patients analyzed had advanced disease (stage III or, in one case, plasma cell leukemia) and cytogenetic analysis (performed in 31 cases) found monosomy 13 in 9 patients. No rearrangement of the RB-1 gene was found by Southern analysis. Absent or greatly reduced RB-1 protein level was found in plasma cells in 4 of the patients (11%), whereas normal levels were seen in the remaining cases. No point mutation in exons 20 to 24 and their flanking introns were found in any of the 35 patients. Three of the 4 patients with absent or reduced RB-1 protein expression had advanced MM (stage III: 2 cases; plasma cell leukemia: 1 case); all 4 patients were resistant to treatment (as compared to 7 of the 31 patients with normal RB-1 protein levels); only one of them was subsequently found to have monosomy 13 (as compared to 9 of the 28 other karyotyped patients). Our findings suggest that abnormalities of the RB-1 gene and its expression are rare in MM. Absent or reduced expression of RB-1 protein was not significantly correlated to monosomy 13 and was not associated with gross rearrangements of the RB-1 gene by Southern analysis or point mutations in exons 20 to 24 of the gene. Reduced expression of RB-1 protein may be associated with advanced disease and poor response to treatment, although larger numbers of patients will be required for more adequate conclusions.

Clinical significance of p53 mutations in newly diagnosed Burkitt's lymphoma and acute lymphoblastic leukemia: a report of 48 cases.

PURPOSE: To correlate the presence of p53 mutations and initial characteristics, response to chemotherapy, and survival in newly diagnosed Burkitt's lymphoma (BL) and Burkitt's acute lymphoblastic leukemia (L3 ALL). PATIENTS AND METHODS: Forty-eight patients with newly diagnosed BL or L3 ALL, most of whom were treated with very intensive regimens, including early CNS disease treatment, were studied. Detection of p53 mutations was made by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 of the gene, and mutations were determined by direct sequencing of exons with abnormal SSCP findings. Comparison of outcome between mutated and nonmutated cases was made in all patients and also after excluding five patients who received therapeutic regimens considered as suboptimal and one patient who died of AIDS while in complete remission (CR), as those six patients had no p53 mutations. RESULTS: A point mutation was found in nine patients (19%), and consisted of a missense mutation in seven and a chain-terminating mutation in two. SSCP, sequence, and cytogenetic analysis strongly suggested that eight of nine patients with mutations had retained the normal p53 allele, which had been lost in the remaining patient. These findings were confirmed by fluorescence-in-situ hybridization (FISH) with a p53-specific probe in two patients, including the one who had lost the normal p53 allele. Unexpectedly, mutations were significantly less frequent in patients with disseminated disease, ie, L3 ALL or stage IV BL (four of 35, 11%), than in more localized forms, ie, BL stage I, II, or III (five of 13, 38%) (P = .03). CR rates were similar in mutated (78%) and nonmutated cases (78%). The actuarial disease-free interval (DFI) after 12 months and actuarial survival rates after 24 months were 49% and 66%, respectively, in patients with mutations, and 73% and 48%, respectively, those without mutations. The differences were not significant. CONCLUSION: Our findings suggest that, contrary to what is seen in most other neoplasias, p53 mutations in newly diagnosed BL and L3 ALL are not associated with extensive tumor mass or poor response to intensive therapeutic regimens. It is hypothesized that this difference with most tumors could be due to the fact that p53 mutations in BL and L3 ALL are generally associated with persistence of a normal residual p53 allele, contrary to what is observed in the majority of tumors.

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.

p16 gene homozygous deletions in acute lymphoblastic leukemia.

The p16 protein is a cyclin inhibitor encoded by a gene located in 9p21, which may have antioncogenic properties, and is inactivated by homozygous p16 gene deletion or, less often, point mutation in several types of solid tumors often associated to cytogenetic evidence of 9p21 deletion. We looked for homozygous deletion and point mutation of the p16 gene in acute lymphoblastic leukemia (ALL), where 9p21 deletion or rearrangement are also nonrandom cytogenetic findings. Other hematologic malignancies including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), and myeloma were also studied. Homozygous deletion of the p16 gene was seen in 9 of the 63 (14%) ALL analyzed, including 6/39 precursor B-ALL, 3/12 T-ALL, and 0/12 Burkitt's ALL. Three of the 7 ALL with 9p rearrangement (including 3 of the 5 patients where this rearrangement was clearly associated to 9p21 monosomy) had homozygous deletion compared to 5 of the 55 patients with normal 9p (the last patient with homozygous deletion was not successfully karyotyped). Single stranded conformation polymorphism analysis of exons 1 and 2 of the p16 gene was performed in 88 cases of ALL, including the 63 patients analyzed by Southern blot. Twenty-six of the cases had 9p rearrangement, associated to 9p21 monosomy in at least 12 cases. A missense point mutation, at codon 49 (nucleotide 164), was seen in only 1 of the 88 patients. No homozygous deletion and no point mutation of the p16 gene was seen in AML, MDS, CLL, and myeloma. Homozygous deletion of interferon alpha genes (situated close to p16 gene in 9p21) was seen in only 3 of the 9 ALL patients with p16 gene homozygous deletion, and none of the ALL without p16 gene homozygous deletion. Our findings suggest that homozygous deletion of the p16 gene is seen in about 15% of ALL cases, is not restricted to cases with cytogenetically detectable 9p deletion, and could have a pathogenetic role in this malignancy. On the other hand, p16 point mutations are very rare in ALL, and we found no p16 homozygous deletions or mutations in the other hematologic malignancies studied.

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.

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.

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.

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.

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.

Rare occurrence of P53 gene mutations in multiple myeloma.

We looked for mutations of exons 5-8 of the P53 gene in bone marrow cell from 37 cases of multiple myeloma, using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing. 25 patients also had cytogenetic analysis. A point mutation, leading to an amino acid change in the P53 protein was found in only one case, involving exon 5. These findings suggest that P53 mutations are very rare in multiple myeloma, and that this disease may be categorized among the few neoplasms where P53 abnormalities have very limited role, if any.

Mutations of the p53 gene in B-cell chronic lymphocytic leukemia: a report on 39 cases with cytogenetic analysis.

Mutations of exons 5 to 8 of the p53 gene were looked for in 39 cases of B-cell chronic lymphocytic leukemia (CLL) using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing. All patients also had cytogenetic analysis. A point mutation, leading to an amino acid change in the p53 protein was found in four cases, involving exon 7 (one case) or exon 8 (three cases). Mutations seemed to predominate in advanced clinical stages (Binet's stage C). All four patients with 17p monosomy had a mutation whereas no mutation was found in the 35 patients with cytogenetically normal 17p. These findings suggest that p53 mutations are relatively rare in B-cell CLL, and largely predominate or may even be restricted to patients with 17p monosomy (who constitute about 5% of all B-cell CLL patients in large published series). In those patients, the mutations may play a role in leukemogenesis through loss of tumor suppressive activity of normal p53 genes.