Allelotype analysis of childhood acute lymphoblastic leukemia.

To identify the genetic events that may play an important role in leukemogenesis of childhood ALL, we report for the first time the allelotyping of childhood ALL. Twenty-four cases of childhood ALL were screened for loss of heterozygosity (LOH) using 101 highly polymorphic microsatellite markers, which are distributed among all autosomal chromosomes. For LOH analysis on both chromosomes 9 and 12, 54 childhood ALL samples were examined. The most frequent allelic loss was found on chromosomal arm 9p, where 20 of 50 (40%) informative samples showed LOH. Moreover, nearly 30% of samples that did not have either homozygous deletions or point mutations of the putative tumor suppressor genes CDKN2/INK4A/p16 and INK4B/p15 on chromosomal arm 9p had LOH at D9S171. Loss of chromosomal arm 12p was also frequent (26%). Mutational analysis suggested that the altered gene on 12p is not the cyclin-dependent kinase inhibitor p27/Kip1, which is also on 12p. Several other regions that had LOH included 1p, 4q, 5p, 6q, 7p, 8p, 9q, 10q, 13q, 17p, 17q, 18q, 19q, and 22q. Of 24 patients, 19 (79%) showed allelic loss on at least one chromosomal arm. Samples of two patients (8%) showed LOH on almost all chromosomes. Fractional allelic loss, calculated for each sample as the total number of chromosomal arms lost/total number of arms with information, showed a median value of 0.04 and a mean of 0.123 (range, 0 to 0.95). This fractional allelic loss is lower than those reported for many solid tumors. This analysis shows the extreme power of LOH analysis using microsatellite markers in childhood ALL.

Frequent loss of heterozygosity on the long arm of chromosome 6: identification of two distinct regions of deletion in childhood acute lymphoblastic leukemia.

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) identified nonrandom chromosomal abnormalities of the long arm of chromosome 6. Most of the alterations are deletions that are thought to be indicative of the presence of a tumor suppressor gene that is mutated on the remaining allele. These observations led us to consider whether 6q loss may contribute to the pathogenesis of childhood ALL. To define further a region containing this gene, we analyzed the loss of heterozygosity (LOH) of chromosome 6 in 113 primary ALL samples with matched normal DNA using 34 highly informative microsatellite markers. LOH was found in 17 (15%) samples at one or more of the loci, and partial or interstitial deletions of 6q were detected in 11 of these tumors. On the basis of these results, we performed a detailed deletional map and identified two distinct regions of deletion. The first region is flanked by D6S283 and D6S302 loci at 6q21-22. The second region is flanked by D6S275 and D6S283 loci at 6q21. Clinical analysis determined that LOH of 6q was demonstrated both in precursor-B cell ALLs (15 of 93; 16%) and in T cell ALLs (2 of 19; 11%). In addition, 19 patients have been studied at diagnosis and relapse; 18 showed the same 6q21-22 structural abnormality at relapse (normal, 16 patients; LOH, 2 patients) as their initial presentation, suggesting, albeit with a small patient sample size, that 6q21-22 deletions may be an initial event in leukemogenesis and may occur less frequently during the progression of childhood ALL. These data suggest the presence of putative tumor suppressor genes on chromosome arm 6q that are important in the development of both T and precursor-B childhood ALLs. Our map provides important information toward cloning putative ALL tumor suppressor genes.

Identification of three distinct regions of deletion on the long arm of chromosome 11 in childhood acute lymphoblastic leukemia.

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) identified deletions of chromosome arm 11q. These observations led us to analyse the loss of heterozygosity (LOH) of chromosome arm 11q in 113 primary childhood ALL samples using 14 microsatellite markers. LOH was found in 18 (16%) patients. Detailed examination identified three distinct regions of deletion. The first region is flanked by D11S901 and D11S1391 at 11q22-23 containing the ATM gene. Mutational analysis suggested that the altered gene in this region is not the ATM gene. The second region is flanked by D11S614 and D11S924 at 11q23 containing the MLL gene. The third region is flanked by D11S1356 and D11S614 at 11q23 containing the MLL gene. All the cases with LOH at MLL locus lacked detectable MLL gene rearrangements. In addition, 20 children have been studied both at initial diagnosis and relapse; none of the individuals who relapsed acquired LOH of 11q, suggesting that 11q deletions were infrequently involved in the progression of childhood ALL. Children with 11q LOH had a good response to induction chemotherapy (P=0.015). These data suggest that alterations of putative tumor suppressor genes on 11q are important events in development of childhood ALL. Our map provides important information toward cloning putative tumor suppressor genes associated with childhood ALL.

T cell receptor Ddelta2Ddelta3 rearrangement: a suitable allele-specific marker for the detection of minimal residual disease in childhood acute lymphoblastic leukemia.

The applicability of T cell receptor (TCR) Ddelta2Ddelta3 junctional regions for the detection of minimal residual disease (MRD) was examined in childhood acute lymphoblastic leukemia (ALL). Southern blot analysis showed a Ddelta2Ddelta3 rearrangement in 22 of 172 (13%) precursor-B ALL. No Ddelta2Ddelta3 rearrangement was identified in 29 T-ALL cases. Three patients exhibited Ddelta2Ddelta3 recombinations in both alleles. Sequence analysis of Ddelta2Ddelta3 junctions revealed extensive diversity due to the random insertion and deletion of nucleotides at the joining site. PCR analysis utilizing allele-specific probes or oligonucleotides generated on the basis of Ddelta2Ddelta3 junctional sequences reached a sufficient sensitivity of 10(-4) to 10(-5) in the majority of cases. In four of 25 (16%) rearranged alleles, however, the 5' heptamer-nonamer recombination signal sequence (RSS) of the Ddelta2 segment had recombined directly to the 3' heptamer-nonamer RSS of the Ddelta3 segment thus generating a so-called signal junction. Respective heptamer-heptamer junctions are not suited to design allele-specific oligonucleotides for the detection of MRD because of their limited diversity and hence specificity.

Immunoglobulin kappa gene rearrangements between the kappa deleting element and Jkappa recombination signal sequences in acute lymphoblastic leukemia and normal hematopoiesis.

The kappa deleting element (kappaDE) located 24 kb downstream of the Ckappa gene segment mediates the deletion of Ckappa and the Jkappa-Ckappa Intron enhancer, which results in allelic exclusion of the immunoglobulin kappa light chain locus. We here report that the kappaDE can recombine to each recombination signal sequence (RSS) flankappaing Jkappa1 to Jkappa5 in normal hematopoiesis. Moreover, usage of the JkappaRSS-kappaDE junctional sequence allows the detection of minimal residual disease in acute lymphoblastic leukemia.

Rapid and reliable detection of N-ras mutations in acute lymphoblastic leukemia by melting curve analysis using LightCycler technology.

We applied a new strategy for the detection of N-ras gene mutations based on LightCycler technology. We designed two sets of amplimers and internal hybridization probes representing N-ras codons 12/13 and codon 61, respectively. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) patients (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were amplified, followed by the analysis of the melting temperatures of the PCR products on the LightCycler. PCR products exhibiting an abnormal melting characteristic were directly sequenced. Sequence analyses unravelled nucleotide substitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-rasmutations (10%) is compatible with previous reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.

Germline configuration of nfkb2, c-rel and bcl3 in childhood acute lymphoblastic leukemia (ALL).

The well-known family of NF-kappaB/Rel transcription factors is a central regulator of growth, differentiation and apoptosis in hematopoietic cell lineages. There is increasing evidence for their role in malignant transformation, especially in lymphomas. To study the possible involvement of NF-kappaB/Rel genes in the development of pediatric acute lymphoblastic leukemia (ALL), DNA samples from 140 patients were examined by Southern blot analysis. All samples revealed germline configuration of nfkb2, c-rel, and bcl3, indicating that structural alterations of these members of the NF-kappaB/Rel family are extremely rare, if existing at all in childhood ALL.

Prospective monitoring of minimal residual disease during the course of chemotherapy in patients with acute lymphoblastic leukemia, and detection of contaminating tumor cells in peripheral blood stem cells for autotransplantation.

A prospective study for detecting minimal residual disease (MRD) was conducted on children with acute lymphoblastic leukemia (ALL). Thirty-nine patients (38 B-lineage ALL, one T-ALL) with TCR delta rearrangements could be followed for 21 to 44 months (mean 30.9 months) excluding four patients who died. One hundred and ninety four bone marrow (BM) samples and 13 peripheral blood stem cell (PBSC) grafts were available for detection of MRD. Initially 34 cases were treated prospectively according to the CCLSG risk-stratified protocols for ALL (ALL874 or ALL911), and five cases according to the other protocols. Conventional chemotherapy was replaced by autologous PBSC transplantation (PBSCT) in five patients, by allogenic BM transplantation (BMT) in one patient, or suspended in another patient. Twenty-nine of 32 children in whom conventional chemotherapy could be continued without interruption remain in complete remission (CR). In 24 of the 29 patients MRD became undetectable within 12 months of their diagnosis. In five cases, BM samples obtained during maintenance therapy exhibited residual leukemia cells, and yet none of them relapsed (mean follow-up period 28.6 months). Our results thus indicate that intensive maintenance therapy for patients with PCR-positive results during consolidation therapy may prevent subsequent relapse. Nine events of relapse were diagnosed in eight patients (five BM, two isolated central nervous system (CNS), one combined BM and CNS, one isolated skin relapse). An increase or a re-emergence of MRD was detected in BM samples obtained from patients prior to BM relapse, but one patient remained in CR despite reappearance of leukemic cells following a PCR-negative status. Monitoring of MRD failed to predict isolated CNS or skin relapse. PBSCT allows high-dose cytoreduction therapy for patients with refractory neoplasia. In our study, leukemic cells were identified in eight of 13 PBSC grafts harvested from five patients. Three of four children who received PBSC grafts containing leukemic cells relapsed within 6 months after PBSCT. Monitoring of MRD as part of quality control of PBSC grafts may ultimately contribute to improvements in PBSCT procedures.

Improved detection of minimal residual leukemia through modifications of polymerase chain reaction analyses based on clonospecific T cell receptor junctions.

Polymerase chain reaction (PCR) techniques utilizing clonospecific T cell receptor (TCR) gamma or delta junctional regions constitute broadly applicable strategies to study the clinical relevance of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) patients. For the majority of cases current PCR protocols allow the reliable detection of one neoplastic cell among 10(4) to 10(6) normal counterparts. Occasionally, however, PCR analysis fails to reach this level of sensitivity. Here we demonstrate by means of three representative ALL cases how modifications of PCR protocols can overcome some of the limitations. Thus usage of biotinylated PCR products of TCR delta junctional regions and their direct application as templates for the generation of clonospecific probes allows the introduction of a selection step and results in a significant reduction of unspecific background signals. According to our experience as well as data from other laboratories we also recommend the usage of synthetic oligonucleotides representing TCR gamma junctions as clone-specific primers for a consecutive round of amplification rather than as clonospecific probes. Both modifications improve and facilitate the detection of MRD in leukemias characterized by TCR gamma and TCR delta recombinations.

Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage.

We analyzed homozygous deletions and mutations of the CDKN2(p16(INK4A)/MTS1) gene, using polymerase chain reaction and Southern blot analysis, in 120 children with acute lymphoblastic leukemia (ALL). Homozygous deletion was found in 17 of 89 (19%) precursor B-ALL patients, in 11 of 24 (46%) T-ALL patients, and in 0 of 7 other phenotype ALL patients. After excluding 28 (23%) patients who showed a homozygous deletion of CDKN2, we found that three patients (3%) had mutation at exon 2 of CDKN2 using PCR-SSCP and sequencing strategy. One had a CGA to TGA nonsense mutation (Arg to stop) at codon 72, one had a 1-bp deletion at codon 117, and the third had a 2-bp deletion at codon 70, resulting in frameshifts in the two latter patients. All three of these patients were T phenotype ALL, and the incidence of mutation in the 24 T-ALL patients examined was 13%. In contrast, no mutation was detected in the remaining patients with precursor-B or other type ALL (0/96). Our results suggest that mutational inactivation of the CDKN2 gene may contribute to the leukemogenic growth, especially in some patients with T-ALL.

Analysis of mutations and expression of GAP-related domain of the neurofibromatosis type 1 (NF1) gene in the progression of chronic myelogenous leukemia.

We investigated mutations in the GTPase activating protein-related domain of the neurofibromatosis type 1 gene (NF1-GRD) and its expression in each phase of chronic myelogenous leukemia (CML). Samples from 45 cases in chronic phase (CP), 41 in acute phase, and four CML cell lines were examined for mutations in the NF1-GRD by single-strand conformation polymorphism (SSCP) analysis and allele specific restriction analysis (ASRA). No mutations were detected in the exon where frequent mutations have recently been reported in human tumors, namely the FLR exon. We also examined for point mutations of the N-ras gene but found no mutations either. In 23 samples from CML cases and four CML cell lines, expression of two types of the NF1-GRD transcripts, type I and type II, were examined by NF1-GRD-specific polymerase chain reaction-based densitometric analysis and by the quantitative assay with coamplification of the NF1-GRD and beta-actin transcripts. Consequently, although expression level of type I transcripts varied among the samples, type II expression was increased in CML cell lines and a minor increase in type II expression was observed in the samples in acute phase compared with CP. However, this difference in type II expression between CP and acute phase was so small that changes of NF1-GRD transcripts as well as NF1-GRD or N-ras mutations might not be responsible for the progression of CML.

Long-term study of the clinical significance of loss of heterozygosity in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in childhood, with a widely variable outcome. Differences in the behavior and prognosis of the leukemia suggest that ALL can be divided into several biologic subgroups. We analyzed the loss of heterozygosity (LOH) of 6q, 9p, 11q and 12p using 31 microsatellite sites to determine their overall frequency and clinical value. We have studied 244 primary ALL samples obtained from the Multicenter Trial ALL-BFM 90 of Childhood ALL group. These patients have now been followed clinically for over 8 years. LOH occurred in 169 (69%) individuals in the following frequencies: 6q, 49 patients (20%); 9p, 97 patients (40%); 11q, 29 patients (12%); 12p, 60 patients (25%). Clinical data showed that those with 6q LOH were younger (P = 0.01) and had lower WBC counts (P = 0.02); patients with 9p LOH more frequently had CNS involvement (P = 0.01) and T cell phenotype (P = 0.0001); individuals with 11q LOH had a good response to induction chemotherapy (P = 0.02); those with 12p LOH were younger (P = 0.005), frequently had precursor B ALL (P = 0.001), and had a longer event-free survival (P = 0.05). Taken together, these data confirm that LOH is a very frequent alteration in childhood ALL.

p53 gene mutations and loss of a chromosome 17p in Philadelphia chromosome (Ph1)-positive acute leukemia.

We screened 23 cases of Philadelphia chromosome (Ph1)-positive acute leukemia (Ph1AL) for loss of a chromosome 17p and mutations in exons 2 to 11 of the p53 gene by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Loss of a distal part of chromosome 17p including loss of a whole chromosome 17 emerged in three cases, among which two were Ph1-positive acute lymphoblastic leukemia (Ph1ALL) with point mutations within the highly conserved region of the p53 gene. Another case of Ph1-positive acute myelogenous leukemia (Ph1AML) also exhibited a p53 point mutation in company with loss of normal p53 allele, although showing normal chromosome 17 homologues. We also performed Southern blot hybridization analysis to examine p53 gene rearrangements in 13 cases of Ph1AL. We found a rearrangement in one case of Ph1ALL and a loss of heterozygosity (LOH) at the p53 locus without any rearrangement in another Ph1ALL. Both cases showed no abnormality within the entire coding region by SSCP analysis. Thus, p53 gene alterations were commonly involved in Ph1AL with loss of a 17p (two point mutations in three cases), while rarely in cases with normal chromosome 17s (one point mutation in 20 cases and one rearrangement in 13 cases). Rare p53 gene alterations in Ph1AL may therefore be related to low incidence of loss of a chromosome 17p.

Homozygous deletions at 9p21 in childhood acute lymphoblastic leukemia detected by microsatellite analysis.

To gain a fuller understanding of the role of deletions of chromosome 9 in the development of childhood acute lymphoblastic leukemia (ALL), we performed detailed deletional mapping of chromosome 9 in 54 primary ALL samples with matched normal DNA using 22 highly polymorphic markers; and this information was combined with our previous data concerning the presence of deletions of CDKN2/INK4A/p16 and CDKN2B/INK4B/p15 in these samples. We have found a very high frequency of loss of heterozygosity (LOH) (31 of 54 cases (57%)) on chromosome arm 9p. As expected, the smallest region of LOH was between D9S1747 and D9S1748 at 9p21, including CDKN2/INK4A/p16, but excluding CDKN2B/INK4B/p15. Homozygous deletions at 9p21 occurred in 23 of 54 (43%) samples (seven of 11 (64%) T-ALL, 16 of 45 (36%) precursor-B ALL). We detected seven cases of homozygous deletions at 9p21 which had not been detected by Southern blot hybridization, showing the power of microsatellite analysis in detecting homozygous deletions. In most cases, homozygous deletions were limited to the region between D9S1747 and CDKN2B/INK4B/p15. We have attempted to determine the mechanism and timing of 9p deletions. Of the 23 samples with homozygous deletions at 9p21, 21 samples had surrounding large LOH. Of the 29 samples with LOH of 9p, homozygous deletion at 9p21 was identified in 22 cases. In addition, six patients have been studied at diagnosis and relapse, all six showed the same 9p21 structure at relapse (normal, three patients; hemizygous deletions, two patients; homozygous deletion, one patient) as their initial presentation. Finally, three patients (homozygous deletion, one patient; hemizygous deletion, two patients) had the IFN-alpha rather than CDKN2/INK4A/p16 deleted. In summary, these data further emphasize the importance of 9p21 loss in the development of childhood ALL.

TEL is one of the targets for deletion on 12p in many cases of childhood B-lineage acute lymphoblastic leukemia.

Abnormalities of the short arm of chromosome 12 including loss of heterozygosity (LOH) and TEL/AML-1 fusion resulting from a t(12;21)(p13;q22) translocation are frequently observed in childhood acute lymphoblastic leukemia (ALL). We investigated 21 DNA samples of childhood ALL which had LOH at 12p13. Rearrangement of TEL was observed in eight cases and another case showed a homozygous deletion of TEL. Two informative samples with TEL rearrangement had a deletion localized to the 5' region of this gene. The deletion in these two cases includes the helix-loop-helix (HLH) domain. This is consistent with the hypothesis that the normal tel can heterodimerize with the TEL/AML-1 gene product and inhibit the transforming capacity of the chimeric protein. Presumably, loss of the HLH of the normal remaining TEL allele abrogates this tumor suppressor-like function. The case with homozygous deletion of TEL is also consistent with this gene having qualities of a tumor suppressor. One unusual case had T-ALL rather than B-lineage ALL and the leukemic cells had rearrangement of TEL, but they did not have an alteration of the remaining TEL allele suggesting that the etiology of this disease may be different. This analysis further emphasizes the importance of loss of the normal TEL allele in childhood precursor B-lineage ALL.

Alterations of the cyclin-dependent kinase inhibitor p19 (INK4D) is rare in hematopoietic malignancies.

Cyclin-dependent kinase inhibitors (CDKIs) can be classified into two groups based on the structure of the proteins. One group includes the p21 (CIP1, WAF1, CAP20), p27 (Kip1), and p57 (Kip2) CDKIs, which contain a homologous amino-terminal cyclin-dependent kinase (cdk) inhibitory domain. The p16 (INK4A), p15 (INK4B), and p18 (INK4C) CDKIs, which have an ankyrin repeat motifs, belong to the other group. The p16 and p15 CDKI genes are very frequently altered in a variety of cancers including hematopoietic malignancies. The p19 (INK4D) gene is a newly cloned CDKI which belongs to the latter group. To determine if p19 genetic alterations play a role in hematopoietic malignancies, we examined DNA from 45 childhood newly diagnosed acute lymphocytic leukemias (ALLs), 30 acute myeloblastic leukemias (AMLs), 10 chronic myelocytic leukemias (CMLs), 45 adult T cell leukemias (ATLs), 70 non-Hodgkin's lymphomas (NHLs), and 20 multiple myelomas (MM) as well as 14 ALL, 20 AML, two ATL, and five lymphoma cell lines. Using Southern blot analysis, one homozygous deletion of the p19 gene was detected in a human immunodeficiency virus (HIV)-related Burkitt-like lymphoma sample. No point mutations in any of the samples were found by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Our investigation suggests that alterations of p19 do not play an important role in the development of most hematopoietic malignancies.

Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.

It is now widely accepted that the detection of minimal residual disease (MRD) has prognostic value in acute leukemia. However clinical MRD studies need standardized techniques. Therefore, several European laboratories have aligned their goals and performed comparative studies to achieve optimization and standardization of MRD techniques. This was achieved via the BIOMED-1 Concerted Action "Investigation of minimal residual disease in acute leukemia: International standardization and clinical evaluation." This report describes the development of PCR primers and protocols for the detection of MRD in acute lymphoblastic leukemia (ALL) using clone-specific junctional regions of immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. A total of 54 primers was developed (1) to amplify rearrangements of the TCRD, TCRG, and IGK (Kde) genes as well as TAL1 deletions; (2) to sequence the junctional regions and breakpoint fusion regions; and (3) to perform MRD detection in bone marrow or peripheral blood samples during follow-up of ALL patients. Protocols were established to identify PCR targets at diagnosis by performing 25 PCR reactions per patient using appropriate positive and negative controls. Standardized protocols were developed for MRD monitoring via single amplification of the PCR target followed by dot blot hybridization with the corresponding patient-specific junctional region probe. In addition, alternative approaches were designed for cases where the target sensitivity of at least 10(-4) was not obtained. The standardization described here of MRD-PCR techniques is essential for the process of translating MRD research into clinical practice.

Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group.

The medium-risk B cell precursor acute lymphoblastic leukemia (ALL) accounts for 50-60% of total childhood ALL and comprises the largest number of relapses still unpredictable with diagnostic criteria. To evaluate the prognostic impact of minimal residual disease (MRD) in this specific group, a case control study was performed in patients classified and treated as medium (or intermediate)-risk according to the criteria of national studies (ALL-BFM 90, DCLSG protocol ALL-8, AIEOP-ALL 91), which includes a good day 7 treatment response. Standardized polymerase chain reaction (PCR) analysis of patient-specific immunoglobulin and T cell receptor gene (TCR) rearrangements were used as targets for semi-quantitative estimation of MRD levels: > or =10(-2), 10(-3), < or =10(-4). Twenty-nine relapsing ALL patients were matched with the same number of controls by using white blood cell count (WBC), age, sex, and time in first complete remission, as matching factors. MRD was evaluated at time-point 1 (end of protocol Ia of induction treatment, ie 6 weeks from diagnosis) and time-point 2 (before consolidation treatment, ie 3 months from diagnosis). MRD-based high risk patients (> or =10(-3) at both time-points) were more frequently present in the relapsed cases than in controls (14 vs 2), while MRD-based low risk patients (MRD negative at both time-points) (1 vs 18) showed the opposite distribution. MRD-based high risk cases experienced a significantly higher relapse rate than all other patients, according to the estimated seven-fold increase in the odds of failure, and a much higher rate than MRD-based low risk patients (OR = 35.7; P= 0.003). Using the Cox model, the prediction of the relapse-free interval at 4 years was 44.7%, 76.4% and 97.7% according to the different MRD categories. MRD-based risk group classification demonstrate their clinical relevance within the medium-risk B cell precursor ALL which account for the largest number of unpredictable relapses, despite the current knowledge about clinical and biological characteristics at diagnosis. Therefore, MRD detection during the first 3 months of follow-up can provide the tools to target more intensive therapy to those patients at true risk of relapse.

Molecular analysis of the secretory phospholipase A2 gene, a candidate of Mom1 gene, in neuroblastomas.

Mice with hereditary intestinal polyposis have mutations of the APC gene which causes formation of multiple polyps. At least one other gene influences the susceptibility for development of polyps in mice, and the locus was named Mom1. The causative gene for the Mom1 locus has recently been cloned and was found to be identical to the secretory type II phospholipase A2 (PLA2S-II) gene. Although the mechanism of contribution of PLA2S-II to formation of polyps is unclear, abnormalities of the PLA2S-II gene contribute to cellular transformation in mice. We speculated that this gene could contribute to tumorigenesis in human neoplasms. The human homologue of this gene maps to 1p35-36.1. Chromosomal deletions involving this region are frequently observed in neuroblastomas. We analyzed 19 neuroblastomas to detect point mutations of the PLA2S-II gene by PCR-single strand conformational polymorphism (SSCP). A polymorphism was detected at codon 32; no point mutations were found in the coding region of the gene. Moreover, in cases that were heterozygous at codon 32, three samples had hemizygous deletion of the gene. Taken together, PLA2S-II is frequently hemizygously deleted, but no point mutations are observed in neuroblastomas.

Molecular analysis of the cyclin-dependent kinase inhibitor genes, p15, p16, p18 and p19 in the myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal blood disorders characterized by dyshematopoiesis with a frequent evolution to acute leukemia. Chromosomal deletions rather than translocations are the predominant karyotypic abnormalities in MDS, suggesting a recessive mechanism in the pathogenesis of MDS, such as inactivation of tumor suppressor genes. A group of cyclin-dependent kinase inhibitors, p15 (INK4B), p16 (INK4A), p18 (INK4C) and p19 (INK4D), are candidate tumor suppressor genes. To determine whether genetic alterations of these genes play an important role in the development and/or progression of MDS, we examined 46 samples from MDS patients by Southern blotting, single-strand-conformation polymorphism (SSCP) using polymerase chain reaction (PCR) and sequencing of DNA. These samples included 13 refractory anemias (RA), four refractory anemias with ringed sideroblasts (RARS), 16 refractory anemias with an excess of blasts (RAEB), eight refractory anemias with an excess of blasts in transformation (RAEB-T) and five chronic myelomonocytic leukemia (CMMoL) samples. Except for allelic polymorphisms or silent point mutations, no alterations of coding regions of these four CDKI genes were identified. In summary, genetic abnormalities of the p15, p16, p18 and p19 genes are rare events in the development and/or progression of MDS.