Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features

A recently described putative tumor suppressor gene, the cyclin- dependent kinase 4 inhibitor (p16), has been shown to be altered by deletions and/or point mutations in various human cancers. To assess the incidence and clinico-biologic correlations of p16 homozygous deletion in hemopoietic tumors, we studied a panel of 244 DNA samples representative of distinct acute (99 cases) and chronic (57 cases) leukemia subtypes, myelodysplastic (22 cases) and myeloproliferative (15 cases) syndromes, and lymphomas (51 cases). A 361-bp probe complementary to the p16 exon 2 gene sequences was generated by polymerase chain reaction and used in Southern blot hybridization against these tumor DNAs. Homozygous deletions of p16 (p16-/-) were detected in 10 of 58 (17%) cases of acute lymphoblastic leukemia (ALL) of either B or T lineage and in no other tumors. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 was also performed in 40 of the 58 ALL cases and in 16 lymphomas. In no cases were point mutations detected. The comparison of clinical features at presentation in p16-/- and in p16 germline ALL cases showed a greater leukemic cell mass (P = .001) and higher white blood cell counts (P = .01) in the former group. Two ALL cases in which diagnostic and relapse DNA samples were available showed p16-/- in both specimens. We conclude that homozygous p16 gene deletions characterize a subset of ALL with features of aggressive disease.     

Homozygous deletions of p16/MTS1 gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia

Fifty-six primary childhood T-cell acute lymphoblastic leukemia (T-ALL) samples and 17 T-ALL cell lines were examined for mutations and homozygous deletions of the p16/MTS1 gene using polymerase chain reaction single-strand conformation polymorphism and Southern blot analysis. Homozygous deletions were found in 22 primary samples (39%) and in 10 cell lines (59%). In contrast, mutations including small deletions and/or insertions were identified in only 4 primary samples (7%) and in 2 cell lines (12%). Mutations included samples (7%) and in 2 cell lines (12%). Mutations included one nonsense mutation at codon 72, one missense mutation at codon 58, one deletion (29 bp from codon 52-61), one insertion (7 bp into codon 50), and two deletion/insertions (codon 63 and intron 1). Four of the six mutations caused subsequent stop codon and presumably produced truncated p16 protein. Our results suggest that p16 gene alterations are involved in the development of T- ALLs and that the inactivation of the p16 gene occurs mainly through homozygous deletions rather than mutations.     

Clinical analysis of adult Philadelphia chromosome-positive acute lymphoblastic leukemia with p16 gene deletion

正Objective To investigate the clinical implications of p16 gene deletion in adult Philadelphia chromosome-positive acute lymphoblastic leukemia(Ph+ALL).Methods Retrospective analysis of clinical,immunophenotypic,cytogenetics,molecular characteristics and prognosis of80 newly diagnosed Ph+ALL patients with p16 deletion.     

High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia

Summary. To determine the incidence of homozygous deletions of the newly identified tumour suppressor gene, CDK4I, molecular genomic DNA analyses by PCR technique were performed on primary neoplastic cells from 22 childhood acute leukaemias obtained at presentation. The blast cells derived in all the analysed cases from bone marrow. We found that none of acute myeloblastic leukaemias (four cases) showed the CDK4I alteration, whereas 6/13 (46%) common acute lymphoblastic leukaemias (ALLs) displayed homozygous deletions. Moreover, and even more important, all the blasts purified from ALLs derived from early lymphoid precursors (three early-T ALLs and two pre-B ALLs) showed the absence of CDK4I gene. When the entire coding sequence of the CDK4I gene from samples without homozygous deletions was analysed by the single-strand conformational polymorphism method, no point mutations were identified. These results demonstrate that CDK4I gene deletions are very frequent and probably early events in childhood acute leukaemias of lymphoid origin and especially in early-T and pre-B ALLs. Moreover, the molecular mechanism of the loss of function of the gene is correlated, at least in childhood ALLs, almost exclusively to deletions and not to point mutations.     

p16INK4A gene homozygous deletions in human acute leukaemias with alterations of chromosome 9

Acute leukaemias are characterized by non-random chromosomal aberrations which are often strictly related to the inactivation of tumour suppressor genes (TSGs). Alterations at the short arm of chromosome 9 have been reported in a remarkable percentage of acute lymphoblastic leukaemias (ALL) and have been suggested to cause the loss of activity of the putative TSG, p16^INK4A (MTS1/CDKN2) gene. In order to evaluate the correlation between this gene inactivation and visible cytogenetic abnormalities, we have investigated p16^INK4A homozygous gene deletions in 10 paediatric acute leukaemias of different cell lineages which demonstrated karyotype aberrations involving chromosome 9. Moreover, the dimension of the genetic alteration was evaluated by studying the loss of heterozygosity of two highly polymorphic markers of chromosome 9p, namely α-interferon (IFNA) and D9S104, and the deletion of 5'-methylthioadenosine phosphorylase (MTAPase) gene. Finally, the deletion of a gene belonging to p16^INK4A family, the p18 gene, was analysed in these acute leukaemias. Our results demonstrated that: (i) the biallelic loss of p16^INK4A gene is strictly related to a specific immunophenotype, namely ALL of T-cell lineage; (ii) no significant correlation exists between alterations at chromosome 9p level and the homozygous deletions of p16^INK4A gene; and (iii) p18 gene was not deleted in the examined cases. These findings suggest a possible correlation between the T-lymphocyte phenotype and the expression of p16^INK4A gene. Moreover, the absence of MTAPase activity seems to be a valuable marker of p16^INK4A gene inactivation, thus indicating that the deleted chromosomal area on 9p21 very frequently involves the MTAPase gene.     

初诊和复发急性髓性白血病p15、p16基因甲基化研究

目的探讨急性髓性白血病(AL)p15、p16、p18、p19基因失活的发生率及与疾病发生、发展、预后的关系。方法用聚合酶链反应(PCR)方法扩增46例患者的p15、p16、p18、p19基因外显子1和外显子2。再用限制性内切酶一PCR方法检测基因甲基化。结果46例患者中。急性淋巴细胞白血病(ALL)19例．11例p15基因失活,10例p16基因失活;急性非淋巴细胞白血病(ANLL)27例．9例p15基因失活,18例P16基因失活;均以甲基化失活为主。所有病例均无p18、p19基因失活。结论在AL发生、发展过程中。p15、p16基因失活主要是由于P15、p16基因甲基化所致。     

p15ink4B and p16ink4 gene inactivation in acute lymphocytic leukemia

Malignant cells from 52 children with acute lymphocytic leukemia (ALL) were investigated for inactivation of the p15ink4B and p16ink4 genes and other genetic alterations on chromosome 9p21. Homozygous deletions of the p15ink4B and/or the p16ink4 genes were detected in 16 cases and a further 9 cases showed evidence of allelic loss either by hemizygous deletion or loss of heterozygosity (LOH) for 9p21 markers. Most cases had loss of both genes, but 5 patients had lost only p16ink4 and 2 cases had homozygous loss of p15ink4B only. Sequence analysis of all exons of p15ink4B and p16ink4 was performed in patients with hemizygous deletions or LOH for 9p21 markers. A frame shift mutation of p16ink4 exon 1 was shown in 1 case, whereas all other clones carried the wild- type sequence of p15ink4B and p16ink4 in the remaining allele. The data suggest that both the p15ink4B and p16ink4 genes can be inactivated in ALL. The existence of a hitherto undefined tumor-suppressor gene on chromosome 9p cannot be ruled out.     

Deletion analysis of p16(INKa) and p15(INKb) in relapsed childhood acute lymphoblastic leukemia

This study aimed at determining the prevalence of INK4 deletions and their impact on outcome in 125 children with acute lymphoblastic leukemia (ALL) at first relapse using real-time quantitative polymerase chain reaction. Patients were enrolled into relapse trials ALL-REZ BFM (ALL-Relapse Berlin-Frankfurt-Münster) 90 and 96. The prevalence of p16(INK4a) and p15(INK4b) homozygous deletions was 35% (44 of 125) and 30% (38 of 125), respectively. A highly significant association of both gene deletions was found with the 2 major adverse prognostic factors known for relapsed childhood ALL: T-cell immunophenotype and first remission duration. There was no correlation between INK4 deletions and probability of event-free survival. These findings argue against an independent prognostic role of INK4 deletions in relapsed childhood ALL.     

Homozygous deletions of the p16 tumor-suppressor gene are associated with lymphoid transformation of chronic myeloid leukemia

The p16 gene, also referred to as MTS1, INK4, CDK4I, or CDKN2, at chromosome 9p21 has recently been described as a tumor suppressor that may be involved in a wide range of tumors. We have used a semiquantitative multiplex polymerase chain reaction assay to search for deletions of the p16 gene in 34 patients with chronic myeloid leukemia in blast crisis (CML BC), 19 patients with acute lymphoblastic leukemia (ALL), and 25 patients with acute myeloid leukemia (AML). Homozygous deletions of p16 exons were found in 5 of 10 (50%) patients with CML in lymphoid BC and in 5 (26%) ALL patients, but in only 1 (2%) case with AML. No deletions were found in CML BC of nonlymphoid phenotype. Comparison of chronic phase DNA or remission DNA with acute leukemia DNA in 5 individuals showed that the p16 deletions were acquired and not inherited, directly implicating these lesions in the pathogenesis of the disease. We conclude that functional elimination of the p16 gene, or a closely mapping gene, is involved in a significant number of patients with CML in lymphoid transformation.     

急性淋巴细胞白血病p73基因甲基化临床意义的研究

目的探讨p73基因甲基化在急性淋巴细胞白血病(ALL)发病机制中的作用。方法对吉林大学第二医院2001~2004年42例ALL患者采用限制性内切酶结合多聚酶链反应方法(REP)检测p73基因第一外显子甲基化情况。结果42例ALL患者甲基化检测结果28.6%(12/42),存在p73甲基化患者化疗前周围血白细胞数及骨髓原始细胞数均高于无p73甲基化患者,而且首次化疗即获完全缓解及平均缓解时间较无甲基化者差异有显著性。结论p73基因甲基化在ALL发病机制中有一定的作用。其检测对估计预后具有临床意义。     

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.     

The bcr gene in Philadelphia chromosome positive acute lymphoblastic leukemia

In chronic myelogenous leukemia (CML) and in a percentage of childhood and adult acute lymphoblastic leukemia (ALL) the Philadelphia (Ph') chromosome is present in the leukemic cells of patients. This chromosome is the result of a reciprocal translocation between chromosomes 9 and 22. In CML the break on chromosome 22 occurs within the major breakpoint cluster region (Mbcr) of the bcr gene. In this study, we report on the examination of DNAs from nine Ph'-chromosome positive ALL patients for rearrangements within the bcr gene using Southern blot analysis. Of nine patients having a karyotypically identifiable Ph'-chromosome, only five exhibited rearrangements of the bcr gene. This could indicate that in ALL, chromosome 22 sequences other than the bcr gene are involved in the Ph'-translocation. Within the group of Ph'-positive ALL patients having a bcr gene breakpoint, a correlation appears to exist between the age of the patient and the location of the breakpoint within the gene: all or the vast majority of pediatric patients analyzed to date do not have a Mbcr breakpoint as found in CML and in adult ALL.     

Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene

The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.