Detection of p53 oncogene in acute-leukemia cells by immunoperoxidase technique

Expression of p53 oncogene in blast cells may have prognostic importance in acute leukemia. Simple and reliable methods which could detect enhanced p53 expression in leukemia cells would be important for follow-up studies of leukemia patients in remission. We used immunoperoxidase (IP) technique with an anti-p53 monoclonal antibody PAb421 to study the expression of p53 in leukemia cells. The expression of p53 was studied in 9 cell lines and 17 de novo acute leukemia (9 acute myeloid leukemia [AML], 8 acute lymphoblastic leukemia [ALL]) patients. The expression of p53 was demonstrated in non-T non-B cells and Burkitt's lymphoma cell lines, but neither in two myeloid leukemia cell lines nor in normal lymphoid cells after mitogenic stimulation. p53 expression was demonstrated in 7 cases (2 AML, 5 ALL) but only in ALL cases the percentage of positive of cells was over 20%. Bone marrow cells from patients were studied also after short-term culture (AML patients); in 1 case the number of PAb421-positive cells rose significantly after culture. These data suggest that IP staining with PAb421 can be used to demonstrate high p53 expression in B cell leukemias.     

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.     

Detection of p53 mutations using nonradioactive SSCP analysis: p53 is not frequently mutated in myelodysplastic syndromes (MDS)

Summary p53 is one of the most frequently mutated genes in human cancers. Since p53 has been implicated in lymphatic and some myeloid leukemias, such as the blastic phase of chronic myelogeneous leukemia, we sought to address the role of p53 gene mutations within exons 4–9 in myelodysplastic syndromes (MDS), a myeloid preleukemic condition. In order to avoid the potential hazard of using radioactive single-strand conformation analysis (SSCP), we used a nonradioactive SSCP method based on the silver stain of small minigels. In cell lines with known point mutations of the p53 gene, aberrant migrating bands were found. Serial dilutions indicated a sensitivity comparable to radioactive methods. Furthermore, a common polymorphism within the 4th exon of the p53 gene was easily detected. However, of 17 primary samples from patients with MDS, none harbored a p53 gene mutation. We conclude that this nonradioactive method can easily be used to screen for p53-gene mutations, and that p53-gene mutations do not play a major role in the pathogenesis of MDS.     

High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21

The AML1 gene, situated in 21q22, is often rearranged in acute leukemias through t(8;21) translocation, t(12;21) translocation, or less often t(3;21) translocation. Recently, point mutations in the Runt domain of the AML1 gene have also been reported in leukemia patients. Observations for mutations of the Runt domain of the AML1 gene in bone marrow cells were made in 300 patients, including 131 with acute myeloid leukemia (AML), 94 with myelodysplastic syndrome (MDS), 28 with blast crisis chronic myeloid leukemia (CML), 3 with atypical CML, 41 with acute lymphoblastic leukemia (ALL), and 3 with essential thrombocythemia (ET). Forty-one of the patients had chromosome 21 abnormalities, including t(8;21) in 6 of the patients with AML, t(12;21) in 8 patients with ALL, acquired trisomy 21 in 17 patients, tetrasomy 21 in 7 patients, and constitutional trisomy 21 (Down syndrome) in 3 patients. A point mutation was found in 14 cases (4.7%), including 9 (22%) of the 41 patients with AML of the Mo type (MoAML) (none of them had detectable chromosome 21 rearrangement) and 5 (38%) of the 13 myeloid malignancies with acquired trisomy 21 (1 M1AML, 2 M2AML, 1 ET, and 1 atypical CML). In at least 8 of 9 mutated cases of MoAML, both AML alleles were mutated: 3 patients had different stop codon mutations of the 2 AML1 alleles, and 5 patients had the same missense or stop codon mutation in both AML1 alleles, which resulted in at least 3 of the patients having duplication of the mutated allele and deletion of the normal residual allele, as shown by FISH analysis and by comparing microsatellite analyses of several chromosome 21 markers on diagnosis and remission samples. In the remaining mutated cases, with acquired trisomy 21, a missense mutation of AML1, which involved 2 of the 3 copies of the AML1 gene, was found. Four of the 7 mutated cases could be reanalyzed in complete remission, and no AML1 mutation was found, showing that mutations were acquired in the leukemic clone. In conclusion, these findings confirm the possibility of mutations of the Runt domain of the AML1 gene in leukemias, mainly in MoAML and in myeloid malignancies with acquired trisomy 21. AML1 mutations, in MoAML, involved both alleles and probably lead to nonfunctional AML1 protein. As AML1 protein regulates the expression of the myeloperoxidase gene, the relationship between AML1 mutations and Mo phenotype in AML will have to be further explored. (Blood. 2000;96:2862-2869)     

Genomic analysis of acute leukemia

Acute leukemia is the commonest childhood cancer and a major cause of morbidity from hematologic malignancies in adults. Acute lymphoblastic leukemia (ALL) is commonest in children, and acute myeloid leukemia (AML) is more frequent in adults. Apart from childhood ALL, the prognosis of acute leukemia is suboptimal, with many patients experiencing relapse, which carries a poor prognosis, or toxicities from nonspecific therapies. Recent years have witnessed great interest in the application of high‐resolution, genome wide approaches to the study of acute leukemia. These studies have identified multiple novel genetic alterations targeting critical cellular pathways that contribute to leukemogenesis, including alterations of genes regulting lymphoid development, tumor suppressors, apoptosis regulators, and oncogenes. These studies have also delineated novel genetic alterations that are associated with prognosis, and have demonstrated substantial evolution in patterns of genetic alterations from diagnosis to relapse, indicating that specific genetic changes determine resistance to therapy in ALL. Overall, fewer recurring alterations have been identified in AML. These studies have demonstrated the power of genome‐wide approaches to identify new lesions in acute leukemia, and suggest that ongoing genomic analyses, including deep resequencing and epigenetic analysis, will continue to yield novel, clinically relevant insights into the pathogenesis of this disease.     

Mutator phenotype in human hematopoietic neoplasms and its association with deletions disabling DNA repair genes and bcl-2 rearrangements.

As mice carrying mutations of the DNA mismatch repair genes MSH2 and MSH6 often develop lymphoid neoplasms, we addressed the prevalence of the replication error (RER(+)) phenotype, a manifestation of an underlying defect of DNA mismatch repair genes, in human lymphoid tumors. We compared microsatellite instability (MSI) at 10 loci in 37 lymphoid tumors, including 16 acute lymphoid leukemias (ALL) and 21 non-Hodgkin's lymphomas (NHL), and in 29 acute myeloid leukemias (AML). Significant differences in MSI prevalence between AMLs and ALLs emerged, and MSI occurrence was more frequent in the NHLs versus AMLs. Indeed, only 3 of 29 (10%) AMLs exhibited MSI, thus confirming its paucity in myeloid tumors, while 10 of 37 (27%) lymphoid tumors, 6 ALLs and 4 NHLs, disclosed an RER(+) phenotype. In 1 ALL patient, the same molecular alterations were observed in correspondence with a relapse, but were not detected during remission over a 14-month follow-up; in another ALL patient, findings correlated with impending clinical relapse. These results suggest that the study of MSI in lymphoid tumors might provide a useful molecular tool to monitor disease progression in a subset of ALLs. To correlate MSI with other known genetic abnormalities, we investigated the status of the proto-oncogene, bcl-2, in the lymphoma patients and found that 4 of 4 NHL patients with MSI carried bcl-2 rearrangements, thus linking genomic instability to enhanced cell survival in NHL; moreover, no p53 mutations were found in these patients. Finally, we addressed the putative cause of MSI in hematopoietic tumors by searching for both mutations and deletions affecting DNA repair genes. A limited genetic analysis did not show any tumor-specific mutation in MLH1 exons 9 and 16 and in MSH2 exons 5 and 13. However, loss of heterozygosity (LOH) of markers closely linked to mismatch repair genes MLH1, MSH2, and PMS2 was demonstrated in 4 of 6 ALLs and 1 of 3 AMLs with MSI. These observations indicate that chromosomal deletions might represent a mechanism of inactivation of DNA repair genes in acute leukemia.     

Mutant p53 in bone marrow stromal cells increases VEGF expression and supports leukemia cell growth

OBJECTIVE: Genetic alterations, including p53 mutations, have been identified in the stroma of solid tumors and are thought be involved in the induction of tumor growth and metastasis. We tested the hypothesis that somatic molecular alterations in bone marrow stromal cells provide a favorable growth environment for leukemic cells. MATERIALS AND METHODS: We established an in vitro model consisting of stroma expressing mutant p53 (Cys135Ser) to study its ability to support growth of cells from a pre-B acute lymphoblastic leukemia (ALL) cell line. Normal and leukemic bone marrow stromal cells were screened for p53 mutations by mutant-specific ELISA, SSCP, and direct sequencing. Secretion of vascular endothelial growth factor (VEGF) was measured by quantitative ELISA. RESULTS: Transfection of stromal cells with mutant p53 increased synthesis of VEGF and supported the growth of leukemic cells. An ELISA-based assay suggested the occurrence of in vivo p53 alterations in bone marrow stromal cells from 2 of 12 ALL patients screened. Direct sequencing of one of these samples revealed a somatic heterozygous p53 gene mutation (Asp49His). This sample secreted more VEGF and provided increased growth support to leukemic cells. The ability of Asp 49His-p53 to increase the expression of VEGF was confirmed with transfection experiments in a p53-null cell line. CONCLUSION: Our findings indicate that genetic alterations, such as p53 mutations, in stromal cells can increase stromal-derived support of leukemia growth. Increased synthesis of pro-angiogenic cytokines, such as VEGF, may constitute one possible pathway by which this process is mediated.     

Refined mapping of genomic rearrangements involving the short arm of chromosome 9 in acute lymphoblastic leukemias and other hematologic malignancies

Deletions of chromosomal band 9p21 have been detected in various tumor types as well as in more than 20% of acute lymphoblastic leukemia (ALL). These deletions frequently include the entire interferon (IFN) gene cluster as well as the methylthioadenosine phosphorylase (MTAP) gene. Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) was proposed as a candidate tumor-suppressor gene on 9p21 because it is frequently deleted in cell lines derived from multiple tumor types. To determine if CDKN2 or another closely related gene on 9p is the target of 9p deletions in ALL and other hematologic malignancies, we analyzed 20 primary patient samples (13 ALL, 2 acute myeloid leukemias [AML], and 5 non-Hodgkin's lymphomas [NHL]) with 9p rearrangements using Southern blot analysis, fluorescence in situ hybridization (FISH), and single- strand conformation polymorphism (SSCP) for alterations of CDKN2. Homozygous deletions of the CDKN2/CDKN2B (p15) region were detected in 10 cases (50%; 6 ALL, 2 AML, and 2 NHL). In 1 additional case, the intensity of the Southern blot band was significantly reduced, suggesting a CDKN2 deletion in a subpopulation of the malignant cells. No CDKN2 or CDKN2B rearrangements were seen. The IFN gene cluster was homozygously deleted in 2 of 15 (13%) analyzed cases, whereas the MTAP gene was deleted in 6 of 15 cases (40%). In addition, hemizygous deletions of the CDKN2 region were identified in 6 ALL cases using interphase FISH. No point mutation of the coding region of CDKN2 was detected by SSCP in these cases. We conclude that CDKN2 is the most frequently homozygously deleted marker on 9p. The absence of point mutations in the coding region of CDKN2 in cases with hemizygous 9p deletions and the frequent codeletion of MTAP, CDKN2B, and other yet unidentified neighboring genes suggest that the simultaneous deletion of these genes may be necessary for the selective growth advantage of malignant cells.     

Adenosine deaminase and ecto-5''-nucleotidase activities in various leukemias with special reference to blast crisis: significance of ecto-5''-nucleotidase in lymphoid blast crisis of chronic myeloid leukemia

Adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'-N) activities were examined in peripheral leukocytes from patients with leukemias, including nine patients with chronic myeloid leukemia (CML) in blast crisis. Four of none cases of CML in blast crisis were myeloid and the remaining lymphoid morphologically. The diagnosis of CML in lymphoid blast crisis was further contributed by the measurement of terminal deoxynucleotidyl transferase (TdT) activity. In all four cases of lymphoid blast crisis and one of myeloid blast crisis, leukemia cells had high 5'-N activity, while there was a little or no detectable activity in those from four cases of myeloid blast crisis and all of CML in chronic phase. ADA activity was high in seven of nine patients with blast crisis. Taken together, leukemia cells from two cases of lymphoid blast crisis had high ADA and 5'-N activities comparable to those in acute lymphocytic leukemia (ALL) cells. In contrast, the enzyme activities of leukemia cells from all but one patient in myeloid blast crisis were in a range similar to acute myeloid leukemia cells. The implications of these findings are as follows: (1) 5'-N may be used as a new biochemical marker of CML in lymphoid blast crisis. (2) Some lymphoid cells of CML in blast crisis have high ADA, 5'-N, and TdT activities and thus are very similar to ALL cells.     

Constitutional trisomy 8p11.21-q11.21 mosaicism: a germline alteration predisposing to myeloid leukaemia

Juvenile myelomonocytic leukaemia (JMML) is a unique myeloproliferative disorder of early childhood. Frequently, mutations in NRAS, KRAS, PTPN11, NF1 or CBL are found in these patients. Monosomy 7 is the most common cytogenetic aberration. To identify submicroscopic genomic copy number alterations, 20 JMML samples were analysed by comparative genomic hybridization. Ten out of 20 samples displayed additional submicroscopic alterations. In two patients, an almost identical gain of chromosome 8 was identified. In both patients, fluorescence in situ hybridization confirmed a constitutional partial trisomy 8 mosaic (cT8M). A survey on 27 cT8M patients with neoplasms showed that 21 had myeloid malignancies, and five of these had a JMML. Notably, the region gained in our cases is the smallest gain of chromosome 8 reported in cT8M cases with malignancies so far. Our results dramatically reduce the critical region to 8p11.21q11.21 harbouring 31 protein coding genes and two non-coding RNAs, e.g. MYST3, IKBKB, UBE2V2, GOLGA7, FNTA and MIR486--a finding with potential implications for the role of somatic trisomy 8 in myeloid malignancies. Further investigations are required to more comprehensively determine how constitutional partial trisomy 8 mosaicisms may contribute to leukaemogenesis in different mutational subtypes of JMML and other myeloid malignancies.     

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.     

Genetic evidence for lineage-related and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia

SHP-2 is a protein tyrosine phosphatase functioning as signal transducer downstream to growth factor and cytokine receptors. SHP-2 is required during development, and germline mutations in PTPN11, the gene encoding SHP-2, cause Noonan syndrome. SHP-2 plays a crucial role in hematopoietic cell development. We recently demonstrated that somatic PTPN11 mutations are the most frequent lesion in juvenile myelomonocytic leukemia and are observed in a smaller percentage of children with other myeloid malignancies. Here, we report that PTPN11 lesions occur in childhood acute lymphoblastic leukemia (ALL). Mutations were observed in 23 of 317 B-cell precursor ALL cases, but not among 44 children with T-lineage ALL. In the former, lesions prevalently occurred in TEL-AML1(-) cases with CD19(+)/CD10(+)/cyIgM(-) immunophenotype. PTPN11, NRAS, and KRAS2 mutations were largely mutually exclusive and accounted for one third of common ALL cases. We also show that, among 69 children with acute myeloid leukemia, PTPN11 mutations occurred in 4 of 12 cases with acute monocytic leukemia (FAB-M5). Leukemia-associated PTPN11 mutations were missense and were predicted to result in SHP-2 gain-of-function. Our findings provide evidence for a wider role of PTPN11 lesions in leukemogenesis, but also suggest a lineage-related and differentiation stage-related contribution of these lesions to clonal expansion.     

Genetic analysis of p53 and RB1 tumor-suppressor genes in blast crisis of chronic myeloid leukemia

Summary We have investigated the involvement of the p53 and RB1 tumor-suppressor genes in 26 cases of chronic myeloid leukemia (CML) blast crisis, including 17 myeloid, eight lymphoid, and one megakaryoblastic crisis. The presence of p53 mutations in exons 5 through 9 was tested by the PCR-single-strand conformation polymorphism (SSCP) assay, followed by PCR-direct sequencing; in addition, loss of heterozygosity (LOH) at 17p13, the site of the p53 gene, was assayed by Southern blot. Given the variability of the mechanisms of inactivation of the RB1 gene in human tumors, a combination of Southern blot and mutational analysis by PCR-SSCP was used. p53 mutations were restricted to one case of myeloid blast crisis, showing a CGCTGC (ArgCys) mutation at codon 283; two additional cases displayed LOH at 17p13 in the absence of p53 mutations. No molecular lesions of the RB1 gene were detected in any of the cases analyzed. These data indicate that inactivation of p53 and RB1 is a rare event in the molecular pathogenesis of CML acute transformation.     

Mutation analysis of CD95 (APO-1/Fas) in childhood B-lineage acute lymphoblastic leukaemia

The CD95 system plays an important role in lymphocyte homeostasis, has been implicated in the development of lymphoid malignancies, exerts a tumour suppressor function, and contributes to drug-induced cytotoxicity. We hypothesized that mutations of CD95 may occur in childhood B-lineage acute lymphoblastic leukaemia (ALL), a disease known for its constitutive resistance towards CD95-mediated apoptosis. We investigated 32 primary B-lineage ALL of childhood and five B-lineage ALL cell lines. All primary leukaemias expressed CD95 and bcl-2 to a variable degree. Most of the leukaemias were resistant towards CD95-mediated apoptosis. However, using SSCP analysis, no mutations in the coding and proximal promoter region could be detected. We conclude that the resistance towards CD95-mediated apoptosis observed in most de novo B-lineage ALL is not caused by mutations of the CD95 death receptor.     

A single monoclonal antibody identifies T-cell lineage of childhood lymphoid malignancies

Immunophenotyping studies with monoclonal antibodies have revealed the heterogeneity of childhood acute lymphoblastic leukemia (ALL) and non- Hodgkin's lymphoma (NHL). The lymphoid malignancies of T-cell lineage are particularly heterogeneous and, until now, no single monoclonal antibody has been found to identify all cases of T-ALL and T-NHL. A monoclonal antibody, 4H9, recognizes an antigen of 40,000 molecular weight on normal and malignant T cells. Thirty-six cases of childhood T- ALL and T-NHL were tested, and in all cases, the malignant blast cells were reactive with 4H9, whereas malignant cells from 61 cases of non-T ALL and NHL were not reactive with 4H9. Monoclonal antibody 4H9 is a sensitive and specific reagent for the identification of childhood T- cell ALL and NHL and should be extremely useful in immunophenotyping studies of lymphoid malignancies.     

Involvement of the cyclin-dependent kinase-4 inhibitor (CDKN2) gene in the pathogenesis of lymphoid blast crisis of chronic myelogenous leukaemia

Summary. Recent data suggest that homozygous deletion of the cyclin-dependent kinase 4 inhibitor gene (CDKN2), a putative tumour suppressor gene located on chromosome 9p21, represents a common genetic event in human cancer. As the molecular basis of the evolution of chronic myelogenous leukaemia (CML) into blast crisis remains largely unknown, we decided to investigate if the occurrence of similar deletions could represent one of the mechanisms underlying the disease progression. Whereas none of 22 chronic phase CML cases examined showed alterations, we found that 3/17 total blast crisis examined (18%) showed a homozygous deletion of the CDKN2 gene. The deletions were restricted to cases of lymphoid blast crisis, being present in 3/8 (40%) of the lymphoid and in none of the nine myeloid cases examined. The fact that the chronic phase DNA obtained at diagnosis in one of the cases lacks the homozygous deletion observed in blast crisis, suggests that the final deletion event took place concomitantly with the progression of the disease. Furthermore, the analysis of polymorphic regions on chromosome 9p21 flanking at both sides the CDKN2 gene, showed that deletions at 9p21 differ between cases and are characterized by a wide range of extensions. A concomitant search for a possible involvement of the p53 tumour suppressor gene in the same series of patients showed mutations of the gene and loss of heterozygosity at 17p only in myeloid blast crisis, suggesting the presence of distinct molecular pathways in the pathogenesis of lymphoid and myeloid blast crisis.     

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.