The Prognostic Significance of P16ink4a/P14arf and P15ink4b Deletions in Adult Acute Lymphoblastic Leukemia

Vol. 5, 1855–1861, July 1999 Clinical Cancer Research 1855

The Prognostic Significance of p16INK4a/p14ARF and p15INK4b Deletions in Adult Acute Lymphoblastic Leukemia

Stefan Faderl, Hagop M. Kantarjian, INTRODUCTION Taghi Manshouri, Chin-Yung Chan, ALLs2 result from clonal proliferation, accumulation, and Sherry Pierce, Kimberly J. Hays, Jorge Cortes, tissue infiltration of neoplastic hematopoietic cells. Disruptions Deborah Thomas, Zeev Estrov, and of the molecular mechanisms facilitating normal cell growth and differentiation frequently result from alterations of cell cycle Maher Albitar1 control (1). Transitions of the eukaryotic cell cycle from G1 Departments of Leukemia [S. F., H. M. K., S. P., J. C., D. T., Z. E.] phase through DNA replication (S phase), G , and cell division and Laboratory Medicine [T. M., C-Y. C., K. J. H., M. A.], The 2 (M phase) are tightly regulated at multiple checkpoints known University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 as restriction points. Progression through these stages is mediated by sequential accumulation of a family of serine-threonine protein kinases called CDKs and cyclins that activate the ki- ABSTRACT nases (2–4). The CDKs are opposed by CDKIs that function like Cytogenetic/molecular abnormalities significantly in- brakes in the cell cycle machinery, assuring the cells’ functional fluence the prognosis of patients with acute leukemia. integrity and readiness to progress across cell cycle restriction Recently, two genes, p16INK4a and p15INK4b, encoding two points, thus preventing uninhibited growth and proliferation cyclin-dependent kinase inhibitor proteins of the INK4 (2, 4, 5). family of Mr 15,000 and 16,000, respectively, have been Several CDKI proteins have been cloned and divided into localized to 9p21. Remarkably, the p16INK4a locus has families by homologies in their amino acid sequence (2). The been found to encode a second protein, p14ARF, known as INK4 family of CDKIs includes p16INK4a (MTS1 and p19ARF in mice, with a distinct reading frame. Like CDKN2A), p15INK4b (MTS2 and CDKN2B), p18INK4c, and p16INK4a, p14ARF is involved in cell cycle regulation, p19INK4d. They share a 90% homology in coding exon 2 and blocking cells at the G1 restriction point through the preferentially inhibit cyclin D-CDK-4/6 complexes (1, 5, 6). activity of MDM-2 and p53. Kamb et al. (7) and Nobori et al. (8) localized the genes coding We studied bone marrow samples of 42 newly diag- for p16INK4a and p15INK4b to chromosomal segment 9p21. nosed and untreated patients with acute lymphoblastic leu- Deletions of p16INK4a and p15INK4b have been identified in kemia for the incidence of deletions of p16INK4a/p14ARF and up to 80% of human leukemia cell lines, with homozygous p15INK4b using Southern blot analysis and determined the deletions as the most frequent mechanism of inactivation and clinical outcome with regard to complete remission (CR) p16INK4a as the primary target for such deletions (7, 8, 9–11). duration, event-free survival, and overall survival. However, analysis of primary leukemia samples revealed lower INK4a ARF We found deletions of p16 /p14 in 17 of 42 rates of detection than those in cell lines (9, 11). patients (40%), with homozygous deletions in 11 of 42 pa- Recently, the p16INK4a locus was found to encode a second, tients (26%) and hemizygous deletions in 6 of 42 patients distinct protein. The mRNA for p14ARF is composed of exons INK4b (14%). The gene for p15 was codeleted in most, but not 1␤, 2, and 3, whereas the mRNA for p16INK4a is derived from INK4a all, cases and was never deleted without deletion of p16 / exons 1␣, 2, and 3. Alternative splicing of exon 1 in p14ARF ARF p14 . No correlation was observed between molecular results in a different reading frame for exons 2 and 3 (12). studies and karyotype abnormalities as determined by con- p14ARF inhibits the expression of oncogene MDM-2, therefore ventional cytogenetics. Furthermore, no difference was preventing MDM-2-mediated inactivation of tumor suppressor found in the CR rate, CR duration, event-free survival, and gene p53 (13). overall survival in patients with homozygous gene deletions Most studies of 9p21 anomalies and molecular analyses for compared to patients with no deletions or loss of only one p16INK4a/p14ARF and p15INK4b were performed in childhood allele. ALL. We reported p16INK4a/p14ARF and p15INK4b deletions in 178 cases of primary adult leukemias (14). Here we report an analysis of p16INK4a/p14ARF and p15INK4b deletions in a series of 42 newly diagnosed adult ALL patients. Received 9/21/98; revised 1/19/99; accepted 3/23/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at The University of 2 The abbreviations used are: ALL, acute lymphoblastic leukemia; CR, Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 72, complete remission; EFS, event-free survival; CDK, cyclin-dependent Houston, TX 77030. E-mail: [email protected]. kinase; CDKI, CDK inhibitor; BM, bone marrow.

Table 1 Incidence of deletions of p16INK4a/p14ARF and p15INK4b in 42 patients with ALL by immunophenotype and comparison with cytogenetic abnormalities Deletiona Immunophenotype p16 p15 Pre-B CallaBTN/AIncidence (%) 9p-(%) Ph (%) Ϫ/ϪϪ/Ϫ 1 7 1 9 (21) Ϫ/Ϫϩ/ϩ 1 1 2 (5) 3/11 (27) 4/11 (36) ϩ/Ϫϩ/Ϫ 4 1 5 (12) ϩ/Ϫϩ/ϩ 1 1 (2) 1/6 (17) ϩ/ϩϩ/ϩ 3 13 5 3 1 25 (60) 1/25 (4) 5/25 (20) a Ϫ/Ϫ, homozygous deletion; ϩ/Ϫ, hemizygous deletion; ϩ/ϩ, no deletion.

PATIENTS AND METHODS whereas intron 1 is significantly more divergent between the INK4a ARF Patient Samples two genes. Exon 2 of p16 /p14 cross-hybridizes to the INK4b BM specimens were obtained from 42 newly diagnosed p15 gene fragment, allowing the detection of separate fragments of p15INK4b upon digestion with BamHI. The relative and untreated patients who presented to the Leukemia Depart- INK4a ARF INK4b ment at The University of Texas M. D. Anderson Cancer Center intensities of the p16 /p14 and p15 gene were between 1985 and 1997. Six of the patients had mature B-cell compared with the control gene on chromosome 11 (MLL) using a Bio-Rad phosphorimager (Hercules, CA). The ratios of ALL, 30 patients displayed markers of pre-B-cell ALL (positive INK4a ARF INK4b for CD10/calla in 25 patients), and 4 patients presented with p16 /p14 and p15 to MLL were considered as 1 in T-lineage ALL. Results of immunophenotyping were not avail- normal samples. The ratios were adjusted accordingly in patient able for two patients (Table 1). The BM samples were obtained samples with reference to normal controls. A ratio of 0.4–0.6 Ͻ with informed consent, and the study was approved by the was considered as a hemizygous deletion, and a ratio of 0.2 Human Experimentation Committee of our institution. was considered as a homozygous deletion (Fig. 1). Cytogenetic Analysis. BM cells were placed in 10 ml of Treatment Ham’s F-10 with 20% FCS to obtain a final concentration of 1–4 ϫ 106 nucleated cells/ml. The cultures were incubated for All but four patients were treated with the “hyper-CVAD” 24 h at 37°C. Standard harvesting and fixation procedures were regimen as described elsewhere (15). The remaining four pa- used, as described elsewhere (17). Up to three slides of each tients (patient 12 from Table 3 and three patients without gene preparation were stained with Gurr’s Giemsa stain and placed in deletions) received VAD (vincristine, Adriamycin, and dexa- a 60°C oven overnight before Giemsa banding. A maximum of methasone) chemotherapy (16). 25 metaphases were analyzed on the Giemsa-stained slides. Two abnormal metaphase cells with identical karyotype anomalies Specimen Collection were required for establishing the diagnosis of a clonal abnor- All specimens were obtained during routine diagnostic mality (17). procedures under approved protocols. Only BM specimens in which leukemic cells exceeded 80% of the population were used for analysis. Low-density cells were separated by Ficoll- Statistical Methods Hypaque gradient centrifugation (Sigma, St. Louis, MO) and The influence of clinical and cytogenetic parameters between washed twice with PBS. Genomic DNA was extracted. groups of patients with homozygous, hemizygous, and no deletions was 2 Genomic DNA with 11q deletions or rearrangement of the MLL evaluated using the ␹ test. Median values were compared using gene was not used. A few cases with deletion of chromosome 11 Kruskal-Wallis test statistics (18). The probability of surviving and remaining in CR was evaluated by Kaplan-Meier analysis (19). in less than 10% of metaphase cells and comparable intensities of 11q23 bands in Southern blot analysis were included. Cases with chromosomal translocations t(11;14)(q12;q32) and t(11;14) RESULTS (q13;q32) were also included as long as the MLL gene was not Incidence of Deletions of p16INK4a/p14ARF and p15INK4b rearranged by Southern blot analysis. in Adult ALL. Table 1 summarizes the incidence of deletions of the p16INK4a/p14ARF and p15INK4b genes in our samples. Laboratory Methods Biallelic deletions of p16INK4a/p14ARF were observed in 11 of Southern Blot Analysis. Detection of p16INK4a/p14ARF 42 (26%) patients. The gene for p15INK4b was codeleted ho- and p15INK4b was performed as reported previously (14). mozygously in nine patients (21%). In no patient could we Briefly, 10 ␮g of genomic DNA were digested with BamHI, demonstrate homozygous deletions of p15INK4b without dele- fractionated on a 0.7% agarose gel, transferred to a nylon tions of p16INK4a/p14ARF. Hemizygous deletions of p16INK4a/ membrane, and hybridized with a 32P-labeled 11q23 probe and p14ARF occurred in six cases (14%) and were associated with a 509-bp genomic DNA fragment, respectively, using standard hemizygous deletions of p15INK4b in five patients (12%). As conditions. The DNA test probe contained 297 bp from exon 2 was the case in patients with homozygous deletions, no hem- and 212 bp of the first intron of p16INK4a/p14ARF. Exon 2 of the izygous deletions of p15INK4b were found without concomitant p16INK4a/p14ARF gene is 90% homologous to the p15INK4b gene, deletions of p16INK4a/p14ARF. Overall, deletions of p16INK4a/

Fig. 1 Representative South- ern blot analysis of p16INK4a/ p14ARF and p15INK4b genes. The MLL gene signal is used as internal control. Het, an example of hemizygous deletions; Hom, an example of homozygous deletions. Normal control and samples that lack deletion are labeled N. Lane M, ␭/HindIII molecular size markers.

Table 2 Clinical characteristics of patients Splenomegaly Hepatomegaly Phϩ Parameter n Agea (range; yrs) WBCa (range ϫ 109/liter) BM blasta (range; %) (%) (%) (%) Homozygous deletions of 11 26 (16–79) 45.7 (13–298.5) 87 (76–98) 3 (27) 1 (9) 4 (36) p16/14 with or without p15 Hemizygous deletions of 6 57 (20–78) 5.1 (3.9–74.9) 92 (74–97) 1 (17) 1 (17) 1 (17) p16/p14 with or without p15 No deletion detected 25 42 (16–79) 8.3 (0.6–89) 81 (24–98) 4 (16) 2 (8) 6 (24) P NSb 0.0005 NS NS NS NS a Median. b NS, not significant. p14ARF and p15INK4b were detected in 40% of our patients: Table 3. In 6 of 17 patients (patients 4, 6, 10, 11, 12, and 16), homozygous deletions were detected in 26% of our patients determination of karyotype was not possible due to insufficient (21% for p15INK4b); and hemizygous deletions were detected in metaphases. The percentage of insufficient metaphases was not 14% of our patients (12% for p15INK4b). significantly different in the group of patients with deletions of Deletions of p16INK4a/p14ARF/p15INK4b were highest p16INK4a/p14ARF/p15INK4b and patients without deletions in among patients with precursor B-cell ALL (40–48% for ho- these two genes (35% versus 32%; P Ͼ 0.05). Two patients mozygous gene deletions and 20–44% for hemizygous dele- (patients 15 and 17) had a diploid karyotype. The remaining tions) and lowest in patients with mature B-cell ALL (17% in nine cases showed complex karyotypic abnormalities involving either group). One of four patients with T-cell ALL showed anomalies of the short arm of chromosome 9 in four patients. homozygous loss of p16INK4a/p14ARF, whereas no deletions of Three of these patients had homozygous deletions of p16INK4a/ p15INK4b could be associated with T-cell immunophenotype. No p14ARF/p15INK4b by Southern blot (Table 1). The 9pϪ abnor- significant difference was found between T-cell immunopheno- malities consisted of del 9(p22) in patients 3 and 7 and 9pϪ in type and deletions of p16INK4a/p14ARF or p15INK4b (P ϭ 0.49 patient 2. The fourth patient had no detectable deletion of and 0.21, respectively; Table 1). p16INK4a/p14ARF/p15INK4b by molecular analysis but showed a Clinical and Cytogenetic Characteristics of Patients 9pϪ abnormality by cytogenetics. No patient with hemizygous with Deletions of p16INK4a/p14ARF and p15INK4b. Clinical gene deletions had 9pϪ anomalies. Overall, abnormalities of the and cytogenetic characteristics were compared between patients short arm of chromosome 9 were detected in 27% of cases with with homozygous deletions, hemizygous deletions, and no de- biallelic deletions of p16INK4a/p14ARF/p15INK4b, in 4% of cases letions. Patients with homozygous deletions of p16INK4a/p14ARF without deletions, and in no cases with hemizygous deletions. or p15INK4b had a significantly higher WBC count when com- The Philadelphia translocation t(9;22)(q34;q22) was found in 4 /No significant differ- of 11 patients (36%) with complete loss of p16INK4a/p14ARF .(0.0005 ؍ pared to the other groups (P ence existed between the groups for age, percentage of BM p15INK4b, in 1 of 6 patients (17%) with hemizygous deletions, blasts at diagnosis, hepatomegaly and splenomegaly, and and in 5 of 25 patients (20%) without deletions (P Ͼ 0.05; presence of the Philadelphia chromosome (Table 2). Tables 1 and 2). Association between Molecular Abnormalities and Cy- Association of Gene Deletions with Clinical Outcome. togenetic Analysis of 9p21. Cytogenetic profiles of patients Clinical outcome was assessed in all 42 patients. Median fol- with deletions of p16INK4a/p14ARF and p15INK4b are shown in low-up was 20 months (range, 6–157 months). For survival

Table 3 Patients with deletions of p16INK4a/p15ARF and p15INK4b: cytogenetic characteristics Patient Age p16 p16 p15 p15 no. (yrs)/sex Lineage Cytogeneticsa hob hec ho he 1 39/F Calla (5)46xx,t(9qϩ;22qϪ);(7)47”,ϩ22q;(2)49”,ϩ?x,ϩ?9qϩ;(2)Phϩadd;(5)dip ϩϪϩϪ 2 26/M Calla (25)46xy,9pϪ;(1)“t(1qϩ;4qϪ);(19)46,Ϫ5Ϫ7ϩder7,t(7pϩ;?)del7,t(7qϪ;?)ϩM;15D ϩϪϩϪ 3 66/M B (34)44x,R(y)(p11;q12),Ϫ8Ϫ9,del9(p22),Ϫ13,der14,t(8;14)(q11;q32),Ϫ16Ϫ21ϩ3M;(1)dip ϩϪϩϪ 4 21/M Calla IM ϩϪϩϪ 5 39/M Calla (10)46xy,Ϫ9,t(9;22)(q34;q11),ϩM;(10)dip ϩϪϩϪ 6 39/M PreB IM ϩϪϩϪ 7 24/F Calla (1)46xx,t(9;22)(q34;q11);(2)47,del9(p22),t(9;22);ϩder22,t(9;22);(8)dip ϩϪϩϪ 8 17/F Calla (8)46xx,t(9;22)(q34;q11)ϩrandom chgs;(3)45,t(9;10)Ϫ20;(13)dip ϩϪϩϪ 9 26/M Calla (20)47xy,t(9;22)(q34;q11),ϩder22,t(9;22) ϩϪϩϪ 10 26/M T IM ϩϪϩϪ 11 50/F Calla IM ϩϪϩϪ 12 48/M Calla IM ϪϩϪϩ 13 78/M N/A (3)46xy,add4(q35),der9,del9(q22),t(9;22)(q34;q11),ϩder22,t(9;22);(4)dip ϪϩϪϩ 14 70/M Calla (11)45x,Ϫy;(2)59Ϫ63xy[ϩ1ϩ6ϩ9ϩ10ϩ11ϩ14ϩ19ϩ22ϩMϩchgs];(8)dip ϪϩϪϩ 15 66/M Calla 46xy ϪϩϪϩ 16 20/F Calla IM ϪϩϪϩ 17 30/F PreB 46xx ϪϩϪϪ a IM, insufficient metaphases; dip, diploid karyotype. b ho, homozygous. c he, heterozygous.

analysis, patients were classified into two groups: (a) those with Table 4 Outcome of patients with homozygous deletions of complete loss of p16INK4a/p14ARF and p15INK4b; and (b) those p16INK4a/p14ARF/p15INK4b with either hemizygous deletions or no deletions. Patient num- CR rate CRDa EFS OS bers were too small for patients with hemizygous deletions to be Parameter (%) (mo) (mo) (mo) analyzed separately. Ten of 11 patients (91%) with biallelic loss Homozygous deletions of 10/11 (91) 10 9 12 of p16INK4a/p14ARF achieved CR, whereas 30 of 31 patients p16/p14/p15 (97%) with loss of one allele or no deletions achieved CR (Table Normal and hemizygous 30/31 (97) 17 14 23 4). No significant differences between the two groups were deletions of p16/p14/p15 P NS NS NS NS found with respect to CR duration (Fig. 2), EFS (Fig. 3), and a overall survival (Fig. 4), although a trend for worse outcome in CRD, complete remission duration; OS, overall survival; NS, nonsignificant. homozygously deleted patients exists.

DISCUSSION Abnormalities of the short arm of chromosome 9 including in children (26, 30–38) and adults (14, 21, 39, 40) with ALL. segment 9p21 occur in ALL with a frequency of 7–13% (9, 14, Our study showed deletions of p16INK4a/p14ARF and p15INK4b in 20, 21). Some authors reported an association between abnor- 40% of the patients. The most frequently deleted gene was malities of 9pϪ and characteristic clinical features such as high p16INK4a/p14ARF with codeletions of p15INK4b in most but not WBC and blast counts at presentation, prominent lymphadenop- all cases. These data confirm p16INK4a/p14ARF as the primary athy and splenomegaly, expression of predominantly T-cell target for inactivation by deletion of 9p21. Takeuchi et al. (38) surface markers, and significantly shorter survival than that of undertook detailed deletional mapping of chromosome 9 by other patients with ALL (22). Other studies did not confirm this microsatellite analysis in 54 children with primary ALL. They association (21, 23–25, 26). Inactivation of genes for p16INK4a/ found loss of heterozygosity on the short arm of chromosome 9 p14ARF and p15INK4b by deletion and intragenic mutations has in 57% of the samples. Similar to data from our study, the been described in a variety of cell lines and primary samples smallest region of loss of heterozygosity included p16INK4a/ from patients with both hematological malignancies and solid p14ARF but not the locus for p15INK4b. Aguiar et al. (41) con- tumors (10, 11, 27–29). The discovery of the p14ARF gene structed a map of deletions at 9p21 using multiplex PCR. arising by alternative splicing within the p16INK4a locus raises Although variable in size, the commonly deleted region in- questions regarding which of these genes represents the target cluded p16INK4a/p14ARF but did not include either p15INK4b or gene of the 9p21 deletion. Loss of both genes, p16INK4a and the IFN-␣ gene cluster. p14ARF, may be an important factor in the biological effects of Importantly, our results demonstrated that most of dele- deletions of 9p21. Most of the previous studies on 9p21 deletions of p16INK4a/p14ARF and p15INK4a are detected molecularly tions do not discuss the loss of p14ARF, although it is most likely and not by conventional cytogenetics. Nine of the 17 patients that deletions of p16INK4a accompany deletions of p14ARF, be- (53%) who had either biallelic or hemizygous loss of p16INK4a/ cause both genes share exons 2 and 3. p14ARF/p15INK4b showed karyotypic abnormalities. In the re- The incidence of homozygous and hemizygous deletions maining cases, cytogenetics revealed diploid karyotypes in two ranges from 20% to more than 70% in most series and is similar (12%) patients or could not be performed due to insufficient

Fig. 2 CR duration.

Fig. 3 EFS.

metaphases in six patients (35%). Only three patients (18%) had (95%). Of 13 cases for whom cytogenetic studies were avail- gene deletions and 9pϪ abnormalities by cytogenetics. In all able, 12 showed no detectable 9p alterations. Faienza et al. (32) three of these patients, deletions of p16INK4a/p14ARF were ho- found no correlation between karyotype and p16INK4a/p14ARF mozygous. In one patient, 9pϪ was found without gene dele- deletions in their series of childhood ALL. Our study confirmed tions. However, this karyotype was present in only a subpopu- these findings in adult ALL and emphasizes the need to perform lation of metaphases that were analyzed from that sample. In molecular studies in patients with ALL at diagnosis to detect concordance with our results, Iolascon et al. (37) analyzed 21 abnormalities that may have an impact on response to therapy children with T-cell ALL, scanning for deletions of p18INK4c and prognosis. and p16INK4a/p14ARF by multiplex PCR. They observed ho- Patients with homozygous loss of p16INK4a/p14ARF and mozygous deletions of p16INK4a/p14ARF in 20 of 21 patients p15INK4b had no worse outcome than patients who had either no

Fig. 4 Overall survival.

deletions or loss of only one allele. CR duration, EFS, and 6. Xiong, Y., Zhang, H., and Beach, D. Subunit rearrangement of the overall survival were not significantly different. However, lon- cyclin-dependent kinases is associated with cellular transformation. ger follow-up will be needed to substantiate these results in Genes Dev., 7: 1572–1583, 1993. adults with ALL. 7. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, Q., Harshman, K., Tavtigian, S. V., Stockert, E., Day, R. S., III, Johnson, B. E., and In an analysis of 79 children with ALL, Heyman et al. (36) INK4a ARF Skolnick, M. H. A cell cycle regulator potentially involved in genesis of found a correlation between inactivation of p16 /p14 / many tumor types. Science (Washington DC), 264: 436–440, 1994. INK4b p15 and lower CR rates and shorter EFS. Deletion of 8. Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., and genetic material from one allele but preservation of a normal Carson, D. A. Deletions of the cyclin-dependent kinase-4 inhibitor gene coding sequence on the remaining allele conferred a similar in multiple human cancers. Nature (Lond.), 368: 753–756, 1994. prognosis to cases without deletions. The importance of this 9. Ogawa, S., Hangaishi, A., Miyawaki, S., Hirosawa, S., Miura, Y., difference in survival between patients with homozygous and Takeyama, K., Kamada, N., Ohtake, S., Uike, N., Shimazaki, C., hemizygous deletions in this study is not understood. It is Toyama, K., Hirano, M., Mizoguchi, H., Kobayashi, Y., Furusawa, S., Saito, M., Emi, N., Yazaki, Y., Ueda, R., and Hirai, H. Loss of the intriguing to think that patients with hemizygous deletions may cyclin-dependent kinase 4-inhibitor (p16; MTS1) gene is frequent in and carry a point mutation in the second or third allele that affects highly specific to lymphoid tumors in primary human hematopoietic either p16INK4a or p14ARF but not both together. More knowl- malignancies. Blood, 86: 1548–1556, 1995. edge of the molecular mechanisms of inactivation and of the 10. Siebert, R., Willers, C. P., Schramm, A., Fossa, A., Gana Dresen, role of each of the genes for p16INK4a, p14ARF, and p15INK4b in I. M., Uppenkamp, M., Nowrousian, M. R., Seeber, S., and Opalka, B. Homozygous loss of the MTS1/p16 and MTS2/p15 genes in lymphoma leukemogenesis will allow us to better understand the impor- and lymphoblastic leukaemia cell lines. Br. J. Haematol., 91: 350–354, tance of genetic events and provide opportunities for new ther- 1995. apies in the future. 11. Ogawa, S., Hirano, N., Sato, N., Takahashi, T., Hangaishi, A., Tanaka, K., Kurokawa, M., Tanaka, T., Mitani, K., Yazaki, Y., and REFERENCES Hirai, H. Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16) gene in human leukemias. Blood, 84: 2431–2435, 1994. 1. Del Sal, G., Loda, M., and Pagano, M. Cell cycle and cancer: critical 12. Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. Alternative events at the G restriction point. Crit. Rev. Oncog., 7: 127–142, 1996. 1 reading frames of the INK4a tumor suppressor gene encode two unre- ink4a 2. Quesnel, B., Preudhomme, C., and Fenaux, P. p16 gene and lated proteins capable of inducing cell cycle arrest. Cell, 83: 993–1000, hematological malignancies. Leuk. Lymphoma, 22: 11–24, 1996. 1995. 3. Clurman, B. E., and Groudine, M. The CDKN2A tumor-suppressor 13. Pomerantz, J., Schreiber-Agus, N., Lie´geois, N. J., Silverman, A., locus: a tale of two proteins. N. Engl. J. Med., 338: 910–912, 1998. Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Han-Woong, L., 4. Della Ragione, F., Mercurio, C., and Iolascon, A. Cell cycle regula- Cordon-Cardo, C., and DePinho, R. A. The Ink4a tumor suppressor gene tion and human leukemias. The role of p16ink4a gene inactivation in the product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibi- development of human acute lymphoblastic leukemia. Haematologica, tion of p53. Cell, 92: 713–723, 1998. 80: 557–568, 1995. 14. Haidar, M. A., Cao, X-B., Manshouri, T., Chan, L. L., Glassman, 5. Serrano, M., Hannon, G. J., and Beach, D. A new regulatory motif in A., Kantarjian, H. M., Keating, M. J., Beran, M. S., and Albitar, M. cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature p16ink4a and p15ink4b gene deletions in primary leukemias. Blood, 86: (Lond.), 366: 704–707, 1993. 311–315, 1995.

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Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 1999 American Association for Cancer Research. The Prognostic Significance of p16INK4a/p14ARF and p15 INK4b Deletions in Adult Acute Lymphoblastic Leukemia

Stefan Faderl, Hagop M. Kantarjian, Taghi Manshouri, et al.

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