Lack of ETV6 (TEL) gene rearrangements or p16INK4A ... - Nature

Leukemia (1997) 11, 979–983  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Lack of ETV6 (TEL) gene rearrangements or p16INK4A /p15INK4B homozygous gene deletions in infant acute lymphoblastic leukemia KW Maloney1, JE Rubnitz2, ML Cleary3, LS Frankel4, N Hakami4, MP Link4, DJ Pullen4 and SP Hunger1 1

Section of Pediatric Hematology/Oncology, Department of Pediatrics, University of Colorado School of Medicine, Denver, CO; 2Department of Hematology/Oncology, St Jude Children’s Research Hospital and the Department of Pediatrics, University of Tennessee Health Sciences Center, Memphis, TN; 3Laboratory of Experimental Oncology, Department of Pathology, Stanford, CA; and 4Pediatric Oncology Group (POG), Chicago, IL, USA

Acute lymphoblastic leukemia (ALL) occurring in infants less than 1 year of age differs clinically and biologically from that observed in older children. Cytogenetically, 11q23 translocations are detected in approximately 50% of infant ALLs and fuse the 11q23 gene HRX with a variety of partner chromosomal loci. Overall, HRX rearrangements are detected molecularly in 70–80% of infant ALLs as compared to 5–7% of ALLs arising in older children. Two recently described molecular abnormalities in childhood ALL are ETV6 gene rearrangements and homozygous deletions of p16INK4A and/or p15 INK4B. Each of these abnormalities occurs in 15–20% of all childhood ALLs, and neither can be accurately identified by routine cytogenetic analyses. The incidence of these genetic abnormalities and their potential relationship to HRX gene status in infant ALL is unknown. Using Southern blot analyses, we determined ETV6 and p16 INK4A/p15INK4B gene status in a cohort of infant ALLs. No ETV6 rearrangements or homozygous deletions (n = 69) or homozygous p16INK4A and/or p15INK4B gene deletions (n = 54) were detected in any of the infant ALLs. Therefore, ETV6 and p16INK4A/p15INK4B do not play a significant role in the pathogenesis of infant ALL, further emphasizing the distinctive biology of this subset of leukemias. Keywords: infant ALL; HRX; ETV6; TEL; AML1; p16 and p15

Introduction Approximately 3% of childhood acute lymphoblastic leukemia (ALL) occurs in infants less than 1 year of age.1 The clinical and biological features observed in infants with ALL as well as their treatment outcome differ remarkably from those of older children.2,3 Infant ALLs often present with hyperleukocytosis and display a notably increased frequency of central nervous system involvement and bulky extramedullary disease.1–4 A T cell phenotype is much less common in infants than in older children.4 The immunophenotypic features of infant B-precursor ALL differ from the phenotype generally observed in older children as many are CD10-negative and co-express myeloid-associated cell surface antigens such as CD15.1,5 Cytogenetic and molecular genetic abnormalities in infant ALL also differ from those observed in older children. Hyperdiploidy with a modal chromosome number of .50 is extremely uncommon in infants, yet occurs in approximately 20% of older children.1,6 Specific reciprocal chromosome translocations which are observed most frequently in older children, such as the t(1;19)(q23;p13) or t(9;22)(q34;q11), are rarely observed in infants with ALL.1,6 In contrast, cytogenetic abnormalities, particularly reciprocal translocations, involving chromosome band 11q23 occur much more frequently in infants than in older children with ALL. Translocations affect-

Correspondence: SP Hunger, UCHSC Campus Box C229, 4200 East Ninth Ave, Denver, CO 80262, USA Received 13 January 1997; accepted 27 February 1997

ing 11q23 are detected by standard cytogenetics in approximately 50% of infant ALLs vs approximately 5% of older children.1,4,5,7–11 Molecular studies have demonstrated that these translocations almost invariably interrupt a gene termed HRX (MLL, ALL-1, HTRX-1), fusing it to one of a variety (more than 25 have been described) of partner chromosomes on the der(11) and encoding HRX protein chimeras postulated to be involved in transcriptional regulation.12,13 Molecular studies from several different groups have revealed that 70–80% of infant ALLs (vs approximately 7% of ALL in older children) contain HRX gene rearrangements and that these abnormalities are associated with an extremely poor outcome following treatment with various chemotherapy regimens (Table 1).5,11,14–17 The outcome of infant ALLs lacking HRX rearrangements has been more variable, with several studies finding such patients to have an excellent treatment outcome while others found their outcome to be inferior to that expected in older children lacking biological high-risk features.5,11,14–17 Over the past several years, two molecular abnormalities have been identified which occur in a significant percentage of childhood ALLs and are difficult to identify by standard cytogenetics. The t(12;21)(p13;q22) is a newly recognized non-random translocation that fuses the chromosome 12 gene ETV6 (TEL) to the chromosome 21 gene AML1, resulting in production of ETV6-AML1 fusion mRNAs and chimeric proteins.18–20 Although rarely observed cytogenetically, this abnormality is detected molecularly in 15–25% of childhood B-precursor ALL and is associated with good risk clinical features and an excellent treatment outcome.19,21–24 Another recurring genetic abnormality in childhood ALL is homozygous deletion of one or both of a pair of closely linked and related tumor suppressor genes (TSGs) located on the short arm of chromosome 9 (9p21): the cyclin-dependent kinase inhibitor genes p16INK4A(MTS1) and p15INK4B (MTS2).25–27 In Table 1 Clinical outcome for HRX-germline and HRX-rearranged infant ALLs

No. HRX-R (%)

EFS % (timepoint)

No. HRX-G (%)

EFS % (timepoint)

Ref.

(70) a (81) (69) (72) a (63) b (76)

15 (46 months) 19 (3 years) 13 (3 years) 28 (2 years) 9 (50 months) 5 (3 years)

9 (30)a 18 (19) 9 (31) 11 (28)a 14 (37)b 10 (24)

80 (46 months) 46 (3 years) 67 (3 years) 100 (2 years) 57 (50 months) 89 (3 years)

14 11 5 15 16 17

21 78 19 29 24 32

HRX-G, HRX-germline; HRX-R HRX-rearranged; EFS, event-free survival. a These two studies are partially overlapping b This study included infants up to 18 months of age. All other studies include only infants less than 1 year of age.

ETV6 and p16INK4A/p15INK4B in infant ALL KW Maloney et al

980

ALL, these genes are predominantly inactivated by homozygous deletion. Hemizygous deletion with point mutation of the retained allele is an uncommon event, leading some to suggest that co-deletion of both genes is specifically selected for as it provides a powerful growth advantage to the cell.28–30 Homozygous deletions of one or more commonly both genes is detected in approximately 15–20% of childhood ALLs.31–34 Deletions appear to occur more frequently among T-lineage than B-lineage childhood ALLs. The independent prognostic import of p16INK4A and p15INK4B deletions remains unresolved and is an area of intensive investigation in many laboratories.31,35 Thus, ETV6–AML1 fusion and homozygous deletion of p16INK4A and/or p15INK4B are two of the most common genetic abnormalities in childhood ALL. As neither can be consistently identified by standard cytogenetics, we undertook a molecular study to determine the incidence of these two genetic abnormalities in infant ALL. Because we were particularly interested in determining the relationship between these abnormalities and HRX gene rearrangements, we analyzed infant ALLs from a large cohort in which HRX gene status has previously been determined and correlated with clinical outcome.11 A major hypothesis that we wished to test was whether ETV6–AML1 fusion might identify a subset of HRXgermline infant ALLs with an excellent prognosis which could allow treatment stratification and obviate the need for more intensive therapies in some infants with ALL.

Materials and methods

Patient specimens

in .95% of cases with ETV6–AML1 fusion.21,22 Following completion of these analyses, the membranes were stripped and co-hybridized with a 360-bp DNA fragment corresponding to p16INK4A exon 233 (a kind gift of Francesco Lo Coco, Rome, Italy) and a commercially available BCR probe (Pr-1; Oncogene Science, Uniondale, NY, USA). This p16INK4A probe also cross-hybridizes with p15INK4B, allowing detection of deletions of either gene. Blots were inspected visually and/or with the aid of a Phosphorlmager (Molecular Dynamics, Santa Clara, CA, USA) to compare the intensities of bands corresponding to p16INK4A and p15INK4B to BCR. Samples in which bands were absent or were less than 10–20% of the intensities of control BCR bands were scored as containing homozygous deletions of the corresponding gene. Appropriate positive (genomic DNA from a healthy individual) and negative (DNA from cell lines known to contain homozygous p16INK4A and p15INK4B gene deletions) controls were included on each blot.

Results and discussion DNA was available from 69 cases of infant ALL for analysis of the status of the ETV6 gene. Of these 69 cases, 55 were previously shown to contain HRX rearrangements and 14 to be HRX-germline. 11 None of the 69 infant ALLs analyzed contained ETV6 gene rearrangements or homozygous deletions (representative results are shown in Figure 1). To ensure that this failure to detect alterations was not due to technical factors, nine cases of randomly selected standard-risk ALL (as defined by the NCI criteria: age 1.00–9.99 years, presenting white blood count ,50 00036) were analyzed in parallel. Of these nine standard-risk childhood ALLs, ETV6 was germline

Previously, Rubnitz et al11 reported the HRX gene status in 96 infants enrolled in one of three consecutive Pediatric Oncology Group (POG) clinical trials. Sufficient DNA and/or frozen leukemic cells for additional molecular analyses was available from a subset of these cases and was used to determine ETV6 (69 cases) and p16INK4A/p15INK4B (54 cases) gene status. Additional childhood ALL samples were obtained from the cell bank of the Children’s Hospital, Denver, CO. All samples were originally collected as part of POG or institutional clinical trials that had been approved by the Institutional Review Boards of individual institutions.

Molecular analyses Genomic DNA was isolated using the Puregene Genomic DNA Isolation Kit (Gentra, Research Triangle Park, NC, USA) exactly as suggested by the manufacturer. High molecular weight DNA (5–10 mg) was digested with BamHI, size-fractionated by electrophoresis in 0.8% agarose gel and transferred to charged nylon membranes (Zeta Probe, Bio-Rad, Hercules, CA, USA). To determine the presence of ETV6 gene rearrangements, the membranes were hybridized using previously described procedures12 with a 466 basepair (bp) SacI/BamHI fragment of the ETV6 cDNA21 (a generous gift of Gerard Grosveld, Memphis, TN, USA) radiolabeled with a32PdCTP using the Random Primers DNA labeling system (Gibco BRL, Gaithersburg, MD, USA) and visualized by autoradiography. This combination of probe and restriction enzyme digest has been demonstrated to detect ETV6 gene rearrangements

Figure 1 Southern blot of infant ALL samples for ETV6 gene rearrangements. The autoradiogram displays Southern blot analyses for ETV6 gene rearrangements in infant ALLs. High molecular weight DNA from infant ALLs was digested with BamHI and probed with an ETV6 cDNA probe. The location of the germline ETV6 band is indicated on the right. Molecular size markers are shown in kilobases on the left. Samples include the K562 cell line, normal DNA from a healthy control and 10 infant ALL specimens. No ETV6 gene rearrangements are observed.


in three, rearranged in four and rearranged with deletion of the other allele in two cases (data not shown), confirming that the procedures employed in this study can accurately identify ETV6 rearrangements and deletions. Following completion of these analyses, 54 of the 69 cases (44 HRX-rearranged, 10 HRX-germline) were analyzed to look for homozygous deletions of either p16INK4A or p15INK4B. None of these 54 cases contained homozygous deletions of either p16INK4A or p15INK4B (representative analyses are shown in Figure 2). As each blot contained control samples with and without co-deletion of p16INK4A and p15INK4B we are confident that no cases with homozygous deletions were missed. In the current study, no infant ALLs were identified which contained ETV6 rearrangements or homozygous deletions or homozygous p16INK4A and/or p15INK4B deletions, further underscoring the unique biological nature of infant ALL. It is unlikely that these findings are due to inadequate sampling as they are based on analysis of 69 (ETV6 rearrangements) and 54 (p16INK4A and/or p15INK4B deletions) cases, a number that closely approximates the total number of infant ALLs occurring each year in the United States (3% of 2000 childhood ALLs, or approximately 60 cases per year1,6). Furthermore, approximately 20% of the infant ALLs studied were HRXgermline and 80% were HRX-rearranged, indicating that our sample was quite representative of the two major currently recognized molecular subtypes of infant ALL. Thus, while it is possible that individual infant ALLs may be identified that contain ETV6 rearrangements or homozygous p16INK4A and/or p15INK4B deletions, it is highly unlikely that either of these genetic abnormalities plays a significant role in the overall pathogenesis of infant ALL.

ETV6–AML1 fusion is detected in 15–25% of ALL occurring in children between 2 and 10 years of age from the United States, Western Europe and Asia.19,21–24,37 In contrast, ETV6– AML1 fusion is extremely uncommon in adults with ALL, being detected in 0/81 Taiwanese adults with ALL38 and 1/118 (the single positive cause was a 19-year-old female) adults with ALL treated on the Western European LALA 94 trial.39 These data taken together with that presented herein suggest that ETV6–AML1 fusion is largely confined to a distinct subset of childhood ALLs. When the current study was undertaken, we hypothesized that ETV6–AML1 fusion might help to identify a subset of HRX-germline infant ALLs with an excellent prognosis, perhaps allowing treatment to be stratified on this basis. This question merits further investigation in other cohorts of infants with ALL, as the occurrence of a small number of cases with ETV6–AML1 fusion among the HRX-germline subset might help to explain the differences in clinical outcome documented for HRX-germline cases in Table 1. A major unresolved question concerning infant ALL is the nature of the initiating leukemogenic event(s) in the 20–30% of cases that are HRX-germline. The current study, in concert with prior data, indicate that this subset does not contain a significant number of cases with genetic events observed commonly in older children: hyperdiploidy, specific non-random reciprocal translocations, ETV6–AML1 fusion, or homozygous p16INK4A and/or p15INK4B deletions. Thus, it appears incorrect conceptually to visualize infant ALL as consisting of a biologically unique subset (HRX-rearranged cases) superimposed on a baseline incidence of ALL that closely resembles that seen in older children. Rather, the data indicate that infant ALL may be composed of two (or more) subsets which are biologically distinct from those observed commonly in older children. A major leukemogenic event, HRX gene fusion, has been identified for one subset. A current challenge is to define the genetic events which underlie the other 20–30% of infant ALLs.

Acknowledgements We thank Gerard Grosveld for the ETV6 probe and Francesco Lo Coco for the p16INK4A exon 2 probe. We also thank Cita Nichols for her work in isolating some of the DNAs utilized in this study, Majilinde Fall for technical assistance, and Lorrie Odom for her work in coordinating the cell bank at The Children’s Hospital in Denver, CO. KWM was supported in part by the Monfort Pediatric Oncology Fellowship and is the recipient of the Greg and Laura Norman Fellowship Award from the National Childhood Cancer Foundation. JER is the recipient of a Career Development Award from the American Society of Clinical Oncology, MLC is a Scholar of the Leukemia Society of America and SPH is the recipient of a BLOOD/ASH Scholar Award. This work was supported by a grant from the Cancer League of Colorado to SPH. Figure 2 Southern blot of infant ALL samples for p16INK4A/p15INK4B gene deletions. The autoradiogram displays Southern blot analyses for p15INK4A/p15INK4B gene deletions in infant ALLs. High molecular weight DNA from infant ALLs was digested with BamHI and probed with both p16INK4A and BCR probes. The location of germline p16INK4A, p15INK4B and BCR bands is indicated on the right. Molecular size markers are shown in kilobases on the left. Samples include the K562 cell line which has homozygous deletions of p16INK4A and p15INK4B, normal DNA from a healthy control and 10 infant ALL specimens. Homozygous p16INK4A or p15INK4B deletions are not present in any of the 10 infant ALLs.

References 1 Pui C-H, Kane JR, Crist WM. Biology and treatment of infant leukemias. Leukemia 1995; 9: 762–769. 2 Reaman G, Zeltzer P, Bleyer WA, Amendola B, Level C, Sather H, Hammond D. Acute lymphoblastic leukemia in infants less than one year of age: a cumulative experience of the Childrens Cancer Study Group. J Clin Oncol 1985; 3: 1513–1521. 3 Crist W, Pullen J, Boyett J, Falletta J, van Eys J, Borowitz M, Jackson

981


982

4

5

6 7

8

9 10

11

12

13

14

15

16

17

18 19

20

J, Dowell B, Frankel L, Quddus F, Ragab A, Vietti T. Clinical and biologic features predict a poor prognosis in acute lymphoid leukemias in infants: a Pediatric Oncology Group study. Blood 1986; 67: 135–140. Ferster A, Bertrand Y, Benoit Y, Boilletot A, Behar C, Margueritte G, Thyss A, Robert A, Mazingue F, Souillet G, Phillippe N, Solbu G, Suciu S, Otten J. Improved survival for acute lymphoblastic leukaemia in infancy: the experience of EORTC-childhood leukaemia cooperative group. Br J Haematol 1994; 86: 284–290. Pui C-H, Behm FG, Downing JR, Hancock ML, Shurtleff SA, Ribeiro RC, Head DR, Mahmond JJ, Sandlund JT, Furman WL, Roberts WM, Crist WM, Raimondi SC. 11q23/MLL rearrangement confers a poor prognosis in infants with acute lymphoblastic leukemia. J Clin Oncol 1994; 12: 909–915. Pui C-H. Childhood leukemias. New Engl J Med 1995; 332: 1618–1630. Chessells JM, Eden OB, Bailey CC, Lilleyman JS, Richards SM. Acute lymphoblastic leukaemia in infancy: experience in MRC UKALL trials. Report from the medical research council working party on childhood leukaemia. Leukemia 1994; 8: 1275–1279. Kaneko Y, Shikano T, Maseki N, Sakurai M, Sakurai M, Takeda Y, Hiyoshi T, Mimaya J-i, Fujimoto T. Clinical characteristics of infant acute leukemia with or without 11q23 translocations. Leukemia 1988; 2: 672–676. Lampert F, Harbott J, Ritterbach J. Cytogenetic findings in acute leukaemias of infants. Br J Cancer 1992; 66 (Suppl. XVIII): S20– S32. Cimino G, Lo Coco F, Biondi A, Elia L, Luciano A, Cronce CM, Masera G, Mandelli F, Canaani E. ALL-1 gene at chromosome 11q23 is consistently altered in acute leukemia of early infancy. Blood 1993; 82: 544–546. Rubnitz JE, Link MP, Shuster JJ, Carroll AJ, Hakami N, Frankel LS, Pullen DJ, Cleary ML. Frequency and prognostic significance of HRX rearrangements in infant acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood 1994; 84: 570–573. Hunger SP, Tkachuk DC, Amylon MD, Link MP, Carroll AJ, Welborn JL, Willman CL, Cleary ML. HRX involvement in de novo and secondary leukemias with diverse chromosome 11q23 abnormalities. Blood 1993; 81: 3197–3203. Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, Le Beau MM, Diaz MO, Rowley JD. Rearrangement of the MLL gene in acute lymphoblastic and acute myleoid leukemias with 11q23 chromosomal translocations. New Engl J Med 1993; 329: 909–914. Chen C-S, Sorensen PHB, Domer PH, Reaman GH, Korsmeyer SJ, Heerema NA, Hammond GD, Kersey JH. Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome. Blood 1993; 81: 2386–2392. Hilden JM, Frestedt JL, Moore RO, Heerema NA, Arthur DC, Reaman GH, Kersey JH. Molecular analysis of infant acute lymphoblastic leukemia: MLL gene rearrangement and reverse transcriptasepolymerase chain reaction for t(4;11)(q21;q23). Blood 1995; 86: 3876–3882. Cimino G, Rapanotti MC, Rivolta A, Lo Coco F, D’Arcangelo E, Rondelli R, Basso G, Barisone E, Rosanda C, Santostasi T, Canaani E, Masera F, Biondi A. Prognostic relevance of ALL-1 gene rearrangement in infant acute leukemias. Leukemia 1995; 9: 391–395. Taki T, Ida K, Bessho F, Kikuchi A, Yamamoto K, Sako M, Tsuchida M, Seto M, Ueda R, Hayashi Y. Frequency and clinical significance of the MLL gene rearrangements in infant acute leukemia. Leukemia 1996; 10: 1303–1307. Romana SP, Le Coniat M, Berger R. t(12;21): a new recurrent translocation in acute lymphoblastic leukemia. Genes Chromos Cancer 1994; 9: 186–191. Romana SP, Poirel H, Leconiat M, Flexor M-A, Mauchauffe M, Jonveaux P, Macintyre EA, Berger R, Bernard OA. High frequency of t(12;21) in childhood B-lineage acute lymphoblastic leukemia. Blood 1995; 86: 4263–4269. Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, BrayWard P, Morgan E, Raimondi SC, Rowley JD, Gilliland DG. Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute

21

22

23

24

25

26 27 28

29 30

31

32

33

34

35

36

37

lymphoblastic leukemia. Proc Natl Acad Sci USA 1995; 92: 4917–4921. Shurtleff SA, Buifs A, Behm FG, Rubnitz JE, Raimondi SC, Hancock ML, Chan GC-F, Pui C-H, Grosveld G, Downing JR. TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia 1995; 9: 1985–1989. Raynaud S, Cave´ H, Baens M, Bastard C, Cacheux V, Grosgeorge J, Guidal-Giroux C, Guo C, Vilmer E, Marynen P, Grandchamp B. The 12;21 translocation involving TEL and deletion of the other TEL allele: two frequently associated alterations found in childhood acute lymphoblastic leukemia. Blood 1996; 87: 2891–2899. Liang DC, Chou TB, Chen JS, Shurtleff SA, Rubnitz JE, Downing JR, Pui C-H, Shih LY. High incidence of TEL/AML1 fusion resulting from a cryptic t(12;21) in childhood B-lineage acute lymphoblastic leukemia in Taiwan. Leukemia 1996; 10: 991–993. Rubnitz JE, Downing JR, Pui C-H, Shurtleff SA, Raimondi SC, Evans WE, Head DR, Crist WM, Rivera GK, Hancock ML, Boyett JM, Buijs A, Grosveld G, Behm FG. TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J Clin Oncol 1997; 15: 1150–1157. Kamb A, Gruis NA, Weaver-Felhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, Johnson BE, Skolnick MH. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994; 264: 436–440. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cellcycle control causing specific inhibition of cyclin D/CDK4. Nature 1993; 366: 704–707. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994; 368: 753–756. Gombart AF, Morosetti R, Miller CW, Said JW, Koeffler HP. Deletions of the cyclin-dependent kinase inhibitor genes p16 INK4A/p15INK4B in non-Hodgkin’s lymphomas. Blood 1995; 86: 1534–1539. Uchida T, Watanabe T, Kinoshita T, Murate T, Saito H, Hotta T. Mutational analysis of the CDKN2 (MTSI/p16INK4A) gene in primary B-cell lymphomas. Blood 1995; 86: 2724–2731. Delmer A, Tang R, Senamaud-Beaufort C, Paterlini P, Brechot C, Zittoun R. Alterations of cyclin-dependent kinase 4 inhibitor (p16INK4A/MTSI) gene structure and expression in acute lymphoblastic leukemias. Leukemia 1995; 9: 1240–1245. Quesnel B, Preudhomme C, Philippe N, Vanrumbeke M, Dervite I, Luc Lai J, Bauters F, Wattel E, Fenaux P. p16 gene homozygous deletions in acute lymphoblastic leukemia. Blood 1995; 85: 657–663. Okuda T, Shurtleff SA, Valentine MB, Raimondi SC, Head DR, Behm F, Curcio-Brint AM, Liu Q, Pui C-H, Sherr CJ, Beach D, Look AT, Downing JR. Frequent deletion of p16INK4A/MTSI and p15INK4B/MTS2 in pediatric acute lymphoblastic leukemia. Blood 1995; 85: 2321–2335. Fizzotti M, Cimino G, Pisegna S, Alimena G, Quartarone C, Mandelli F, Pelicci PG, Lo Coco F. Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor (p16) gene in acute lymphoblastic leukemia and association with adverse prognostic features. Blood 1995; 85: 2685–2690. Ohnishi H, Kawamura M, Ida K, Sheng XM, Hanada R, Nobori T, Yamamori S, Hayashi Y. Homozygous deletions of p16/MTS1 gene are frequent but mutations are infrequent in childhood Tcell acute lymphoblastic leukemia. Blood 1995; 86: 1269–1275. Heyman M, Rasool O, Brandter LB, Liu Y, Grande´r D, So¨derha¨ll S, Gustavsson G, Einhorn S. Prognostic importance of p15INK4B and p16 INK4A gene inactivation in childhood acute lymphocytic leukemia. J Clin Oncol 1996; 14: 1512–1520. Smith M, Arthur D, Camitta B, Carroll AJ, Crist W, Gaymon P, Gelber R, Heerema N, Korn EL, Link M, Murphy S, Pui C-H, Pullen J, Reaman G, Sallan SE, Sather H, Shuster J, Simon R, Trigg M, Tubergen D, Uckun F, Ungerleider R. Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia. J Clin Oncol 1996; 14: 680–681. Nakao M, Yokota S, Horiike S, Taniwaki M, Kashima K, Sonoda Y, Koizumi S, Takaue Y, Matsushita T, Fujimoto T, Misawa S. Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia. Leukemia 1996; 10: 1463–1470.


38 Shih L-Y, Chou T-B, Liang D-C, Tzeng Y-S, Rubnitz JE, Downing JR, Pui C-H. Lack of TEL–AML1 fusion transcript resulting from a cryptic t(12;21) in adult B lineage acute lymphoblastic leukemia in Taiwan. Leukemia 1996; 10: 1456–1458.

39 Raynaud S, Mauvieux L, Cayuela JM, Bastard C, Bilhou-Nabera C, Debuire B, Bories D, Boucheix C, Charrin C, Fiere D, Gabert J. TEL/AML1 fusion gene is a rare event in adult acute lymphoblastic leukemia. Leukemia 1996; 10: 1529–1530.

983