Translocations Involving Chromosome i2pii-13, Methotrexate Metabolism, and Outcome in Childhood B-Progenitor Cell Acute Lymphoblastic Leukemia: A Pediatric Oncology Group Study’

Children with B-progenitor cell acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTh) and MTX polyglutamates (MTXPGs) appear to have a good prognosis. This has been attributed to increased sensitivity oftheir blast cells to MTX. However, the proportion of children who are cured of Bprogenitor cell acute lymphoblastic leukemia exceeds the number whose lymphobbasts accumulate high MTXPG 1evels. We report that lymphoblasts from patients with <50 chromosomes who have translocations that involve the short arm of chromosome i2 accumulate low levels of MTXPGs. These patients appear to have an excellent survival because none of 14 patients with transbocations affecting i2p has relapsed, 26-79 months following diagnosis. INTRODUCTION Contemporary multiagent chemotherapy regimens cure substantially more than half of all children over 1 year of age with pro-B ALL3 (1). The antifol MTX is a mainstay of most regimens (1-9). It is administered intrathecally, alone or with other agents, to prevent central nervous system leukemia; as a 24-h infusion during intensification therapy; and i.m. or p.o., once a week, during continuation treatment. Several other agents are commonly used to treat childhood ALL, including vincristine, prednisone, L-asparaginase, and 6-mercaptopurine. For patients at higher risk of treatment failure, cyclophosphamide, anthracycines, 13-D-arabinofuranosylcytosine, and epipodophyllotoxins have been added in an attempt to increase the cure rate (2, 3, 5, 10). MTX is converted to MTXPGs, containing up to seven ‘y-linked glutamyl residues, by the enzyme FPGS (1 1). Accumulation of MTXPGs in lymphoblasts in vitro correlates with cell ploidy and is highest in hyperdipboid lymphoblasts, measured either as >50 chromosomes or as a DI of > 1.16 (l2-14). Hyperdiploidy is associated with a favorable outcome in patients who are treated with regimens that emphasize MTX (4), Received 3/14/97; revised 9/30/97; accepted 10/8/97. 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. I This research was supported in part by the following National Cancer Institute grants: CA-33587, CA-25408, CA-32053, CA-03 161 , CA29139, CA-53490, CA-33625, CA-29691, CA-28841, CA-31566, CA52317, and CA-15989. In addition, this work was supported by grants from the National Cancer Institute of Canada, the Cancer Research Society. and the McGill University-Montreal Children’s Hospital Research Institute, and it was assisted by the Penny Cole Fund, the Fast Foundation, the InterService Clubs Council Telethon of Stars, the Lamplighters Children’s Cancer-Leukemia Association, the Children’s Cancer Fund, the Debbie Saunders Fund, the Friends of Andc, the Midwest Athletes against Childhood Cancer Fund, the American Lebanese Syrian Associated Charities, and the American Cancer Society. 2 To whom requests for reprints should be addressed, at Pediatric Oncology Group, 645 North Michigan Avenue, Suite 910, Chicago, IL 60611. Phone: (312)482-9944; Fax: (312)482-9460. 3 The abbreviations used arc: pro-B ALL, B-progenitor cell acute lymphoblastic leukemia; MTX, methotrexate; MTXPG, MTX polyglutamate, containing 2-6 total glutamates; FPGS, folate polyglutamate synthetase; DI, DNA index; EFS, event-free survival; trl2pl 1-13, chromosome l2pll-l3 translocation; P0G. Pediatric Oncology Group; del(l2p), chromosome l2p deletion. 4 V. M. Whitehead, M-J. Vuchich, A. J. Carroll, S. J. Lauer, D. Mahoney, J. J. Shuster, C. Payment, P. A. Koch, J. J. Akabutu, T. Bowen, B. A. Kamen, Y. Ravindranath, A. Emami, G. P. Beardslcy, D. J. Pullcn, and B. Camitta. Accumulation of methotrexate polyglutamates, ploidy and trisomies of both chromosomes 4 and 10 in lymphoblasts from children with B-progenitor ccl! acute lymphobbastic leukemia: a Pediattic Oncology Group Study, submitted for publication, 1997. Research. on October 2, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 184 MTXPGs and Translocations Affecting 12p suggesting that the extent of MTXPG accumulation in bymphoblasts may be important to the effectiveness of MTX treatment. Indeed, children with pro-B ALL, whose lymphoblasts at diagnosis accumulated high levels of both MTX and MTXPGs in vitro, experienced a superior EFS, compared with children whose bymphoblasts did not (15). Moreover, the accumulation of higher levels of MTXPGs in lymphobbasts in vivo was associated with greater antileukemic effects (14). The proportion of children with pro-B ALL whose lymphoblasts accumulate high levels of MTXPGs is less than the number expected to be cured with contemporary chemotherapy. In reviewing a series of patients whose lymphoblasts had <50 chromosomes but whose age and WBC count suggested a favorable prognosis, we noted that lymphoblasts from many of them failed to accumulate high levels of MTXPGs. Here, we describe a group of patients with trl2p! !-13s who had lymphoblasts that accumulated low levels of MTXPGs. There has been no recurrence of leukemia in these patients after a considerable follow-up, suggesting that they have a good prognosis. MATERIALS AND METHODS Between April 1989 and September 1994, children from designated POG institutions were registered on P00 8901, a limited-institution POG study that was undertaken to measure the accumulation of MTXPGs in bymphoblasts in vitro. This analysis involves a subgroup of 95 of these children with pro-B ALL who had both a lymphoblast MTXPG level determined at diagnosis and numerical or structural cytogenetic abnormalities.4 There were 50 boys and 45 girls. Their median age at diagnosis was 4.4 years (range, 1.3-18.6 years), and their median white cell count at diagnosis was 13.0 X 109/liter (range, 0.7-315.2 x l09/liter). Forty patients were hyperdipboid, with >50 chromosomes, and 55 were nonhyperdiploid, with <50 chromosomes. Patients were treated on one of several successive contemporary front-line POG therapeutic protocols (POG 8602, POG 8698, POG 8699, P00 9005, or POG 9006). Written informed consent was obtained both for the laboratory studies and for the treatment protocols according to Institutional Review Board requirements. Data on patients 3, 7, 8, 1 1, and 12 (Table 1) have been published previously (12). Pro-B ALL (early pre-B and pre-B cell) was diagnosed by standard POG criteria (16). Karyotype analysis was carried out in the POG Cytogenetics Reference Laboratory at the University of Alabama (Birmingham, AL), as well as in the Baylor Cancer Cytogenetics Laboratory (Houston, TX), the Cytogenetics Laboratory at the University of Texas Southwestern Medical Center (Dallas, TX), and the Cytogenetics Laboratory at the Medical College of Wisconsin (Milwaukee, WI). The DI was determined at St. Jude Children’s Research Hospital (Memphis, TN; Ref. 17). Lymphobbasts were separated by Ficoll-Paque sedimentation from a 1-2-mb bone marrow aspirate sample obtained at diagnosis. Samples were judged satisfactory if the percentage blasts was 75% or more or the initial white cell count was 25.0 X l09lliter or greater. Cells were washed three times with HBSS. Five million lymphoblasts were incubated with 1 .0 iM [3H]MTX in 2.0 ml of RPM! 1640 containing 10% fetal bovine serum in a P-35 culture dish in 5% C02-95% 02 for 24 h at 37#{176}C. Following incubation, cells were washed three or four times with HBSS to remove extracellubar [3H]MTX. Extracts were prepared by addition of trichloroacetic acid or by boiling for 5 mm. MTX and MTXPGs were separated using highperformance liquid chromatography. These techniques are described in more detail elsewhere (12). Results were judged valid ifthe lymphoblast MTXPG level was > 100 pmolJlO9 cells or the predominant MTXPG had five glutamyl residues. A lymphoblast MTXPG bevel of >500 pmol/l09 cells was defined as high, based on biological advantage (14, 15), whereas a level >1500 pmol/l09celbs was arbitrarily defined as very high.4 This level yields a better patient distribution than the previous “very high” bevel of >2000 pmol/l09 cells used (12). Results are means of triplicate determinations. The two-sided Wilcoxon test (18) was used for comparison of MTXPG levels between groups. Because the vast majority of P00 institutions were not part of this pharmacology study, this patient series represents a very small fraction of the patients on the aforementioned studies. Yet a!! institutions were required to submit bone marrow samples to the University of Alabama or other approved cytogenetics laboratories (see above) for karyotyping. The prognostic significance of cytogenetic abnormalities affecting l2p are being investigated as part of a much larger series that will be reported in the near future. Therefore, no statistical comparison of outcome based on l2p in this series was deemed to be valid. RESULTS trl2pll-13s. A total of 14 (15%) of the 95 patients had trl2pl 1-13 as a feature of the principal cytogenetically abnormal clone (Table 1 ). Translocations are reported as involving one or more of the bands l2pl 1, l2pl2, and l2pl3 in different patients and may represent different break sites. They have been grouped together for this analysis. There were seven boys and seven girls. Their median age was 4.4 years (range, 2.1-10.6 years), and their median initial white cell count was 20.3 X 109/liter (range, 3.8-260. 1 X 109/liter). One (2.5%) of the 40 hyperdipboid patients had 61 chromosomes, a DI of 1.34, and tn 2pl 1-1 35 (Table 1 , patient 14). His lymphoblast MTXPG level was 1727 pmolll09 cells, falling in the very high range. Thirteen (24%) of the 55 nonhyperdiploid patients had 45-47 chromosomes, a DI of 1.00, and trl2pl l-13s (Table 1). All had MTXPG levels of <500 pmoL/l09 cells, that is, in the low range. The median value was 167 pmol/109 cells (range, 41-468 pmol/l09 cells; Table 1 and Fig. 1). del(12p)s. There were three patients with del(l2p). They had <50 chromosomes and a DI of 1.00 (Table 1, patients 15-17). They had lymphoblast MTXPG bevels of 804, 597, and 396 pmolIlO9 cells. In addition to del(l2p), patient 16 had a complex four-way transbocation involving chromosome 12, but this involved 12q rather than l2p. Patient 17 had a minor clone with a t(l2;l5)(pll;q13). Transbocations Not Involving i2pii-i3. Twenty-three (42%) of the 55 patients with <50 chromosomes had one or more translocations, none of which involved l2pl 1-13 (Fig. 1). The median MTXPG level in these patients was 261 pmolll09 cells, with a range of 31-1832 pmol/l09 cells. Seven (30%) of these 23 patients had high lymphoblast MTXPG levels, including two with very high bevels. Comparing patients with translocations, there was a trend toward higher MTXPG levels in those without versus those with trl2pl l-l3s (P = 0.093). Translocations not involving 12pi 1-13 were present in bymResearch. on October 2, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from