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From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1992 80: 1316-1323
Accumulation of high levels of methotrexate polyglutamates in
lymphoblasts from children with hyperdiploid (greater than 50
chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric
Oncology Group study
VM Whitehead, MJ Vuchich, SJ Lauer, D Mahoney, AJ Carroll, JJ Shuster, DW Esseltine, C
Payment, AT Look and J Akabutu
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Accumulation of High Levels of Methotrexate Polyglutamates in Lymphoblasts
From Children With Hyperdiploid ( >50 chromosomes) B-Lineage Acute
Lymphoblastic Leukemia: A Pediatric Oncology Group Study
ByV.M. Whitehead, M.J. Vuchich, S.J. Lauer, D. Mahoney, A.J. Carroll, J.J. Shuster, D.W. Esseltine, C. Payment,
A.T. Look, J. Akabutu, T. Bowen, L.D. Taylor, B. Camitta, and D.J. Pullen
Hyperdiploidy (>50 chromosomes, or a DNA index >1.16)
confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute
lymphoblastic leukemia whose lymphoblasts at diagnosis
accumulate high levels of methotrexate (MTX) and MTX
polyglutamates (MTXPGs) in vitro experience a better eventfree survival than those whose lymphoblasts do not (Blood
76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (>50 chromosomes) accumulated high levels of MTXPGs (1,095 and 571 t o 2,346 pmol/lO9 cells [median and 25%
t o 75% intraquartile range]). These levels were higher than
those in B-lineage lymphoblasts from 19 children with other
aneuploidy (326 and 159 t o 775 pmol/lOg cells) and 15
children with diploidy (393 and 204 t o 571 pmol/109 cells)
(P = .0015). Chromosomal trisomies in hyperdiploid cases
were highly nonrandom. Chromosome 9 was not one of the
chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis.
The correlation between MTXPG level and percentage of
S-phase cells was weak, suggesting that increased levels of
MTXPGs could not be attributed t o elevated proportions of
cells in active DNA synthesis. The ability of hyperdiploid
lymphoblasts t o accumulate high levels of MTXPGs may
increase their sensitivity t o MTX cytotoxicity, accounting in
part for the improved outlook for hyperdiploid patients
treated with regimens that emphasize MTX as a primary
component of continuation therapy.
o 1992b y The American Society of Hematology.
M
most active antileukemic drugs for this disease, accounting
in part for the success that has been a ~ h i e v e d .It~ is
administered intravenously in intermediate- or high-dose
infusions during consolidation therapqP-6;orally or intramuscularly once or twice a week, together with daily oral
6-mercaptopurine, during continuation therapy’; and intrathecally to prevent central nervous system l e ~ k e m i a . ~
MTX is a substrate for the enzyme folate polyglutamate
synthetase (FPGS), which converts folates to folate polyglutamates,8 the natural coenzyme forms of folic acid. Whereas
the major species of natural folate polyglutamates in cells
contain 5 to 7 gamma-linked glutamyl residues, MTX
polyglutamates (MTXPGs) in nonleukemic cells contain
predominantly 3 glutamyl residues (MTXGIU~).~
In contrast, lymphoblasts from children with B-lineage ALL
accumulate predominantly MTXPGs with 5 glutamyl residues (MTXGIu5).’O
MTXPGs, particularly those with more than 3 glutamyl
residues (long-chain MTXPGs), are retained in cells for
long intervals, inhibiting DNA ~ y n t h e s i s . MTXPGs
~ ~ - ~ ~ are
equipotent with MTX as inhibitors of dihydrofolate reductase (DHFR).15J6 In addition, MTXPGs inhibit other
folate-requiring enzymes, including thymidylate synthase17J8
and the transformylases required for purine ~ y n t h e s i s , ~ ~
which are not inhibited by MTX. These features indicate
that MTXPGs, particularly long-chain forms, are more
broadly active as antifolates than is MTX itself. Indeed,
their formation in cells appears critical to MTX cytotoxicity.20-22
A number of host and disease features of childhood
B-lineage ALL have important prognostic implications.
Among these are age and sex, the initial white blood cell
count (WBC), and the blast cell immunophenotype. Cytogenetic abnormalities, both numeric and structural, have been
recognized as additional prognostic feature^.^^-^^ Of the
major ploidy subgroups studied to date, hyperdiploidy,
defined as > 50 chromosome^^^-^^ or by a DNA index (DI)
>1.16 measured by flow c y t ~ m e t r y , ~has
~ ” ~the most
ORE THAN HALF of all children with B-lineage
acute lymphoblastic leukemia (ALL) can be cured
with combined systemic and intrathecal chemotherapy.’V2
The folate antagonist methotrexate (MTX) is one of the
From the Penny Cole Hematology Research Laboratory and the
Department of Pediatrics, McGiN University-Montreal Children’s
Hospital Research Institute, Montreal, Quebec, Canada; the Department of Pediatrics, Midwest Children’s Cancer Center and Children’s
Hospital of Wisconsin, Medical College of Wnconsin, Milwaukee, Wl;
the Departments of Pediatrics and Pathology, Baylor College of
Medicine, Houston, T X the Laboratory of Medical Genetics, University of Alabama at Birmingham, A L ; the Department of Statistics and
the Pediam‘c Oncology Group Statistical m c e , University of Florida,
Gainesville, FL; the Department of Hematology-Oncology, St Jude
Children’sResearch Hospital and Department of Pediatrics, University
of Tennessee College of Medicine, Memphis, TN; the Department of
Pediatrics, Cross Cancer Center, University of Alberta, Edmonton,
Alberta, Canada; the Department of Pediatrics, Alberta Children’s
Hospital, University of Alberta, Calgary, Alberta, Canada; and the
Department of Pediatrics, University of Mississippi Medical Center,
Jackson, MS.
Submitted October 30, 1991; accepted May I I , 1992.
Supported by grants from the National Cancer Institute of Canada,
the National Cancer Institute, and the National Institutes of Health
(CA-33587, CA-32053, CA-03161, CA-25408, CA-29139, CA-31566,
CA-21765, CA-30969), and by the McGill University-MontrealChildren’s Hospital Research Institute, 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 fiends of Ande,
the Midwest Athletes against Childhood Cancer (MACC Fund), and
the American Lebanese Syrian Associated Charities (ALSAC).
Address reprint requests to V.M. Whitehead, MD, Pediatric Oncology Group, 4949 WPine Blvd, St Louis, MO 63108.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
O 1992 by The American Society of Hematology.
0006-4971f92/8005-OO11$3.00/0
1316
Blood, Vol80, No 5 (September 1). 1992: pp 1316-1323
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
METHOTREXATE POLYGLUTAMATESAND PLOIDY IN ALL
1317
favorable prognosis. The hyperdiploid > 50 group accounts
for a quarter of all patients with B-lineage ALL (see
S e ~ k e r - W a l k eand
r ~ ~Pui et a13*for recent reviews).
Recently, we found that children with B-lineage ALL
whose lymphoblasts accumulated high levels of both MTX
and MTXPGs in vitro experienced better 5-year event-free
survival (EFS) than did those with lower MTX and/or
MTXPG levels.39We now report that lymphoblasts from
children with hyperdiploid ( > 50 chromosomes) B-lineage
ALL accumulate high to very high levels of MTXPGs.
PATIENTS AND METHODS
Patient characteristics. Between April 1989 and January 1991, 1
to 2 mL of bone marrow was obtained with informed consent and
ethical review committee approval from 80 children with B-lineage
ALL at the time of diagnostic bone marrow aspiration. Patients
were entered onto POG 8901, a limited-institution Pediatric
Oncology Group (POG) study of in vitro MTX metabolism.
B-lineage (early pre-B and pre-B cell) ALL was determined by
standard POG criteria.40 Forty-eight of the 80 patients had
successful evaluation of both MTXPG accumulation by lymphoblasts in vitro and karyotype analysis.
Karyotype analysis was performed in the POG cytogenetics
reference laboratory at the University of Alabama at Birmingham
and in the Baylor Cancer Cytogenetics Laboratory. Patients with 51
to 65 chromosomes with and without structural abnormalities were
classified as hyperdiploid. Those with 45 to 47 chromosomes with
structural abnormalities were classified as other aneuploid. Those
with a normal karyotype were designated diploid. One patient had
near-haploidy and was excluded from analysis. The clinical features of the 47 patients are presented in Tables 1through 3.
Forty patients had flow cytometric analysis of cellular DNA
content and determination of percentage of S-phase ~ e l l s . 3A~DI,
defined as the DNA content of leukemic compared with normal
Go/G1 cells, was calculated as an approximation of chromosome
ploidy. A DI of > 1.16 usually corresponds to 53 or more chromosomes.34
Sample acquisition and handling. Bone marrow was collected in
heparin, mixed with an equal volume of Hanks’ Balanced Salt
Solution (HBSS) lacking phenol red and NaHC03, pH 7.4 (Flow
Laboratory, Toronto, Ontario, Canada), and 4.0 mL layered onto
3.0 mL of Ficoll-Paque (Pharmacia Fine Chemicals, Montreal,
Quebec, Canada). After centrifugation at 1,500g for 30 minutes,
the mononuclear cells were separated, washed with HBSS, and
counted. Five million lymphoblasts were incubated in 2.0 mL
modified Eagle’s minimal essential medium (MEM; Flow Laboratory), to which was added 0.67 mmol/L glycine, 0.037 mmol/L
adenosine, 0.04 mmol/L thymidine, 26 mmol/L NaHC03, 1.25
mmol/L pyruvate, 8.3 mmol/L dextrose, and 0.025 mmol/L ferric
nitrate, and containing 10% newborn calf serum (GIBCO Co,
Burlington, Ontario, Canada) and 1.0 p,mol/L 3‘,5’-7- 3H-MTX
(Moravek Co, Brea, CA) for 24 hours in a P-35 culture dish (Falcon
Co,Pointe Claire, Quebec, Canada) at 37°C in 5% C02-95% 02.39
After incubation, cells were washed three times in 5.0 mLHBSS to
remove extracellular MTX and then suspended in 2.0 mL HBSS.
Trichloracetic acid was added to cells to a final concentration of
10% and the mixture was frozen at -20°C.
Extracts were shipped to the reference laboratory at McGill
University at room temperature and were stable. Samples were
centrifuged and MTX and MTXPGs in the supernatant were
concentrated on a SepPak cartridge (Waters Associates, Milford,
MA), eluted from it in 2.0 mL methanol, dried under N2,
resuspended in 200 IJ.L5.0 mmol/L tetrabutylammonium phosphate (TBAP) and 10.0 mmol/L potassium phosphate buffer, pH
5.5, and separated and quantitated using high performance liquid
Table 1. Cytogenetic Findings, DI. Percentage of S-Phase (% SI Cells, and MTXPG Levels in Lymphoblasts From Children With Hyperdiploid
(>50 chromosomes) B-Lineage ALL
Patient
No.
Age
(yr)
WBC
Sex
(xlOg/L)
DI
1
Pre-B
4.3
M
4.4
-
2
Pre-B
5.1
M
5.0
1.17
3
Earlypre-B
8.9
F
7.0
1.20
4
5
6
Earlypre-B
Earlypre-B
Early pre-B
2.2
2.0
3.0
M
M
17.0
9.9
4.3
1.16
1.13
1.20
7
8
9
10
11
12
13
Earlypre-B
Earlypre-B
Pre-B
Earlypre-B
Earlypre-B
Earlypre-B
Earlypre-B
2.7
3.5
5.5
8.2
3.8
1.3
6.1
4.6
79.0
2.8
18.6
1.5
73.2
3.0
1.22
1.21
1.19
1.10
1.19
1.19
ALL
Cells
Total
MTXPGs
(pmol/lOgcells)
-
2,167
%S
Karyotype
(no. of metaphases)
56,X,-Y,+X,+4,+6,+7,+10,+14,+17,+18,+21,+21,+21,d~~(1)
(q21q44)(3)/46,XY(11)
53,XY,+X,+8,+10,+18,+21,+21,de1(5)(q33),del(l3)(q13q14),
+der(5)t(1;5)(q21q35)(8)/54,XY,+X,+8,+10,+11,+18,+21,+21,
10.2
320
del(5)(q33),del(l3)(ql3q14),+der(5)t(l;5)(q21;q35)(3)/46,XY(9)
F
M
F
F
F
M
M
F
-
56,XX,+X,-3,+5,+7,+8,+10,+
14,+ 18,+21,+21,+22,t(8;14)
(411;q32),+der(3)t(3;8)(~27.q13)(5)/46,XX(11)
54,XY,+X,+4,+5,+ lo,+ 14,+ 17,+ 18,+21(4)/46,XY( 16)
51,XY,+X,+6,+ 14,+21,+21(18)/46,XY(2)
56,XX,+X,+4,+6,+ lo,+ 14,+ 17,+ 18,+ 18, +21,+21(8)/57,XX,
+X,+4,+6,+ lo,+ lo,+ 17,+ 18,+ 18,+21,+21 ,+der( 14)t(1;14)
(q23;q32)(5)/46,XX(5)
56,XY,+X,+4,+6,+8,+
lo,+ 14,+ 17,+ 18,+21.+21(15)
55,XX,+X,+4.+6,+8,+10,+14,+17,+21,+21(15)
56,XX,+X,+4,+6,+8,
lo,+ 14,+ 14,+ 18,+ 18,+21(14)/46,XX(6)
51,XX,+X,+14,+21,+21,de1(6)(ql3q21),del(9)(pl3),del(l l)(q14)(17)
56,XY. X, +4, +6, 10, 14, + 14, 17,+ 21, + 21, 21(9)/46,XY(10)
55,XY, X, +4, +6, + lo,+ 14, + 17, 18,+ 21, +2 1(19)/46,XY (11)
+
+
+
+ +
+
+
+
63,XX,+2,+5,+6,+7,+8,+10,+11,+12,+14,+17,+18,+21,+21,+22,
+der(4)t(l;4)(4pter+4q35: :1ql2+lqter),+2mar( 10)/63,XX,- 1,
+2,+5,+6,+7,+8,+10,+11,+12,+14,+17,+18,+21,+21,+22,
+dup(l )(q31+q41 ),+der(4)t( 1;4)(4pter+4q35:: 1q 12-1 qter),
+2mar(3)/46,XX(7)
Age and WBC values are at diagnosis.
12.6
3,287
7.9
18.3
16.1
626
798
2,526
22.9
2,047
1,439
14.0
22.7
8.0
3.9
-
1,095
516
4,343
422
851
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1318
WHITEHEAD ET AL
Table 2. Cytogenetic Findings, DI, Percentage of S-Phase Cells (% S), and MTXPG Levels in Lymphoblasts From Children With Other Aneuploid
B-Lineage ALL
Patient
?'OS
Sex
WBC
(xlOg/L)
14
15
16
Pre-B
Early pre-B
Earlypre-B
5.1
3.1
5.0
M
M
M
9.4
16.0
116.0
1.oo
1.oo
1.oo
17
18
19
20
Pre-B
Earlypre-B
Pre-B
Earlypre-B
8.6
4.1
5.2
5.2
F
F
M
1.oo
1.00
M
216.0
3.7
9.3
45.0
21
22
Pre-B
Non-T,non-B
2.4
3.5
M
F
144.0
5.9
1.00
23
Earlypre-B
10.0
F
172.0
1.oo
47,XX,del(l3)(ql4),t(8;14)(ql
l;q32).+der(l4)t(8;14)(qll;q32)(14)/
12.1
46,XX(6)
24
25
26
Earlypre-B
Pre-B
Earlypre-B
6.0
5.9
3.9
F
M
F
12.9
10.3
3.8
1.oo
1.oo
1.oo
27
28
29
30
31
32
Early pre-B
Earlypre-B
Earlypre-B
Pre-B
Earlypre-B
Pre-B
6.4
0.1
9.2
1.3
6.8
2.1
F
F
F
M
F
F
36.0
122.8
21.9
34.6
10.4
63.9
1.oo
1.oo
1.oo
47,XX,+ 1O,t(15;20)(ql5;p12)(8)/46,XX(12)
47,XY,- 13,+2l,+der(l3)t(l;l3)(q21;q14)(5)/46,XY(10)
46,XX.- 11,- 12,dup(l)(q21q32),del(5)(q22q33),de1(6)(ql5),
t(5;12)(q15;p13),+der(12)t(l2;?)(p13).+m,XX(16)
45,X,-X,?de1(6)(ql3q21)(4)/46,XX(5)
46,XX,t(4; 1l)(q21;q23)(16)/46,XX(4)
47,XX,- 12,-21 ,+der(l2)t(l2;?)(pl2;?),+mar,+ring(?)(20)
46,XY,t(l; 19)(q23;~13)(2)/46,XY(18)
46,XX,t(5; 14)(q31;q32)(10)/46,XX(12)
46,XX. - 12,+der( 12)t(l2;?)(pl1;?)(3)/46,XX(21)
No.
ALL
Age
(yr)
DI
Kawotype
(no.of metaphases)
1.oo
-
1.oo
1.oo
Cells
46,XY,dup(l)(q21q44)(7)/46,XY(13)
46,XY.- 12,t(6; 12)(p21;p12),+der(l2)t(6; 12)(p21;pl2)(17)/46,XY(3)
46,XY,del(3)(pl4),inv(14)(ql lq32)(11)/46,XY,inv(14)(ql1q32)(6)/
46#XY(5)
46,XX,t(4; 11)(q21;q23)(4)/46,XX(6)
47,XX,+21,t(l1;15)(ql l;q15),?t(13;22)(q13;ql3)(6)/46,XX(17)
46,XY,de1(6)(ql3q23)(2)/46,XY(6)
46,XY,-8,de1(6)(ql5),del(l l)(q2lq23),?t(lO;l2)(q24;p13),+der(9)
t(9;?)(q34;?)(5)/46,XY,-9,del( 1l)(q2lq23),+der(9)t(9;?)(q34;?)
(3)/46,XY(13)
46,XY,t(7;9)(ql l;q21)(13)/46,XY(7)
47,XX.- 1,-8,+21 ,+der(l )t(l ;?)(p36;?),+der(8)t(8;?)(pll;?)(2)/46,
XX(28)
Total
MTXPGs
(pmolllO~cells)
8.5
19.4
1.4
1,118
279
775
3.1
5.5
122
190
245
162
3.5
11.7
-
1,698
159
1,220
10.7
24.6
4.9
536
658
63
6.3
6.6
2.3
773
1,426
154
326
39 1
49
4.2
3.6
Age and WBC values are at diagnosis.
chromatography (HPLC, Waters Associates) as previously de~cribed.'~
Levels of MTXPGs > 500 pmol/109 cells were considered to be
while levels >2,000 pm01/109 cells were
considered to be very high.
Because the ability of lymphoblasts to accumulate MTX and
metabolize it to MTXPGs in vitro decreases if incubation is
delayed more than 24 hours, incubation of fresh bone marrow
samples with MTX was performed in the reference laboratory
(McGill University) and in four new laboratories: the Clinical
Immunology Laboratory, Montreal Children's Hospital, McGill
University; Midwest Children's Cancer Center, Milwaukee, WI;
Baylor College of Medicine, Houston, T X and the University of
Alberta, Edmonton, Alberta, Canada.
Establishment of this methodology in these new laboratories
involved a written protocol, frequent telephone calls, and exchange
and testing of culture media and buffers with the reference
Table 3. Cytogenetic Findings, DI, Percentage of S-Phase Cells (YOS), and MTXPG Levels in Lymphoblasts From Children With Diploid
B-LineageALL
Patient
No.
ALL
Age
(vr)
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Early pre-B
Early pre-B
Early pre-B
Early pre-B
Early pre-B
Pre-B
Early pre-B
Pre-B
Early pre-B
Pre-B
Early pre-B
Early pre-B
Pre-B
Early pre-B
Pre-B
9.7
10.5
7.1
3.5
5.8
6.6
2.8
4.2
10.7
3.5
5.7
3.0
3.5
4.9
6.2
Age and WBC values are at diagnosis.
Sex
M
M
F
F
M
F
M
F
M
M
M
M
F
F
F
WBC
( X 1091~)
1.6
3.2
16.0
9.7
318.0
17.0
49.0
3.6
5.2
17.0
2.3
112.8
4.9
3.8
5.2
DI
1.oo
1.00
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
1.oo
Karyotype
(no. of
metaphases)
46,XY
46,XY
46,XX
46,XX
46,XY
(20)
(20)
(21)
(20)
46,xx
(22)
(30)
(20)
(4)
(33)
(20)
(20)
(20)
(7)
(20)
46,XY
46,xx
46,xy
46,XY
46,XY
46,XY
46,XX
46,XX
46,XX
(20)
%s
Cells
13.0
1.1
6.5
7.9
5.1
5.1
10.1
19.5
6.6
6.7
11.1
4.9
11.6
13.1
Total
MTXPGs
(pmol/l09 cells)
87
223
793
571
284
237
96
726
441
197
204
819
481
393
542
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1319
METHOTREXATE POLYGLUTAMATES AND PLOIDY IN ALL
laboratory. It was validated by new laboratories demonstrating the
ability to incubate L5178Y mouse leukemia cells successfully with
3H-MTX. Successful incubation resulted in uptake of 2,000 to
4,000 pmol MTX and MTXPGs/109 cells and formation of MTXPGs containing predominantly 4 glutamyl residues. For this study,
patient results were judged valid if the lymphoblast MTXPG level
was > 100 pmol/109 cells and/or the predominant MTXPG had 5
glutamyl residues.
Quality control and validation studies by the reference laboratory demonstrated that culture medium containing 3H-MTX was
stable for 2 years when stored at -20°C. Exposure of lymphoblasts
to HBSS buffer at alkaline pH during washing before cell incubation, an interval of 30 minutes, markedly decreased MTX uptake
and MTXPG formation during the subsequent 24 hours of incubation. High levels of MTX, 5 to 20 times those encountered in the
previous
were present in some samples from new laboratories. This was due to suboptimal removal of HBSS from the cell
pellet during washing. With removal of all but 50 to 100 pL of
buffer, elevated MTX levels were no longer noted. Nevertheless, a
fourth wash was added to ensure effectiveness of washing of later
samples. No difference in levels or chain-length distribution of
MTXPGs was seen when samples were split and washed optimally
and suboptimally, demonstrating that measurement of MTXPGs
was not affected by high levels of MTX in the sample.
Five samples were shipped to the reference laboratory in less
than 24 hours and were incubated successfully with 3H-MTX
(patients no. 5, 7, 19, 25, and 39; Tables 1 through 3). Despite
apparent technical success, the use of several laboratories to
measure lymphoblast MTX and MTXPG levels in the present
study is likely to have introduced variables not present in the
earlier single institution
Statistical methods. Associations between MTXPG levels and
quantitative covariates were conducted via the Spearman correlation analysis.4l Associations between MTXPG levels and qualitative factors (sex and diagnosis) were conducted by the Wilcoxon
rank sum test."2 Regression analysis comparing percentage of cells
in S-phase and MTXPG levels was conducted by standard methods." Unless otherwise stated, P values are based on two-tailed
tests. Because of the skewed nature of the data regarding MTXPGs
(Fig 1) and other parameters, medians and intraquartile (25% to
75%) ranges are reported, rather than means and standard
deviations.
RESULTS
Karyotypic findings and results of incubation of lymphoblasts with 1.0 kmol/L 3H-MTX were available in 48 of the
80 patients with B-lineage ALL from whom bone marrow
samples were obtained at the time of diagnosis. High MTX
levels were present in 19 of these, due to incomplete
removal of extracellular MTX from washed cells (see
Patients and Methods). Therefore, lymphoblast MTX levels could not be analyzed in these patients. However,
MTXPG levels were available in these 48 patients and were
the subject of this analysis. Patients were divided on the
basis of ploidy into 13 with hyperdiploidy, 19 with other
aneuploidy, and 15 with diploidy. The 48th patient was a
black male, aged 3.1 years, with early p r e - h e l l ALL who
had a near-haploid karyotype, comprised of two clones (27,
X, +Y, +14, +18, +21 and 54, XY, +X, +Y, +14, +14,
+18, +18, +21, +21 present in 5 and 8 of 13 cells analyzed,
respectively), and a DI of 0.57. His initial WBC was 35.3 x
109/L and his lymphoblast levels of MTX and MTXPG
were 189 and 2,645 pmol/109 cells, respectively, with the
predominant MTXPG having 5 glutamyl residues.
Patients with hyperdiploid ALL. There were 13 patients
with a hyperdiploid clone containing > 50 chromosomes
(Table 1). Four patients had translocations in addition to
hyperdiploidy (patients no. 2, 3, 6, and 13) and four had
structural changes other than translocations (patients no. 1,
2, 10, and 13). Eleven of the 13 patients had high lymphoblast MTXPG levels of > 500 pmol/109 cells, and 5 patients
had very high MTXPG levels of > 2,000 pmol/109 cells (Fig
1). The median MTXPG level was 1,095 pmol/109 cells and
the 25% to 75% intraquartile range was 571 to 2,346
pmol/lO9 cells.
Chromosome trisomies were nonrandom, with preferential involvement of several specific chromosomes (X, 4, 6,
10, 14, 17, 18, and 21; Table 1). Although none of the
patients had Down's syndrome, two or more extra copies of
chromosome 21 were present in the leukemic cells of 11
patients. Trisomies were not observed of chromosome 9, on
which the gene for FPGS is located.44 Eight patients had
trisomies of both chromosomes 4 and 10.
The DI was > 1.16 in 8 of the 11patients in whom it was
measured (Table 1). Of these eight patients, six had
MTXPG levels > 500 and four had levels > 2,000 pmol/109
cells.
Patients with other aneuploid ALL. Nineteen patients
had other aneuploidy by cytogenetic analysis (Table 2).
Seven patients showed multiple abnormalities of chromosomal structure and number. Six patients had 47 chromosomes and one had 45 chromosomes. Fifteen patients had
translocations. Other abnormalities included inversions,
deletions, duplications, derivatives, and markers (Table 2).
The median and intraquartile range of MTXPG levels for
these 19 patients were 326 and 159 to 775 pmol/109 cells.
5'
0
4 '
0
30
0
Hyperdiploid
Other
Diploid
Aneuploid
Fig 1. Levels of MTXPGs in lymphoblasts in B-lineage childhood
ALL by ploidy. Groups are hyperdiploid (51 to 65 chromosomes with
and without structural changes); other aneuploid (45 to 47 chromosomes with structural changes); and diploid (46 normal chromosomes). Bars are median levels.
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1320
WHITEHEAD ET AL
Eight patients had MTXPG levels >500 pmol/109 cells.
None had levels > 2,000 pmol/109 cells (Fig 1).
Patients with diploidALL. Fifteen patients had a normal
diploid karyotype. Their clinical and diagnostic features are
shown in Table 3. The median and intraquartile range of
MTXPG levels were 393 and 204 to 571 pmol/109 cells. Five
patients had MTXPG levels >500 pmol/109 cells. None
had levels > 2,000 pmol/109 cells (Fig 1).
M?XPG levels and ploidy. Three-way comparison of
MTXPG levels in hyperdiploid (A) versus other aneuploid
(B) versus diploid (C) ALL showed a significant relationship (P = .0015). This was true as well for two-way comparisons: (A) versus (B), P = .002; (A) versus (C), P < .001;
and (A) versus (BC), P < .001. A similar relationship was
found between high MTXPG levels and DI > 1.16 (P = .009,
three-way comparison). Despite the strong correlation
between hyperdiploidy and increased accumulation of MTXPGs in lymphoblasts in vitro, there were no significant
correlations between MTXPG levels and age (P = .18), sex
(P = .85), WBC (P = 30) and diagnosis (early pre-B v
pre-B ALL) ( P = .62), or with hemoglobin level ( P = .35)
and platelet count (P = .97) (not shown) at diagnosis.
Relation of MTXPG chain-length to ploidy. The distribution of MTXPGs by chain-length in lymphoblasts from
patients with hyperdiploid, other aneuploid, and diploid
ALL is shown in Table 4. The predominant form was
MTXGlus in all ploidy groups. Long-chain MTXPGs accounted for more than 60% of total MTXPGs. There was a
trend to shorter chain-length MTXPGs in hyperdiploidy,
based on percent MTXGlu5 (P = .025, three-way comparison; P = .063, hyperdiploid v not). No significant difference
was found comparing percent MTXGh4+5+6by ploidy.
MTXPG level and percentage of S-phase cells. The accumulation of high levels of MTXPGs in hyperdiploid lymphoblasts might be explained by their increased proliferative
activity. The relationship of proliferative activity of the
leukemic cells to the extent of accumulation of MTXPGs in
lymphoblasts was assessed by comparing these measurements in the 40 patients in whom both determinations were
available. There was a positive ordinal relationship between
these two measures (P = .0012 by Spearman association).
However, there was far too much scatter to consider either
variable as a useful predictor of the other. For example, on
the basis of ordinary linear regression, only 7% of the
variation of one measure could be explained by the other
(R2 = 7%). Of several transformations of the data attempted, the highest R2 value was 16%, which occurred
when the log of MTXPG levels were plotted against percentage of cells in S-phase (Fig 2). These findings indicated
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Total MTXPGs (pmol/109 cells X 10 -3)
Fig 2. Relationship of the percent S-phase cells to the log of the
MTXPG level in lymphoblasts in childhood 6-lineage ALL. Ploidy
groups are hyperdiploid (e),other aneuploid (0).
and diploid (A).
that the elevated MTXPG levels could not be explained by
the proliferative function of the leukemic cell population.
DISCUSSION
Recent studies have highlighted the prognostic significance of cytogenetic abnormalities in childhood ALL.23-32
A
striking finding has been the good prognosis associated with
h y p e r d i p l ~ i d y ? ~ -Pui
~ ~ ,et~ ~ - ~ reported
~
on 138 children
with hyperdiploidy with 4 years of follow-up. They suggested an 80% EFS for those with numeric changes only
and 60% EFS for those with both numeric and structural
changes. Rivera et a145 reported 86% 4-year EFS in 74
children with hyperdiploid ALL. MTX was a major component of continuation and central nervous system therapy in
many of these s t ~ d i e s . ~ 3 , ~ , ~ ~ , ~ ~ , ~ ~ , ~ ~
Interestingly, Fletcher et a146found no favorable prognostic role for hyperdiploidy. They reported an overall 5-year
EFS of 78% for 165 patients. For hyperdiploid, pseudodiploid, and diploid subgroups, 5-year EFS were SO%, 73%,
and 81%, respectively. They attributed their excellent
results in these and other cytogenetic groups to intensive
chemotherapy, which included MTX during induction,
intensification, and sanctuary therapy.47
Hyperdiploidy, measured by flow cytometry and expressed as a DI > 1.16, also carries an excellent prognosis
relative to other ploidy g r o ~ p s . Look
~ ~ - et
~ ~a148compared
results in children who received intrathecal MTX plus
high-dose MTX infusions with those who received cranial
radiation therapy and sequentially administered pairs of
drugs as intensification therapy. The beneficial effect of
hyperdiploidy was much greater in those who received
Table 4. Distributionof MTXPGs by Chain-Length and Ploidy
KaryoWpe
Hyperdiploid
Other aneuploid
DipIoid
No. of
Samples
13
19
15
MTXGIu. (%)*
Glu2
12 (7-14)
9 (4-13)
12 (10-19)
Glul
27 (18-31)
19 (14-24)
22 (14-25)
Glu.
Glus
Glus
27 (24-30)
25 (21-27)
25 (23-30)
34 (26-40)
41 (35-48)
31 (26-45)
3 (1-5)
4 (2-7)
2 (0-4)
Values are median and 25% to 75% intraquartile ranges. Sums of median percents do not equal 100.
*MTXGIU~.~
are MTXPGs with 2 to 6 glutamyl residues.
GlUa+~+s
61 (57-73)
74 (61-83)
63 (58-72)
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
METHOTREXATE POLYGLUTAMATES AND PLOIDY IN ALL
parenteral intensification therapy with MTX, with a 5-year
EFS of 82% (95% confidence limits, 65% to 98%). This
analysis suggested that MTX may be important to the cure
of hyperdiploid ALL. Recently, Trueworthy et a1 showed
that a DI > 1.16 was the most important single prognostic
feature defining excellent outcome in children with ALL
treated with a regimen containing intensive MTX treatment during continuation
Further, Pui et a1
showed that a DI > 1.16 was the single persisting prognostic
factor that defined success of treatment after discontinuation of ~ h e m o t h e r a p y .Again,
~~
MTX was an important
component of therapy of these patients.34-36*4n,49
To confirm and extend our previous findings linking
combined lymphoblast MTX and MTXPG levels with
EFS,39 we compared MTXPG levels in hyperdiploid with
those in other aneuploid and diploid lymphoblasts. Unfortunately, MTX levels could not be analyzed (see Patients and
Methods), resulting in an incomplete picture of MTX
metabolism. However, hyperdiploid patients had significantly higher MTXPG levels (P= .0015) than patients with
other aneuploid and with diploid lymphoblasts (Fig 1).
Furthermore, nearly half of the patients with hyperdiploidy
had very high MTXPG levels, ie, > 2,000 pmol/109 cells, a
feature unique to these patients (Fig 1). These findings
provide additional evidence linking MTX metabolism in
lymphoblasts at diagnosis with response to multidrug antileukemic therapy in which MTX is a prominent component.
Patient follow-up is too short in the present study to permit
comparison of findings with therapeutic outcome.
There was a trend to shorter MTXPG chain-lengths in
hyperdiploidy (Table 4). If confirmed in a larger series of
patients, this finding might reflect altered substrate concentrations or FPGS activity.n
The mechanism for these high and very high MTXPG
levels is unknown. Trisomies of chromosomes in our patients with hyperdiploidy were nonrandom. Frequent trisomies were similar to those reported by others.37MTXPGs
are synthesized by FPGS, which is encoded by a gene
located on chromosome 9." Whereas trisomy of chromosome 9 was present in other patients with hyperdipl~idy?~
it
was not present in any of our 13 patients (Table 1).
Therefore, there is no evidence that an increased FPGS
gene dosage explains the increased accumulation of MTXPGs in hyperdiploid ALL. Harris et a1 have shown that
children with B-progenitor cell ALL with trisomies of both
1321
chromosomes 4 and 10 have a very low risk of treatment
failure.50 Eight of our patients had these two trisomies
(Table 1).Of these, seven had high MTXPG levels and four
had very high MTXPG levels. It is tempting to speculate
that genes whose products promote FPGS gene expression
may be located on one or both of these chromosomes.
Proliferating cells accumulate higher levels of MTXPGs
than do resting
As previously
we
found an increase in percent S-phase cells in hyperdiploid
compared with other aneuploid and diploid blasts (Fig 2). If
this increase in percent S-phase cells reflects increased
proliferative activity, rather than slower passage of blasts
through S - p h a ~ e ?then
~ increased proliferative rate may
contribute to increased accumulation of MTXPGs. However, the association was weak, suggesting that other factors
exist and play a more prominent role. Some of these factors
may be identified by measuring the rate of blast cell uptake
of MTX, the intracellular level of MTX, and the activities
of FPGS and gammaglutamyl hydrolases in lymphoblasts.
Gammaglutamyl hydrolases are enzymes that hydrolyze
folate and MTX polyglutamates to mono glut am ate^.^^^^^
Several mechanisms have been advanced to explain why
hyperdiploidy confers a better prognosis than pseudodiploidy or diploidy. An increase in the number of cells in
S-phase due to either increased proliferative rate or increased residence of cells in S-phase may make these blast
cells more sensitive to cell-phase-specific drugs, such as
vincristine, 6-mercaptopurine, and MTX. Hyperdiploid
cells have a tendency toward terminal differentiation and
therefore are more sensitive to corticosteroid treatment.35
Tsuchiya et aIs6 have suggested that extra chromosomes
may moderate the tumorigenicity of malignant cells, either
through the action of putative antioncogenes or by altering
the balance of the number of chromosomes with and
without oncogenes.
The present findings, together with the clinical findings
of Look et ala and the observation by Whitehead et a139
linking high lymphoblast MTX and MTXPG levels and
improved EFS, suggest an additional possibility. It is likely
that the ability of hyperdiploid lymphoblasts to accumulate
high and very high levels of MTXPGs makes them particularly sensitive to MTX cytotoxicity. This may explain in part
the apparent improved outlook for hyperdiploid patients
treated with regimens emphasizing MTX as a primary
component of continuation therapy.
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