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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1643-1650
Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A
Mechanism for Ploidy but not Lineage Differences in Methotrexate
Accumulation
By
Vladimir M. Belkov,
Eugene Y. Krynetski,
John D. Schuetz,
Yuri Yanishevski,
Eric Masson,
Susan Mathew,
Susana Raimondi,
Ching-Hon Pui,
Mary V. Relling, and
William E. Evans
From the St Jude Children's Research Hospital, Memphis, TN; and the
University of Tennessee, Memphis, TN.
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ABSTRACT |
Methotrexate (MTX) is one of the most active and widely used agents
for the treatment of acute lymphoblastic leukemia (ALL). To elucidate
the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in
hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of
the reduced folate carrier (RFC) was assessed by reverse
transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a
60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with
nonhyperdiploid ALL (median, 2.1; P < .0006), but no
significant difference between nonhyperdiploid B-lineage and T-lineage
ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21)
were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene
copy number, MTX-PG accumulation was compared in ALL blasts isolated
from 121 patients treated with either low-dose MTX (LDMTX; n = 60) or
high-dose MTX (HDMTX; n = 61). After LDMTX, MTX-PG accumulation was
highest in hyperdiploid B-lineage ALL with 4 copies of chromosome 21 (P = .011), but MTX-PG accumulation was not significantly
related to chromosome 21 copy number after HDMTX (P = .24).
These data show higher RFC expression as a mechanism for greater MTX
accumulation in hyperdiploid B-lineage ALL and indicate that lineage
differences in MTX-PG accumulation are not due to lower RFC expression
in T-lineage ALL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
METHOTREXATE (MTX) is one of the most
active and widely used medications for the treatment of childhood acute
lymphoblastic leukemia (ALL).1,2 Recent studies have shown
significant lineage and ploidy differences in the intracellular
accumulation and metabolism of MTX in ALL blasts,3
providing insights into mechanisms underlying prognostic differences in
these ALL subtypes. Intracellular accumulation of MTX and MTX
polyglutamate can be an important determinant of event-free survival in
ALL patients.4 After entering cells via the reduced folate
carrier (RFC) and by passive diffusion at higher extracellular
concentrations, MTX is metabolized to polyglutamylated metabolites.
These active MTX polyglutamates (MTX-PG) inhibit dihydrofolate
reductase, thymidylate synthase, and enzymes involved in de novo purine
synthesis and are retained in cells longer than the parent drug
(MTX).1,2,5 It has been shown that B-lineage lymphoblasts
accumulate higher intracellular concentrations of active MTX-PG
compared with T-lineage ALL3 and that this is related in
part to higher activity of folylpolyglutamate synthetase (FPGS) in
B-lineage lymphoblasts.6 Among patients with B-lineage ALL,
those with hyperdiploid (>50 chromosomes) ALL accumulate higher
MTX-PG in their leukemia cells compared with nonhyperdiploid
ALL.3 However, the mechanism of higher MTX-PG accumulation
in hyperdiploid ALL has not been elucidated, although it has been shown
that FPGS activity does not differ between hyperdiploid and
nonhyperdiploid B-lineage ALL.6 Because the great majority
(~97%) of hyperdiploid ALL blasts have 3 or 4 copies of chromosome
21,7 we hypothesized that hyperdiploid blasts have
increased expression of the RFC, which is located on human chromosome
21 (21q22.2-q22.3),8 and that chromosome 21 copy number is
associated with RFC mRNA expression and MTX accumulation in
hyperdiploid ALL. The present study was therefore undertaken to assess
the relation between chromosome 21 copy number and (1) the level of RFC
mRNA expression and (2) in vivo MTX-PG accumulation in leukemia cells
of children with newly diagnosed ALL.
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MATERIALS AND METHODS |
Human subjects.
The diagnosis of B-lineage and T-lineage ALL was made using
immunological criteria, as previously described.9 To
determine MTX-PG accumulation, ALL blasts were isolated from bone
marrow aspirates obtained from consecutively treated children with
newly diagnosed ALL, 44 hours after MTX treatment. After providing
informed consent, patients enrolled on St Jude Total-XIIIA protocol
were randomized to initial single agent therapy with either low-dose (180 mg/m2 administered orally as 30 mg/m2
every 6 hours 6 times) or high-dose (1,000 mg/m2 administered intravenously as a 24-hour
infusion) MTX, plus leucovorin rescue, as previously
described.3 ALL blasts were isolated by Ficoll gradient
separation, and MTX-PG concentrations were measured in 5 × 106 blasts by a high-performance liquid chromatography
(HPLC) radioenzymatic assay as previously
described.3 ALL ploidy and chromosome 21 copy number were
determined by cytogenetic analysis, as previously described.7
To determine RFC mRNA expression, ALL blasts were isolated from either
bone marrow aspirates or peripheral blood obtained from patients
enrolled on the TOTAL-XIIIB protocol, either before treatment (n = 26)
or within 48 hours of starting chemotherapy (n = 3). RNA was isolated
from ALL blasts within 6 hours of being obtained from patients (or cell
culture) and then analyzed for RFC expression by the reverse
transcription-polymerase chain reaction (RT-PCR) method
described below. Informed consent was obtained from the patient's
parents or guardian according to IRB guidelines.
Human leukemia cell lines.
The CEM/MTX and K500E/MTX cell lines with impaired MTX uptake and the
RFC wild-type K562/wt cell lines were generous gifts from Dr L. Matherly (Karmanos Cancer Institute, Detroit,
MI).10,11 CCRF-CEM cells were purchased from ATCC
(Rockville, MD). The CEM/T-cell line with impaired MTX RFC transport
was a generous gift from Dr J. Bertino (Memorial Sloan Kettering, New
York, NY).12 NALM6 cells were purchased from
DSMZ (Braunschweig, Germany).
Total RNA preparation.
Total RNA from cell lines and patient's lymphoblasts were
isolated using TRI REAGENT from Molecular Research Center, Inc
(Cincinnati, OH). Typically, 1 mL of TRI REAGENT solution was
used to isolate total RNA from 5 to 10 × 106 cells.
The yield of total RNA varied from 5 to 10 µg total RNA per 1 × 106 cultured cells and from 1 to 2 µg total RNA per 1 × 106 lymphoblasts from patients.
Preparation of RFC mRNA external standard.
All oligonucleotides used as primers for PCR were synthesized in the
Center for Biotechnology at St Jude Children's Research Hospital
(Memphis, TN). Primers RFC617 (5'-CCAAGCGCAGCCTCTTCTTCAACC) and
RFC949 (5'-CCAGCAGCGTGGAGGCAGCATCTGCC)13 were used to
generate a fragment corresponding to nucleotides 617-949 of the human
RFC cDNA by RT-PCR (numeration based on the complete RFC cDNA
sequence10). The synthesized DNA fragment was directly
cloned into pCR2.1 vector (Invitrogen, San Diego, CA) and sequenced in
both directions. Acc I treatment followed by self-ligation gave
a plasmid with a 58-bp deletion within the 617-949 fragment of cDNA.
The resulting fragment was excised by EcoRI treatment, purified
by electrophoresis in an agarose gel, isolated by QIAquick Gel
extraction kit from QIAgen (Santa Clarita, CA), and cloned into the
plasmid pGEM7 with a previously inserted polyadenylate stretch
(A30) in the multiple cloning site. Orientation of the
inserted fragment was determined by restriction analysis with
Acc I and Xba I. The insert was completely sequenced in
both directions, and an external standard for RT-PCR (stRNA) with 58 ribonucleotides deleted was obtained using T7 RNA polymerase from
RiboMax Large Scale RNA production system from Promega (Madison, WI)
and purified using Oligotex direct mRNA Midi/Maxi kit from QIAgen.
stRNA concentration was measured by UV absorbance (GeneQuant; Pharmacia
Biotech, Cambridge, UK).
RFC mRNA quantitation by competitive RT-PCR.
Serial 10-fold dilutions (1:1 to 1:105) of stRNA were made
to obtain PCR signals comparable to those obtained for RFC using mRNA
from the MTX-sensitive human leukemia cell line, CCRF-CEM/wt. An
aliquot of stRNA was added to 1 µg of total RNA and isolated either
from cell lines or lymphoblasts from patients, and this mixture was
converted to cDNA as follows. The reaction mixture (20 µL) containing
50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2,
10 mmol/L dithiothreitol, 3 µg of random primers from
GIBCO BRL Life Technology (Gaithersburg, MD), and 1 U of RQ1 RNAse-free
DNAse I (Promega) was incubated at 37°C for 30 minutes and at
75°C for 5 minutes to inactivate DNAse I and then chilled for 3 minutes on ice. Two hundred units (1 µL) of Moloney murine leukemia
virus reverse transcriptase (SuperScript II) from GIBCO BRL Life
Technology was added and the mixture was incubated at 42°C for 50 minutes. The final PCR amplification mixture contained buffer J (PCR
Optimizer kit) from Invitrogen [60 mmol/L Tris-HCl (pH 9.5), 15 mmol/L
(NH4)2SO4, 0.4 mmol/L
MgCl2], 0.25 µg of each primer, 1 U Taq DNA
polymerase (Promega), and 2 µL cDNA template. The amplification
program was initiated according to the "Hot start" protocol as
suggested by the manufacturer: 5 µL 10 mmol/L dNTPs and 5 µCi
[ -32P] dCTP (10 µCi/µL) from Amersham (Arlington
Heights, IL) were added at 80°C, followed by the denaturation step
at 94°C for 2 minutes. PCR was performed as follows: 30 cycles at
94°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes followed by a final cycle at 72°C for 7 minutes. The PCR
products were purified by QIAquick PCR purification kit (QIAgen) and
separated by gel electrophoresis at 300 V in 5% nondenaturating PAAG
gel (4°C), the gel was dried under vacuum at 80°C for 1 hour
and visualized using a Molecular Dynamics PhosphorImager (Molecular
Dynamics, Sunnyvale, CA), and the amount of
32P-labeled RFC fragments was quantified by
"ImageQuaNT" software (ver 4.2a; Molecular Dynamics).
To estimate a suitable range of RNA concentrations and number of PCR
cycles with good RFC signal and minimal nonspecific products, we
determined conditions in which the amount of the PCR product increased
linearly with the number of PCR cycles in logarithmic coordinates.14 An excellent correlation
(r2 = .94) was achieved between the amount
of PCR product and the number of cycles performed (data not shown).
Amplification of  -actin as an internal standard.
To amplify -actin mRNA fragment, primers BA-67
(5'-GGGAGAGCGGGAAATCGTGCGTGACATT) and BA-68
(5'-GATGGAGTTGAAGGTAGTGGCGTG) were used as described
previously.15 Because -actin mRNA is much more abundant
in cells than RFC mRNA, the cDNA template was diluted 1,000-fold to
obtain a signal from -actin comparable to that of RFC mRNA. PCR of
-actin, separation, and quantification of PCR products were
performed under the conditions described above.
Northern blotting.
To validate RFC mRNA quantification by RT-PCR, RFC mRNA levels were
determined by Northern analysis of human leukemia cell lines. Samples
of total RNA (40 µg per lane, 8.1 µL) were incubated at 50°C
for 1 hour with 8.1 µL of 6 mol/L glyoxal, 24 µL of dimethyl sulfoxide (DMSO), and 4.5 µL of 0.1 mol/L sodium
phosphate buffer (pH 7.0). After adding loading buffer, samples were
electrophoresed in 1.4% agarose with 10 mmol/L sodium phosphate (pH
7.0) at 60 V with buffer circulation. Separated products were
transferred to the nylon membrane Hybond-N+ (Amersham), and the
membrane was washed in 6× SSC, dried at room temperature for 30 minutes, and baked at 80°C for 30 minutes under vacuum. The RFC
mRNA probe, 32P-labeled by Rediprime from Amersham, was a
fragment of approximately 1,000 bp of the 3' end of the RFC
coding region. The probe was hybridized overnight in Rapid-hyb buffer
from Amersham and then washed at 65°C with 2× SSC for 15 minutes, 2× SSC with 0.1% sodium dodecyl sulfate
(SDS) for 30 minutes, and then 0.1× SSC for 30 minutes. Signals were visualized by PhosphorImager and
quantified by "ImageQuaNT" software, as described above.
Fluorescence in situ hybridization (FISH).
A BAC clone containing the human RFC gene was used to perform FISH
analysis of metaphase chromosomes from lymphoblasts isolated from two
patients with B-lineage ALL, one containing 4 copies of chromosome 21 and the other containing 2 copies of chromosome 21, with 1 involved in
a 12;21 translocation (ie, ETV6-CBFA2 fusion). To isolate the
RFC probe, human genomic DNA from Molt4 cells was cloned into the
PBeloBacII vector (Genome Systems, St Louis, MO). The library was
screened by hybridization with the RFC EST cDNA (Genbank Accession no.
R87517) containing a 2,034-bp cDNA insert. One positive clone was
isolated and further characterized by hybridization with the RFC cDNA
and the RFC promoter. For in situ hybridization, the RFC probe was
labeled with biotin 11-dUTP by nick translation (Life Technologies,
Inc, Gaithersburg, MD). As a control probe for chromosome 21, digoxigenin-labeled 21q22.3-ter DNA was used (Oncor, Gaithersburg, MD).
Slides were denatured in 70% formamide for 2 minutes and dehydrated.
The RFC probe was denatured for 5 minutes at 70°C and preannealed
for 10 minutes at 37°C. The control probe was prewarmed to
37°C, and the probes were mixed and applied to the denatured slide.
Slides were placed in a humidified chamber and hybridized at 37°C
overnight. Posthybridization washes were performed at 45°C in 50%
formamide three times for 5 minutes each, followed by two 2× SSC
washes at room temperature. The probes were detected using fluorescein
isothiocyanate (FITC)-avidin (RFC) and Rodamine-labeled
antidigoxigenin (21q22.3 probe). The slides were stained with 4',
6-diamidino-2-phenylindole (DAPI) and the cells were analyzed using an
Olympus microscope and an image capturing system (Vysis Inc, Downer's
Grove, IL). For the second case, the digoxigenin-labeled Coatasome
12-chromosome probe (Oncor) was denatured for 10 minutes at 70°C
and preannealed for 2 hours at 37°C. The biotin-labeled RFC probe
was denatured separately and hybridized together on the denatured
slide. Probes were detected using the Rhodamine-labeled antidigoxigenin
and FITC-avidin, and the slide was stained with DAPI.
Statistical analysis.
Pearson coefficient was used to assess the correlation between RFC mRNA
measured in the same cells by different methods. The difference in RFC
mRNA between hyperdiploid and nonhyperdiploid B-lineage ALL was
evaluated using the Mann-Whitney U test. The relation between in vivo
MTX-PG1-7 accumulation and chromosome 21 copy number was
evaluated separately in ALL patients treated with low-dose and
high-dose MTX. To adjust for differences in extracellular MTX
concentrations among patients treated with the same dose of MTX, the
ratio of intracellular MTX-PG (picomoles per 109 blasts) to
extracellular steady-state plasma MTX concentration (Cpss)
was also assessed. The shape of data distribution was fitted by normal
and log-normal functions and the quality of the fit was assessed by
 2 test. For log-normally distributed data, a logarithmic
transformation was applied before parametric analyses (ie, all MTX-PG
datasets were log-transformed for analysis). Standard deviations of
log-normal data were calculated from log-transformed values and are
thus symmetrical when depicted on log-scale graphs. The amount of RFC mRNA and MTX-PG accumulation among groups with different chromosome 21 copy number were compared using analysis of variance (ANOVA), followed
by the Tukey multiple comparison test for unequal sample sizes.
Computations were performed with STATISTICA, version 5.1 (StatSoft,
Inc, Tulsa, OK), and P < .05 was considered statistically significant.
| |
RESULTS |
Estimation of RFC mRNA by RT-PCR versus Northern analysis.
As shown in Fig 1, there was good agreement
between the level of RFC mRNA in human leukemia cell lines when
determined by RT-PCR and Northern analysis, using either competitive
RT-PCR with external standard (r2 = .77, P = .0016) or RT-PCR with -actin as the internal
standard (r2 = .69, P = .002). With these methods, the amount of RFC mRNA in the
transport-deficient CEM/T cells was approximately 30% to 60% lower
than CCRF-CEM/wt, and the CEM/MTX line (with a mutant RFC gene) had a
20% to 70% higher RFC mRNA level compared with CCRF-CEM/wt. The RFC
mRNA amounts in K562/wt and MTX-resistant K500E/MTX cells were
comparable to each other.
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| Fig 1.
Concordance among three methods of RFC mRNA
quantification. (A) Comparison of Northern blotting and competitive
RT-PCR using stRNA as an external standard. (B) Comparison of Northern
blotting and RT-PCR using -actin as an internal standard. (C)
Comparison of competitive RT-PCR using stRNA and RT-PCR using
-actin. The amount of RFC mRNA from the CCRF-CEM/wt cell line was
assigned a value of 100%. The RFC mRNA amount from other
cell lines was calculated as a percentage of CCRF-CEM/wt. CCRF-CEM/wt
( ), CEM/T ( ), CEM/MTX ( ), Nalm6 ( ), K562/wt ( ),
K500E/MTX ( ).
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RFC mRNA in lymphoblasts from patients.
RFC expression was determined in lymphoblasts isolated from 29 newly
diagnosed children with ALL, 26 of them before treatment and 3 at 44 hours after treatment with MTX. In 6 patients studied both before and
44 hours after treatment, there was not a significant difference in RFC
mRNA at the two time points (data not shown). The 29 patients were
selected from newly diagnosed patients entered on the Total
XIIIB protocol (between April 1997 and December 1997 and
in June and July 1998), including all available children with T-lineage
ALL (n = 8), all with hyperdiploid (>50 chromosomes) B-lineage ALL (n = 7), and a comparable number of patients with nonhyperdiploid
B-lineage ALL (n = 14). None of these patients had Down's syndrome
(ie, germline trisomy 21). There was a 60-fold range in RFC mRNA
expression in leukemia cells isolated from these patients. Using RFC
mRNA expression in CCRF-CEM/wt cells as a reference value of 1.0, patients with hyperdiploid B-lineage ALL had significantly higher
levels of RFC mRNA expression (median, 11.3) compared with those with
nonhyperdiploid ALL (median, 2.1; P < .0006), with no
significant difference between nonhyperdiploid B-lineage (median, 1.4)
and T-lineage (median, 4.8) ALL (Fig 2A). As depicted in Fig 2B, there was a relation between the level of RFC
mRNA and chromosome 21 copy number in B-lineage ALL lymphoblasts. ANOVA
showed significant differences (P < .002) in the relative amounts of RFC mRNA among the three patient groups: B-lineage with 2 (median, 1.35), 3 (median, 6.7), or 4 (median, 12.8) copies of
chromosome 21 (Fig 2B). Pairwise comparisons using the Tukey test
showed that B-lineage ALL with 4 copies of chromosome 21 had
significantly higher levels than B-lineage with 2 copies (P = .004), whereas the other groups did not differ significantly (P > .3). Seven of 10 lymphoblast samples with greater than 2 copies of
chromosome 21 were hyperdiploid (>50 chromosomes). As depicted in Fig
2B, the three nonhyperdiploid samples had the lowest RFC mRNA levels
among those with greater than 2 copies of chromosome 21.
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| Fig 2.
RFC mRNA in ALL blasts from newly diagnosed patients. (A)
Relative RFC mRNA expression in nonhyperdiploid T-lineage lymphoblasts
(n = 8), nonhyperdiploid B-lineage lymphoblasts (n = 14), and
hyperdiploid B-lineage lymphoblasts (n = 7). Diamonds (, )
depict lymphoblasts isolated from peripheral blood before treatment,
squares (, ) depict lymphoblasts from bone marrow before
treatment, and circles () depict lymphoblasts from bone marrow 44 hours after MTX treatment. Shaded symbols depict nonhyperdiploid blasts
and solid symbols depict hyperdiploid blasts (>50 chromosomes).
Horizontal lines depict the median values in each group. (B) RFC mRNA
expression in B-lineage ALL blasts with either 2 (n = 11), 3 (n = 5), or 4 (n = 5) copies of chromosome 21. Symbols are the same as in
(A). Among lymphoblasts with 3 copies of chromosome 21, the 2 samples
with the lowest RFC expression have less than 50 chromosomes, and the
lowest value had a translocation involving the long arm of 1 copy of
chromosome 21 and the short arm of chromosome 12 (ie, a 12;21
[p13;q22] translocation) resulting in the ETV6-CBFA2 fusion.
Similarly, among those with 4 copies of chromosome 21, the sample with
the lowest RFC expression had less than 50 chromosomes.
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As shown in Fig 3A, FISH of chromosomes
from a hyperdiploid ALL blast with 4 copies of chromosome 21 showed 4 copies of the RFC gene located telomeric to the human
chromosome 21q22.3-ter probe. Figure 3B documents that the RFC
gene is translocated with the ETV6 gene on the long arm of
chromosome 21 in the 12;21 translocation that produces the
ETV6-CBFA2 fusion.
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| Fig 3.
FISH of a human RFC probe with chromosomes from B-lineage
ALL blasts. (A) is from a hyperdiploid ALL blast with 4 copies of
chromosome 21. The green signal is from the RFC gene probe and the red
signal from a chromosome 21q22.3-ter probe. (B) is from a
nonhyperdiploid ALL blast with 2 copies of chromosome 21, one of which
is involved in a 12;21 translocation. The green signal is from the RFC
gene probe and the red signal is from a chromosome 12 probe (Coatasome
12).
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MTX-PG accumulation and chromosome 21 copy number.
In vivo MTX-PG concentrations were measured in ALL blasts isolated from
bone marrow aspirates in a total of 140 patients, 121 with B-lineage
ALL and 19 with T-lineage ALL. As previously reported,3
patients with T-lineage ALL had significantly lower MTX-PG accumulation
when compared with nonhyperdiploid B-lineage ALL after either high-dose
(P < .03) or low-dose MTX (P < .001). Because
essentially all patients with T-lineage ALL have nonhyperdiploid lymphoblasts with only 2 copies of chromosome 21 (18 of 19 T-lineage ALL were nonhyperdiploid), all analyses of the relation between chromosome 21 copy number and MTX-PG accumulation were restricted to
B-lineage ALL. For patients treated with low-dose MTX, there were
statistically significant differences in MTX-PG among patients whose
ALL blasts had 2 (mean, 499 pmol/109 cells; n = 43), 3 (mean, 581 pmol/109 cells; n = 7), or 4 (mean, 1,064 pmol/109 cells; n = 10) copies of chromosome 21 (P < .011 by ANOVA for log-transformed values;
Fig 4). Likewise, differences in
MTX-PG/Cpss were significant (P < .003) among
patients treated with low-dose MTX (Fig 5).
Pairwise comparisons using the Tukey test showed that those with 4 copies of chromosome 21 had significantly higher MTX-PG accumulation
when compared with those with 2 copies (P = .044 for MTX-PG and
P = .024 for MTX-PG/Cpss), whereas other pairwise
comparisons among chromosome copy number groups were not significant.
In contrast, after high-dose MTX treatment (Figs 4 and 5), there was
not a significant difference among ALL blasts with 2 (mean, 1,309 pmol/109 cells; n = 42), 3 (mean, 1,839 pmol/109 cells; n = 10) or 4 (mean, 2,217 pmol/109 cells; n = 9) copies of chromosome 21 (P = .24 for MTX-PG and P = .18 for MTX-PG/Cpss by
ANOVA). There were 7 patients with B-lineage ALL treated with low-dose
MTX in whom in vivo MTX-PG accumulation in ALL blasts and RFC mRNA
levels were measured in the same sample. In these patients, the
Spearman rank order correlation for RFC mRNA and the relative amount of
MTX-PG accumulation at 44 hours was R = .678 (P = .09),
with higher RFC mRNA associated with greater MTX-PG accumulation. This
association was not evident in patients treated with HDMTX
(P = .6; n = 4).
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| Fig 4.
Relation between MTX-PG concentrations in ALL blasts and
chromosome 21 copy number. (A) Data for B-lineage ALL blasts isolated
from bone marrow at 44 hours after low-dose MTX treatment of 60 children (n = 43, 7, and 10 for 2, 3, and 4 copies of chromosome 21, respectively). (B) Data for B-lineage ALL blasts isolated from 61 patients at 44 hours after treatment with high-dose MTX (n = 42, 10, and 9 for 2, 3, and 4 copies of chromosome 21, respectively). ()
Mean values; the boxes depict the standard errors (SE) of the mean; and
the bars depict the range of ±1 standard deviation (SD) in each
group. The SE and SD are symmetrical because they were calculated from
log-transformed values of log-normal data.
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| Fig 5.
Relation between the ratio of intracellular MTX-PG to
extracellular plasma MTX concentration (Cpss) versus
chromosome 21 copy number in ALL blasts. (A) Data for B-lineage ALL
blasts isolated from bone marrow at 44 hours after low-dose MTX
treatment of 60 children (n = 43, 7, and 10 for 2, 3, and 4 copies of
chromosome 21, respectively). (B) Data from B-lineage ALL blasts
isolated from bone marrow at 44 hours after high-dose MTX treatment in
61 children (n = 42, 10, and 9 for 2, 3, and 4 copies of chromosome
21, respectively). () Mean values; the boxes depict the standard
errors of the mean; and bars depict the range of ±1 standard
deviation in each group.
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Among patients treated with low-dose MTX, there were 2 hyperdiploid
B-lineage cases whose ALL blasts had only 2 copies of chromosome 21, yet these 2 patients had significantly higher MTX-PG accumulation (989 and 2,187 pmol/109 cells) when compared with
nonhyperdiploid B-lineage ALL with 2 copies of chromosome 21 (n = 41;
median MTX-PG, 517 pmol/109 cells; P = .028).
| |
DISCUSSION |
Previous studies from our laboratory and others have established that
MTX-PG accumulation is greater in hyperdiploid B-lineage ALL when
compared with nonhyperdiploid B-lineage or T-lineage ALL, both in
vivo3 and ex vivo.4 Because increased
intracellular accumulation of MTX-PG has been associated with greater
antileukemic effects,4,16 this may explain, in part, the
favorable prognosis of children with hyperdiploid B-lineage
ALL17 and offer insights for developing alternative
treatment strategies for different subtypes of ALL.
The present study has identified a novel mechanism for higher
intracellular concentrations of MTX-PG in hyperdiploid ALL, showing
significantly higher expression of the RFC in these leukemic lymphoblasts. Furthermore, the present study showed a relation between
chromosome 21 copy number and both the level of RFC mRNA expression and
the level of MTX-PG accumulation in hyperdiploid ALL blasts, with the
highest level of expression and MTX-PG accumulation in hyperdiploid
lymphoblasts with 4 copies of chromosome 21. The human RFC gene has
been mapped to chromosome 21q22.2-q22.3,8 suggesting a
gene-dose effect for RFC expression in these lymphoblasts. Because
hyperdiploid B-lineage ALL blasts almost always have at least 1 extra
copy of chromosome 21 (97% in one large series7), this
appears to be a common mechanism for increased MTX-PG accumulation in
this favorable subgroup of childhood ALL. It is known that, in
individuals with Down's syndrome, trisomy 21 is associated with
overexpression of a number of genes on this chromosome, including cystathionine synthase,18,19 phosphoribosylglycinamide
synthetase, and phosphoribosylaminoimidasole synthetase.20
Constitutive overexpression of the RFC gene in all cells of patients
with Down's syndrome may explain why these individuals are more
susceptible to MTX toxicity,21 a hypothesis that remains to
be investigated. It is also interesting that, among lymphoblasts with
greater than 2 copies of chromosome 21, the nonhyperdiploid samples (n = 3) had lower RFC mRNA than samples that were hyperdiploid (n = 7; medians, 3.4 v 11.3). This finding is also consistent with
higher MTX-PG accumulation in patients who had hyperdiploid blasts with only 2 copies of chromosome 21 (n = 2) compared with nonhyperdiploid blasts with two chromosomes 21 (n = 41; P = .028).
One nonhyperdiploid sample with 3 copies of chromosome 21 had a 12;21
translocation involving 1 copy of chromosome 21. This is a reciprocal
translocation that fuses the long arm of chromosome 21 (q22) to the
short arm of chromosome 12 (p13), resulting in the ETV6-CBFA2
fusion. It is not known whether translocation of the RFC gene
(22q22.2-q22.3), which maps close to the CBFA2 gene, alters its
expression, but these cells had the lowest level of RFC mRNA among all
samples with greater than 2 copies of chromosome 21.
Given the low abundance of RFC mRNA, we developed a PCR-based technique
that allows quantitation of RFC mRNA in a relatively small number of
leukemia cells (1 × 106) that differs from published
methods.13,22 This method permits assessment
of RFC expression using an aliquot of patient cells that is not
sufficient to quantitate RFC mRNA by Northern analysis. To enhance the
accuracy of RFC mRNA measurements, we prepared an artificial RFC RNA
fragment with a deletion of 58 ribonucleotides and used it as an
external standard. This competitive RT-PCR method permitted estimation
of RFC mRNA in both cultured cell lines and patient lymphoblasts with
good precision (coefficient of variation of 19.9% within
day and 26.5% between days).
The current studies with cultured human leukemia cell lines indicate
that MTX-transport-deficient CEM/T cells are resistant to MTX, at
least in part due to decreased RFC mRNA (30% to 60% lower compared
with CCRF-CEM/wt). In contrast, the transport-deficient CEM/MTX cells
had a high level of RFC mRNA, which is not unexpected, because the RFC
gene in this MTX-resistant cell line contains inactivating mutations,
resulting in substitution of Ser-127 by Asn, or a 4-bp (CATG) insertion
at position 191, generating a frame shift and a premature stop
codon.23 It was previously reported that, in the
transport-defective L1210 murine leukemia cell line, the RFC gene
contains a G429 C429 mutation,
resulting in an amino acid substitution (Ala-130 Pro).24 In both CEM/MTX and transport-deficient L1210
cells, these mutations are located in a very homologous and highly
conserved region in the predicted fourth transmembrane domain of the
protein. We found no difference in RFC mRNA level in the MTX-resistant K500E/MTX cells compared with the parent MTX-sensitive K562/wt cells,
suggesting that the RFC gene in these cells may also contain inactivating mutations. These data indicate that either decreased RFC
expression or inactivating mutations in the RFC gene are potential mechanisms for MTX resistance, although neither has yet been identified in primary leukemia cells isolated from patients. In contrast, this is
the first report of increased RFC expression as a mechanism for
enhanced sensitivity to MTX in a genetically defined subtype of ALL
(ie, hyperdiploid ALL).
It is recognized that mechanisms other than RFC expression may also
contribute to greater MTX-PG accumulation in hyperdiploid ALL, such as
increased FPGS activity and (or) decreased  glutamyl hydrolase (GGH)
activity.25,26 However, in contrast to lineage differences
in FPGS activity,6,25 we did not find a difference in FPGS
activity in hyperdiploid versus nonhyperdiploid B-lineage ALL,3 whereas the activity of GGH has not been investigated in these subtypes of childhood ALL. The human FPGS gene has been mapped
to chromosome 9q34.127 and the human GGH gene to chromosome 8q12.23-13.1,28 and these chromosomes are not commonly
present in increased (or decreased) copy number in hyperdiploid ALL
(ie, 20% have an extra chromosome 9 and 34% an extra chromosome
8).7 Two lines of evidence from the current study indicate
that at least one mechanism in addition to an RFC gene-dose effect
contributes to ploidy differences in MTX-PG accumulation. First, in
patients treated with low-dose MTX, MTX-PG concentrations were
significantly higher (P = .028) in hyperdiploid blasts with
only 2 copies of chromosome 21 compared with nonhyperdiploid blasts
with 2 copies of chromosome 21, although the small number of patients
in the former group limits the certainty of this finding. Second, RFC mRNA levels were higher in the 7 hyperdiploid samples with greater than
2 copies of chromosome 21 compared with the 3 nonhyperdiploid samples
with greater than 2 copies of chromosome 21 (Fig 2B). It is plausible
that hyperdiploid blasts have greater expression of selected
transcription factors, leading to overexpression of genes such as RFC,
a hypothesis requiring further investigation.
It is interesting that the relation between in vivo MTX-PG accumulation
and chromosome 21 copy number was statistically significant after
treatment with low-dose MTX, but did not reach statistical significance
(P < .24) after treatment with high-dose MTX (Figs 4 and 5).
With the doses of MTX evaluated in the current study, the mean
steady-state MTX plasma concentrations were approximately 0.9 µmol/L
with low-dose MTX treatment and approximately 12 µmol/L with
high-dose MTX.3 It is known that MTX accumulation at lower plasma concentrations is more dependent on the level of RFC expression and function, whereas MTX entry into lymphoblasts occurs by additional mechanisms (eg, passive diffusion) at high extracellular MTX
concentrations.29,30 It is also possible that intracellular
metabolism to MTX-PG via FPGS is saturated at the higher intracellular
MTX concentrations produced by high-dose MTX, providing another
explanation for why lymphoblasts with extra copies of chromosome 21 do
not accumulate significantly higher MTX-PG after high-dose MTX, in
contrast to low-dose MTX (Figs 4 and 5). Because MTX-PG accumulation in
B-lineage lymphoblasts with either 2, 3, or 4 copies of chromosome 21 was higher after high-dose MTX compared with low-dose MTX, there
appears to be a rationale for using high-dose MTX in all children with B-lineage ALL. However, the current results indicate that hyperdiploid B-lineage ALL with extra copies of chromosome 21 may be adequately treated with relatively lower doses than nonhyperdiploid B-lineage or
T-lineage ALL. Because the mechanisms responsible for lineage and
ploidy differences in MTX-PG accumulation are not the same, it is
probable that the optimal dose of high-dose MTX will differ for
specific subtypes of childhood ALL,31 a hypothesis
currently under investigation.
| |
ACKNOWLEDGMENT |
The authors thank Drs G. Rivera, R. Riberio, J.T. Sandlund, J. Rubnitz,
and F. Behm and all other individuals involved in the treatment of
these patients; N. Kornegay for her expertise in database management
and quality control; E.T. Melton, M. Needham, M. Chung, L. McNinch, E. Su, E. Ye, Y. Chu, and A. Atkinson for excellent technical assistance;
our research nurses, S. Ring, L. Walters, T. Kuehner, and M. Edwards;
Drs Joseph Bertino and Larry Matherly for providing cell lines; and
most importantly, the patients and parents who volunteered to
participate in this study.
| |
FOOTNOTES |
Submitted April 16, 1998; accepted October 12, 1998.
Supported in part by National Institutes of Health Grants No. R37
CA36401 and R01 CA78224, by Cancer Center Grant No. CA21765, by a
Center of Excellence grant from the State of Tennessee, and by American
Lebanese Syrian Associated Charities.
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
1734 solely to indicate this fact.
Address reprint requests to William E. Evans, PharmD, St
Jude Children's Research Hospital, 332 N Lauderdale St, Memphis,
TN 38105; e-mail: william.evans{at}stjude.org.
| |
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Top
Abstract
Introduction