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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 259-266
Prednisolone Resistance in Childhood Acute Lymphoblastic
Leukemia: Vitro-Vivo Correlations and Cross-Resistance to Other
Drugs
By
G.J.L. Kaspers,
R. Pieters,
C.H. Van Zantwijk,
E.R. VanWering,
A. Van Der Does-Van Den Berg, and
A.J.P. Veerman
From the Department of Pediatric Hematology/Oncology, University
Hospital Vrije Universiteit, Amsterdam; and Dutch Childhood Leukemia
Study Group, The Hague, The Netherlands.
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ABSTRACT |
As an important determinant of response to
chemotherapy, accurate measurement of cellular drug resistance may
provide clinically relevant information. Our objectives in this study
were to determine the relationship between in vitro resistance to
prednisolone (PRD) measured with the colorimetric
methyl-thiazol-tetrazolium (MTT) assay, and (1) short-term clinical
response to systemic PRD monotherapy, (2) long-term clinical outcome
after combination chemotherapy within all patients and within the
subgroups of clinical good and poor responders to PRD, and (3) in vitro
resistance to 12 other drugs in 166 children with newly diagnosed acute
lymphoblastic leukemia (ALL). The 12 clinical poor PRD responders had
ALL cells that were median 88-fold more in vitro resistant to PRD than
131 good responders (P = .013). Within all patients,
increased in vitro resistance to PRD predicted a significantly worse
long-term clinical outcome, at analyses with and without stratification for clinical PRD response, and at multivariate analysis (P  .001). Within both the clinical good and poor responder subgroups,
increased in vitro resistance to PRD was associated with a worse
outcome, which was significant within the group of clinical good
responders (P < .001). LC50 values, ie, lethal
concentrations to 50% of ALL cells, for PRD and each other drug
correlated significantly with those of all other 12 drugs, with an
average correlation coefficient of 0.44 (standard deviation 0.05). The
highest correlations were found between structurally related drugs. In
conclusion, in vitro resistance to PRD was significantly related to the
short-term and long-term clinical response to chemotherapy, the latter
also within the subgroup of clinical good responders to PRD. There was
a more general in vitro cross-resistance between anticancer drugs in
childhood ALL, although drug-specific activities were recognized.
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INTRODUCTION |
THEORETICALLY, CELLULAR drug resistance
together with pharmacokinetics and the regrowth potential of minimal
residual leukemic cells determines the clinical outcome after
chemotherapy in leukemia. We have therefore adapted the short-term
total cell kill methyl-thiazol-tetrazolium (MTT) assay for accurate
measurements of cellular drug resistance in childhood and adult acute
lymphoblastic leukemia (ALL) and nonlymphoblastic (ANLL) leukemia
samples.1-4 Current combination chemotherapy regimens
generally do not allow for single agent vitro-vivo comparisons.
However, the German Berlin-Frankfurt-Münster (BFM)-ALL Group introduced a therapeutic window in its
protocols, with 1 week of systemic predniso(lo)ne (PRD) monotherapy as
an initial treatment of all patients.5 It appeared that the
short-term clinical response to PRD was related to the long-term
clinical outcome after combination chemotherapy, and in current
BFM-based protocols, initial clinical response to PRD is one of the
factors for risk-group stratification.6
The therapeutic window enabled a study on the relation between the in
vitro antileukemic activity of PRD and the clinical response to an
initial 1 week PRD therapy in children with newly diagnosed ALL. We
also studied the relationship between in vitro PRD resistance and
clinical outcome after combination chemotherapy, especially within the
subgroups of clinically good and poor PRD responders. Finally, we
studied the in vitro cross-resistance patterns between PRD and 12 other
drugs. The relationship between resistance to each of the single drugs
and a drug resistance profile and long-term clinical outcome after
combination chemotherapy has been described separately.7
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MATERIALS AND METHODS |
Patients and patient samples.
Children (age 0 to 18 years) with non-B ALL, newly diagnosed between
1989 and 1994 were eligible. Bone marrow (BM) and peripheral blood (PB)
samples, and smears of 306 patients were sent by local institutions to
the Dutch Childhood Leukemia Study Group (DCLSG) laboratory for
confirmation of the diagnosis ALL, French-American-British (FAB) classification8 and immunophenotyping as
described previously.9 The DCLSG also calculated the BFM
risk factor (based on peripheral leukemic cell count and liver and
spleen size.10 Fresh samples with sufficient cells of 190 children were sent by the DCLSG laboratory to the research laboratory
for pediatric hematology/oncology of the University Hospital Vrije
Universiteit in Amsterdam for in vitro drug resistance testing,
successfully performed in 166 of 190 (87%) children. We have recently
described this process and consequences of selection of cases in a
previous study.7 The selected group of patients tested for
in vitro drug resistance has a significantly higher leukemic cell
burden, more often concerns T-cell immunophenotype, and has a worse
long-term clinical outcome than the total group of ALL patients.
Moreover, assay failures more often concern DNA hyperdiploid
cases.7 Characteristics of the 166 patients are shown in
Table 1. Results for PRD of 93 of these 166 samples have been used in a previous preliminary meeting report limited
to the short-term response to PRD only,11 while 152 of the
166 samples have also been used in our study on drug resistance versus
long-term clinical outcome after combination chemotherapy (excluding in
that study the 14 patients who were not treated according to protocol
after the systemic PRD monotherapy).7 Hyperdiploidy was
defined as a DNA index of 1.16 to 1.35, nonhyperdiploidy as a DNA index
of <1.16 or >1.35, as determined by flow cytometry.12
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Table 1.
Characteristics of 166 Children With Newly Diagnosed ALL
in Whom In Vitro Drug Resistance Was Successfully
Assessed
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In vitro drug resistance.
This was measured with the cell culture MTT assay.2 BM and
PB ALL samples were evaluated together because they do not differ in
drug resistance.13 Fresh (noncryopreserved) leukemic cells were cultured in RPMI 1640 (Dutch modification; GIBCO, Uxbridge, UK)
containing fetal calf serum and other supplements.2
Thirteen drugs were tested, each at six concentrations, as described
previously.13 Methotrexate was not included in the panel
because this drug is not cytotoxic to human leukemia samples in
nonclonogenic assays.2,13 Leukemic cells were incubated with each drug at each concentration in duplicate in wells of microculture plates at 37°C in humidified air with 5%
CO2. Six wells contained leukemic cells in drug-free medium
to determine the control cell survival and the percentage of leukemic
cells after culture. Six wells contained medium only to blank the
spectrophotometer. After 4 days, 10 µL (5 mg/mL) MTT salt (Sigma
Chemical Corp, St Louis, MO) was added for 6 hours. MTT is reduced to
colored formazan crystals by living cells only. The crystals were
dissolved with 100 µL acidified isopropanol, and formazan production
was quantitated using a spectrophotometer at 562 nm. The optical
density (OD) is linearily related to the cell number.14
Leukemic cell survival (LCS) was calculated at each drug concentration
by the equation LCS = (OD treated well/mean OD control wells) × 100%. The drug concentration lethal to 50% of the ALL cells, the
LC50, was used as measure of resistance. Samples were considered
evaluable if the drug-free control wells contained  80% leukemic
cells before and 70% leukemic cells after 4 days of culture
(determined by morphology and occasionally in case of doubt, by
immunology) and if the control OD at day 4 exceeded 0.050. The MTT
assay gives reliable results under these conditions.2,15
The coefficient of variation of the OD of the control wells in the
successful assays is median 5.2% (range, 0.9% to 15.3%). The
intraassay variation (duplicates) and interassay variation (repeated
testing of frozen sample) in LC50 values for all drugs are well within
one dilution step.16
Treatment.
Patients were treated according to DCLSG protocols (ALL-VII and
ALL-VIII). All patients first received a 1-week systemic monotherapy with PRD (60 mg/m2/day), and one injection with
methotrexate (the dose being age-dependent) intrathecally at day 1 of
the PRD window. Patients were then stratified into one of three risk
groups, according to several factors including BFM risk factor,
immunophenotype, extramedullary disease, karyotype, clinical response
to PRD (if poor, high risk), and achievement of complete remission
(CR).
Treatment outcome.
The results of treatment were evaluated at the DCLSG operations office.
Routine BM, PB, and cerebrospinal fluid examinations were performed at
the central laboratory of the DCLSG during chemotherapy and up to 3 years after cessation of therapy. Patients were divided into clinical
PRD poor responders (1,000 leukemic cells/µL PB at day 8) and good
responders (<1,000 leukemic cells/µL PB at day 8) by
protocol definition, independent of the number of leukemic cells/µL
PB at the start of this treatment. CR was defined as less than 5%
leukemic blasts in representative BM containing megakaryocytes and
granulocytic precursors with some degree of maturation, and no
manifestation of leukemia elsewhere. Failure to achieve CR after
induction chemotherapy (induction failure) was considered an event at
day 0. Early death was defined as death before completion of induction
therapy. Disease-free survival (DFS) was defined as the time from
diagnosis to induction failure or relapse (leukemia-related events).
For estimation of DFS, toxic deaths in remission were censored at the
time of occurrence and early deaths at day 0, and only patients
eligible and treated according to one of the two DCLSG protocols were
included. Patients who were disease-free were censored at the time of
latest follow-up as evaluated at this planned analysis. The relation
between in vitro PRD resistance or clinical PRD response and long-term
clinical outcome was studied only within the 152 patients without
protocol violation after the systemic PRD monotherapy.
Statistics.
Differences in distribution of variables were tested with the
Mann-Whitney U (MWU) test or the  2 test. Estimates of
the probability (p) of DFS (with standard errors [SE]) were
calculated according to the Kaplan-Meier product limit
analysis.17 Because toxic and early deaths presumably are
unrelated to cellular drug resistance, in contrast to induction failures and relapses, results of DFS analysis are shown. Univariate and multivariate statistical comparisons of outcome were conducted by
proportional hazard Cox regression analysis, after stratification for
risk group or clinical PRD response where appropriate.18 The model for multivariate analysis included the conventional prognostic factors age, BFM risk factor, immunophenotype, DNA ploidy,
clinical PRD response, and in vitro drug resistance. Information on
karyotype was not available for the majority of patients and therefore
was not included in this analysis. Cross-resistance patterns were
studied by correlating the LC50 values for different drugs, using the
Spearman test. Means were calculated with standard deviations (SD) for
the correlation coefficients. The analyses were two-tailed at a
significance level of 5%.
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RESULTS |
In vitro resistance to PRD versus short-term clinical response to PRD.
The clinical response to the window therapy with PRD was known in 156 patients. Differences between the good and poor responders are outlined
in Table 2. Poor responders more often were
male (although not significantly), had a higher leukemic cell burden (P = .024) and more often an unfavorable (pro-B or T-cell)
immunophenotype (P < .001). The 12 (8%) clinical poor
responders to PRD had significantly (P = .013) higher LC50
values for PRD (median, 130.9; range, 0.43 to >2,000 µg/mL) than
the 131 good responders (median, 1.49; range, <0.06 to >2,000)
tested successfully for PRD resistance. However, 17 (13%) of the 131 good responders had less than 1,000 leukemic cells/mm3 PB
at day 0, but were by definition classified as clinical good responders. After omitting these 17 patients from the analysis to allow
a more meaningful comparison, the 12 in vivo poor responders (10%) had
a 97-fold higher median LC50 for PRD than the 114 good responders
(P = .006, Fig 1 and
Table 2). Relapses occurred in five of these 17 patients, and all five
were highly in vitro PRD-resistant with LC50 values 250 µg/mL,
compared with a median LC50 value of 2.1 µg/mL for the remaining 12 patients. Overall, events did not occur significantly more frequent in
the group of clinical poor PRD responders (P = .08, Table
2), and the far majority of relapses were seen within the group of
clinical good PRD responders.
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| Fig 1.
Distribution of LC50 values as a measure of in vitro
resistance to PRD within potentially clinically poor PRD responders
(blasts at day 0 1,000/µL of peripheral blood) in clinical good
(blasts day 8 of <1,000/µL, n = 114) and clinical poor (blasts
day 8 of 1,000/µL, n = 12) responders to a 1-week systemic PRD
monotherapy (plus one intrathecal injection with methotrexate at day 1)
in childhood ALL.
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In vitro resistance to PRD versus long-term clinical outcome after
combination chemotherapy.
The extent of in vitro resistance to PRD was known in 153 of the 166 patients. Table 3 outlines the differences
between in vitro PRD sensitive (LC50 < 150 µg/mL) and in
vitro PRD-resistant (LC50 150 µg/mL) patients. This definition of
sensitivity versus resistance is based on our previous retrospective
study.19 The in vitro resistant group more often had pro-B
or T-cell ALL (P = .007), and less often DNA hyperdiploidy
(P = .03). In vitro PRD-resistant patients more often had a
clinical poor response to PRD then in vitro PRD sensitive patients
(P = .04). Finally, the frequency of no CR and relapses was
higher in the in vitro PRD-resistant subgroup (P = .002).
Patients were further classified as in vitro highly sensitive (LC50
<0.1 µg/mL), intermediately sensitive (LC50 0.1 to 150 µg/mL), or
in vitro resistant (LC50 150 µg/mL) to PRD according to the
definition in our retrospective study.19 In the total patient-group, in vitro PRD resistance was related to the long-term clinical outcome after combination chemotherapy at a median follow-up of 46 months (range, 17 to 79) for patients at risk, at univariate analysis both without any stratification and with stratification for
risk-group or clinical response to the PRD monotherapy (P < .001), and at multivariate analysis (P = .001, Table 4). The pDFS progressively decreased
from highest (100%) in those patients with highly sensitive ALL-cells
to lowest (42%, SE 10%) in those with resistant ALL-cells. This has
been described by us in detail previously.7 However, within
the subgroup of clinical good PRD responders, again, the extent of in
vitro PRD resistance was related to the clinical outcome
(Fig 2). Within this subgroup, the pDFS at
3 years was 100% for the in vitro PRD highly sensitive patients, 79%
(SE, 5%) for the intermediately sensitive patients, and 43% (SE,
11%) for the in vitro PRD-resistant patients (P < .001 at
univariate, risk-group stratified analysis, and P = .002 at
multivariate analysis, Table 4). Apparently, relapses that had not been
predicted by the clinical response to PRD were predicted by the in
vitro antileukemic activity of PRD. Within the small subgroup of
clinical poor PRD responders, none of the patients had ALL cells that
were highly sensitive to PRD in vitro. At 3 years, the pDFS was 60%
(SE 22%) for in vitro intermediately sensitive patients (n = 6) and
33% (SE 19%) for the remaining group of in vitro resistant patients
(P = .29). Similarly, LC50 values for PRD were median sevenfold
lower in the six patients who did achieve CR and did not suffer a
relapse (median, 32.7 µg/mL) than in the remaining six clinical poor
responders who did not achieve CR (n = 1) or relapsed (median, 229 µg/mL), although this again was not a statistically significant
difference.
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Table 4.
Multivariate Analysis of the Relationship Between
Several Potential Prognostic Factors and the Probability of
Leukemia-Related Events in the Total Group of Children With ALL and
Within the Subgroup of Clinical Good PRD Responders
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| Fig 2.
Relationship between in vitro PRD resistance (cut-off
values for sensitivity and resistance 0.1 and 150 µg/mL,
respectively) and long-term clinical outcome after combination
chemotherapy in childhood ALL, within (A) the total group of clinical
good PRD responders (15 highly sensitive, 79 intermediately sensitive, and 30 resistant to PRD in vitro), and (B) the group of clinical poor
PRD responders (none highly sensitive, six intermediately sensitive,
and six resistant to PRD in vitro). The numbers in the figures along
the x-axis indicate the patients at risk for a relapse at the different
time points.
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In vitro cross-resistance patterns.
LC50 values of the different drugs could be correlated for 104 up to
142 samples, except for ifosfamide. For the latter drug, being included
in the drug panel later on in the study, this was possible for 48 to 60 samples. There was a significant cross-resistance between all drugs
(Table 5). However, cross-resistance
between structurally related drugs was more pronounced, eg, for the
glucocorticoids, the vinca-alkaloids, the anthracyclines, and the
thiopurines. There was a relatively high degree of cross-resistance
between the epipodophyllotoxin teniposide (VM-26) and the three
anthracyclines tested, and between the vinca-alkaloids and the
anthracyclines. For prednisolone, the correlation coefficients ranged
from 0.30 (vindesine and ifosfamide) to 0.84 (dexamethasone), with an
average of 0.44 (SD 0.14). For the other drugs, the mean correlation
coefficient ranged from 0.38 (l-asparaginase) to 0.53 (doxorubicin),
with an overall mean correlation coefficient of 0.44 (SD 0.05).
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Table 5.
Correlation Between LC50 Values in 166 Childhood ALL
Samples Expressed by Spearman's Rank Correlation Coefficients
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DISCUSSION |
Cellular drug resistance is an important cause of the ultimate failure
of chemotherapy. Therefore, the accurate measurement of cellular drug
resistance on primary clinical specimens is of clinical importance. For
that purpose, we have adapted the short-term semiautomated colorimetric
MTT assay for its use on clinical specimens of children and adults with
ALL and ANLL and have shown the clinical relevance of the results
obtained so far.3,4,7,16,19-23 With regard to
glucocorticoids, we showed that unfavorable subgroups as defined by age
(infants or >10 years at diagnosis), immunophenotype (pro-B, T-cell
ALL), leukemia type (ANLL), or disease status (relapses) are relatively
resistant to PRD in vitro, and that the extent of in vitro PRD
resistance is related to the long-term clinical outcome after
combination chemotherapy.24,25 In the present study, again
pro-B and T-cell immunophenotype was associated with a relative PRD
resistance, both clinically and in vitro. The reason for this is
unknown, but it is not related to decreased numbers of glucocorticoid
receptors.24 Rather, it seems that ALL cells of these
immunophenotypes show a more general drug resistance, because we showed
that pro-B and T-cell ALL samples were also more resistant to a large
number of drugs other than glucocorticoids in comparison with common
and pre-B ALL samples.20,23
In this study on 166 untreated childhood ALL samples, we have shown
that in vitro PRD resistance is significantly related to the short-term
response to a systemic 1 week PRD monotherapy (Fig 1) and to the
clinical outcome after combination chemotherapy (Fig 2), and that these
ALL cells have a general cross-resistance, with drug type-specific
patterns (Table 5). The relationship between in vitro PRD resistance
and clinical outcome was significant also within the subgroup of
clinical good PRD responders, in which the far majority of relapses
occurred (Fig 2). Apparently, in vitro PRD resistance predicts relapses
not identified by the clinical response to the PRD window. Within the
subgroup of 12 clinical poor PRD responders, in vitro PRD-resistant
patients also had a worse outcome than the in vitro sensitive patients,
but this was not significant in this low number of cases. Therefore,
this study is inconclusive with respect to the prognostic significance of in vitro PRD resistance within the subgroup of clinical poor PRD
responders.
We previously showed that in addition to PRD, the extent
of in vitro resistance to asparaginase and vincristine was also
significantly related to the long-term clinical outcome in childhood
ALL. In that study, we also showed that combining the results for these three drugs provided a drug resistance profile, which was the single
most important prognostic factor at multivariate analysis, and a
(slightly) better prognostic indicator than in vitro resistance to each
of the single drugs.7 A drawback of the previous and present study is that karyotype was unknown in most patients and could
not be included in the multivariate analyses. Although we did show that
combinations with two drugs can be tested in vitro in the MTT
assay,26 it does not seem feasible to mimick clinical combination chemotherapy in in vitro cellular drug resistance testing.
Clinical poor PRD responders were nearly 100-fold more resistant to PRD
in vitro than the good responders, but there were many clinical good
PRD responders who were relatively resistant to PRD in vitro. There are
several possible explanations for this. First, 17 patients were not
actually tested clinically for their PRD responsiveness because they
had less than 1,000 blasts/µL blood before the start of the systemic
PRD monotherapy. Of interest, five of these 17 patients relapsed, and
all five had ALL cells highly resistant to PRD in vitro. Second, the
systemic PRD monotherapy was accompanied by one intrathecal injection
with methotrexate at day 1. It is well-known that methotrexate
delivered intrathecally has systemic antileukemic
activity27,28 up to inducing a tumor-lysis syndrome.29 The Children's Leukemia Cooperative Group
recently reported that the percentage of clinical good responders to
PRD alone was 70% and increased to 90.4% if methotrexate had been delivered before day 2 of the therapeutic window.30
Preliminary data of our laboratory (not shown) showed that clinically
good responders to systemic PRD plus intrathecal methotrexate with in
vitro PRD-resistant ALL cells were not resistant to methotrexate in
vitro, and there was no significant in vitro cross-resistance between
PRD and methotrexate. Therefore, methotrexate may indeed have
contributed to the good clinical response in this group of patients. In
addition to the systemic antileukemic activity of intrathecal methotrexate itself, a synergistic interaction with PRD may
contribute to a good clinical response in the case of relatively
PRD-resistant ALL cells in vitro. Third, while our in vitro assay tests
for cell death, counting the absolute number of blasts in the
peripheral blood may speculatively be influenced by a temporary
redistribution of the blasts under the influence of PRD. This has been
described to occur for normal lymphocytes.31 Finally, some
of the assay results may have been unreliable, although we do apply
strict rules for evaluability. Whatever the explanation, in vitro PRD
resistance was the single most important prognostic factor at
multivariate analysis with respect to long-term clinical outcome after
combination chemotherapy within the total group and within the clinical
good PRD responders.
Part of the clinical poor PRD responders were in vitro intermediately
(never highly) sensitive to PRD. In these cases, unfavorable pharmacokinetics may have caused a relatively low exposure of the ALL
cells to PRD, contributing to the poor clinical response ("pharmacokinetic resistance"). In addition, an antagonistic
interaction between PRD and methotrexate in individual patients may
have occurred. Finally, it should be noted that the applied arbitrary
definition of in vitro PRD resistance versus sensitivity was based on
our previous retrospective study on the relationship between in vitro PRD resistance and long-term clinical outcome after combination chemotherapy19 and not on a proper single agent vitro-vivo
comparison of the antileukemic activity of PRD. However, it seems
appropriate to consider patients with ALL cells that are highly or
intermediately sensitive to PRD in vitro as relatively sensitive to PRD
clinically, in view of the 70% clinical response rate to single agent
PRD in the study of the Children's Leukemia Cooperative
Group.30
ALL cells were cross-resistant to all types of drugs, although not
strongly, with an average correlation coefficient for PRD of 0.44. The
extent of cross-resistance was more pronounced between structurally
related drugs. Specific drug targets may be more accessible in ALL
cells of certain patients than in others, and mechanisms of resistance
affecting specific types of drugs may be operative. Regarding the
latter, P-glycoprotein is unlikely to be a major cause of resistance in
untreated childhood ALL.25 The lung-resistance protein,
which may alter the subcellular drug distribution, is a
candidate.32 We speculate that the general cross-resistance
pattern that we observed is in agreement with our current knowledge on
apoptosis and a trigger-independent common pathway leading to cell
death.
In conclusion, in vitro PRD resistance is related to the short-term
response to systemic PRD monotherapy. This has been previously reported
for children and adults,33-35 although some could not confirm this.36-38 In vitro PRD resistance is also related
to the long-term clinical response to combination chemotherapy, even within the subgroup of clinical good PRD responders and was the single
most important prognostic factor at multivariate analysis. This may
indicate the essential contribution of glucocorticoids to the
successful chemotherapy of ALL and may also be partly explained by the
cross-resistance between PRD and other drugs. ALL cells show a general
cross-resistance to all drugs, but drug-specific patterns can be
recognized. Cross-resistance patterns of new drugs can be tested in
vitro. The use of noncross-resistant drug combinations may offer
therapeutic advantage.39 In vitro drug resistance can be
used as a prognostic factor, and in vitro PRD resistance adds
prognostic value to that of the clinical PRD response. Within the
setting of the BFM-ALL protocols, it seems rational to include in vitro
drug resistance testing as second stratification factor, after a first
stratification based on the clinical response to PRD.
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FOOTNOTES |
Submitted August 18, 1997;
accepted March 2, 1998.
Supported by the Dutch Cancer Society (IKA 89-06, Amsterdam, The
Netherlands) and by the project VONK (VU Onderzoek Naar Kinderkanker, Amsterdam, The Netherlands).
Address reprint requests to G.J.L. Kaspers, MD, PhD, Department of
Pediatric Hematology/Oncology, University Hospital Vrije Universiteit,
De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank F.R. Rosendaal (Departments of Clinical Epidemiology
and Hematology, University Hospital Leiden, The Netherlands) for
statistical assistance. The Dutch Childhood Leukemia Study Group
(DCLSG) provided the patient samples. Board members of the DCLSG are H. Van Den Berg, M.V.A. Bruin, J.P.M. Bökkerink, P.J. Van Dijken, K. Hählen, W.A. Kamps, F.A.E. Nabben, A. Postma, J.A. Rammeloo, I.M.
Risseeuw-Appel, A.Y.N. Schouten-Van Meeteren, G.A.M. De Vaan, E. Th
Van't Veer-Korthof, A.J.P. Veerman, M. Van Weel-Sipman, and R.S.
Weening.
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Abstract
Introduction
Abstract