In vitro cellular drug sensitivity at diagnosis is correlated to minimal residual disease at end of induction therapy in childhood acute lymphoblastic leukemia

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Leukemia Research 33 (2009) 46–53

In vitro cellular drug sensitivity at diagnosis is correlated to minimal residual disease at end of induction therapy in childhood acute lymphoblastic leukemia Gudmar Lönnerholm a,∗ , Ingrid Thörn b , Christer Sundström b , Britt-Marie Frost a , Jonas Abrahamsson c , Mikael Behrendtz d , Jesper Heldrup e , Stefan Jacobsson f , AiHong Li g , Tor Olofsson h , Anna Porwit i , Stefan Söderhäll j , Rolf Larsson k , Erik Forestier l a

h

Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden b Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden c Qween Silvias Children’s Hospital, Gothenburg, Sweden d Department of Pediatrics, University Hospital, Linköping, Sweden e Department of Pediatrics, Lund University Hospital, Lund, Sweden f Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden g Department of Biosciences, Clinical Genetics, University of Umeå, Sweden Division of Hematology & Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden i Department of Pathology, Karolinska University Hospital, Stockholm, Sweden j Department of Pediatric Oncology, Karolinska University Hospital, Stockholm, Sweden k Department of Medical Sciences, Section of Pharmacology, University Hospital, Uppsala, Sweden l Department of Clinical Sciences, Paediatrics, University of Umeå, Sweden Received 29 February 2008; received in revised form 3 June 2008; accepted 7 June 2008 Available online 17 July 2008

Abstract Leukemic cells from 85 children with newly diagnosed precursor B-lineage ALL were tested for in vitro drug sensitivity to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. There was a significant correlation between MRD day 29 and in vitro sensitivity to prednisolone (p < 0.001) and doxorubicin (p = 0.017), drugs administered during induction therapy. In patients with t(12;21) (n = 20), in vitro sensitivity to doxorubicin was an independent factor for MRD <0.1% (p = 0.031; R2 = 0.66). Thus, data show that in vitro drug sensitivity at diagnosis is correlated to cell kill during induction therapy as measured by MRD day 29. © 2008 Elsevier Ltd. All rights reserved. Keywords: Acute lymphoblastic leukemia; Childhood; In vitro drug resistance; MRD; t(12 ;21); Prednisolone; Doxorubicin

1. Introduction Minimal residual disease (MRD) has evolved as an independent prognostic parameter in childhood acute lym∗ Corresponding author at: Department of Women’s and Children’s Health, University Children’s Hospital, SE-751 85 Uppsala, Sweden. Tel.: +46 18 6115896; fax: +46 18 6115853. E-mail address: [email protected] (G. Lönnerholm).

0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.06.012

phoblastic leukemia (ALL) and is currently used in several clinical trials for treatment stratification [1–5]. In vitro measurement of drug resistance in leukemic cells obtained at diagnosis has also shown promise in the prediction of clinical outcome in selected groups of patients [6–9], but only two small retrospective studies have investigated the relationship between in vitro drug resistance and MRD. In both studies a weak correlation was found between resistance to prednisolone and MRD during and after induction therapy

G. Lönnerholm et al. / Leukemia Research 33 (2009) 46–53

[10,11]. No such correlation was observed for the other drugs used in induction. The aim of the present investigation was to study the correlation between cellular drug sensitivity at diagnosis and cell kill during induction therapy, as measured by MRD. Bone marrow samples were prospectively collected from children with newly diagnosed ALL, who then received treatment with a common Nordic protocol using doxorubicin, prednisolone and vincristine as induction therapy. Leukemic cells were tested by the fluorometric microculture cytotoxicity assay (FMCA), a rapid and reproducible method for determination of in vitro drug sensitivity [12,13]. MRD was determined by real time quantitative PCR (RQ-PCR) at treatment days 29, 50, and 106, and at day 15 in a subgroup of patients. The results are the first to show a clear correlation between cellular drug sensitivity at diagnosis and the effect of induction therapy.

2. Patients and methods Leukemic cells from the bone marrow of children (aged 1–17 years) with newly diagnosed precursor B-lineage ALL, collected during 2002–2005, were used in this study. All six Swedish centers for pediatric oncology participated, entering patients consecutively in a prospective manner. The diagnosis was established at a pediatric oncology center by analysis of bone marrow aspirates including morphology, immunophenotype, and cytogenetics of the leukemic cells. Immunophenotypes were defined according to EGIL [14]. Chromosome banding analyses of bone marrow and/or peripheral blood samples were performed using standard methods. The definition and description of clonal abnormalities followed the recommendations of ISCN [15]. All karyotypes have been centrally reviewed. Fluorescence in situ hybridization (FISH) and/or reverse-transcriptase polymerase chain reaction (RT-PCR) analyses were applied to identify ETV6/RUNX1[t(12;21) (p13;q22)] [16–18]. The children were treated according to the NOHPO (Nordic Society for Paediatric Haematology and Oncology) ALL 2000 protocol. All patients received identical treatment during the first 4 weeks, including daily oral prednisolone 60 mg/m2 , i.v. vincristine 2 mg/m2 (maximum dose 2.5 mg) days 1, 8, 15, and 22, i.v. doxorubicin 40 mg/m2 days 1 and 22, and intrathecal methotrexate 12 mg days 1, 8, and 15. In addition, patients with WBC above 50 × 109 l−1 received increasing doses of prednisolone during a prephase of 3–7 days. Patient characteristics and clinical follow-up data were obtained from annual reports submitted by the treating clinicians to the Nordic registry at the Childhood Cancer Research Unit in Stockholm, and the last follow-up was December 2007. 2.1. FMCA procedure Bone marrow aspirates were collected in heparinized glass tubes, kept at room temperature, and sent by mail or through international express delivery companies. As a rule they reached the in vitro sensitivity laboratory in Uppsala for processing within 24–36 h. Leukemic cells were prepared by 1.077 g/ml Ficoll-Isopaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation. The

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median viability, as determined by Trypan-blue exclusion test, was 95% and FMCA was performed only when the viability was ≥70%. The median proportion of lymphoblasts after separation was 90% and FMCA was performed only when this proportion was ≥70%. FMCA is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate (FDA) to fluorescein by cells with intact plasma membranes and has been described in detail previously [13,19–22]. Drugs were tested in triplicate. Six wells without drugs served as controls and six wells containing culture medium only served as blanks. The results are presented as survival index (SI), defined as fluorescence in test wells/fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of the drug. Cytotoxic drugs were obtained from commercial sources and tested at empirically derived cut-off concentrations, adopted from previous studies of leukemic cells and chosen to produce a large scatter of SI values among the samples [12]: asparaginase 1 U/ml, cytarabine 0.5 ␮g/ml, dexamethasone 1.4 ␮g/ml, doxorubicin 0.5 ␮g/ml, etoposide 5 ␮g/ml, prednisolone 50 ␮g/ml, vincristine 0.5 ␮g/ml, and 6-thioguanine 10 ␮g/ml. 2.2. MRD detection DNA was extracted from leukemic blasts in diagnostic and follow-up bone marrow samples separated on 1.077 g/ml FicollIsopaque. In the diagnostic samples, junction regions of IG/TCR rearranged genes were identified by PCR amplification with consensus primers and subsequently sequenced [23–25]. Allele specific oligonucleotides (ASO) complementary to the identified junction regions were designed. Preferable two different target genes were selected in each leukemia case. Real time quantitative PCR was performed with consensus probes and primer [26–30]. Minimal residual disease was identified in the bone marrow aspirates at days 15, 29, 50, and 106 by use of the clone specific sequences giving the highest quantitative range and sensitivity [31]. Results are presented as percentage of leukemic cells of total number of cells (leukemia copies/albumin copies) in the sample investigated [32]. All followup samples were run as triplets with pooled mononuclear cells as background, and with the standard curve in duplicate. The analyses were performed at five different laboratories (Umeå, Uppsala, Stockholm, Göteborg and Lund), and samples were exchanged to minimise inter-laboratory variability (details to be published separately). 2.3. Statistical analysis Non-parametric methods were used throughout. Differences in variable distribution were tested with the Mann–Whitney U test, Kruskal–Wallis H test or the chi-square test. The Spearman correlation coefficient was used to examine relationships between continuous variables, and logistic regression analysis to test the relative importance of selected parameters for a defined event. For the calculation of odds ratios by regression analysis, the SI values of relevant drugs were divided by 10. The SPSS 14 software package was used for the calculations. All analyses were two-tailed and the level of statistical significance was set at p < 0.05. Local ethics committees approved the study.

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G. Lönnerholm et al. / Leukemia Research 33 (2009) 46–53 Table 1 Baseline data for included patients compared to not included patients

3. Results Samples from 125 patients for whom MRD data at day 29 was available were sent at diagnosis for test of in vitro cellular drug resistance, and FMCA was successfully accomplished in 85 samples. Reasons for failure included delivery failure (n = 3), insufficient total cell count (n = 6), inadequate proportion of lymphoblasts (<70%) before the test (n = 6), or after 72 h incubation (n = 5), excessively low signal in controls compared with blanks, i.e. signal-to-noise ratio <5 (n = 15), and control CV of >30% (n = 5). Thus, the technical success rate for samples with a sufficient number of blast cells was 85 out of 110 (77%). When baseline data for patients with successful and unsuccessful FMCA were compared, no significant differences were found (not shown). Table 1 shows baseline data for included patients with successful FMCA and RQ-PCR, compared to all other children with precursor Blineage ALL diagnosed in Sweden during the study period. The cohort of not included patients also comprises the 40 patients with unsuccessful FMCA mentioned above. There were no significant differences (p > 0.2 for all parameters shown). MRD values are presented in seven clinically relevant groups (Table 2): ≥25%, 5 to <25%, 1 to <5%, 0.1 to <1%, <0.1% (samples where a sensitivity of 0.1% was reached), 0.01 to <0.1%, and <0.01%. For statistical analyses demanding numerical values, negative samples were denoted a value just below the detection limit, e.g. negative samples with a detection limit of <0.1% were denoted an MRD value of 0.09%. 3.1. Day 15 For 50 of the 85 patients, samples had been taken for MRD analysis on day 15. There was a statistically significant correlation between cellular drug resistance at diagnosis and MRD day 15 for drugs administered during the two first weeks of treatment, i.e. prednisolone (Fig. 1), doxorubicin, and vincristine, but also for other drugs (see Table 3). 3.2. Day 29 A significant correlation between cellular drug resistance at diagnosis and MRD day 29 was found for all drugs tested,

Included patients

Not included patients

No. of patients Male/female Age median (range)

85 49/36 5.5 (1.0–16.7)

159 90/69 5.0 (1.0–17.0)

WBC <10 10 to <50 ≥50 Missing

51 (60%) 18 (21%) 14 (16%) 2 (2%)

88 (56%) 50 (31%) 21 (13%)

25 (29%) 20 (23%) 4 (5%) 4 (5%) 2 (2%) 3 (4%) 3 (4%) 2 (2%)

56 (35%) 24 (15%) 2 (1%) 7 (4%) 6 (4%) 3 (2%) 4 (3%) 3 (2%) 3 (2%) 24 (15%) 18 (11%) 9 (6%)

Cytogenetics High hyperploidy t(12;21) Dic (9;20) Down t(9;22) t(1;19) amp (21q22) 11q23 rearr Hypodiploidy Other clonal abnormality Normal karyotype No result Events CCR Relapse Death in CCR Resistant disease Induction failure SMN

12 (14%) 9 (11%) 1 (1%) 69 (81%) 13 (15%) 3 (4%)

131 (82%) 18 (11%) 3 (2%) 2 (1%) 4 (3%) 1 (1%)

Not included patients are all children ≥1 year of age diagnosed with precursor B-lineage ALL in Sweden during the study period, who were not successfully investigated by FMCA and RQ-PCR. There were no significant differences between included and not included patients. WBC, white blood cell count; CCR, continuous complete remission; SMN, secondary malignancy.

except etoposide and vincristine. As shown in Table 3, the rho values for the days 15 and 29 data were similar for most drugs, while p-values were lower day 29, reflecting the higher number of samples tested for MRD at this point in time. The notable exception was vincristine. Fig. 2A and B show the correlation between MRD day 29 and cell survival after in vitro exposure to prednisolone and doxorubicin. Since several studies have shown, that patients with MRD values below 0.1% after induction therapy have a low risk of relapse, we divided the patient material in two groups

Table 2 Levels of minimal residual diseases (MRD) days 15, 29, 50, and 106 MRD level

Day 15 no. (%)

Day 29 no. (%)

Day 50 no. (%)

Day 106 no. (%)

≥25% 5 to <25% 1 to <5% 0.1 to <1% <0.1% 0.01 to <0.1% <0.01%

6 (12) 8 (16) 11 (22) 9 (18) 3 (6) 5 (10) 8 (16)

1 (1.2) 9 (10.6) 9 (10.6) 16 (18.8) 5 (5.9) 15 (17.6) 30 (35.3)

0 0 0 5 (6.3) 5 (6.3) 12 (15) 58 (72.5)

0 0 0 1 (1.3) 8 (10.7) 3 (4) 63 (84)

Total

50

85

80

75

G. Lönnerholm et al. / Leukemia Research 33 (2009) 46–53

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Fig. 1. Percent cell survival (SI) after exposure to prednisolone 50 ␮g/ml in diagnostic samples grouped by minimal residual disease (MRD) levels day 15 of induction therapy. The box-and-whisker plot shows median, first and third quartiles; whiskers extend to the highest and lowest value, excluding outliers, which are denoted individually.

using that cut-off value for regression analyses (see Table 2). For this purpose, patients were also collapsed into 3 groups according to cytogenetics: t(12;21), high hyperploidy, and all other. In multivariate logistic regression analysis, including in vitro sensitivity to prednisolone, sex, age, white blood cell count (WBC) at diagnosis, and cytogenetics, the only independent factor for MRD <0.1% day 29 was sensitivity to prednisolone (p < 0.001; R2 = 0.22). A 10% difference in SI value, i.e. 10% higher cell survival after in vitro exposure to prednisolone, increased the odds ratio for MRD ≥0.1% by 1.56 (95% confidence interval 1.22–1.99). Similar results were obtained for doxorubicin (p = 0.007; R2 = 0.14). Analysis within cytogenetic subgroups was only meaningful for the two largest groups (cf Table 1). In 20 children with t(12;21), there was a significant correlation between MRD day 29 and in vitro sensitivity to prednisolone and doxoruTable 3 In vitro drug sensitivity correlated to minimal residual disease (MRD)

Asparaginase Cytarabine Dexamethasone Doxorubicin Etoposide Prednisolone Vincristine 6-Thioguanine

MRD day 15 (n = 50)

MRD day 29 (n = 85)

Correlation coefficient

p-Value

Correlation coefficient

p-Value

0.34 0.27 0.40 0.31 0.19 0.33 0.33 0.33

0.034 0.057 0.004 0.037 0.22 0.023 0.019 0.025

0.35 0.34 0.42 0.26 0.18 0.39 0.16 0.24

0.005 0.002 <0.001 0.017 0.13 <0.001 0.14 0.033

Cell survival (SI values) of individual cases were correlated to MRD levels.

bicin (Fig. 3), as well as to dexamethasone, etoposide and 6-thioguanine. As shown in Table 4, the strongest correlation was found for doxorubicin. In multivariate logistic regression analysis, including in vitro sensitivity to doxorubicin, sex, age, and WBC count at diagnosis, drug sensitivity to doxorubicin was the only independent factor for MRD <0.1% (p = 0.031; R2 = 0.66). A 10% difference in SI value, i.e. 10% higher cell survival after exposure to doxorubicin, increased the odds ratio for MRD ≥0.1% by 3.57 (95% CI 1.12–11.3). In 25 children with high hyperploidy, defined as main modal number >50 chromosomes, no relation between MRD day 29 and in vitro sensitivity was found for any drug. 3.3. Days 50 and 106 As expected, MRD values decreased with duration of treatment. At day 106 only one patient had an MRD of 0.1 to <1%, and three had MRD values of 0.01 to <0.1% (Table 2). Table 4 In vitro drug sensitivity correlated to minimal residual disease (MRD) day 29 in patients with t(12;21) (n = 20)

Asparaginase Cytarabine Dexamethasone Doxorubicin Etoposide Prednisolone Vincristine 6-Thioguanine

Correlation coefficient

p-Value

0.30 −0.19 0.65 0.71 0.64 0.64 0.21 0.46

0.25 0.44 0.002 0.001 0.003 0.002 0.37 0.041

Cell survival (SI values) of individual cases were correlated to MRD levels.

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G. Lönnerholm et al. / Leukemia Research 33 (2009) 46–53

Fig. 2. Cell survival in diagnostic samples after exposure to (A) prednisolone 50 ␮g/ml, (B) doxorubicin 0.5 ␮g/ml in relation to MRD day 29. See also legend to Fig. 1.

Due to the low number of MRD-positive samples, correlation analysis with in vitro sensitivity data was not meaningful.

4. Discussion A number of studies have demonstrated that MRD measured at the end of induction therapy has prognostic impact on survival in childhood ALL. In the current NOPHO ALL 2000 protocol, MRD data are collected on days 29, 50, and

106, and on day 15 at some centres, but data are not used for stratification of patients. We focus here on day 29 data because they reflect the cell kill of a limited number of drugs given during induction therapy. Furthermore, the prognostic impact of MRD measured after 4–5 weeks of therapy is well documented [1,3–5]. Included in the present study were 85 out of 244 children (35%) diagnosed with precursor Blineage ALL at participating centres during the study period. Main reasons for not being included were, that MRD for practical reasons was evaluated by flow cytometry rather than by

G. Lönnerholm et al. / Leukemia Research 33 (2009) 46–53

Fig. 3. Cell survival in diagnostic samples after exposure to (A) prednisolone 50 ␮g/ml, (B) doxorubicin 0.5 ␮g/ml in patients with t(12;21).

PCR, or unsuccessful FMCA. We found no indications that the included patients in any respect differed compared to the greater cohort not investigated by FMCA and PCR. Our finding of a correlation between prednisolone sensitivity at diagnosis and MRD day 29 agrees well with the observation that the short-term response to prednisolone monotherapy was significantly correlated to in vitro sensitivity for the drug [33]. As expected, the in vitro sensitivity data for dexamethasone were very similar to those for prednisolone. The detection limit for most PCR analyses was 0.01% or lower, but for a few patient samples the limit was 0.1%. Several studies have shown that patients with MRD val-

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ues below 0.1% after induction therapy have a low risk of relapse (see above), and we therefore divided our patient material in two groups, using that cut-off value. Sensitivity to prednisolone was the only independent factor for MRD <0.1% day 29 in the multivariate analysis including sex, age, WBC at diagnosis, and cytogenetics. However, the predictive value was relatively low (R2 = 0.22), indicating that other factors, e.g. individual pharmacokinetics and cytogenetic subgroup, might have influenced the MRD levels. In vitro sensitivity to doxorubicin, another component of the induction therapy, also showed a correlation to MRD day 29. The same was true for a number of drugs not used in induction therapy: asparaginase, cytarabine, and 6-thioguanine. This probably reflects the cross-resistance between many anticancer drugs that has been observed both clinically and in in vitro tests in childhood ALL [12,33,34]. The cross-resistance phenomenon precluded the introduction of prednisolone and doxorubicin in the same multivariate analysis, and their relative importance for cell kill during induction therapy is therefore difficult to evaluate. No significant correlation was found between MRD day 29 and in vitro sensitivity to vincristine. There was, however, a statistically significant correlation between cellular drug resistance and MRD day 15 for vincristine, prednisolone, and doxorubicin, suggesting that all three drugs administered during the first week of treatment were of importance for very early cell kill. We have no explanation for the unexpected difference between day 15 and day 29 data for vincristine, but MRD analysis was optional at day 15 and data were available for only 59% of the patients. Cytogenetic aberrations, which identify distinct leukemia subgroups with differing response to therapy, correlate strongly to clinical outcome [3,17]. In children with t(12;21) there was a significant relation between MRD day 29 and in vitro sensitivity to several drugs. The strongest correlation was found for doxorubicin, and in multivariate analysis sensitivity to doxorubicin was the only independent factor for MRD <0.1% day 29, with a high predictive value (R2 = 0.66). In children with high hyperploidy, on the other hand, no correlation between MRD and in vitro sensitivity was found for any drug. This may reflect that leukemias with high hyperploidy constitute a heterogeneous group, containing subgroups with varying drug sensitivity. Three independent studies have shown that drug resistance profiles can identify patients at higher risk of early treatment failure but are less able to predict late relapses [7–9]. This fits well with the present finding that drug sensitivity correlates to the clearance of malignant cells from the bone marrow during induction therapy. Late relapses may develop from leukemic cells hidden in sanctuary sites or from cells unresponsive to chemotherapy by dormancy, and other factors than drug sensitivity might then be of importance, e.g. the ability of the immune system to recognise and eradicate these cells.

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In view of previous and present findings, what is the future role of in vitro sensitivity testing in the management of childhood ALL? A crucial question is whether or not drug testing adds prognostic information to what is achieved from analysis of MRD. The follow-up is too short, and our patient material is probably too small, to answer this question. Prospective studies on a large number of uniformly treated patients, in which both techniques are performed and evaluated with regard to clinical outcome, are required to study also less common cytogenetic aberrations. Such studies are presently undertaken by the Dutch (DCOG) and the German COALL groups [9], and further data collection is ongoing also in the NOPHO group. A great difficulty in interpreting data from in vitro drug testing in relation to clinical outcome has been the fact that the treatment of ALL includes many drugs in various combinations over a long period of time. The present study demonstrates that analysis of MRD offers new possibilities to study the effect of drugs administered during induction therapy. Our present study also illustrates the fact, that ALL is a heterogeneous disease, where analysis of data should take into account the influence of cytogenetic aberrations. In spite of a relatively small number of patients, our analysis of patients with t(12;21) revealed a correlation between drug sensitivity and MRD day 29. While prednisolone showed the strongest correlation to MRD for the whole group of ALL patients, doxorubicin sensitivity correlated strongest to MRD within the t(12;21) subgroup. This is interesting in view of the finding, that cells from t(12;21) patients have an increased sensitivity to doxorubicin [22]. High throughput techniques should be used to clarify the mechanisms by which cytogenetic aberrations are linked to specific drug sensitivity profiles in childhood ALL, with the ultimate goal of finding new therapies that allow a more specific therapy than today [34–36]. In conclusion, our data show a significant correlation between in vitro cellular drug sensitivity at diagnosis and cell kill during induction therapy as measured by MRD day 29. Further studies are needed to clarify whether or not drug testing adds prognostic information to what is achieved from analysis of MRD.

Conflict of interest No conflicts of interest to report.

Acknowledgement This study was supported by the Swedish Children’s Cancer Foundation. Contributions. GL, EF: main authors, data interpretation. BMF, RL: in vitro sensitivity testing. IT, CS, SJ, AL, TO, AP: MRD analyses and data interpretation, manuscript work. JA, MB, JH, SS: sample collection, clinical data reports, manuscript work.

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