ARTICLE

Predicting Resistance or Response to Chemotherapy by Proton Magnetic Resonance Spectroscopy in Neuroblastoma

Magnus Lindskog, Christian Spenger, Jüri Jarvet, Astrid Gräslund, Per Kogner

Affiliations of authors: Childhood Cancer Research Unit, Department of Woman and Child Health, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden (ML, PK); MR-Centre, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden (CS); Astra Zeneca R & D, Södertälje, Sweden (CS); Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden (JJ, AG)

Correspondence to: Magnus Lindskog, Childhood Cancer Research Unit, Q6:05, Astrid Lindgren Children’s Hospital, Karolinska Hospital, S-171 76 Stockholm, Sweden (e-mail: magnus.lindskog{at}kbh.ki.se)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background: We previously showed that proton magnetic resonance spectroscopy (1H-MRS) enables estimation of neuroblastoma tumor viability. Here we investigated if 1H-MRS can predict response or resistance to chemotherapy in neuroblastoma. Methods: Neuroblastoma cell lines with various drug sensitivities were treated with cytotoxic drugs (cisplatin, etoposide, and irinotecan) and examined by 1H-MRS. Viability was assessed by trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Nude rats carrying drug-sensitive or drug-resistant neuroblastoma xenografts were treated for 4 days with irinotecan (n = 11) or saline (n = 11) and were examined with 1H-MRS at 4.7 T before and during treatment. The Wilcoxon matched-pairs test was used to test statistical significance of difference within treatment groups. Independent groups were compared using the Mann–Whitney U test. Correlation was assessed with Spearman’s rank correlation. All statistical tests were two-sided. Results: Cytotoxic drug treatment of drug-sensitive SH-SY5Y neuroblastoma cells resulted in increased methylene and polyunsaturated fatty acid resonances and decreased choline resonance. The methylene/choline ratio correlated with cell death (rs = .94, P<.001) and was increased in cisplatin-treated drug-sensitive (SH-SY5Y, IMR-32) but not drug-resistant [SK-N-BE(2), SK-N-FI, SK-N-AS] cell lines. No changes were observed in SK-N-BE(2) cells treated with irinotecan or cisplatin, whereas circumvention of the resistance by arsenic trioxide treatment led to lipid accumulation and choline depletion. Irinotecan therapy of rats carrying drug-sensitive xenografts caused the methylene/choline ratio of tumors to increase eightfold after 3 days (95% confidence interval [CI] = fivefold to 12-fold; P = .005 compared with pretreatment spectra at day 0) and caused tumors to regress statistically significantly on day 10 compared with pretreatment volume on day 0 (difference = –60%, 95% CI = –12% to -100%, n = 6; P = .012). The methylene/choline ratio of nonregressing drug-resistant xenografts was unaffected. No differences were observed after saline treatment. Conclusions: Response or resistance to chemotherapy is accurately predicted by 1H-MRS in experimental neuroblastoma models in vivo.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Despite aggressive treatment, children with high-risk neuroblastoma remain difficult to cure (1). Repeated assessment of tumor response may help to optimize their treatment. Although standard imaging methods provide useful information on the size of the tumor and its relationship to adjacent anatomical structures, they do not accurately differentiate between viable tumor tissue, necrosis, and fibrosis. Additional information about the biologic activity of the tumor could allow early treatment modification. Because magnetic resonance imaging (MRI) is routinely used for tumor evaluation in neuroblastoma (2), does not involve ionizing radiation, is widely available, and is comparatively cheap, an magnetic resonance–based method to assess treatment efficacy would be of value.

Proton nuclear magnetic resonance spectroscopy (1H-MRS) provides detailed biochemical information about the tumor and can be performed in the same session as a diagnostic MRI (3). 1H-MRS is routinely performed in many adult and pediatric neurooncology centers (4,5). MRS of neuroblastoma was originally proposed by Maris et al. (6), who demonstrated 31P-MRS to be useful for disease monitoring. The potential of MRS methods, in particular 31P-MRS for assessment of pediatric extracranial tumors, was recently reviewed (7). 1H-MRS, however, is more widely available and shows greater sensitivity because of the high water (1H) content of the human body. Biologically interesting metabolites accessible with 1H-MRS include choline compounds and mobile lipids, both of which are commonly elevated in malignant tumors (810). Mobile lipids have been associated with apoptosis and necrosis (1113). We recently showed that 1H-MRS can detect metabolic changes in neuroblastoma xenografts after antiangiogenic treatment (14).

In the present study, we used experimental models of neuroblastoma to investigate whether 1H-MRS can detect early responses to cytotoxic treatment and predict the level of response or resistance. A panel of human neuroblastoma cell lines exhibiting various degrees of drug resistance or sensitivity was thus studied by 1H-MRS in vitro and, after establishment of xenografts, in vivo. The changes in spectral characteristics in response to treatment with clinically relevant chemotherapeutic drugs were monitored and compared with the chemosensitivity of the respective cell lines and with in vivo xenograft growth.


    METHODS
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Drugs and Chemicals

The cytotoxic drugs cisplatin, etoposide, and irinotecan were obtained from the Karolinska Hospital Pharmacy and stored in accordance with the instructions of the manufacturer. Arsenic trioxide (As2O3; Sigma Chemicals, Täby, Sweden) was dissolved in 1M NaOH and kept as a stock solution for up to 14 days. For cell culture experiments, the respective drugs were dissolved directly in the cell culture medium to achieve the desired concentrations.

Cell Lines and Culture Conditions

The human neuroblastoma cell lines cell lines SH-SY5Y, SK-N-BE(2), SK-N-FI, SK-N-AS, and IMR-32 were maintained at 37 °C in a humidified 95% air–5% CO2 atmosphere in Dulbecco’s modified Eagle minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU of penicillin G per milliliter, and 100 µg of streptomycin (Gibco BRL, Paisley, Scotland, UK) per milliliter. Cultures were free from mycoplasma, as verified by DNA staining. Of the cell lines used, SK-N-BE(2) and IMR-32 harbor MYCN amplification (15,16), the SK-N-BE(2) cell line harbors a mutant p53 (17), the SH-SY5Y and IMR-32 lines have normal p53 status (18), and the SK-N-AS cell line harbors a p73 deletion (19). The level of sensitivity or resistance of the respective cell lines to cytotoxic treatment was evaluated using the tetrazolium salt–based colorimetric assay (20).

In Vitro Growth Inhibition and Cell Death Assays

Cells were plated in 96-well plates (1 to 4 x 104 cells/well) and allowed to attach overnight. After incubation with cytotoxic agents for the desired period of time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/mL) was added, and the cells were further incubated for 3 hours in the dark before formazan crystals were dissolved by adding 150 µL of isopropanol with HCl (3.3 mL of 37% HCl per liter of isopropanol). The absorbance was measured spectrophotometrically at 595 nm. The absorbance from empty wells was used to calculate the background absorbance. The cytotoxic response was evaluated after 24, 48, and 72 hours, respectively.

1H-MRS In Vitro

Prior to preparation of samples for 1H-MRS, cells were counted under a microscope, and the number of viable cells was determined by their ability to exclude trypan blue. Cells were washed twice with phosphate-buffered saline, suspended in 600 µL of phosphate-buffered saline with 10% D2O, and placed on ice until data acquisition. Five-millimeter Shigemi tubes were filled with 5 x 107 cells, and samples were analyzed on a 500-MHz Bruker spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany) at 25 °C. The residual H2O signal at approximately 4.75 ppm was suppressed by low-power presaturation. The acquisition parameters included the following: 90° pulse, 1.5-second repetition time, 256 or 512 repetitions (depending on desired signal-to-noise ratio), acquisition time of 1.64 seconds, and spectral width of 5 KHz. Following acquisition, spectra were Fourier transformed and carefully phased if necessary. The resonances were assigned based on the known chemical shifts ({delta}) of major structural groups such as methylene, methyl, and choline, as previously described (14). The relative content of metabolites was estimated by peak area integration using XWINNMR software (version 3.1; Bruker) after appropriate baseline correction. The methyl resonance ({delta} = 0.9 ppm) was used as an internal reference, on the basis of a previous report showing that this peak does not undergo major changes during apoptosis of cancer cells in vitro (21). To compare spectra between treatment groups, we calculated metabolic ratios for a number of metabolites in each spectrum by dividing the integrated area under the curve for a relevant MRS peak by the integrated area under the curve for either the choline peak ({delta} = 3.2 ppm) or by the methyl peak ({delta} = 0.9 ppm) in the same spectrum. Metabolic ratios (expressed in arbitrary units) were compared between treatment groups, using sample sizes as indicated. Changes in cell viability during spectroscopic analysis were negligible, as confirmed by rescanning cell suspensions after 4 hours of storage on ice (data not shown).

Neuroblastoma Xenograft Model

Male nude rats (HsdHan and RNU-rnu; Harlan, Horst, The Netherlands) (n = 30) 5 to 6 weeks of age were used for establishment of xenografts as previously described (14). The animal experiments described in this report were approved by the regional ethics committee for animal research in accordance with the Animal Protection Law (SFS 1988:534), the Animal Protection Regulation (SFS 1988:539), and the Regulation for the Swedish National Board for Laboratory Animals (SFS 1988:541).

A total of 20 animals were injected with SH-SY5Y cells, and 10 animals received SK-N-BE(2) cells. Tumors were palpable within 2 to 4 weeks in 27 of 30 animals. The remaining three animals (all SH-SY5Y) were excluded from further analyses. Tumors were measured with a digital caliper every other day, and tumor volume was calculated as length x width2 x 0.44 (22). Of the animals carrying SH-SY5Y xenografts, five rats were evaluated in an initial separate pilot study to gain insight in chemotherapy-induced spectroscopic changes and were treated with irinotecan (using the schedule described) and examined by 1H-MRS before and after 2 and 3 days of chemotherapy. These five rats were not included in the comparative analyses of independent treatment groups. The remaining rats with SH-SY5Y xenografts (n = 12) were randomized to either treatment with irinotecan (5 mg/kg of body weight, in 2 mL of saline, n = 6) or sham-treatment with saline only (n = 6). Animals carrying SK-N-BE(2) xenografts were likewise randomized to treatment with either irinotecan (5 mg/kg, in 2 mL of saline, n = 5) or saline (n = 5). Treatment was administered by daily intraperitoneal injections for 4 consecutive days and was then discontinued, followed by continued monitoring of tumor size for an additional 6 days (total, 10 days). One tumor in each animal was monitored by repeated 1H-MRS and volume measurements. To allow pretherapy 1H-MRS examination in all animals, treatment began at individualized time points. To adjust for interindividual differences in pretreatment tumor volumes, a tumor volume index was calculated for each xenograft tumor by dividing the calculated tumor volume at each time point by the tumor volume measured at start of treatment (day 0). In each treatment group, the final tumor volume index was calculated at the end of the study (day 10). The response to therapy was thus assessed by evaluating the effect of treatment on the mean tumor volume index in each treatment group, with an index of 1 indicating stable volume (no tumor growth or regression), an index less than 1 indicating decreased tumor volume, and an index greater than 1 reflecting tumor growth.

1H-MRS In Vivo

In vivo MRI and 1H-MRS examinations were performed using a 4.7-T magnet, as previously described. For 1H-MRS, a short echo time of 20 ms was used to allow detection of a maximum number of chemical groups. All animals were subjected to repeated single-voxel 1H-MRS examinations, with the first examination carried out before start of treatment (day 0) and later examinations after 2 days and 3 days of treatment. Six of the animals with SH-SY5Y tumors (irinotecan, n = 3; saline, n = 3) were subjected to additional 1H-MRS examinations on day 1 (24 ± 6 hours after the first injection) of treatment. We were careful to place the volume of interest as precisely as possible from one 1H-MRS examination to another so that the same part of a tumor would always be sampled. The relative content of metabolites was estimated by peak area integration after appropriate baseline correction. Metabolite values were corrected for tissue water content as obtained from non–water-suppressed spectra (eight averages) and expressed in arbitrary units (a.u.) with the mean relative choline content of untreated tumors (at day 0) set to 100 a.u.

Statistical Analyses

The Mann–Whitney U test was used to test statistical significance of differences in metabolic ratios obtained by 1H-MRS, differences in cell survival, and differences in mean tumor volume of independent groups. Correlation analysis was done by Spearman’s rank correlation. The Wilcoxon matched-pairs test was used to test the statistical significance of in vivo spectral alterations between measurements within the same tumors. All statistical tests were two-sided. A P value of less than .05 was considered to be statistically significant.


    RESULTS
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Metabolic Response of Neuroblastoma Cells to Cytotoxic Drug Treatment as Detected With 1H-MRS

Cytotoxic drug (irinotecan, cisplatin, etoposide) treatment of SH-SY5Y cells, using concentrations corresponding to 80%–90% inhibitory concentrations (IC80–90), was associated with changes in the signal intensity of several metabolites, as detected with 1H-MRS. Examples of time-dependent effects in response to irinotecan are shown in Fig. 1, A. Resonances of methylene groups from mobile lipids ({delta} = 1.3 ppm) and resonances from polyunsaturated fatty acid ({delta} = 2.8 ppm) increased in a time- and dose-dependent manner relative to the methyl resonance ({delta} = 0.9 ppm) (Fig. 1, A and data not shown). Conversely, choline compounds decreased upon cytotoxic challenge of SH-SY5Y cells (Fig. 1, A). 1H-MRS of SH-SY5Y cells treated for 72 hours with cytotoxic drugs (irinotecan or cisplatin) demonstrated that the mean methylene/methyl ratio of these treated cells was 3.6 (n = 7), and that of untreated cells was 1.7 (n = 10) (difference = 1.9, 95% CI = 1.3 to 2.6; P<.001). The mean choline/methyl ratio was statistically significantly lower in treated (0.4, n = 7) than in untreated (2.1, n = 10) (difference = –1.7, 95% CI = –2.3 to –1.3; P<.001). The 1H-MRS spectral changes in SH-SY5Y cells treated with chemotherapy were similar irrespective of the drug chosen to induce cell death (irinotecan, cisplatin, and etoposide). Spectra from treated cells could thus be analyzed as a single entity and compared with spectra obtained from untreated SH-SY5Y cells.



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Fig. 1. Proton magnetic resonance spectroscopy (1H-MRS) of neuroblastoma cells after chemotherapy. A) Time-dependent 1H-MRS changes in response to irinotecan treatment. 1H-MRS (500 MHz, 256 averages) spectra from bottom to top: cells incubated without (untreated) or with irinotecan (2.5 µg/mL) for 24, 48, or 72 hours. Spectra were scaled to give a constant height of the 0.9 ppm methyl resonance (23). Cho = choline compounds. B) Effects of cytotoxic treatment (48 hours incubation) on the mean 1H-MRS methylene/choline ratio. Untreated, n = 11; cis, cisplatin (5 µM, n = 3); VP-16, etoposide (1 µM, n = 3); irino, irinotecan (2.5 µg/mL, n = 4). Bars indicate upper 95% confidence intervals. C) Correlation between methylene/choline ratio and cell death (trypan blue positivity) after cytotoxic drug treatment, rs = .94, P<.001 (Spearman’s rank correlation, two-sided).

 
Methylene/Choline Ratio as Indicator of Cytotoxic Response

Pooled 1H-MRS spectra obtained from SH-SY5Y cells treated with a cytotoxic drug demonstrated a statistically significantly higher average methylene/choline resonance intensity ratio (14.5, n = 10) than that of untreated cells (0.89, n = 11) (difference = 13.6, 95% CI = 9.0 to 18.2; P<.001) (Fig. 1, B). In contrast, the mean methylene/choline ratio (2.5, 95% CI = 1.8 to 3.1, n = 8) of SK-N-BE(2) cells treated with a cytotoxic drug (cisplatin or irinotecan) in concentrations corresponding to the IC90 for SH-SY5Y cells was not statistically significantly different from the corresponding ratio obtained from untreated SK-N-BE(2) cells (2.4, 95% CI = 1.6 to 3.2, n = 6). Also, SK-N-BE(2) cells showed no increase in the proportion of nonviable cells in response to cytotoxic drugs under these experimental conditions. Comparison of drug-sensitive SH-SY5Y cells with drug-resistant SK-N-BE(2) cells after treatment with cytotoxic drugs (cisplatin, irinotecan) thus demonstrated a 5.8-fold higher methylene/choline ratio in the sensitive compared with the resistant cells (95% CI = 3.1-fold to 8.5-fold increase; P<.001).

Notably, cisplatin or etoposide at high (nonpharmacologic) concentrations (40 and 20 µM, respectively) was able to overcome the drug resistance of SK-N-BE(2) cells. Under these conditions, 1H-MRS demonstrated increased methylene intensity and depletion of total choline in both SH-SY5Y and in SK-N-BE(2) cells (data not shown).

The methylene/choline ratio, previously reported to reflect the viability of neuroblastoma tumor tissue (14), was statistically significantly nonlinearly correlated with cell death after cytotoxic treatment of SH-SY5Y cells in vitro (rs = .94, P<.001) (Fig. 1, C). The observed relationship can be described using an exponential fit with the equation y = 0.51 e0.030x.

Cisplatin as Model Drug To Validate 1H-MRS for Response Prediction

1H-MRS effects and dose–response relationships of five neuroblastoma cell lines treated with cisplatin were analyzed (Fig. 2). In the drug-sensitive SH-SY5Y and IMR-32 cell lines, treatment with cisplatin was associated with a marked increase in methylene ({delta} = 1.3 ppm) and polyunsaturated fatty acid ({delta} = 2.8 ppm) resonances and with a decrease in total choline ({delta} = 3.2 ppm) (Fig. 2, A). In contrast, spectra obtained from cells lines with drug-resistant phenotypes [SK-N-AS, SK-N-FI, SK-N-BE(2)] after treatment with cisplatin revealed no apparent metabolic changes compared with those obtained from corresponding untreated cells (Fig. 2, A and data not shown). The methylene/choline intensity ratios were elevated in SH-SY5Y and IMR-32 cells treated with cisplatin compared with those of untreated cells (Fig. 2, B), whereas it was unchanged in drug-resistant cell lines [SK-N-AS, SK-N-FI, SK-N-BE(2)]. Survival assays confirmed the statistically significant differences in the sensitivity to cisplatin between the metabolically responding cell lines and those without apparent 1H-MRS response (Fig. 2, C). However not significantly different, the magnitude of increase in methylene/choline ratio seemed somewhat more variable in cisplatin-treated IMR-32 cells than in cisplatin-treated SH-SY5Y cells (Fig. 2, B), despite the absence of a statistically significant difference in cisplatin toxicity between these two cell lines (Fig. 2, C). Two of the cell lines with relatively cisplatin-resistant phenotypes [SK-N-AS and SK-N-BE(2)] experienced a 40% growth inhibition in response to 5 µM cisplatin (Fig. 2, C). In terms of shape, these resistant cell lines appeared more rounded than their untreated counterparts but were not detached and appeared viable (data not shown). This cytostatic effect of cisplatin was thus not reflected in the 1H-MRS spectra. However, 1H-MRS was able to accurately predict complete response (>90% inhibition) of SH-SY5Y and IMR-32 cells to cisplatin in vitro and, based on the absence of substantial spectroscopic changes, was able to identify a group of poor or partial responders [SK-N-AS and SK-N-BE(2), and SK-N-FI]. On the basis of 1H-MRS findings, cells with a low level of response (SK-N-FI, 25% inhibition) could not be accurately separated from cells showing partial response (SK-N-AS, 45% inhibition) to cisplatin.



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Fig. 2. Proton magnetic resonance spectroscopy (1H-MRS) and survival of cytotoxic drug–sensitive and –resistant neuroblastoma cell lines. A) 1H-MRS of four neuroblastoma cell lines incubated without (NT) or with cisplatin (cis, 5 µM) for 48 hours. B) Effects of cisplatin on the methylene/choline ratios of sensitive and resistant cell lines (spectra in panel A). Mean values of three independent experiments with upper 95% confidence intervals are shown. C) Dose-dependent toxicity [by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] of cisplatin (48 hours) in the five neuroblastoma cell lines. Graph shows mean values of three independent experiments with upper 95% confidence intervals.

 
As2O3 Treatment, 1H-MRS Effects, and Cell Death in p53-Mutated Neuroblastoma Cells Resistant to Conventional Chemotherapy

As2O3 induces apoptosis of promyelocytic leukemia and solid-tumor cell lines, including neuroblastoma, in a non–p53-dependent manner (2427). SK-N-BE(2) cells are highly sensitive to As2O3 (25). Accordingly, treatment of these cells with As2O3 (8 µM) was associated with a pronounced (–70%) decrease in cell viability (data not shown). To determine if this sensitivity could be detected by 1H-MRS, we subjected SK-N-BE(2) cells to 1H-MRS after treatment with As2O3. 1H-MRS showed reduced choline intensity, increased polyunsaturated fatty acid, and increased methylene resonances, all after normalization to the methyl resonance (Fig. 3).



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Fig. 3. Proton magnetic resonance spectroscopy (1H-MRS) of SK-N-BE(2) cells treated with As2O3. A) 1H-MRS of SK-N-BE(2) cells treated with or without As2O3 (8 µM, 72 hours). The experiment was performed in duplicate and repeated twice. Cho = choline; PUFA = polyunsaturated fatty acid. B) Methylene/choline ratios obtained by 1H-MRS of SK-N-BE(2) cells incubated with irinotecan (irino) (2.5 µg/mL, 48 hours), As2O3 (8 µM, 72 hours), or without drug (NT). Mean values of four experiments with upper 95% confidence intervals are shown.

 
In Vivo Prediction of Neuroblastoma Xenograft Response by 1H-MRS

Irinotecan is highly effective against neuroblastoma xenografts (28,29) and was therefore used for in vivo validation of the 1H-MRS findings in two different xenograft models [SH-SY5Y and SK-N-BE(2)]. In vitro, the SH-SY5Y cells were sensitive to irinotecan (IC90 = 2.5 µg/mL), whereas the SK-N-BE(2) cells were strikingly resistant (IC50 > 50 µg/mL), as shown by tetrazolium salt–based colorimetric assay after 48 hours (data not shown). Treatment with irinotecan was associated with choline depletion and increased lipid methylene resonance in SH-SY5Y cells (Fig. 1, A), whereas 1H-MRS of SK-N-BE(2) cells treated with irinotecan demonstrated no differences compared with untreated cells (data not shown). Time-dependent 1H-MRS changes in an SH-SY5Y xenograft treated with irinotecan are shown in Fig. 4, A. Substantial changes in several peak intensities normalized to spectroscopic tissue water were observed in SH-SY5Y treated with irinotecan compared with pretreatment spectra. These metabolic changes included a statistically significantly increased methylene resonance intensity 2 days (difference = 318%, 95% CI = 135% to 506%; P = .028) and 3 days (difference = 361%, 95% CI = 196% to 426%; P = .035) after initiation of therapy. The mean polyunsaturated fatty acid resonance ({delta} = 2.8 ppm) intensity was statistically significantly increased after 3 days of irinotecan treatment compared with pretreatment measures (difference = 13.3-fold, 95% CI = 5.1-fold to 21.5-fold; P = .036). The signal intensity of total choline (relative to unsuppressed water) was highly variable in tumors before the start of irinotecan therapy (on average, 100 a.u., 95% CI = 25 to 175 a.u., n = 6). However, tumor total choline decreased in all SH-SY5Y tumors subjected to treatment with irinotecan. After 3 days of this treatment, total choline (relative to unsuppressed water) had decreased to 33 a.u. (95% CI = 0.23 to 0.43 a.u., yielding a difference of –67% [95% CI = –98% to –3%]) compared with pretreatment values (P = .03).



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Fig. 4. Proton magnetic resonance spectroscopy (1H-MRS) and growth of cytotoxic drug–sensitive and –resistant xenograft tumors after short-term treatment with irinotecan. A) Typical time course of in vivo 1H-MRS spectral changes in an SH-SY5Y xenograft tumor before and during treatment with irinotecan (5 mg/kg of body weight/day). The same spectral parameters, including the same voxel size, were used for repeated examinations. Stimulated echo acquisition (STEAM) sequence, echo time 20 ms, 512 repetitions. Cho = choline compounds; PUFA = polyunsaturated fatty acids; methylene (CH2 groups from lipids and possible contribution from lactate). B) In vivo tumor methylene/choline resonance intensity ratio as a function of treatment duration. The ratio of the 1.3 ppm resonance (lipid methylene groups), to the 3.2 ppm resonance (choline compounds) was calculated for each time point and compared with the corresponding ratio for the same voxel before start of therapy (ratio normalized to 1 at day 0). The relative methylene/choline ratios (mean values with upper 95% confidence intervals) for the different treatment groups are shown as fold change of the pretreatment values. Open circles, SH-SY5Y xenografts, irinotecan 5 mg/kg/day intraperitoneally (ip), n = 11; closed circles, SH-SY5Y xenografts, saline daily ip, n = 6; open triangles, SK-N-BE(2) xenografts, irinotecan 5 mg/kg/day ip, n = 5. C) Patterns of in vivo tumor response to irinotecan. The mean tumor volume indexes in the respective treatment groups are shown on a log scale for the time points indicated. Bars = upper 95% confidence intervals. Closed triangles represent SK-N-BE(2) xenografts treated with saline, n = 5. Other symbols are as described for panel B.

 
The methyl resonance ({delta} = 0.9 ppm) did not change with irinotecan treatment (P = .35) and was therefore used as an internal reference. The mean methylene/methyl ratio of the SH-SY5Y tumors increased statistically significantly, compared with pretreatment spectra, in response to irinotecan (difference = 473%, 95% CI = 141% to 805%; P = .012). This ratio was not affected by saline treatment. After 3 days of treatment, the mean methylene/methyl ratio was statistically significantly higher in the irinotecan group (mean = 4.6) than in the saline group (mean = 1.7) (difference = 2.9, 95% CI = 1.2 to 4.6; P = .03).

1H-MRS Methylene/Choline Ratio In Vivo in Response to Irinotecan Treatment

The mean tumor methylene resonance intensity (relative to unsuppressed water) of SH-SY5Y xenografts treated with saline for 3 days was not statistically significantly different from methylene values obtained before the start of treatment (difference = –9%, 95% CI = –86% to 68%). Consequently, after 3 days of treatment, the tumor methylene resonance intensity (normalized to spectroscopic tissue water) was statistically significantly higher in SH-SY5Y tumors in the irinotecan-treated group compared with SH-SY5Y tumors in the saline-treated group (difference = 470%, 95% CI = 340% to 600%; P = .002). No statistically significant change in the tumor choline resonance intensity was seen after 3 days of saline treatment compared with pretreatment measurements (difference = 15%, 95% CI = –56% to 86%). After 3 days, the tumor total choline (normalized to spectroscopic tissue water) was statistically significantly lower in SH-SY5Y tumors treated with irinotecan than in saline-treated control tumors (difference = –48%, 95% CI = –95% to –5%; P = .047). A statistically significantly increased methylene/choline ratio was observed in SH-SY5Y xenografts treated with irinotecan after 1 day (mean = 2.9, difference = 465%, 95% CI = 189% to 741%; P = .030), 2 days (mean = 3.0, difference = 476%, 95% CI = 337% to 615%; P = .028), and 3 days (mean = 5.3, difference = 858%, 95% CI = 539% to 1177%; P = .005) of daily injections, all values compared with pretreatment spectra on day 0 (mean = 0.6) (Fig. 4, B). In contrast, the mean methylene/choline ratio did not change in response to saline treatment (mean = 0.6 after 3 days) compared with corresponding pretreatment value (mean = 0.8 on day 0) (difference = –25%, 95% CI = –100% to 78%; P = .24) (Fig. 4, B). The methylene/choline ratio was statistically significantly higher in the irinotecan-treated group (mean = 5.0, n = 6) than in the saline-treated group (mean = 0.5, n = 6) of SH-SY5Y tumors after 3 days of treatment (difference = 10-fold higher, 95% CI = 5.6-fold to 14.4-fold; P = .004). The polyunsaturated fatty acid/choline ratio was statistically significantly increased in SH-SY5Y tumors after 2 days (difference = 871%, 95% CI = 325% to 1400%; P = .046) and 3 days of irinotecan treatment (difference = 1237%, 95% CI = 505% to 1967%; P = .018) compared with pretreatment spectra obtained from the same tumors (n = 10). Saline treatment had no effect on this ratio (P = .80).

Irinotecan treatment of animals carrying SK-N-BE(2) xenografts did not induce substantial changes in the tumor lipid (methylene and polyunsaturated fatty acid) or choline content. The mean methylene/choline ratio of SK-N-BE(2) xenografts did not change in response to treatment with irinotecan (P = .65) (Fig. 4, B). The difference in the methylene/choline ratio between SH-SY5Y and SK-N-BE(2) xenografts treated with irinotecan for 3 days was statistically significant (P<.001) (Fig. 4, B).

1H-MRS To Predict Regression of Neuroblastomas and Identify Resistant Tumors Early After Initiation of Chemotherapy In Vivo

Figure 4, C shows the effects of respective therapy on tumor volume change as monitored repeatedly from start of therapy for SH-SY5Y and SK-N-BE(2) xenografts, respectively. The tumor volume of SH-SY5Y xenografts treated with irinotecan started to decrease from day 4, and at day 10, the mean tumor volume in this group of six rats was 1.0 mL, corresponding to a 60% decrease compared with pretreatment volumes at day 0 (2.6 mL) (difference = –60%, 95% CI = –12% to 100%; P = .012). The mean tumor volume of SK-N-BE(2) xenografts treated for 3 days with irinotecan was not statistically significantly different on day 10 compared with pretreatment volumes (Fig. 4, C). A day 10 comparison of the groups of animals previously treated for 3 days with irinotecan showed the tumors of the six rats with SH-SY5Y xenografts to be statistically significantly smaller (1.0 mL, 95% CI = 0 to 2.2 mL) than those of rats with SK-N-BE(2) tumors (2.8 mL, 95% CI = 0.4 to 5.3; P = .02). In saline-treated rats with xenografts derived from either cell line, tumors increased exponentially in volume at similar growth rate (Fig. 4, C). Thus, tumors characterized by early changes in the lipid and choline homeostasis (SH-SY5Y) after a short period of irinotecan therapy (3 days) later regressed statistically significantly (after 10 days), whereas the absence of early statistically significant changes on 1H-MRS after irinotecan (day 3) predicted resistance to irinotecan [SK-N-BE(2) xenografts].


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study demonstrates that changes in the lipid and choline homeostasis monitored by 1H-MRS predict response to chemotherapy in experimental neuroblastoma. In particular, the methylene/choline ratio (14) seems to be a useful predictor of the level of response to cytotoxic treatment in vivo. On the basis of 1H-MRS findings, it was possible to group neuroblastoma cell lines as highly responsive or relatively resistant to cisplatin (Fig. 2), an agent often used in clinical neuroblastoma protocols (30,31). Importantly, in chemoresistant SK-N-BE(2) cells (17), substantial 1H-MRS changes of lipids or choline were not observed after treatment with chemotherapy (Figs. 2 and 3), whereas circumvention of the resistance by As2O3 treatment resulted in reduced choline and increased lipid resonances (Fig. 3). These results suggest that 1H-MRS may be suitable for predicting the magnitude of response or resistance of an individual tumor to standard chemotherapy and for early assessment of response to experimental salvage therapy in chemotherapy-resistant neuroblastoma. To our knowledge, this study provides the first experimental evidence that the presence or absence of lipid and choline changes detected by 1H-MRS shortly after administration of chemotherapy can predict the magnitude of response or resistance of a human cancer.

Tumor p53 status, in addition to other mechanisms of anticancer drug resistance, is an important determinant of cellular response to DNA-damaging agents (32,33). Mutated p53 is associated with a poor response to irinotecan in a xenograft model of colorectal carcinoma (34). In the present study, SK-N-BE(2) xenograft tumors with mutated p53 did not respond metabolically to irinotecan in vivo and failed to regress despite therapy (Fig. 4, B and C). Interestingly, p53-independent Bax-mediated cell death induced by As2O3 (27) was associated with a spectroscopic response similar to that of cytotoxic drug–induced cell death in SK-N-BE(2) cells (Fig. 3). The SK-N-FI and SK-N-AS cell lines, both of which are resistant to cisplatin and are nonresponders on 1H-MRS, display high expression and functional activity of P-glycoprotein and a highly active multidrug-resistance–related protein 1, respectively (35). SK-N-AS, in addition, harbors a deletion of p73, an important target gene of p53 (19).

Increased 1H-MRS lipid methylene resonance can be detected in lymphoma and carcinoma cell lines after cytotoxic treatment that is statistically correlated with the extent of apoptotic cell death (12,21,36). In the present study, xenograft growth or regression was chosen as the primary endpoint, and no attempts were made to analyze early histologic changes. However, we previously reported a statistically significant inverse correlation between the in vivo methylene/choline ratio and the tumor viability of neuroblastoma xenografts (14). In addition, here we observed an increase in the methylene/methyl ratio in response to chemotherapy, in agreement with findings in other tumor models (11,21).

The role of 1H-MRS signals from polyunsaturated fatty acids in malignancy was pioneered by J. M. Hakumaki et al. (37), who demonstrated increased levels in rat gliomas in vivo during gene therapy–mediated apoptosis. They proposed a functional role for phospholipase A2 in the occurrence of these lipids. A recent report suggests that damage to mitochondria, which are known to be rich in polyunsaturated fatty acids, may be the cause of increased 1H-MRS–visible lipids and lipid droplet formation (38).

The metabolic events detectable with 1H-MRS in response to cytotoxic treatment depend on the magnitude of the tumor response, as shown in the present study. The mechanism of action of the drug is not crucial to the predictive value of 1H-MRS analysis because anthracyclines (21), platinum compounds, topoisomerase inhibitors, and pyrimidine analogues (13), in addition to more experimental regimens such as gene therapy (39) or As2O3 treatment, all result in the accumulation of 1H-MRS–visible lipids in cancer cells. However, certain anticancer drugs do not cause lipid accumulation, as shown for methotrexate in vitro (13). Treatment of neuroblastoma cells with the synthetic retinoid fenretinide (N-[4-hydroxyphenyl]retinamide), which induces a mixed mode of cell death (40), is not associated with lipid accumulation as assessed by 1H-MRS (M. Lindskog, unpublished observation).

Decreased choline intensity on 1H-MRS may reflect tumor cell death (11) and has been associated with treatment response of human brain tumors (9,10). However, choline homeostasis may be differentially affected when novel experimental treatment approaches are used (39,41). Therefore, preclinical evaluation of the metabolic response to an experimental therapy should preferably precede application of 1H-MRS for clinical response assessment. Nevertheless, 1H-MRS may be widely applicable for therapeutic monitoring and early identification of good and poor responders among cancer patients because the 1H-MRS response is not specific for a particular histologic origin of the tumor but appears to provide general surrogate markers of cytotoxicity.

1H-MRS has several limitations, including the need for macroscopic tumors (typically >1 cm3) and vulnerability to magnetic field disturbances due to patient motion. The data may be of inferior quality when pronounced tissue heterogeneity exists in the sample. Tissue heterogeneity may, however, be circumvented by two- or three-dimensional multivoxel spectroscopy (spectroscopic imaging) (42). The absence of substantial spectroscopic changes in SK-N-BE(2) xenografts despite growth stabilization indicate that 1H-MRS may be limited to detection of pronounced cytotoxicity and prediction of tumor regression, whereas disease stabilization might not be accurately predicted. This limitation is also supported by the absence of 1H-MRS findings in cell lines responding partially to cisplatin in vitro (Fig. 2).

A substantial proportion of neuroblastoma patients are diagnosed with tumors that are not completely resectable and are metastatic, and/or the patients experience local relapses (43,44). In patients who have unresectable primary tumors, response to chemotherapy is associated with both progression-free and overall survival (44). In addition to neuroblastoma, early identification of poor and good responders to treatment may be of importance in other solid childhood tumors (45,46). In children with recurrent brain tumors, 1H-MRS assessment of tumor choline provides predictive information (10). Because high-risk neuroblastomas are commonly metastatic, whole-body scanning with 123I-MIBG (metaiodobenzylguanidine) scintigraphy or bone scan (technetium), rather than localized imaging, is often used for diagnostic workup (47,48). Our findings, however, indicate that localized 1H-MRS may be advantageous for early treatment evaluation in neuroblastoma patients.

Although experimental, the present study strongly suggests that 1H-MRS is likely to provide biochemical surrogate markers of neuroblastoma treatment response, obtainable through standard magnetic resonance scanners. Preliminary results from an ongoing pilot study at our institution suggest that 1H-MRS examinations are feasible and informative in children with neuroblastoma or other solid tumors.

In conclusion, we have shown that response or resistance to chemotherapy can be accurately predicted by 1H-MRS in experimental neuroblastoma in vivo. Trials to validate this application in children with neuroblastoma and other solid tumors are warranted.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Supported in part by grants from the Children’s Cancer Foundation, Mary Béve’s Childhood Cancer Foundation, Stockholm Cancer Society, and the Swedish Cancer Society.

We are grateful to Dr. Frida Ponthan, Dr. Tomas Klason, and Dr. Peter Damberg for valuable discussions and technical assistance and to Ms. Lena Klevenvall and Ms. Lotta Elfman for kind and skillful assistance in the laboratory.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Manuscript received March 23, 2004; revised July 28, 2004; accepted August 5, 2004.


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