Preclinical report 415
Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumor cells Erik Berglunda,b,*, Sarojini Jayantha Kumari A. Ubhayasekerad,*, Fredrik Karlssona,b, Pinar Akcakayac, Warunika Aluthgedarad, Jan A˚hlena,b, Robin Fro¨boma, Inga-Lena Nilssona,b, Weng-Onn Luic, Catharina Larssonc, Jan Zedeniusa,b, Jonas Bergquistd and Robert Bra¨nstro¨ma,b Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-offunction mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure
Introduction The majority of gastrointestinal stromal tumors (GISTs) express constitutively activated forms of either the c-KIT (a stem cell factor receptor) or the platelet-derived growth factor alpha (PDGFRA) receptor tyrosine kinase proteins [1,2]. Underlying gain-of-function mutations appear early in the tumor formation, and imatinib mesylate is a tyrosine kinase inhibitor (TKI) that binds to the intracellular ATP-binding site of the c-KIT or the PDGFRA receptor [3–5]. Inhibition of c-KIT and PDGFRA causes a rapid block in tumor signal transduction and, consequently, tumor regression. The drug also inhibits ABL and the BCR-ABL fusion protein observed
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib c levels in GIST patients. Anti-Cancer Drugs 25:415–422 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:415–422 Keywords: gastrointestinal stromal tumor, imatinib, intracellular concentration a Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, bDepartment of Breast and Endocrine Surgery, Karolinska University Hospital, cDepartment of Oncology–Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm and dDepartment of Chemistry, Biomedical Center, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
Correspondence to Erik Berglund, MD, Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital L1:03, SE-171 76 Stockholm, Sweden Tel: + 46 73 918 12 33; fax: + 46 8 331587; e-mail: [email protected] *Erik Berglund and Sarojini Jayantha Kumari A. Ubhayasekera contributed equally to the writing of this article. Received 25 June 2013 Revised form accepted 28 November 2013
in Philadelphia chromosome-positive chronic myeloid leukemia (CML) patients. GISTs are highly resistant to conventional radiotherapy and chemotherapy [6], and the introduction of TKIs in the treatment of advanced and metastatic GISTs has improved patient survival and outcome markedly [7,8]. Imatinib is also used in the neoadjuvant and adjuvant setting, and recently published data support a 3-year adjuvant treatment regime with a lower risk of tumor recurrence [9]. However, even though imatinib and other TKIs have revolutionized the treatment of GIST, local recurrence, metastasis, and tumor resistance still remain major therapeutic challenges [10]. Imatinib leads to disease control in B85% of patients with advanced GIST, but resistance to the drug develops in most patients within 2–3 years on therapy. As clinical experience with imatinib consistently grows, it becomes DOI: 10.1097/CAD.0000000000000069
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416 Anti-Cancer Drugs 2014, Vol 25 No 4
clearer that different resistance patterns exist. Primary, or early, TKI resistance is observed within the first 3 months of therapy. If GISTs progress after more than 6 months of an initial response or stable disease, they are classified as having a secondary or an acquired resistance. There are several mechanisms leading to primary resistance. The most important is the mutational status of KIT and PDGFRA. Another mechanism is drug metabolism, which affects imatinib plasma levels and thus the clinical response; low imatinib plasma concentrations, less than 1100 ng/ml, are associated with a decreased progressionfree survival [11,12]. Therefore, customizing drug dosing can influence both patient outcome and reduce the frequency of adverse effects [13,14]. Mechanisms of delayed resistance are most commonly secondary mutations in KIT or PDGFRA in addition to the initial mutation, and other postulated genetic mechanisms [11]. It is not known whether the plasma concentration of imatinib correlates with the intracellular concentration of the drug in GIST. Drug transporters play a crucial role in uptake and drug efflux at the cellular level, and they have been implicated as a cause of resistance in many diseases [15]. The uptake of imatinib is an active process [16], and the function is exerted on the intracellular part of the tyrosine kinase protein. A possible explanation for some GISTs developing resistance to TKIs is the interpatient variability in drug transporter activity and expression that leads to decreased intracellular concentrations. Both efflux and uptake transporters in imatinib pharmacokinetics and pharmacodynamics are suggested to be potentially involved in secondary drug resistance in CML [17]. It has not yet been clarified how imatinib is transported into and out of GIST cells, and whether such actions may be contributory mechanisms of resistance. In this study, a novel protocol for the measurement of intracellular concentrations of imatinib, using an in-vitro and an in-vivo system of GIST cells, was developed.
Materials and methods Materials
All chemicals and reagents used in this study were of analytical grade. The internal standard trazodone (C19H22ClN5O) and methanol were obtained from Sigma Aldrich (Stockholm, Sweden). All other chemicals and reagents were purchased from Merck, Eurolab (Stockholm, Sweden) unless otherwise stated. Water was deionized and osmosed with a Milli-Q purification system (Millipore, Bedford, Massachusetts, USA). Imatinib mesylate (C29H31N7OCH4SO3) was kindly donated by Novartis (Basel, Switzerland).
vided by Professor Jonathan Fletcher at Brigham and Women’s Hospital (Boston, Massachusetts, USA). GIST882 is an imatinib-sensitive GIST cell line derived from a patient with an untreated metastatic human GIST expressing a KIT allele with a homozygous exon 13 missense mutation, encoding a K642E mutant c-KIToncoprotein [18]. GIST882 was verified as described previously [19] and maintained in RPMI-1640 medium, supplemented with 15% fetal bovine serum (FBS), 0.25 mg/ml L-glutamine, 100 U/ml penicillin G, 100 mg/ml streptomycin-sulfate, and 0.21 mg/ml amphotericin B at 5% CO2 and 371C. GIST48 is an imatinib-resistant cell line established from a GIST that progressed, after an initial clinical response, during imatinib therapy. GIST48 has a primary homozygous exon 11 missense KIT mutation (V560D) and a heterozygous secondary exon 17 (kinase activation loop) mutation (D820A) [20]. Cells were cultured routinely in F10 HAM medium (Cat no. 31550; Gibco, Grand Island, New York, USA) supplemented with 15% FBS, MITO + serum extender (Cat no. 355006; BD Biosciences, San Jose, California, USA), Bovine Pituitary Extract (Cat no. 354123; BD Biosciences), 0.25 mg/ml L-glutamine, 100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, and 0.21 mg/ml amphotericin B at 5% CO2 and 371C. Cells were passaged weekly and cultured in 175 cm2 flasks. The human embryonic kidney 293 (HEK-293) cell line was obtained from System Biosciences (Cat no. LV900A-1). Cells were maintained in DMEM (Cat no. 41965-039; Invitrogen), supplemented with 10% FBS, 2 mmol/l L-glutamine, and 100 U/ml penicillin G. Clinical sample collection and preparation
Three newly treated patients at the unit for endocrine and sarcoma surgery, Karolinska University Hospital (Stockholm, Sweden), were included in this study. Patients were eligible in cases of advanced GIST, as characterized by CD117 positivity, for which neoadjuvant daily treatment with 400 mg imatinib is indicated. GIST1 was a wild-type small intestine GIST, imatinib treated for 16 months before surgical resection. GIST2 was a gastric GIST, with a c-KIT exon 11 mutation, and was imatinib treated for 11 months before surgery, and GIST3 was a gastric GIST harboring an exon 18 mutation in the PDGFRA receptor (Table 1). Tumor and reference adipose tissues were excised from each case at the time of surgery. Two peripheral blood samples were drawn at the same time as the tumor was removed or the blood supply to the tumor was discontinued. Blood plasma was then prepared by centrifugation and snap frozen to – 801C together with tumor and reference tissues until further analysis. The local ethics committee at Karolinska University Hospital approved the study, and all three patients provided their signed informed consent.
Cell culture
KIT mutation analysis and detection of CD117 expression in GIST48
Two GIST cell lines were used to examine the intracellular concentration of imatinib mesylate, both generously pro-
Evaluation of KIT status and expression of the cell surface marker CD117 in GIST882 were verified as described
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Intracellular imatinib in GIST Berglund et al. 417
Table 1
Patient characteristics
Sex Age at operation (years) Primary tumor site TNM stage [21] Time on imatinib before surgery (months) Daily imatinib dose (mg) RECIST criteria [22] FDG-PET uptake Before imatinib During imatinib Largest tumor size (cm) Size range (cm) Number of tumors Necrosis GIST subtype Mutational status Immunohistochemical studies Ki-67 Microscopic treatment response
GIST1
GIST2
GIST3
Male 63 Small intestine IV 16 400 PD
Male 83 Stomach II 11 400 PR
Male 65 Stomach IV 12 400 PR
NA NA 10 5–10 3 > 50% Spindle cell Wild type CD117 + , DOG1 – 15% No
Medium–high Low 11.5 na 1 < 50% Spindle cell c-kit, exon 11 CD117 + , DOG1 + < 1% Yes
Low (necrotic) NA 8 0.5–8 3 < 50% NA PDGFRA, exon 18 CD117 + , DOG1 + < 1% Yes
FDG-PET, fluorodeoxyglucose-PET; GIST, gastrointestinal stromal tumor; na, not applicable; NA, not available; PD, progressive disease; PR, partial response; RECIST guidelines, Response Evaluation Criteria in Solid Tumors Guidelines.
previously [19]. The same protocols were used for verification purposes of the GIST48 cell line. In brief, an automated Maxwell 16 DNA purification system kit AS1020 (Promega Corporation, Madison, Wisconsin, USA) was used for purification of genomic DNA from the GIST48 cell line. Cell lysate was transferred to the Maxwell cartridge and eluted genomic DNA concentration and purity was determined in a NanoDrop spectrophotometer ND-1000 (Thermo Scientific, Wilmington, Delaware, USA). PCR and sequencing were carried out to detect mutations in exons 9, 11, 13, and 17 of the KIT gene, as described previously by Sihto et al. [23]. Mutations were referenced from http://www. ensembl.org and http://www.sanger.ac.uk/genetics/CGP/cosmic/. For evaluation of CD117 (c-KIT) expression on GIST48 cells, the Miltenyi Biotec’s protocol was used according to the manufacturer’s instructions. The HEK-293 cell line was used as a negative control. Cells were incubated with either an allophycocyanin-coupled antibody against human CD117 (Cat no. 130 091 733), obtained from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), or an allophycocyanin-coupled mouse IgG1 (Cat no. 345818) antibody, obtained from Becton Dickinson GmbH (Franklin Lakes, New Jersey, USA), for isotype control experiments. Cell fluorescence was determined by flow cytometry (Gallois instrument; Beckman Coulter, Brea, California, USA). Imatinib incubation
GIST882 and GIST48 cells were cultured for four to five days in 175 cm2 flasks until near total confluence. Imatinib mesylate (Glivec, formerly STI571; a gift from Novartis Pharmaceuticals, Basel, Switzerland) was dissolved and diluted in cell culture medium, supplemented with only 1% FBS to minimize protein binding of imatinib [24,25] to final concentrations of 1100 and 3300 ng/ml.
For the in-vitro experiments, the cell culture medium was removed and the cell monolayers were washed carefully. Imatinib mesylate was added to GIST882 and GIST48 cells at final concentrations of 1100 and 3300 ng/ml and incubated for 3 h. As the active imatinib uptake is temperature dependent [16], all incubations were carried out in a humidified incubator at 5% CO2 and 371C. The supernatant was removed and the cells were rewashed carefully with prewarmed Dulbecco’s PBS (Cat no. 14190). Subsequently, 15 ml trypsin/EDTA was added to each flask for 7 min to detach the cells. The content from each flask was transferred to a Falcon tube and centrifuged for 5 min at 1000 rpm. After centrifugation and removal of the supernatant, the cell pellet was snap frozen to – 801C before imatinib concentration analysis by liquid chromatography–mass spectrometry (time-offlight) [LC-MS (TOF)]. Imatinib and trazodone stock solutions (1 mg/ml) and working solutions (1, 10, and 100 mg/ml) were prepared in methanol for LC-MS (TOF) analysis. The stock solutions were kept at – 801C before use. Calibration standards for the imatinib analysis in plasma samples were prepared by adding appropriate amounts of the working solutions to blank plasma (200 ml). Quantification of imatinib uptake by LC-MS (TOF)
Homogenized cell culture and tumor tissue (50 mg) sample preparation consisted of two steps: liquid–liquid extraction and enrichment before analysis by an LC-MS. In brief, each cell culture and tumor sample was introduced in glass tubes (DURAN 12 100 mm with Teflon screw-cap) with 20 ml of trazodone as an internal standard (IS) in methanol (10 mg/ml). Then, 0.5 ml of methanol was added to each tube and homogenized with a 3 mm probe (Sonics Vibra Cell; Sonics & Materials Inc., Newtown, Connecticut, USA) at 30% amplitude
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418 Anti-Cancer Drugs 2014, Vol 25 No 4
for 1 min. The samples were vortexed for 10 min and centrifuged at 41C for 15 min at 14 000g. The resulting supernatant was transferred to 1.5 ml Eppendorf tubes, the tubes were blown down with a stream of pure nitrogen, and the resulting dried residues were reconstituted with 200 ml methanol : water (20 : 80, v/v) before the LC-MS analysis. The final solution was transferred to an HPLC autosampler vial and 2 ml was injected into the LC-MS system. The LC-MS system consisted of an Agilent model 1100 Autosampler (Agilent Technologies, Palo Alto, California, USA), an Agilent 1100 Quaternary pump, and an Agilent ZORBAX C18 (5 mm, 2.1 50 mm) reversed-phase analytical column controlled by Agilent ChemStation software (Agilent Technologies, Palo Alto, California, USA). The mobile phase runs at a linear gradient containing 0.1% formic acid in both methanol and water, with the flow rate starting from 0.2 ml/min. The total run time of the LC program was 14 min. The chromatographic system was coupled to a TOF mass spectrometer (Agilent Technologies, Santa Clara, California, USA) equipped with electrospray ionization. The detection was performed in a positive ion mode. The ionization potential was 3800 V and the ion source temperature was 3001C. The nebulizing gas was used for electrospray ionization at the rate of 15 psi. The voltages fixed at the fragmentor, skimmer, and octopole guides were 225, 60, and 250 V, respectively. The ion pulser at the TOF analyzer was set at the measurement frequency of 2 cycles/s. Peak lists were achieved using the molecular feature extractor software ‘MassHunter’ (Agilent Technologies, Santa Clara, California, USA). Extraction of total ion chromatogram for imatinib and trazodone was performed with m/z ranges 494.15–494.3 and 372.1–372.2, respectively. The calibration curve for the analysis of imatinib in plasma was prepared by plotting the peak area ratio of imatinib/peak area of IS versus each imatinib concentration. The linearity was determined by linear regression analysis. The correlation coefficient of linear regression (r2) of imatinib was 0.9940. Quantification of imatinib (area response) in cell culture and tissue samples was carried out using the internal standard. The imatinib concentration of each measured cell culture sample is given in ng per mg protein. Samples were run three times to ensure accurate measurements. An in-house validated method (Dot-it-Spot-it protein assay, http://dot-it-spot-it.com/; Maple Stone AB, Uppsala, Sweden) was used to quantify the protein content.
prevent protein degradation. After homogenization, the samples were incubated for 1 h at 41C with mild agitation. Consequently, the cell lysates were clarified by centrifugation for 30 min (10 000g at 41C) using a Sigma 2K15 ultracentrifuge (Sigma Aldrich). The supernatant was collected and further processed. The Dot-it-Spot-it protein assay (http://dot-it-spot-it.com/; Maple Stone AB) was used for the protein assay according to the instructions provided. Detection sheets, carbon black detection solution, and washing solution were included. The sample (1 ml) was dispensed on the detection sheets in replicates and dried rapidly. The sheets were then placed in a large well with 1 ml of detection solution and incubated for 5 min, followed by 5 min incubation in 1 ml of washing solution. The absorbent sink was removed from the sheets. The sheets were then dried and mounted on the scanning template, which was detected by an Epson Expression 1600 Pro scanner (Epson, Long Beach, California, USA). The intensity of blackness was quantified in each dedicated spot on the image with Image J (http://rsbweb.nih.gov/ij/). Protein concentrations were estimated by comparing the sample results with the results from a calibration curve using Rodbard curve fitting in Image J. Statistical analysis
Statistical significance was determined using an unpaired Student’s t-test, and P-values less than 0.05 were considered significant. Data are expressed as means±SEM.
Results Mutational analysis and CD117 expression
Sequencing of KIT exons 9, 11, 13, and 17 in the GIST48 cell line confirmed the presence of the primary homozygous exon 11 missense mutation (V560D) and the heterozygous secondary exon 17 (kinase activation loop) mutation (D820A) (Supplemental digital content 1, http://links.lww.com/ACD/A51). Confirmation of the GIST48 phenotype verified high expression of the cell surface marker CD117 in 99% of GIST48 cells, compared with a low expression in the HEK-293 cell line (Supplemental digital content 2, http://links.lww.com/ACD/A52). Sequencing of KIT and CD117 expression analysis in GIST882 has been performed previously in GIST882, which verified the GIST882 genotype and phenotype [19]. The GIST1 tumor showed mutations in neither KIT nor PDGFRA exons, thereby classified as a wild-type GIST tumor. GIST2 contained two point mutations in exon 11: c.1695G > T and c.1696G > T (V559F), whereas GIST3 contained an exon 18 mutation in the PDGFRA receptor.
Protein determination
The protein precipitates were homogenized for 60 s using a probe with a 0.5 ml lysis buffer (10 mmol/l Tris-HCl pH 7.4, 0.15 mol/l NaCl, 1 mmol/l EDTA, and PBS containing 1% octyl-b-D-glucopyranoside). Protease inhibitor cocktail (10 ml) was added during the sample preparation to
Imatinib uptake into GIST cells
Intracellular concentrations of imatinib were examined in an in-vitro system using two GIST cell lines: GIST882 and GIST48. All experiments were conducted in triplicate to prove repeatability of the results. The
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Intracellular imatinib in GIST Berglund et al. 419
Table 2
Recovery of imatinib in cell culture media
Fig. 1
Concentration of imatinib (ng/ml) 1100
3300
16 000
ND ND
1013±42 1119±33
2114±21 2320±17
14 000
Media contain 1% fetal bovine serum. Results based on repeated experiments (n = 3). ND, not detectable.
linearity of the extraction method is excellent and the limit of detection is 5 ng/ml [26]. The average recovery of imatinib in the extracted cell culture media containing 1% FBS was high and reproducible (Table 2). Imatinib could not be detected in cells from flasks incubated without imatinib (negative control) for 3 h. After a 3-h exposure to therapeutic doses of imatinib in the supernatant, 1100 and 3300 ng/ml, the GIST882 cell samples contained 4789±116 and 14295± 559 ng imatinib per mg protein, respectively, whereas the GIST48 cell samples contained 3522±111 and 4427± 171 ng imatinib per mg protein, respectively (Fig. 1). Hence, GIST882 cells contained significantly higher imatinib levels at both incubated concentrations compared with GIST48 cells (n = 3, P < 0.01 and P < 0.001, respectively). Patient samples
Tumor tissue was collected and prepared as described in the Materials and methods section. Two peripheral blood samples were drawn intraoperatively from each patient as close as possible to tumor resection or tumor circulation discontinuation. The plasma levels in GIST1, GIST2, and GIST3 are shown in Fig. 2a. One peripheral tumor specimen was taken from the GIST1 target lesion, whereas pieces were collected from three locations from GIST2: peripherally, centrally, and from a cystic region. Tumor tissue was collected peripherally and centrally in the GIST3 primary target lesion and from two metastatic lesions. Adipose tissue was taken from all three patients as reference tissue, showing lower imatinib concentrations compared with the accumulation in tumor tissue. A summary of imatinib concentration in adipose tissue and tumor samples is presented in Fig. 2b. Histological and immunostaining verification
All GIST tumors (GIST1, GIST2, and GIST3) were reviewed histopathologically in hematoxylin–eosin (HTX) staining and examined immunohistochemically, determining their CD117 and DOG1 expressions (Fig. 3).
Discussion When imatinib was launched onto the market in 2001 for the treatment of malignant metastatic and unresectable GISTs, it effects were considered a ‘miracle’ by many professionals and patients [7]. However, although this
∗∗∗
∗∗
∗∗∗
12 000 ng imatinib/mg protein
GIST882 medium GIST48 medium
0
10 000 8000 ∗
6000 4000 2000 0 GIST882
GIST48
1100 ng/ml imatinib 3300 ng/ml imatinib Intracellular concentration of imatinib in imatinib-sensitive (GIST882) and imatinib-resistant (GIST48) cells after 3 h of incubation to 1100 and 3300 ng/ml (n = 3). The intracellular concentrations of imatinib after exposure of GIST48 and GIST882 cells to 1100 and 3300 ng/ml imatinib for 3 h show that GIST882 contains imatinib in significantly larger quantities. Data are presented as average±SEM. Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, statistical significance.
treatment leads to disease control in B85% of patients, imatinib resistance develops over time, requiring either higher imatinib doses or a switch to a second-line or sometimes even a third-line TKI. In this project, we developed a protocol for evaluation of intracellular imatinib concentrations by studying in-vitro systems of imatinib-sensitive and imatinib-resistant GIST cell lines. The recovery of imatinib in cell culture medium containing 1% FBS was high, and intracellular concentrations of imatinib after exposing GIST cells to therapeutic doses of 1100 and 3300 ng/ml imatinib were detectable and highly reproducible. These imatinib concentrations were selected to correspond to clinically relevant doses. In addition to imatinib estimation in the cell lines, we have measured imatinib concentration in clinical samples from three patients: in blood, adipose, and tumor tissue. Results indicate that the method of determination of imatinib concentration in plasma is highly reproducible, and has a negligible intersample variation. Samples were taken from adipose tissue and tumor tissue and imatinib levels proved to be much lower in the former, whereas imatinib levels varied with the location within the tumor. The levels appeared to be highest in the tumor periphery. To enable comparisons between plasma and tissue concentrations, a plasma density factor (1020 g/) ([27])
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420 Anti-Cancer Drugs 2014, Vol 25 No 4
B75% of GISTs [30–32], implying a possible role in drug transport. Although imatinib has been shown to be a substrate for both ABCB1 and ABCG2 (breast cancer resistance protein) [33], a protein involved in drug transport in the gut epithelium, the latter seems to play no major role in GIST [31].
Fig. 2
(a)
3
3000
2
2000 1500
1
1000
ng imatinib / mg plasma
ng imatinib/ml plasma
2500
500 0
0 GIST1
ng imatinib/mg wet tissue
(b)
GIST2
40
GIST3
(VI) Adipose tissue Tumor tissue
30 (V) 20
(II)
10 (I)
(XI) (III)
(IV)
(IX)
(X)
(VII)(VIII) 0 GIST1
GIST2
GIST3
Imatinib concentration in clinical samples. (a) The imatinib levels in the two plasma samples from GIST1 were 1838±79 and 1873±55 ng/ml, from GIST2, 2504±35 and 2471±42 ng/ml, and from GIST3, 643±24 and 628±18 ng/ml. (b) Imatinib levels in GIST1 were 3.58±0.17 in fat (I) and 10.1±0.20 ng/mg wet tissue in the tumor periphery (II). In GIST2, the imatinib levels were 5±0.16 in fat (III), 6.32±0.27 in the center of the tumor (IV), 23.42±0.79 in the cystic region (V), and 36.56±0.24 ng/mg wet tissue in the tumor periphery (VI). In GIST3, the imatinib levels were 0.36±0.01 in fat (VII), 0.40±0.06 in a peritoneal metastasis (VIII), 4.64±0.25 in a necrotic central tumor piece (IX), 6.44±0.15 in the tumor periphery (X), and 8.52±0.30 ng/mg wet tissue in a cystic metastatic lesion from the spleen (XI). All samples were read three times for accurate measurements.
was used to convert plasma levels into ng imatinib/mg plasma, Fig. 2a. This verifies an accumulation of imatinib in the tumor tissue in all presented cases compared with plasma, which fits well with previously pharmacokinetic studies, where the volume of distribution (Vz/f) for imatinib is 366±19 l [28]. This study includes a small number of clinical samples from three patients to merely show a methodological proof-of-concept. The efflux of drugs was not evaluated. However, a welldocumented efflux pump is the p-glycoprotein (ABCB1 or MDR1), a member of the ATP-binding cassette transport superfamily, associated with multidrug resistance [29]. Both ABCB1 and ABCC1 are expressed in
Although preclinical data suggest that cellular overexpression of ABCB1 leads to decreased intracellular levels of imatinib [34,35], it is not known whether this has functional consequences in GIST cells. Neither is it known whether imatinib treatment induces overexpression of this or other drug transporters in GIST cells. It has also been reported that imatinib is likely to be transported into cells by the OCT-1 influx protein, a member of the solute carrier superfamily (SLC) [16]. This was shown in CML cell lines and the tumoral expression of OCT-1 in CML patients has been correlated with patient outcome. Similar drug transporter correlations are scarce for tumor cells from GIST patients. From a clinical perspective, analysis of intracellular imatinib could be of great interest in selected cases. If the technique can be scaled down and allows concentration estimates from fine needle aspirations, analysis of the drug concentration in close vicinity to its point-of-action is perhaps biologically more relevant than plasma-level measurements, especially as, here, we show a tumordependent and location-dependent imatinib accumulation. Furthermore, patients who are on a standardized dose of 400 mg imatinib daily and do not respond, while mutation analysis indicates that the tumor is expected to be sensitive, may have altered uptake mechanisms. In these cases, an early dose increase or change of TKI may be beneficial. Analysis of intracellular imatinib could, in these respects, potentially complement current follow-up strategies, therapeutic plasma monitoring, and FDG-PET to better reflect the tumor biology.
Conclusion Our data show a novel, feasible, and reproducible approach to measurement of intracellular levels of imatinib in an in-vivo and in-vitro setting. The concentration difference between the two GIST cell lines also indicates that there may be important drug transporters operating in imatinib-resistant GIST cells. The variation in imatinib concentration between plasma, tissue, and different sites within the tumor highlights the importance of further clinical studies to quantify drug transporter expression and measurements of intracellular imatinib levels in GIST tissues to understand their possible impact on pharmacodynamics.
Acknowledgements The authors acknowledge Maria Lo¨nnberg for her kind help with the protein determination (Analytical Chemistry, Department of Chemistry, Biomedical Center and
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Intracellular imatinib in GIST Berglund et al. 421
Fig. 3
GIST1
GIST2
HTX
(a)
CD117
(b)
DOG1
(c)
Histopathological examination and patterns of CD117 and DOG1 staining. (a) Spindle morphology with more vacuolization in GIST2 than in GIST1, reviewed in HTX staining. (b) Strong staining for CD117 in both tumors. (c) DOG 1 with a strong membranous pattern in GIST2, negative in GIST1. Scale bars represent 100 and 50 mm in overview images and magnified insertions, respectively.
Science for Life Laboratory, Uppsala University, Uppsala, Sweden). The Swedish Research Council, the Novo Nordisk Foundation, the Swedish Cancer Society, funds from Karolinska Institutet, the Swedish Society of Medicine (Bengt Ihre grant), the Tore Nilsson Foundation, the Thuring Foundation, the Jeansson Foundation, Magn. Bergvall Foundation, the Cancer Research Foundations of Radiumhemmet, and VR Grant (621-2011-4423) at Uppsala University are gratefully acknowledged for financial support. Financial support was also provided through the regional agreement on medical training and
clinical research (ALF) between the Stockholm County Council and Karolinska Institutet. Conflicts of interest
There are no conflicts of interest.
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Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumor cells Erik Berglunda,b,*, Sarojini Jayantha Kumari A. Ubhayasekerad,*, Fredrik Karlssona,b, Pinar Akcakayac, Warunika Aluthgedarad, Jan A˚hlena,b, Robin Fro¨boma, Inga-Lena Nilssona,b, Weng-Onn Luic, Catharina Larssonc, Jan Zedeniusa,b, Jonas Bergquistd and Robert Bra¨nstro¨ma,b Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-offunction mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure
Introduction The majority of gastrointestinal stromal tumors (GISTs) express constitutively activated forms of either the c-KIT (a stem cell factor receptor) or the platelet-derived growth factor alpha (PDGFRA) receptor tyrosine kinase proteins [1,2]. Underlying gain-of-function mutations appear early in the tumor formation, and imatinib mesylate is a tyrosine kinase inhibitor (TKI) that binds to the intracellular ATP-binding site of the c-KIT or the PDGFRA receptor [3–5]. Inhibition of c-KIT and PDGFRA causes a rapid block in tumor signal transduction and, consequently, tumor regression. The drug also inhibits ABL and the BCR-ABL fusion protein observed
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib c levels in GIST patients. Anti-Cancer Drugs 25:415–422 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:415–422 Keywords: gastrointestinal stromal tumor, imatinib, intracellular concentration a Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, bDepartment of Breast and Endocrine Surgery, Karolinska University Hospital, cDepartment of Oncology–Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm and dDepartment of Chemistry, Biomedical Center, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
Correspondence to Erik Berglund, MD, Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital L1:03, SE-171 76 Stockholm, Sweden Tel: + 46 73 918 12 33; fax: + 46 8 331587; e-mail: [email protected] *Erik Berglund and Sarojini Jayantha Kumari A. Ubhayasekera contributed equally to the writing of this article. Received 25 June 2013 Revised form accepted 28 November 2013
in Philadelphia chromosome-positive chronic myeloid leukemia (CML) patients. GISTs are highly resistant to conventional radiotherapy and chemotherapy [6], and the introduction of TKIs in the treatment of advanced and metastatic GISTs has improved patient survival and outcome markedly [7,8]. Imatinib is also used in the neoadjuvant and adjuvant setting, and recently published data support a 3-year adjuvant treatment regime with a lower risk of tumor recurrence [9]. However, even though imatinib and other TKIs have revolutionized the treatment of GIST, local recurrence, metastasis, and tumor resistance still remain major therapeutic challenges [10]. Imatinib leads to disease control in B85% of patients with advanced GIST, but resistance to the drug develops in most patients within 2–3 years on therapy. As clinical experience with imatinib consistently grows, it becomes DOI: 10.1097/CAD.0000000000000069
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416 Anti-Cancer Drugs 2014, Vol 25 No 4
clearer that different resistance patterns exist. Primary, or early, TKI resistance is observed within the first 3 months of therapy. If GISTs progress after more than 6 months of an initial response or stable disease, they are classified as having a secondary or an acquired resistance. There are several mechanisms leading to primary resistance. The most important is the mutational status of KIT and PDGFRA. Another mechanism is drug metabolism, which affects imatinib plasma levels and thus the clinical response; low imatinib plasma concentrations, less than 1100 ng/ml, are associated with a decreased progressionfree survival [11,12]. Therefore, customizing drug dosing can influence both patient outcome and reduce the frequency of adverse effects [13,14]. Mechanisms of delayed resistance are most commonly secondary mutations in KIT or PDGFRA in addition to the initial mutation, and other postulated genetic mechanisms [11]. It is not known whether the plasma concentration of imatinib correlates with the intracellular concentration of the drug in GIST. Drug transporters play a crucial role in uptake and drug efflux at the cellular level, and they have been implicated as a cause of resistance in many diseases [15]. The uptake of imatinib is an active process [16], and the function is exerted on the intracellular part of the tyrosine kinase protein. A possible explanation for some GISTs developing resistance to TKIs is the interpatient variability in drug transporter activity and expression that leads to decreased intracellular concentrations. Both efflux and uptake transporters in imatinib pharmacokinetics and pharmacodynamics are suggested to be potentially involved in secondary drug resistance in CML [17]. It has not yet been clarified how imatinib is transported into and out of GIST cells, and whether such actions may be contributory mechanisms of resistance. In this study, a novel protocol for the measurement of intracellular concentrations of imatinib, using an in-vitro and an in-vivo system of GIST cells, was developed.
Materials and methods Materials
All chemicals and reagents used in this study were of analytical grade. The internal standard trazodone (C19H22ClN5O) and methanol were obtained from Sigma Aldrich (Stockholm, Sweden). All other chemicals and reagents were purchased from Merck, Eurolab (Stockholm, Sweden) unless otherwise stated. Water was deionized and osmosed with a Milli-Q purification system (Millipore, Bedford, Massachusetts, USA). Imatinib mesylate (C29H31N7OCH4SO3) was kindly donated by Novartis (Basel, Switzerland).
vided by Professor Jonathan Fletcher at Brigham and Women’s Hospital (Boston, Massachusetts, USA). GIST882 is an imatinib-sensitive GIST cell line derived from a patient with an untreated metastatic human GIST expressing a KIT allele with a homozygous exon 13 missense mutation, encoding a K642E mutant c-KIToncoprotein [18]. GIST882 was verified as described previously [19] and maintained in RPMI-1640 medium, supplemented with 15% fetal bovine serum (FBS), 0.25 mg/ml L-glutamine, 100 U/ml penicillin G, 100 mg/ml streptomycin-sulfate, and 0.21 mg/ml amphotericin B at 5% CO2 and 371C. GIST48 is an imatinib-resistant cell line established from a GIST that progressed, after an initial clinical response, during imatinib therapy. GIST48 has a primary homozygous exon 11 missense KIT mutation (V560D) and a heterozygous secondary exon 17 (kinase activation loop) mutation (D820A) [20]. Cells were cultured routinely in F10 HAM medium (Cat no. 31550; Gibco, Grand Island, New York, USA) supplemented with 15% FBS, MITO + serum extender (Cat no. 355006; BD Biosciences, San Jose, California, USA), Bovine Pituitary Extract (Cat no. 354123; BD Biosciences), 0.25 mg/ml L-glutamine, 100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, and 0.21 mg/ml amphotericin B at 5% CO2 and 371C. Cells were passaged weekly and cultured in 175 cm2 flasks. The human embryonic kidney 293 (HEK-293) cell line was obtained from System Biosciences (Cat no. LV900A-1). Cells were maintained in DMEM (Cat no. 41965-039; Invitrogen), supplemented with 10% FBS, 2 mmol/l L-glutamine, and 100 U/ml penicillin G. Clinical sample collection and preparation
Three newly treated patients at the unit for endocrine and sarcoma surgery, Karolinska University Hospital (Stockholm, Sweden), were included in this study. Patients were eligible in cases of advanced GIST, as characterized by CD117 positivity, for which neoadjuvant daily treatment with 400 mg imatinib is indicated. GIST1 was a wild-type small intestine GIST, imatinib treated for 16 months before surgical resection. GIST2 was a gastric GIST, with a c-KIT exon 11 mutation, and was imatinib treated for 11 months before surgery, and GIST3 was a gastric GIST harboring an exon 18 mutation in the PDGFRA receptor (Table 1). Tumor and reference adipose tissues were excised from each case at the time of surgery. Two peripheral blood samples were drawn at the same time as the tumor was removed or the blood supply to the tumor was discontinued. Blood plasma was then prepared by centrifugation and snap frozen to – 801C together with tumor and reference tissues until further analysis. The local ethics committee at Karolinska University Hospital approved the study, and all three patients provided their signed informed consent.
Cell culture
KIT mutation analysis and detection of CD117 expression in GIST48
Two GIST cell lines were used to examine the intracellular concentration of imatinib mesylate, both generously pro-
Evaluation of KIT status and expression of the cell surface marker CD117 in GIST882 were verified as described
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Intracellular imatinib in GIST Berglund et al. 417
Table 1
Patient characteristics
Sex Age at operation (years) Primary tumor site TNM stage [21] Time on imatinib before surgery (months) Daily imatinib dose (mg) RECIST criteria [22] FDG-PET uptake Before imatinib During imatinib Largest tumor size (cm) Size range (cm) Number of tumors Necrosis GIST subtype Mutational status Immunohistochemical studies Ki-67 Microscopic treatment response
GIST1
GIST2
GIST3
Male 63 Small intestine IV 16 400 PD
Male 83 Stomach II 11 400 PR
Male 65 Stomach IV 12 400 PR
NA NA 10 5–10 3 > 50% Spindle cell Wild type CD117 + , DOG1 – 15% No
Medium–high Low 11.5 na 1 < 50% Spindle cell c-kit, exon 11 CD117 + , DOG1 + < 1% Yes
Low (necrotic) NA 8 0.5–8 3 < 50% NA PDGFRA, exon 18 CD117 + , DOG1 + < 1% Yes
FDG-PET, fluorodeoxyglucose-PET; GIST, gastrointestinal stromal tumor; na, not applicable; NA, not available; PD, progressive disease; PR, partial response; RECIST guidelines, Response Evaluation Criteria in Solid Tumors Guidelines.
previously [19]. The same protocols were used for verification purposes of the GIST48 cell line. In brief, an automated Maxwell 16 DNA purification system kit AS1020 (Promega Corporation, Madison, Wisconsin, USA) was used for purification of genomic DNA from the GIST48 cell line. Cell lysate was transferred to the Maxwell cartridge and eluted genomic DNA concentration and purity was determined in a NanoDrop spectrophotometer ND-1000 (Thermo Scientific, Wilmington, Delaware, USA). PCR and sequencing were carried out to detect mutations in exons 9, 11, 13, and 17 of the KIT gene, as described previously by Sihto et al. [23]. Mutations were referenced from http://www. ensembl.org and http://www.sanger.ac.uk/genetics/CGP/cosmic/. For evaluation of CD117 (c-KIT) expression on GIST48 cells, the Miltenyi Biotec’s protocol was used according to the manufacturer’s instructions. The HEK-293 cell line was used as a negative control. Cells were incubated with either an allophycocyanin-coupled antibody against human CD117 (Cat no. 130 091 733), obtained from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), or an allophycocyanin-coupled mouse IgG1 (Cat no. 345818) antibody, obtained from Becton Dickinson GmbH (Franklin Lakes, New Jersey, USA), for isotype control experiments. Cell fluorescence was determined by flow cytometry (Gallois instrument; Beckman Coulter, Brea, California, USA). Imatinib incubation
GIST882 and GIST48 cells were cultured for four to five days in 175 cm2 flasks until near total confluence. Imatinib mesylate (Glivec, formerly STI571; a gift from Novartis Pharmaceuticals, Basel, Switzerland) was dissolved and diluted in cell culture medium, supplemented with only 1% FBS to minimize protein binding of imatinib [24,25] to final concentrations of 1100 and 3300 ng/ml.
For the in-vitro experiments, the cell culture medium was removed and the cell monolayers were washed carefully. Imatinib mesylate was added to GIST882 and GIST48 cells at final concentrations of 1100 and 3300 ng/ml and incubated for 3 h. As the active imatinib uptake is temperature dependent [16], all incubations were carried out in a humidified incubator at 5% CO2 and 371C. The supernatant was removed and the cells were rewashed carefully with prewarmed Dulbecco’s PBS (Cat no. 14190). Subsequently, 15 ml trypsin/EDTA was added to each flask for 7 min to detach the cells. The content from each flask was transferred to a Falcon tube and centrifuged for 5 min at 1000 rpm. After centrifugation and removal of the supernatant, the cell pellet was snap frozen to – 801C before imatinib concentration analysis by liquid chromatography–mass spectrometry (time-offlight) [LC-MS (TOF)]. Imatinib and trazodone stock solutions (1 mg/ml) and working solutions (1, 10, and 100 mg/ml) were prepared in methanol for LC-MS (TOF) analysis. The stock solutions were kept at – 801C before use. Calibration standards for the imatinib analysis in plasma samples were prepared by adding appropriate amounts of the working solutions to blank plasma (200 ml). Quantification of imatinib uptake by LC-MS (TOF)
Homogenized cell culture and tumor tissue (50 mg) sample preparation consisted of two steps: liquid–liquid extraction and enrichment before analysis by an LC-MS. In brief, each cell culture and tumor sample was introduced in glass tubes (DURAN 12 100 mm with Teflon screw-cap) with 20 ml of trazodone as an internal standard (IS) in methanol (10 mg/ml). Then, 0.5 ml of methanol was added to each tube and homogenized with a 3 mm probe (Sonics Vibra Cell; Sonics & Materials Inc., Newtown, Connecticut, USA) at 30% amplitude
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418 Anti-Cancer Drugs 2014, Vol 25 No 4
for 1 min. The samples were vortexed for 10 min and centrifuged at 41C for 15 min at 14 000g. The resulting supernatant was transferred to 1.5 ml Eppendorf tubes, the tubes were blown down with a stream of pure nitrogen, and the resulting dried residues were reconstituted with 200 ml methanol : water (20 : 80, v/v) before the LC-MS analysis. The final solution was transferred to an HPLC autosampler vial and 2 ml was injected into the LC-MS system. The LC-MS system consisted of an Agilent model 1100 Autosampler (Agilent Technologies, Palo Alto, California, USA), an Agilent 1100 Quaternary pump, and an Agilent ZORBAX C18 (5 mm, 2.1 50 mm) reversed-phase analytical column controlled by Agilent ChemStation software (Agilent Technologies, Palo Alto, California, USA). The mobile phase runs at a linear gradient containing 0.1% formic acid in both methanol and water, with the flow rate starting from 0.2 ml/min. The total run time of the LC program was 14 min. The chromatographic system was coupled to a TOF mass spectrometer (Agilent Technologies, Santa Clara, California, USA) equipped with electrospray ionization. The detection was performed in a positive ion mode. The ionization potential was 3800 V and the ion source temperature was 3001C. The nebulizing gas was used for electrospray ionization at the rate of 15 psi. The voltages fixed at the fragmentor, skimmer, and octopole guides were 225, 60, and 250 V, respectively. The ion pulser at the TOF analyzer was set at the measurement frequency of 2 cycles/s. Peak lists were achieved using the molecular feature extractor software ‘MassHunter’ (Agilent Technologies, Santa Clara, California, USA). Extraction of total ion chromatogram for imatinib and trazodone was performed with m/z ranges 494.15–494.3 and 372.1–372.2, respectively. The calibration curve for the analysis of imatinib in plasma was prepared by plotting the peak area ratio of imatinib/peak area of IS versus each imatinib concentration. The linearity was determined by linear regression analysis. The correlation coefficient of linear regression (r2) of imatinib was 0.9940. Quantification of imatinib (area response) in cell culture and tissue samples was carried out using the internal standard. The imatinib concentration of each measured cell culture sample is given in ng per mg protein. Samples were run three times to ensure accurate measurements. An in-house validated method (Dot-it-Spot-it protein assay, http://dot-it-spot-it.com/; Maple Stone AB, Uppsala, Sweden) was used to quantify the protein content.
prevent protein degradation. After homogenization, the samples were incubated for 1 h at 41C with mild agitation. Consequently, the cell lysates were clarified by centrifugation for 30 min (10 000g at 41C) using a Sigma 2K15 ultracentrifuge (Sigma Aldrich). The supernatant was collected and further processed. The Dot-it-Spot-it protein assay (http://dot-it-spot-it.com/; Maple Stone AB) was used for the protein assay according to the instructions provided. Detection sheets, carbon black detection solution, and washing solution were included. The sample (1 ml) was dispensed on the detection sheets in replicates and dried rapidly. The sheets were then placed in a large well with 1 ml of detection solution and incubated for 5 min, followed by 5 min incubation in 1 ml of washing solution. The absorbent sink was removed from the sheets. The sheets were then dried and mounted on the scanning template, which was detected by an Epson Expression 1600 Pro scanner (Epson, Long Beach, California, USA). The intensity of blackness was quantified in each dedicated spot on the image with Image J (http://rsbweb.nih.gov/ij/). Protein concentrations were estimated by comparing the sample results with the results from a calibration curve using Rodbard curve fitting in Image J. Statistical analysis
Statistical significance was determined using an unpaired Student’s t-test, and P-values less than 0.05 were considered significant. Data are expressed as means±SEM.
Results Mutational analysis and CD117 expression
Sequencing of KIT exons 9, 11, 13, and 17 in the GIST48 cell line confirmed the presence of the primary homozygous exon 11 missense mutation (V560D) and the heterozygous secondary exon 17 (kinase activation loop) mutation (D820A) (Supplemental digital content 1, http://links.lww.com/ACD/A51). Confirmation of the GIST48 phenotype verified high expression of the cell surface marker CD117 in 99% of GIST48 cells, compared with a low expression in the HEK-293 cell line (Supplemental digital content 2, http://links.lww.com/ACD/A52). Sequencing of KIT and CD117 expression analysis in GIST882 has been performed previously in GIST882, which verified the GIST882 genotype and phenotype [19]. The GIST1 tumor showed mutations in neither KIT nor PDGFRA exons, thereby classified as a wild-type GIST tumor. GIST2 contained two point mutations in exon 11: c.1695G > T and c.1696G > T (V559F), whereas GIST3 contained an exon 18 mutation in the PDGFRA receptor.
Protein determination
The protein precipitates were homogenized for 60 s using a probe with a 0.5 ml lysis buffer (10 mmol/l Tris-HCl pH 7.4, 0.15 mol/l NaCl, 1 mmol/l EDTA, and PBS containing 1% octyl-b-D-glucopyranoside). Protease inhibitor cocktail (10 ml) was added during the sample preparation to
Imatinib uptake into GIST cells
Intracellular concentrations of imatinib were examined in an in-vitro system using two GIST cell lines: GIST882 and GIST48. All experiments were conducted in triplicate to prove repeatability of the results. The
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Intracellular imatinib in GIST Berglund et al. 419
Table 2
Recovery of imatinib in cell culture media
Fig. 1
Concentration of imatinib (ng/ml) 1100
3300
16 000
ND ND
1013±42 1119±33
2114±21 2320±17
14 000
Media contain 1% fetal bovine serum. Results based on repeated experiments (n = 3). ND, not detectable.
linearity of the extraction method is excellent and the limit of detection is 5 ng/ml [26]. The average recovery of imatinib in the extracted cell culture media containing 1% FBS was high and reproducible (Table 2). Imatinib could not be detected in cells from flasks incubated without imatinib (negative control) for 3 h. After a 3-h exposure to therapeutic doses of imatinib in the supernatant, 1100 and 3300 ng/ml, the GIST882 cell samples contained 4789±116 and 14295± 559 ng imatinib per mg protein, respectively, whereas the GIST48 cell samples contained 3522±111 and 4427± 171 ng imatinib per mg protein, respectively (Fig. 1). Hence, GIST882 cells contained significantly higher imatinib levels at both incubated concentrations compared with GIST48 cells (n = 3, P < 0.01 and P < 0.001, respectively). Patient samples
Tumor tissue was collected and prepared as described in the Materials and methods section. Two peripheral blood samples were drawn intraoperatively from each patient as close as possible to tumor resection or tumor circulation discontinuation. The plasma levels in GIST1, GIST2, and GIST3 are shown in Fig. 2a. One peripheral tumor specimen was taken from the GIST1 target lesion, whereas pieces were collected from three locations from GIST2: peripherally, centrally, and from a cystic region. Tumor tissue was collected peripherally and centrally in the GIST3 primary target lesion and from two metastatic lesions. Adipose tissue was taken from all three patients as reference tissue, showing lower imatinib concentrations compared with the accumulation in tumor tissue. A summary of imatinib concentration in adipose tissue and tumor samples is presented in Fig. 2b. Histological and immunostaining verification
All GIST tumors (GIST1, GIST2, and GIST3) were reviewed histopathologically in hematoxylin–eosin (HTX) staining and examined immunohistochemically, determining their CD117 and DOG1 expressions (Fig. 3).
Discussion When imatinib was launched onto the market in 2001 for the treatment of malignant metastatic and unresectable GISTs, it effects were considered a ‘miracle’ by many professionals and patients [7]. However, although this
∗∗∗
∗∗
∗∗∗
12 000 ng imatinib/mg protein
GIST882 medium GIST48 medium
0
10 000 8000 ∗
6000 4000 2000 0 GIST882
GIST48
1100 ng/ml imatinib 3300 ng/ml imatinib Intracellular concentration of imatinib in imatinib-sensitive (GIST882) and imatinib-resistant (GIST48) cells after 3 h of incubation to 1100 and 3300 ng/ml (n = 3). The intracellular concentrations of imatinib after exposure of GIST48 and GIST882 cells to 1100 and 3300 ng/ml imatinib for 3 h show that GIST882 contains imatinib in significantly larger quantities. Data are presented as average±SEM. Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001, statistical significance.
treatment leads to disease control in B85% of patients, imatinib resistance develops over time, requiring either higher imatinib doses or a switch to a second-line or sometimes even a third-line TKI. In this project, we developed a protocol for evaluation of intracellular imatinib concentrations by studying in-vitro systems of imatinib-sensitive and imatinib-resistant GIST cell lines. The recovery of imatinib in cell culture medium containing 1% FBS was high, and intracellular concentrations of imatinib after exposing GIST cells to therapeutic doses of 1100 and 3300 ng/ml imatinib were detectable and highly reproducible. These imatinib concentrations were selected to correspond to clinically relevant doses. In addition to imatinib estimation in the cell lines, we have measured imatinib concentration in clinical samples from three patients: in blood, adipose, and tumor tissue. Results indicate that the method of determination of imatinib concentration in plasma is highly reproducible, and has a negligible intersample variation. Samples were taken from adipose tissue and tumor tissue and imatinib levels proved to be much lower in the former, whereas imatinib levels varied with the location within the tumor. The levels appeared to be highest in the tumor periphery. To enable comparisons between plasma and tissue concentrations, a plasma density factor (1020 g/) ([27])
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420 Anti-Cancer Drugs 2014, Vol 25 No 4
B75% of GISTs [30–32], implying a possible role in drug transport. Although imatinib has been shown to be a substrate for both ABCB1 and ABCG2 (breast cancer resistance protein) [33], a protein involved in drug transport in the gut epithelium, the latter seems to play no major role in GIST [31].
Fig. 2
(a)
3
3000
2
2000 1500
1
1000
ng imatinib / mg plasma
ng imatinib/ml plasma
2500
500 0
0 GIST1
ng imatinib/mg wet tissue
(b)
GIST2
40
GIST3
(VI) Adipose tissue Tumor tissue
30 (V) 20
(II)
10 (I)
(XI) (III)
(IV)
(IX)
(X)
(VII)(VIII) 0 GIST1
GIST2
GIST3
Imatinib concentration in clinical samples. (a) The imatinib levels in the two plasma samples from GIST1 were 1838±79 and 1873±55 ng/ml, from GIST2, 2504±35 and 2471±42 ng/ml, and from GIST3, 643±24 and 628±18 ng/ml. (b) Imatinib levels in GIST1 were 3.58±0.17 in fat (I) and 10.1±0.20 ng/mg wet tissue in the tumor periphery (II). In GIST2, the imatinib levels were 5±0.16 in fat (III), 6.32±0.27 in the center of the tumor (IV), 23.42±0.79 in the cystic region (V), and 36.56±0.24 ng/mg wet tissue in the tumor periphery (VI). In GIST3, the imatinib levels were 0.36±0.01 in fat (VII), 0.40±0.06 in a peritoneal metastasis (VIII), 4.64±0.25 in a necrotic central tumor piece (IX), 6.44±0.15 in the tumor periphery (X), and 8.52±0.30 ng/mg wet tissue in a cystic metastatic lesion from the spleen (XI). All samples were read three times for accurate measurements.
was used to convert plasma levels into ng imatinib/mg plasma, Fig. 2a. This verifies an accumulation of imatinib in the tumor tissue in all presented cases compared with plasma, which fits well with previously pharmacokinetic studies, where the volume of distribution (Vz/f) for imatinib is 366±19 l [28]. This study includes a small number of clinical samples from three patients to merely show a methodological proof-of-concept. The efflux of drugs was not evaluated. However, a welldocumented efflux pump is the p-glycoprotein (ABCB1 or MDR1), a member of the ATP-binding cassette transport superfamily, associated with multidrug resistance [29]. Both ABCB1 and ABCC1 are expressed in
Although preclinical data suggest that cellular overexpression of ABCB1 leads to decreased intracellular levels of imatinib [34,35], it is not known whether this has functional consequences in GIST cells. Neither is it known whether imatinib treatment induces overexpression of this or other drug transporters in GIST cells. It has also been reported that imatinib is likely to be transported into cells by the OCT-1 influx protein, a member of the solute carrier superfamily (SLC) [16]. This was shown in CML cell lines and the tumoral expression of OCT-1 in CML patients has been correlated with patient outcome. Similar drug transporter correlations are scarce for tumor cells from GIST patients. From a clinical perspective, analysis of intracellular imatinib could be of great interest in selected cases. If the technique can be scaled down and allows concentration estimates from fine needle aspirations, analysis of the drug concentration in close vicinity to its point-of-action is perhaps biologically more relevant than plasma-level measurements, especially as, here, we show a tumordependent and location-dependent imatinib accumulation. Furthermore, patients who are on a standardized dose of 400 mg imatinib daily and do not respond, while mutation analysis indicates that the tumor is expected to be sensitive, may have altered uptake mechanisms. In these cases, an early dose increase or change of TKI may be beneficial. Analysis of intracellular imatinib could, in these respects, potentially complement current follow-up strategies, therapeutic plasma monitoring, and FDG-PET to better reflect the tumor biology.
Conclusion Our data show a novel, feasible, and reproducible approach to measurement of intracellular levels of imatinib in an in-vivo and in-vitro setting. The concentration difference between the two GIST cell lines also indicates that there may be important drug transporters operating in imatinib-resistant GIST cells. The variation in imatinib concentration between plasma, tissue, and different sites within the tumor highlights the importance of further clinical studies to quantify drug transporter expression and measurements of intracellular imatinib levels in GIST tissues to understand their possible impact on pharmacodynamics.
Acknowledgements The authors acknowledge Maria Lo¨nnberg for her kind help with the protein determination (Analytical Chemistry, Department of Chemistry, Biomedical Center and
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Intracellular imatinib in GIST Berglund et al. 421
Fig. 3
GIST1
GIST2
HTX
(a)
CD117
(b)
DOG1
(c)
Histopathological examination and patterns of CD117 and DOG1 staining. (a) Spindle morphology with more vacuolization in GIST2 than in GIST1, reviewed in HTX staining. (b) Strong staining for CD117 in both tumors. (c) DOG 1 with a strong membranous pattern in GIST2, negative in GIST1. Scale bars represent 100 and 50 mm in overview images and magnified insertions, respectively.
Science for Life Laboratory, Uppsala University, Uppsala, Sweden). The Swedish Research Council, the Novo Nordisk Foundation, the Swedish Cancer Society, funds from Karolinska Institutet, the Swedish Society of Medicine (Bengt Ihre grant), the Tore Nilsson Foundation, the Thuring Foundation, the Jeansson Foundation, Magn. Bergvall Foundation, the Cancer Research Foundations of Radiumhemmet, and VR Grant (621-2011-4423) at Uppsala University are gratefully acknowledged for financial support. Financial support was also provided through the regional agreement on medical training and
clinical research (ALF) between the Stockholm County Council and Karolinska Institutet. Conflicts of interest
There are no conflicts of interest.
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