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Section of Endocrine Surgery (K.K., M.S., C.S., C.P., G.P., B.N.), Division of Experimental Surgery (G.H.), Department of Surgery, Division of General Surgery, and Department of Internal Medicine I/Oncology (M.R.), and Section of Clinical Biometrics (M.M.), Department of Medical Computer Sciences, University of Vienna Medical School, A-1090 Vienna, Austria; Children’s Cancer Research Institute (A.W.), St. Anna Children’s Hospital, A-1090 Vienna, Austria; and Department of Pathophysiology (V.S., R.P.), University of Graz Medical School, A-8010 Graz, Austria
Address all correspondence and requests for reprints to: Klaus Kaczirek, M.D., Section of Endocrine Surgery, Department of Surgery, Division of General Surgery, University of Vienna, Medical School, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: klaus.kaczirek{at}akh-wien.ac.at.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Eight MTC cell lines were established from seven patients with MTC. IC50 values were calculated from dose-response relationships using cell counts and a formazan dye assay (WST-1).
IC50 values were 3.5 ± 1.2 nmol/liter for CPT and 8.2 ± 1.9 nmol/liter for paclitaxel. Dacarbazine showed no reduction of cell proliferation at concentrations 10-fold higher than clinically achievable.
Given peak plasma concentrations of 65 ± 20 nmol/liter for CPT and 1 µmol/liter for paclitaxel, these promising in vitro results provide a basis for the performance of clinical trials in patients with advanced MTC.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In 70–80% of cases, MTC occurs sporadically. The remaining 20–30% are inherited in an autosomal-dominant pattern and may occur in three distinct clinical settings: multiple endocrine neoplasia syndrome 2A, multiple endocrine neoplasia syndrome 2B, and familial MTC. Surgical removal of all neoplastic tissue in the neck by total thyroidectomy, central and bilateral (functional) neck dissection, is the only potentially curative treatment in localized disease. However, at the time of initial diagnosis, at least one quarter of patients has distant metastases (3). Thirty to 50% of patients with residual or recurrent disease die within 10 yr of diagnosis (4). To date, there is no established adjuvant treatment for these patients. Radiotherapy may be of some benefit for local/regional tumor control in patients with postoperative gross residual disease or inoperable tumors (5), but an intensive search for new alternative treatment strategies is warranted.
Despite active study, no chemotherapeutic agents have proved to be markedly effective in the management of MTC. In vitro investigation of new therapeutic strategies has been limited by the availability of only one continuous human MTC cell line, the TT line, which was derived from a hereditary MTC with an exon 11, codon 634 mutation (6).
The establishment of eight continuous human MTC cell lines representing different local tumor stages, growth dynamics, immunocytochemical characteristics, ages of patients, and sporadic or hereditary forms (7) provides the tools for a variety of further in vitro research in this field.
Camptothecin (CPT), an inhibitor of topoisomerase I (the parent substance of irinotecan, used for in vitro investigations) and paclitaxel, a taxane with antimicrotubular action, have never been investigated in the treatment of MTC. Both substances have shown successful results combined with reduced toxic side effects in the treatment of various solid tumors, such as colorectal, breast, ovarian, and small-cell lung cancer. Positive results in the therapy of MTC may be expected.
The aim of the present study was to assess the in vitro activity of CPT and paclitaxel in comparison with dacarbazine (DTIC), as a control with no known clinical benefit, using eight human MTC cell lines.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Eight continuous cell lines were established from seven patients with biochemically proven and histologically confirmed MTCs. Six patients had sporadic tumors, and one patient (cell line GSJO) had hereditary MTC (mutation at exon 14, codon 804; amino acid exchange Val>Met, GTG>ATG). Cells were cultured from primary tumors (lines GSJO and SHER-I) or lymph node metastases (MTC-SK, SINJ, BOJO, RARE, GRS IV, and GRS V). The lines GRS IV and GRS V comprise cultures of lymph node metastases of one patient at two different operation times. Three patients (lines MTC-SK, BOJO, and GRS IV/GRS V) had generalized disease at the time of operation and died 4 (line GRS IV/GRS V), 14 (line BOJO), and more than 50 months (line MTC-SK) after the primary operation. The remaining four patients have been alive for 27, 28, 30, and 146 months of follow-up, respectively. All of them have biochemically residual/recurrent disease, and three have measurable distant metastatic disease. Table 1
summarizes the clinical and genetic data and gives the passage numbers of the cultures tested in this study.
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Cell cultivation
Tumor tissue obtained during primary surgery and/or reoperation was transported in Ham’s F12 medium (Biochrom AG-Seromed, Berlin, Germany) containing 100 IU penicillin/ml and 100 µg streptomycin/ml. Directly after transportation, tissue was treated for 20 min with PBS containing 1000 IU penicillin/ml and 1000 µg streptomycin/ml. Previously described techniques for primary and continuous cultivation of MTC were applied (8, 9). Briefly, tissue was dissociated mechanically, and erythrocyte lysis buffer was applied for 15 min. The cell suspension was centrifuged and resuspended in Ham’s F12 medium, supplemented with 10% fetal bovine serum (PAA Laboratories, Exton, PA), 100 IU penicillin, and 100 µg streptomycin/ml medium at an estimated cell number of 3–5 x 105 cells/ml and incubated at 37 C in a 5% CO2 and 95% humidified atmosphere. Cultures were maintained in vented T-12.5, T-25, and T-150 culture flasks (Costar, Cambridge, MA). Stromal fibroblasts were separated from tumor cells by repeated selective adhesion and selective detachment treatment (10). Antibiotics were omitted after a few passages. All cell lines grew anchorage independent as suspensions of single cells as well as spheroid aggregates. Cultures have been tested at regular intervals to exclude mycoplasma contamination (Hoechst dye 33258, Sigma, Vienna, Austria). All cell lines were tested by immunocytochemistry, electron microscopy, in situ hybridization, and tumorigenicity as described recently (7, 11). Each cell line retained characteristic features of MTC. Additionally, RET proto-oncogene mutation status was determined in all sporadic cell lines. No mutations at exons 10, 11, 13, 14, 15, and 16 were found in all cell lines.
Chemosensitivity tests
CPT (parent substance of irinotecan, Camptosar, used for in vitro investigations), paclitaxel (Taxol), and DTIC were obtained from Sigma. Stock solutions of the compounds were prepared in 70% ethanol or dimethyl sulfoxide and diluted more than 100-fold for chemosensitivity assays. Cells were counted (CASY-1 Cell Size/Analyzer Counter, Schärfe System, GmbH, Reutlingen, Germany) and transferred to the wells of microtiter plates (96 wells, Greiner, Kremsmunster, Austria) in a cell density of 2 x 104 cells/well in 100 µl medium. Appropriate dilutions of test compounds were added to a total volume of 200 µl/well, and the plates were incubated for 24, 48, and 72 h (48, 96, and 168 h for DTIC where the active metabolite 5-amino-imidazole 4-carboxamide is slowly formed in vitro) under cell culture conditions. Solvent controls (ethanol or dimethyl sulfoxide) were added in all tests. Cell viability was assessed using a modified tetrazolium dye assay (cell proliferation reagent WST-1, Roche Molecular Biochemicals, Vienna, Austria) and measurement of the reduced formazan at 450 nm (ELISA, Eurogenetics, Tessenderlo, Belgium) after 1, 2, or 3 d of incubation for CPT/paclitaxel and 2, 4, or 7 d of incubation for DTIC. Simultaneously, cell counting was performed (CASY-1 Cell Size/Analyzer Counter, Schärfe) as a control toxicity end-point parameter. Each test was performed in triplicate, and mean values were used for further calculations.
IC50 values were calculated by linear regression analysis of the obtained dose-response curves.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The cytotoxic/cytostatic effects of CPT and paclitaxel became apparent after a minimum of 24 h of incubation and increased with longer incubation times. Shorter exposure times (10 and 18 h) did not show any cytotoxic or cytostatic effect on all tested cell lines. Results are therefore not reported.
There were no significant differences in sensitivity to CPT and paclitaxel between the tested cell lines.
IC50 values were calculated after 3 d of incubation with CPT or paclitaxel, respectively. For CPT, IC50 values ranged from 1.4 nmol/liter (cell line SHER I, passage number 17) to 6.0 nmol/liter (cell line MTC-SK, passage number 210). For paclitaxel, IC50 values ranged from 6.0 nmol/liter (cell line GSJO, passage number 11) to 11.2 nmol/liter (cell line GRS V, passage number 24). Mean IC50 values were 3.5 ± 1.2 nmol/liter for CPT and 8.2 ± 1.9 nmol/liter for paclitaxel. Table 2 shows the calculated IC50 values at day 3 for CPT and paclitaxel for all tested cell lines.
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and 2 depict the mean dose-dependent fractions of viable cells after 1, 2, and 3 d of incubation with CPT or paclitaxel and give the exact mean viability values.
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For DTIC no consistent dose-response relationships could be determined after 2, 4, and 7 d of incubation at doses of 0–100 µg/ml (0–550 µmol/liter).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In this study, eight cell lines of seven patients with different tumor stages, sporadic and hereditary forms of MTC, were tested. Two of these patients (cell lines BOJO, GRS) had a very aggressive clinical course and died of tumor generalization 4 and 14 months, respectively, after initial diagnosis/operation. One additional patient had tumor generalization at the time of initial operation but a slower progressive clinical course and died more than 50 months postoperatively. The remaining four patients have been alive for 27, 28, 30, and 146 months of follow-up, respectively. All of them have biochemically residual/recurrent disease, and three have evidence of distant metastases. Benefit of adjuvant treatment can therefore be expected in all seven of these patients.
Drugs investigated in various trials include Adriamycin (generic, doxorubicin), bleomycin, carboplatin, cisplatin, cyclophosphamide, DTIC, epirubicin, etoposide, 5-fluorouracil (5-FU), methotrexate, streptozocin, vincristine, and vindesine (5, 12). Response rates have been partial and short-lasting (only a few months) in most reports.
DTIC used as monotherapy showed complete response in one case report of MTC (13). Various combinations of DTIC + 5-FU (14, 15, 16), DTIC + 5-FU + epirubicin (17), or DTIC + cyclophosphamide + vincristine (18) have been applied in a limited number of patients and showed complete response rates in 3% and partial response rates in 24% (5). No benefit could be demonstrated regarding survival.
We therefore chose DTIC as a control substance with limited clinical value.
CPT and paclitaxel have never been investigated in the treatment of MTC but are in clinical use and have shown promising results in the treatment of colorectal, breast, ovarian, and small-cell lung cancer. Most important side effects include severe diarrhea, hematotoxicity, and acute cholinergic syndrome for CPT and hematotoxicity, hypersensitivity reactions, peripheral neuropathy, and bradycardia for paclitaxel.
Before these substances are used in new indications, in vitro cytotoxicity testing is mandatory. However, human MTC is one of the most difficult cancers to grow in cell culture. For two decades, the only commercially available MTC cell line has been the TT line (7, 19). Results of studies performed on this line can only rarely be transformed into clinical practice. In vitro cytotoxicity studies performed on long-term cultures of MTC cells (20, 21, 22) are difficult to interpret because of unstable cell-culture conditions, which may confound results of viability measurements. The establishment of eight further continuous MTC cell lines provides useful experimental material (7). Cell lines were derived from patients with different tumor stages, clinical behavior, and immunocytochemical characteristics. This underlines the heterogeneity of the cell lines.
These novel lines grow anchorage independent, which is different from the TT cell line. We therefore excluded results of cytotoxicity testing in the TT line from the current study.
Comparison of the IC50 values of chemotherapeutics obtained in vitro with clinically achievable peak plasma concentrations provides a rough estimate of the expected clinical sensitivity. The in vivo values of tumor cells may be lower during prolonged application of a drug or higher due to lower tissue concentration, diminished tumor cell accessibility, and adverse effects caused by the presence of serum or other factors.
Achievable peak plasma concentrations are 65 ± 20 nmol/liter for CPT derivative topotecan (23), 1 µmol/liter for paclitaxel (24), and 47.3 ± 3.3 µmol/liter for DTIC. We found all cell lines to be sensitive to CPT and paclitaxel. Mean IC50 values at day 3 were 3.6 ± 0.9 nmol/liter for CPT (5.5% of peak plasma concentration) and 8.1 ± 2.7 nmol/liter for paclitaxel (0.8% of peak plasma concentration). At IC50 concentrations, both substances showed the same cytotoxic/cytostatic potential after 1, 2, or 3 d of incubation.
DTIC was applied for a maximum of 7 d in a dosage of up to 550 µmol/liter, which is 11-fold above the clinically achievable peak plasma concentration. No cytotoxic/cytostatic potential could be determined among all cell lines.
Resistance of MTC to chemotherapy was attributed to expression of the multidrug resistance mdr1 gene (25) encoding a transmembrane glycoprotein p-170 that antagonizes intracellular accumulation of cytotoxic agents of natural derivation. Because all cell lines were highly sensitive to CPT and paclitaxel, p-glycoprotein 170 was not determined in the tested cell lines.
With all the limitations of in vitro testing, our results suggest a potential clinical effect of CPT and paclitaxel and provide a basis for the design of clinical trials for patients with advanced MTC.
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Footnotes |
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Abbreviations: CPT, Camptothecin; DTIC, dacarbazine; 5-FU, 5-fluorouracil; MTC, medullary thyroid carcinoma.
Received July 29, 2003.
Accepted January 27, 2004.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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