Investigational New Drugs

, Volume 25, Issue 5, pp 417–423 | Cite as

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

  • Axel-Rainer Hanauske
  • Ulrike Eismann
  • Olaf Oberschmidt
  • Heike Pospisil
  • Steve Hoffmann
  • Hartmut Hanauske-Abel
  • Doreen Ma
  • Victor Chen
  • Paolo Paoletti
  • Clet Niyikiza


Aim of the study

mRNA expression of genes involved in the mechanism of action of pemetrexed was correlated with in vitro chemosensitivity of freshly explanted human tumor specimens.

Experimental design

Chemosensitivity to pemetrexed was studied in soft-agar. Multiplex rtPCR experiments for reduced folate carrier (RFC), folate receptor-α (FR-α), folylpolyglutamate synthetase (FPGS), thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), mrp4, and mrp5 were performed in parallel. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies.


In 61 samples, low levels of TS, GARFT, DHFR, and mrp4 gene expression significantly correlated with chemosensitivity to pemetrexed. Optimization analyses demonstrated threshold values of 144 copies for TS and six copies for mrp4 relative to 104 copies of β-actin.


These results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.


Pemetrexed disodium Gene expression Freshly explanted human tumors Thymidilate synthase Dihydrofolate reductase Glycinamide ribonucleotide formyltransferase Mrp4 Mrp5 Predictive testing 



We are grateful for the financial support of Eli Lilly & Co. Indianapolis, Indiana, USA.


  1. 1.
    Vogelzang NJ, Rusthoven JJ, Symanowski J, et al. (2003) Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 21:2636–2644PubMedCrossRefGoogle Scholar
  2. 2.
    Hanna N, Shepherd FA, Fossella FV, et al. (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy. J Clin Oncol 22:1589–1597PubMedCrossRefGoogle Scholar
  3. 3.
    Shih C, Habeck LL, Mendelsohn LG, et al. (1998) Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidin-based antifolate LY231514 (MTA). Adv Enzyme Regul 38:135–52PubMedCrossRefGoogle Scholar
  4. 4.
    Westerhof GR, Schornagel JH, Kathmann I, Jackman AL, Rosowsky A, Forsch RA, Hynes JB, Boyle FT, Peters GJ, Pinedo HM, et al. (1995) Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity. Mol Pharmacol 48(3):459–471PubMedGoogle Scholar
  5. 5.
    Habeck LL, Mendelsohn LG, Shih C, Taylor EC, Colman PD, Gossett LS, Leitner TA, Schultz RM, Andis SL, Moran RG (1995) Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs. Mol Pharmacol 48(2):326–333PubMedGoogle Scholar
  6. 6.
    Hanauske AR (2004) The role of Alimta in the treatment of malignant pleural mesothelioma: an overview of preclinical and clinical trials. Lung Cancer 45(Suppl 1):121–124CrossRefGoogle Scholar
  7. 7.
    Hanauske AR, Dittrich C, Otero J. Overview of phase I/II pemetrexed studies. Oncology 2004 (Williston Park);18:18–25PubMedGoogle Scholar
  8. 8.
    Niyikiza C, Hanauske AR, Rusthoven JJ, et al. (2002) Pemetrexed safety and dosing strategy. Semin Oncol 29:24–29PubMedGoogle Scholar
  9. 9.
    Schultz RM, Chen VJ, Bewley JR, et al. (1999) Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs. Semin Oncol 26(Suppl 6):68–73PubMedGoogle Scholar
  10. 10.
    Wang Y, Zhao R, Goldman ID (2003) Decreased expression of the reduced folate carrier and folylpolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukaemia cell line. Biochem Pharmacol 65(7):1163–1170PubMedCrossRefGoogle Scholar
  11. 11.
    Sigmond J, Backus HH, Wouters D, Temmink OH, Jansen G, Peters GJ (2003) Induction of resistance to the multitargeted antifolate pemetrexed (Alimta) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression. Biochem Pharmacol 66(3):431–438PubMedCrossRefGoogle Scholar
  12. 12.
    Giovannetti E, Mey V, Nannizzi S, et al. (2005) Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells. Mol Pharmacol 68:110–118PubMedGoogle Scholar
  13. 13.
    Kim JH, Lee KW, Jung Y, et al. (2005) Cytotoxic effects of pemetrexed in gastric cancer cells. Cancer Sci 96:365–371PubMedCrossRefGoogle Scholar
  14. 14.
    Vogel M, Hilsenbeck SG, Depenbrock H, et al. (1993) Preclinical activity of taxotere (RP56976, NSC628503) against freshly explanted clonogenic human tumor cells: comparison with taxol and conventional antineoplastic agents. Eur J Cancer 29A(14):2009–2014PubMedCrossRefGoogle Scholar
  15. 15.
    Hanauske AR, Hilsenbeck SG, von Hoff DD (2004) Human tumor screening. In: Teicher BA, Andrews PA (eds) Cancer drug discovery and development: Anticancer drug development guide: Preclinical screenings, clinical trials, and approval. 2nd edition. Humana, Totowa, NJ, USA pp 63–76Google Scholar
  16. 16.
    R Development Core Team (2003) R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical ComputingGoogle Scholar
  17. 17.
    The R project for statistical computing
  18. 18.
    Gomez H, Santillana S, Vallejos C, et al. (2006) A phase II trial of pemetrexed in locally advanced breast cancer: clinical response and association with molecular target expression. Clin Cancer Res 12(3):832–838PubMedCrossRefGoogle Scholar
  19. 19.
    Ardalan B, Dang Z (1996) Thymidylate synthase gene expression in normal and malignant colorectal tissues: relation to in vivo response and survival. Proc Am Assoc Cancer Res 37:201Google Scholar
  20. 20.
    Bathe O, Francesci D, Livingstone A, Moffatt FL, Tian E, Ardalan B (1999) Increased thymidylate synthase gene expression in liver metastases from colorectal carcinoma: implications for chemotherapeutic options and survival. Cancer J Sci Am 5:34–40PubMedGoogle Scholar
  21. 21.
    Cascinu S, Achsele C, Barni S, et al. (1999) Thymidylate synthase protein expression in advanced colon cancer: correlation with site of metastasis and the clinical response to leucovorin-modulated bolus 5-fluorouracil. Clin Cancer Res 5:1996–1999PubMedGoogle Scholar
  22. 22.
    Johnston PG, Lenz HJ, Leichman CG, et al. (1995) Thymidylate synthase gene and protein expression correlate and are associated with response to 5-FU in human colorectal and gastric tumors. Cancer Res 55:1407–1412PubMedGoogle Scholar
  23. 23.
    Lenz HJ, Leichman CG, Danenberg KD, et al. (1996) Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol 14:176–182PubMedGoogle Scholar
  24. 24.
    Paradiso A, Simone G, Petroni S, et al. (2000) Thymidylate synthase and p53 primary tumour expression as predictive factors for advanced colorectal cancer patients. Br J Cancer 82:560–567PubMedCrossRefGoogle Scholar
  25. 25.
    Salonga D, Danenberg KD, Johnson M, et al. (2000) Colorectal tumors responding to 5-Fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 6:1322–1327PubMedGoogle Scholar
  26. 26.
    Sympath J, Adachi M, Hatse S, Naesens L, Balzari J, Flatley RM, Metherly LH, Schuetz JD (2002) Role of MRP4 and MRP5 in biology and chemotherapy. AAPS PharmSci 4:E14CrossRefGoogle Scholar
  27. 27.
    Reid G, Wielinga P, Zelcer N, De Haas M, Van Deemter L, Wijnholds J, Balarini J, Borst P (2003) Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 63:1094–1103PubMedCrossRefGoogle Scholar
  28. 28.
    Pratt S, Shepard RL, Kandasamy RA, Johnston PA, Perry W III, Dantzig AH (2005) The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites. Mol Cancer Ther 4:855–863PubMedCrossRefGoogle Scholar
  29. 29.
    Pratt s, Chen V, Perry WI 3rd, Starling JJ, Dantzig AH (2006) Kinetic validation of the use of carboxydichlorofluoresceine as a drug surrogate for MRP5-mediated transport. Eur J Pharm Sci 27:524–532PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Axel-Rainer Hanauske
    • 1
    • 5
  • Ulrike Eismann
    • 1
  • Olaf Oberschmidt
    • 1
  • Heike Pospisil
    • 2
  • Steve Hoffmann
    • 2
  • Hartmut Hanauske-Abel
    • 3
  • Doreen Ma
    • 4
  • Victor Chen
    • 4
  • Paolo Paoletti
    • 4
  • Clet Niyikiza
    • 4
  1. 1.Asklepios KlinikHamburgGermany
  2. 2.Zentrum für BioinformatikUniversität HamburgHamburgGermany
  3. 3.UMDNJ-New Jersey Medical SchoolNewarkUSA
  4. 4.Eli Lilly & CompanyIndianapolisUSA
  5. 5.HamburgGermany

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