CORRESPONDENCE

Re: Potential Use of Imatinib in Ewing’s Sarcoma: Evidence for In Vitro and In Vivo Activity

Elena Tamborini, Lorena Bonadiman, Veronica Albertini, Marco A. Pierotti, Silvana Pilotti

Affiliation of authors: E. Tamborini, L. Bonadiman, V. Albertini, S. Pilotti (Laboratory of Experimental Molecular Pathology, Department of Pathology), M. A. Pierotti (Experimental Oncology Department), Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.

Correspondence to: Silvana Pilotti, M.D., Laboratory of Experimental Molecular Pathology, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy (e-mail: silvana.pilotti{at}istitutotumori.mi.it).

We read with interest the article by Merchant et al. (1) concerning the presence of activated tyrosine kinases, KIT and platelet-derived growth factor receptor {beta}, in Ewing’s sarcoma cell lines. In this article, the authors demonstrate that imatinib interferes with the growth of Ewing’s sarcoma cell lines in vitro and in vivo, thus identifying Ewing’s sarcoma as a possible candidate for this pharmacologic therapy.

Our preliminary results on the molecular characterization of surgical specimens of primary and metastatic Ewing’s sarcomas fully support the authors’ preclinical data, giving us the opportunity to confirm that the findings observed on cell lines (1,2) parallel those observed on parent tumors (1).

We used reverse transcription–polymerase chain reaction (RT–PCR) to investigate 52 Ewing’s sarcoma specimens (35 EWS-FLI1 type 1, eight EWS-FLI1 type 2, four EWS-FLI1 alternative transcripts, and five EWS-ERG) carrying the specific translocations and found that KIT and stem cell factor (SCF) mRNAs were expressed in 40 (77%) of the 52 specimens. Immunoprecipitation experiments performed on 10 specimens to detect KIT and phosphorylated KIT protein, with sufficient frozen material available, complemented the molecular results. Specimens with negative results from RT–PCR were weakly positive for KIT protein, whereas specimens with positive results from RT–PCR were also positive for phosphorylated KIT receptor protein (Fig. 1Go). Our results thus indicate that a percentage of Ewing’s sarcomas express the KIT tyrosine kinase activated by the autocrine loop.



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Fig. 1. Expression of KIT protein and phosphorylated KIT protein in Ewing’s sarcoma specimens. Tumors that expressed both KIT and stem cell factor (SCF) mRNA were as follows: lane C+ = the positive control, NIH3T3 cell line expressing an autophosphorylated KIT receptor (with deletion of codon 559); lane C– = normal skin; lanes 1 and 5 = EWS-FLI1 type 2 specimen; lane 3 = EWS-ERG specimen; lane 6 = EWS-FLI1 type 1 specimen. Tumors that were negative for both KIT and SCF mRNAs were as follows: lane 2 = EWS-FLI1 type 1 specimen; lane 4 = EWS-FLI1 specimen, where exon 10 of EWS was fused with exon 6 of FLI1. Top: KIT phosphorylation. Equal amounts of total protein were immunoprecipitated with a monoclonal antibody specific for KIT receptor (Ab-3 NeoMarkers; Lab Vision, Union City, CA) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% gel. The membrane was incubated with an anti-phospho-tyrosine antibody (Clone 4G; Upstate Biotechnology, Lake Placid, NY) to detect proteins with phosphorylated tyrosine residues. Bottom: KIT expression. The same membrane was stripped and incubated with a polyclonal KIT antibody (c-19; Santa Cruz Biotechnology, Santa Cruz, CA). KIT receptor was expressed in those specimens that expressed KIT mRNA.

 
Although KIT tyrosine kinase might not be the driving force in these tumors, the results of Merchant et al. indicate that KIT is essential for growth and survival of these tumors, as demonstrated by cell growth inhibition after imatinibdependent KIT inactivation. These results, coupled with findings from small-cell lung carcinoma cell lines (3) showing that activation of the KIT tyrosine kinase-dependent autocrine loop may be inhibited by imatinib, further reinforce the notion that the action of this drug is not restricted to KIT activation achieved through gain-of-function mutations, as in the gastrointestinal stromal tumor model. As pointed out in the accompanying editorial (4), imatinib may not be useful in patients with Ewing’s sarcoma because of the high plasma drug levels needed that are not clinically achievable. However, the indication that a KIT-mediated autocrine loop in Ewing’s sarcoma might represent a therapeutic target suggests that modification of imatinib to increase its efficacy for this tyrosine kinase receptor could overcome this problem. Alternatively one could envisage the development of new drugs able to effectively inhibit the various pathogenetically activated forms of KIT, such as PP1 (5), but without clinical side effects. Thus, the preclinical results of Merchant et al. and our data on surgical specimens are particularly relevant because they provide the molecular and biochemical foundation for a new target for Ewing’s sarcoma therapies.

NOTES

Supported by grants from Ministero della Sanita, Ricerca Finalizzata 2001 Italy, and by AIRC 2001 (Associazione Italiana per la Ricerca sul Cancro) grant 420.198.122.

Marco A. Pierotti and Silvana Pilotti are senior co-authors.

REFERENCES

1 Merchant MS, Woo CW, Mackall CL, Thiele CJ. Potential use of imatinib in Ewing’s Sarcoma: evidence for in vitro and in vivo activity. J Natl Cancer Inst 2002;94:1673–9.[Abstract/Free Full Text]

2 Ricotti E, Fagioli F, Garelli E, Linari C, Crescenzio N, Horenstein AL, et al. c-kit is expressed in soft tissue sarcoma of neuroectodermic origin and its ligand prevents apoptosis of neoplastic cells. Blood 1998;91:2397–405.[Abstract/Free Full Text]

3 Krystal GW, Honsawek S, Litz J, Buchdunger E. The selective tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth. Clin Cancer Res 2000;6:3319–26.[Abstract/Free Full Text]

4 Druker BJ. Taking aim at Ewing’s sarcoma: is KIT a target and will imatinib work? J Natl Cancer Inst 2002;94:1660–1.[Free Full Text]

5 Tatton L, Morley GM, Chopra R, Khwaja A. The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases. J Biol Chem 2003;278:4847–53.[Abstract/Free Full Text]


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