Imatinib Is Not a Potential Alternative Treatment for Uterine Leiomyosarcoma
Authors: César Serrano, Carlos Mackintosh, David Herrero, Ana S. Martins, Teresa Hernández, José Pérez-Fontán, Angustias Pérez, Eugenio Serrano, Alberto Orfao, Agustín Bullón, Mar Abad, Enrique de Alava
Journal: Clinical Cancer Research (2005)

To the Editor: We read with great interest the article written by Raspollini et al. (1). The authors studied a series including 32 cases of uterine leiomyosarcomas, which showed a 50% rate of positive immunostaining for KIT. Based on these results,the authors suggested the possibility of carrying on ananti–tyrosine kinase treatment for this neoplasm (1). Our study aims to validate these results on a series including 18 uterine leiomyosarcoma cases, focusing on investigation of KIT signaling pathway activation. Previous approval of the Institutional Review Board was obtained.We first did a tissue microarray using representative areas of paraffin-embedded uterine leiomyosarcomas, including four 0.6 mm cores for each case. Two gastrointestinal stromal tumors (GIST) were used as a positive control. Immunostaining for KIT using two antibodies from different sources (DakoCytomation, Carpinteria, CA A4502, 1:400; BioGenex, San Ramon, CA T595, prediluted) used following manufacturers' instructions, was negative in all our uterine leiomyosarcoma series, showing strong positivity in the mast cells infiltrating many samples of uterine leiomyosarcoma (Fig. 1A), which served as an internal control for the immunostaining. Accordingly, flow cytometric studies done on cell suspensions of the uterine leiomyosarcoma depicted in Fig. 1A showed a small cell KIT+ population with a characteristic mast cell immunophenotype (CD45+/FCRIgEI+, data not shown; Fig. 1C). Other uterine leiomyosarcoma case showed lack of a KIT+ cell population (Fig. 1D). One case of GIST served as an external positive control, showing strong and extensive immunoreactivity for KIT as shown by immunohistochemistry (Fig. 1B) and flow cytometry (Fig. 1E).Genomic DNA was extracted from paraffin-embedded tissues of the 18 uterine leiomyosarcomas, and DNA sequencing for exons 11 and 9 of c-kit was negative for mutations (data not shown). Moreover, DNA sequencing for exon 12 of PDGFRA, another tyrosine kinase receptor associated with the KIT signaling pathway, also showed lack of mutations.For a therapy for uterine leiomyosarcomas based on KIT receptor signaling blockade to be effective, constitutive phosphorylation of KIT should be present. To assess whether this was the case in our series of uterine leiomyosarcomas, we extracted proteins from frozen tissue corresponding to four cases of uterine leiomyosarcomas and one GIST, all of them included in the previously studied tissue microarray. Western blot, with an anti–c-KIT antibody (A4502, 1:400, DakoCytomation), showed a strongly positive band for the GIST and a weakly positive band in one uterine leiomyosarcoma, which we attributed to mast cell staining (Fig. 2). Nevertheless, Western blot for phospho–719Tyr-KIT (Cell Signaling, Inc., Beverly, MA) was positive only in the GIST but negative in all four uterine leiomyosarcomas (Fig. 2), indicating this particular residue, which is key for the KIT pathway activation (2), was not phosphorylated. Moreover, Western blot for anti–phosphotyrosine residues (Upstate Biotechnology, Lake Placid, NY) after immunoprecipitation with KIT showed the analogous results: a strongly positive control band in the GIST and absence of band in uterine leiomyosarcoma cases, indicating the lack of any phosphorylated tyrosine residues in KIT protein (Fig. 3).KIT receptor overexpression in uterine leiomyosarcoma remains controversial (3–8). KIT is a tyrosine kinase receptor that has been involved in the molecular pathogenesis of several tumor types (GIST, seminoma, small cell lung carcinoma, endometrial carcinomas, ovarian epithelial carcinomas, etc.; ref. 9) and, more recently, of uterine leiomyosarcomas (1). KIT, however, needs to be phosphorylated to start its signaling cascade. We have shown that the uterine leiomyosarcomas included in this study show no phosphorylation of KIT, as shown by Western blot and immunoprecipitation in the absence of either kit or PDGFR mutations. The antibody for total KIT we used in the Western blot/immunoprecipitation studies as well as one of those used in the immunohistochemical studies (DakoCytomation A4502) has been recently shown to be the most sensitive and specific antibody (less background staining) in a comparative study done with seven different KIT antibodies using tissue microarrays (10). Our study shows that validation of a new therapeutic target requires a combined technical approach. Our results also indicate that KIT is probably not involved in uterine leiomyosarcoma pathogenesis, and that KIT is not a very likely target for anti–tyrosine kinase drug therapy in uterine leiomyosarcoma.In response:We thank Serano and colleagues (1) for allowing us to point out the question of c-KIT expression in uterine leiomyosarcomas. In our previous study (2), we observed a positive staining in more than 50% of the 32 analyzed cases (Fig. 1), but, in addition, we already observed in the negative leiomyosarcoma cases the positive staining of the mast cells inside the tumor (Fig. 2). We scored as positive only cases showing at least 20% moderate to strong membranous and/or cytoplasmic positivity. Other than the expected positivity in mast cells, we did not observe background or nonspecific staining of adjacent uterine parenchyma. In our study, a section containing a gastrointestinal stromal tumor case strongly positive for c-KIT served as the positive external control with each run. Mast cells within the tumor served as positive internal control. In addition, each tumor was also stained with Giemsa to identify mast cells for comparison with the c-KIT results. A negative control sample was included in each run by omitting the primary antibody and yielded no signal. Conversely, Serano and colleagues did not show positive cases in their series of 18 uterine leiomyosarcomas.Some reasons may explain the different results: first, the different antibodies used as reported in other studies (3); second, differences may be due to the employment of a system of antigen retrieval with microwave pretreatment; and third, we reported the Fédération Internationale des Gynaecologistes et Obstetristes stage of the disease, whereas this is not described in the Serano's report.It is of interest that in previous studies we reported no mutation of exon 11 (2), exon 9 (4), and exon 12 (data not published) in the immunohistochemically c-KIT–positive uterine leiomyosarcoma cases. Conversely, Serano and colleagues (1) investigated the molecular assessment of exons 9 and 11 in four immunohistochemically c-KIT–negative uterine leiomyosarcoma cases showing lack of mutation, thus confirming our molecular results.This therefore suggests that uterine leiomyosarcomas lack the mutations described in gastrointestinal stromal tumors. Perhaps, unlike in gastrointestinal stromal tumor, KIT is not the most important factor in the pathogenesis of uterine leiomyosarcoma.