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Imatinib Is Not a Potential Alternative Treatment for Uterine Leiomyosarcoma

Centro de Investigación del Cancer Universidad de Salamanca-CSIC, Salamanca, Spain

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).

View larger version (81K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Expression of KIT receptor. A, representative immunohistochemical study of KIT receptor in paraffin-embedded tissue of a uterine leiomyosarcoma included in the tissue array (anti-CD117, DakoCytomation A4502) showing lack of KIT immunostaining in the tumor cells and a mast cell with a strong staining with KIT, which served as a positive internal control. Inset, typical plasma membrane staining pattern (x400; inset, x1,000). B, a 0.6 mm core from a GIST used as a positive control for KIT immunostaining. We can observe spindle tumor cells with eosinophilic fibrillary cytoplasm and ovoid vesicular nuclei. The inset allows us to appreciate strong and extensive immunoreactivity with a plasma membrane/cytoplasm pattern. C–E, illustrative bivariate dot plot histograms of immunophenotypic expression of c-kit (CD117) on two uterine leiomyosarcomas (C and D) and a GIST (E), as analyzed by multiparameter flow cytometry. As illustrated in (E), high CD117 expression was observed in the GIST tumor, whereas CD117 was either absent or expressed in a minor proportion of all cells (<1%) of the two uterine leiomyosarcomas (LMS1 and LMS2, C and D, respectively). Interestingly, CD117+ cells from the GIST tumor were CD45 negative; those from the tumor displayed in (C) were CD45+/FCRIgEI+, a phenotype highly suggestive of a mast cell origin (data not shown).

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).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression and functionality of KIT receptor. Western blotting was done using four frozen LMS samples previously included in the tissue array; a frozen sample from a GIST also included in the tissue array was used as a positive control. 4B42 is a Ewing tumor sample and LAN1 is a neuroblastoma cell line. Top, to assess phosphorylation of KIT, we incubated the membrane with an antibody for the phosphorylated form of KIT p-KIT 719Tyr. An intense band for the GIST was observed with no expression in any LMS. A673 and TC71, two Ewing tumor cell lines used as a positive control, were also positive. All LMS were negative. Middle, stripping and reincubation with anti–total KIT showed a strong 145 kDa band in the GIST and Ewing tumor cell lines, and a very faint one in LMS1. Bottom, ß-actin was used as a loading control.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Functionality of KIT receptor. Immunoprecipitation studies were done in the same samples used in Fig. 2. Western blotting using an antibody for phosphotyrosine residues showed a strong 145 kDa band only in the GIST sample, whereas no uterine leiomyosarcoma was positive. That means there is no constitutive activation of KIT pathway signaling in uterine leiomyosarcomas. TC-71 and SK-ES-1 are two Ewing tumor cell lines. 4B42 is a Ewing tumor sample.

References

1. Raspollini MR, Amunni G, Villanucci A, et al. c-Kit expression in patients with uterine leiomyosarcomas: a potential alternative therapeutic treatment. Clin Cancer Res 2004;10:3500–3.[Abstract/Free Full Text]
2. Ueda S, Mizuki M, Ikeda H, et al. Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration. Blood 2002;99:3342–9.[Abstract/Free Full Text]
3. Wang L, Felix JC, Lee JL, et al. The proto-oncogene c-kit is expressed in leiomyosarcomas of the uterus. Gynecol Oncol 2003;90:402–6.[CrossRef][Medline]
4. Rushing RS, Shajahan S, Chendil D, et al. Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT. Gynecol Oncol 2003;91:9–14.[Medline]
5. Leath CA III, Straughn JM Jr, Conner MG, et al. Immunohistochemical evaluation of the c-kit proto-oncogene in sarcomas of the uterus: a case series. J Reprod Med 2004;49:71–5.[Medline]
6. Hornick JL, Fletcher CD. Immunohistochemical staining for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Am J Clin Pathol 2002;117:188–93.[CrossRef][Medline]
7. Oliva E, Young RH, Amin MB, Clement PB. An immunohistochemical analysis of endometrial stromal and smooth muscle tumors of the uterus: a study of 54 cases emphasizing the importance of using a panel because of overlap in immunoreactivity for individual antibodies. Am J Surg Pathol 2002;26:403–12.[CrossRef][Medline]
8. Klein WM, Kurman RJ. Lack of expression of c-kit protein (CD117) in mesenchymal tumors of the uterus and ovary. Int J Gynecol Pathol 2003;22:181–4.[Medline]
9. Gibson PC, Cooper K. CD117 (KIT): a diverse protein with selective applications in surgical pathology. Adv Anat Pathol 2002;9:65–9.[CrossRef][Medline]
10. Went PT, Dirnhofer S, Bundi M, et al. Prevalence of KIT expression in human tumors. J Clin Oncol 2004;22:4514–22.[Abstract/Free Full Text]

Department of Human Pathology and Oncology, School of Medicine, University of Florence, Viale GB Morgagni 85, 50134 Florence, Italy

Milena Paglierani and Gian Luigi Taddei

Department of Human Pathology and Oncology, School of Medicine, University of Florence, Florence, Italy

Gianni Amunni and Alessandro Villanucci

Department of Gynecology, Perinatology and Reproductive Medicine, School of Medicine, University of Florence, Florence, Italy

Pamela Pinzani and Lisa Simi

Department of Clinical Physiopathology, School of Medicine, University of Florence, Florence, Italy

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.

View larger version (131K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A representative example of a uterine leiomyosarcoma that showed an intense c-KIT immunostaining in the cytoplasm of the tumor cells.

View larger version (123K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. c-KIT–positive inside control which was the mast cells in a c-KIT–negative uterine leiomyosarcoma.

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.

References

1. Serrano C, Mackintosh C, Herrero D, et al. Imatinib is not a potential alternative treatment for uterine leiomyosarcoma. Clin Cancer Res 2005;11:4977–9.[Free Full Text]
2. Raspollini MR, Amunni G, Villanucci A, et al. c-KIT expression in patients with uterine leiomyosarcomas. A potential alternative therapeutic treatment. Clin Cancer Res 2004;10:3500–3.[Abstract/Free Full Text]
3. Rushing RS, Shajahan S, Chendil D, et al. Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT. Gynecol Oncol 2003;91:9–14.[Medline]
4. Raspollini MR, Pinzani P, Simi L, et al. Letter to the Editor: "Uterine leiomyosarcomas express KIT protein but lack mutation(s) in exon 9 of c-KIT" Gynecol Oncol. In press.

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