Gynecologic Oncology 101 (2006) 363–366www.elsevier.com/locate/ygyno

Case Report

Reclassification of a tubal leiomyosarcoma as an eGIST by molecularevaluation of c-KIT

Rosemary Foster a,⁎, Steven Solano a, Jennifer Mahoney a, Arlan Fuller b,Esther Oliva c, Michael V. Seiden a

a Division of Hematology/Oncology, Massachusetts General Hospital, Boston, MA 02114, USAb Division of Gynecologic Oncology, Massachusetts General Hospital, Boston, MA 02114, USA

c Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA

Received 11 October 2005Available online 24 January 2006

Abstract

Background. Extragastrointestinal stromal tumors (eGISTs) are rare mesenchymal-derived tumors arising outside of the GI tract. eGISTs areoften histologically confused with leiomyosarcoma. Distinction between eGIST and leiomyosarcoma is critical because of the uniqueresponsiveness of eGISTs to the molecularly targeted agent imatinib.

Case. Awoman presented with a history of tubal spindle cell tumor that was initially diagnosed and treated as a leiomyosarcoma. Because ofminimal response to sarcoma directed chemotherapy, the possibility that the tumor was in fact an eGIST was investigated and supported byimmunohistochemical and mutational analyses of the c-Kit receptor tyrosine kinase. The patient currently has stable disease control on imatinibfor the last 18 months.

Conclusions. The possibility of eGIST should be considered in the differential diagnosis of tumors with a spindle cell morphology in thegynecologic tract especially when involving the ovary, fallopian tube, or uterine serosa.© 2005 Elsevier Inc. All rights reserved.

Keywords: Sarcoma; c-KIT mutation; eGIST; Imatinib

Introduction

The successful management of metastatic mesenchymaltumors of the gynecologic organs is limited by the relativeradiation and chemotherapy resistance of these tumors.Recently, imatinib has demonstrated highly effective anti-tumor activity in the treatment of gastrointestinal stromaltumors (GISTs) [1] highlighting the importance of distinguish-ing this tumor from leiomyosarcoma, the most commonmalignant mesenchymal tumor in the gynecologic tract, whichis significantly less responsive to targeted therapies.

Imatinib is a small molecule inhibitor that specifically targetsseveral tyrosine kinases. The c-KIT gene encodes a type IIIreceptor tyrosine kinase (Kit) that is expressed in 90–95% ofGISTs [2]. Gain of function c-KIT mutations are characteristic

⁎ Corresponding author. Fax: +1 617 726 6974.E-mail address: Rfoster1@partners.org (R. Foster).

0090-8258/$ - see front matter © 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ygyno.2005.12.022

of GISTs with most mutations occurring in exon 11 [3]. Exons9, 13, and 17 are less frequent sites of mutation [4,5]. Kittyrosine kinase activity is effectively inhibited by imatinibwhich has emerged as a highly effective therapy for GISTs [1].As the name implies, the majority of GISTs arise in thegastrointestinal tract although a minority arises at extragas-trointestinal sites [6]. These eGISTs occasionally involvegynecologic organs and hence should be considered as analternative diagnosis in women diagnosed with a leiomyosar-coma particularly when tumors involve the fallopian tube, theovary, or the serosa of the uterus.

Case report

A 51-year old female presented to our institution in 2000shortly after undergoing a total vaginal hysterectomy andbilateral oophorectomy. At the time of the primary surgicalprocedure, the fallopian tubes were left in situ due to excessive

Fig. 1. Hematoxylin and eosin staining of formalin fixed paraffin embedded specimens of the tumor reveals long fascicles of spindle tumor cells that closely mimic theappearance of leiomyosarcoma (left panel). At higher power (center panel), the tumor cells show prominent cytoplasmic vacuoles and mild to moderate cytologicatypia. The tumor cells also demonstrate strong and diffuse staining for the Kit tyrosine kinase receptor following immunohistochemical analysis (right panel).

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bleeding. Post-operative evaluation at our institution revealedan adnexal mass that on exploration was a large tubal-basedtumor with a small solitary metastasis on the bowel. The tumorwas diagnosed as a grade 2 leiomyosarcoma arising in the rightfallopian tube with evidence of a solitary small bowelmetastasis. The patient was treated with platinum andadriamycin and 45 cGy of pelvic radiotherapy followingcomplete surgical excision of the tumor. In 2003, the patientpresented with a peritoneal recurrence that was completelyresected and consolidated with intraperitoneal cisplatin therapy.Thirteen months later, another peritoneal recurrence wasdetected by CT scan and was resected. At that time, the

Fig. 2. Exon 9 amplified from germline and tumor DNA samples was analyzed by agbp fragment. In contrast, the exon 9 products derived from the tumor comprised bothfirst lane is a 100 bp marker ladder. Sequence analysis of the total exon 9 PCR produ

possibility of eGIST was considered and the patient initiatedimatinib therapy in May, 2004. She currently remains free ofdisease after 18 months of treatment.

Pathology results

The tumor involved the outer half of the wall of the rightfallopian tube and compressed the fallopian tube lumenresulting in secondary hydrosalpinx. The tumor measured11 × 8 × 4 cm and had a polypoid configuration with irregularinfiltrating borders. It was densely cellular and composed ofalternating areas of short or long fascicles of spindle cells with

arose gel electrophoresis (A). The germline-derived product is the expected 307the expected 307 bp fragment and a slightly larger second fragment (arrow). Thects from both germline and tumor genomic DNA revealed a heterozygous 6 bp

365R. Foster et al. / Gynecologic Oncology 101 (2006) 363–366

relatively abundant eosinophilic cytoplasm, many of which hadintracytoplasmic vacuoles (Fig. 1). The nuclei were oval toelongated, showing small or absent nucleoli, coarse chromatin,and moderate to severe degree of cytologic atypia. Mitoticactivity averaged 7 mitosis/10 high power fields. Areas ofnecrosis were seen. The tumor cells were intensively anddiffusely positive for c-Kit, an immunostain performed at thetime of the second recurrence of tumor. Staining for smoothmuscle markers including desmin and smooth muscle actin andS-100, as well as for estrogen and progesterone receptors, wasnegative. The small intestine nodule resected at the time of theright salpingectomy measured approximately 0.5 × 0.5 cm andshowed histologic features similar to those seen in the maintumor.

Molecular analysis of c-KIT

To evaluate c-KIT in the tumor, DNA was isolated fromformalin fixed paraffin embedded tumor sections using theQIAamp DNA minikit (Qiagen) following the manufacturer'sspecifications. Germline DNA was isolated from whole bloodusing a standard phenol:chloroform extraction method. PCRamplification of c-KIT exons 9 and 11 was carried out usingpreviously described primers and reaction conditions. DNAsequence analysis of the purified PCR products was carried outby the MGH DNA Sequencing Core Facility. No exon 11 c-KITmutations were detected in either the tumor or germline DNAsamples. The results from sequence analysis of exon 9 areshown in Fig. 2.

The exon 9 PCR product derived from the tumor samplecomprised two bands which could be resolved by agarose gelelectrophoresis (panel A) and the PCR products were directlysequenced. Comparison of c-KIT exon 9 sequence from thegermline and tumor DNA samples revealed a heterozygoussix base pair insertion specific to the tumor sample (panel B).This mutation has been described previously and results in analanine–tyrosine duplication at amino acids 501–502 in theextracellular domain of the Kit receptor. This findingsupports the reclassification of the tubal leiomyosarcoma asan eGIST.

Discussion

eGISTs are relatively uncommon tumors, comprising about5% of all GISTs [6]. Over the past 3 years, cases of GISTsoutside the gastrointestinal tract have been reported in thescrotum, bladder, ovary, omentum, pharynx, inguinal herniasac, hypochondrium, pancreas, and rectovaginal septum [7–15].This is the first case reported of a GIST arising in the fallopiantube.

It is important to note that until recently most GISTs wereclassified as smooth muscle tumors, more frequently asleiomyosarcomas since the two tumor types show overlappingmorphologic features [16]. It is also important to note thatalthough leiomyosarcomas may be positive for c-Kit and GISTsmay express smooth muscle markers, leiomyosarcomas do notharbor c-KIT mutations [17,18].

It has been well established that somatic mutation of the c-KIT gene is associated with the pathogenesis of GISTs and islikely to be the initiating oncogenic event in tumordevelopment [2]. The most frequently described mutationsare found in exon 11 which comprises the juxtamembraneregion of the Kit receptor. Recent protein modeling analyseshave demonstrated that mutations in this region of thereceptor disrupt the normal Kit autoinhibition conformationresulting in constitutive activation of receptor tyrosine kinaseactivity [19]. Exon 9 of the c-KIT gene encodes themembrane proximal extracellular domain of the Kit receptorand is a less frequent target of mutation in GISTs. Weanalyzed both exon 11 and exon 9 of the c-KIT gene in thepatient's germline and tumor-derived DNA and detected apreviously described heterozygous six base pair insertion inexon 9 that was restricted to the tumor sample. The exon 9insertion mutation results in duplication of alanine andtyrosine residues at amino acids 501–502. Biochemicalanalyses of the mutant protein have determined that theAla–Tyr duplication results in ligand independent autopho-sphorylation of the Kit receptor that may be due to disruptionof an anti-dimerization domain [4].

In a recent study, Reith and coworkers noted that severalpathologic factors studied in conventional GISTs were alsoassociated with higher risk for adverse outcome in eGISTs.These factors included high cellularity, N2 mitoses/10 highpower fields, and presence of necrosis [6]. Our case displayedtwo of these three features, namely more than 2 mitoses/10 highpower fields and necrosis. Furthermore, several studies haveaddressed potential correlations between specific Kit mutations,responsiveness to imatinib, and progression free survival.Patients with a c-KIT exon 11 mutation have a better responserate and longer interval of progression free survival incomparison to patients with mutations in c-KIT exon 9 [1,5].In May 2004, the patient described in this case report initiatedimatinib therapy at 400 mg per day. Although recent studieshave supported higher doses of imatinib, a trial of 600 mg perday in this patient was unsuccessful due to nausea. Shecontinues on the 400 mg daily dose of imatinib.

Accumulating evidence suggests that imatinib therapy canbecome less effective over time. The development of imatinibresistance is a common occurrence in the clinical managementof GISTs although the exact mechanism of drug failure isunclear [20]. Recent work has focused on the analysis ofpotential differences in the activation of downstream signalingpathways by various mutant forms of the Kit receptor as a firststep in identifying alternative molecular targets in GISTs [21].The development of therapies directed against proteinsdownstream of Kit may allow effective treatment of imatinibresistant treatments. Similarly, the use of tyrosine kinaseinhibitors with inhibitory mechanisms distinct from those ofimatinib has proven valuable. Clinical studies have demon-strated effective activity of the novel tyrosine kinase inhibitorSU11248 (Sutent™) in the treatment of imatinib resistant GISTs[22,23]. The most recent CT and PET scan findings obtainedfrom the patient described in these studies suggest some mildprogression of pelvic disease after 18 months of imatinib

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therapy. She is currently being screened for therapy withSU11248 (Sutent).

Acknowledgments

The authors acknowledge Dr. Neil Horowitz for suggestingwe consider evaluating c-Kit expression in this tumor at the timeof tumor recurrence. Laboratory studies were supported in partfrom ongoing support from the OCEAN Foundation at MGH.

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