KIT (CD117)-Positive Breast Cancers Are Infrequent and Lack KIT Gene Mutations
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178 Vol. 10, 178–183, January 1, 2004 Clinical Cancer Research KIT (CD117)-Positive Breast Cancers Are Infrequent and Lack KIT Gene Mutations Ronald Simon,1 Soti Panussis,1 Robert Maurer,2 ations in malignant tumors are of high interest because KIT is Hanspeter Spichtin,3 Kathrin Glatz,1 one of the targets of the tyrosine kinase inhibitor imatinib Coya Tapia,1 Martina Mirlacher,1 Alex Rufle,1 mesylate (STI571; Glivec). STI571 has initially been shown to be effective in the treatment of chronic myeloid leukemia, where Joachim Torhorst,1 and Guido Sauter1 it targets the BCR-ABL fusion protein (7). More recently, 1 Institute of Pathology, University of Basel, Basel, Switzerland; significant treatment responses were also seen in patients with 2Institute of Pathology, City Spital Triemli, Zu¨rich, Switzerland; and 3Institute of Clinical Pathology, Basel, Switzerland advanced KIT-positive gastrointestinal stroma tumors (8) and dermatofibrosarcoma protuberans (9). It is hoped that other KIT-positive tumors may also benefit from STI571 therapy. ABSTRACT In the breast, KIT protein was found in normal breast Purpose: KIT (CD117) is a transmembrane tyrosine epithelium, nonneoplastic breast diseases (10), and in 1–13% of kinase representing a target for STI571 (Glivec) therapy. cancers (10–12). Despite a relatively low prevalence of KIT Some KIT-overexpressing solid tumors have responded fa- expression, breast cancer may be an important candidate for vorably to STI571, potentially because of the presence of STI571 therapy because of its high frequency. Several clinical KIT-activating mutations. trials are now investigating the effect of STI571 on KIT-positive Experimental Design: To investigate the epidemiology malignancies of various origins (13–17). It has been suggested of KIT overexpression and mutations, we investigated a that the response rate may be particularly high in KIT-express- series of 1654 breast cancers. All tumors were analyzed by ing tumors that also harbor activating KIT mutations (18–24). immunohistochemistry in a tissue microarray format. The rate of KIT mutations in breast cancer has never been Results: KIT expression was always present in normal studied. Collecting such information would require the analysis breast epithelium. However, cancer analysis revealed the of a very large number of tumors if the prevalence of KIT only 43 of 1654 (2.6%) tumors were KIT-positive. KIT positivity is as low as the 1% suggested by one recent study expression was more frequent in medullary cancer (9 of 47 (10). In this project we took advantage of a preexisting tissue positive; 19.1%) than in any other histological tumor sub- microarray (TMA) containing samples from Ͼ2000 breast can- type (P < 0.001). KIT expression was significantly associ- cer patients to identify a subset of KIT-positive breast cancers. ated with high tumor grade (P 0.0001) but unrelated to pT < These tumors were then sequenced to screen for mutations at and pN categories or patient survival. Mutation analysis of multiple exons of the KIT gene. exons 2, 8, 9, 11, 13, and 17 was negative in 10 KIT-positive tumors. Conclusions: Overall, our data show that a high level of MATERIALS AND METHODS KIT expression occurs infrequently in breast cancer. KIT- Breast Cancer TMA. A total of 2197 patients with a positive breast cancers may not reflect “KIT up-regulation” median age of 62 (range, 26–101) years were evaluated retro- because KIT is also expressed in normal breast epithelium. spectively. Raw survival data were either obtained from the The lack of KIT mutations also argues against the therapeu- cancer registry of Basel or collected from the patients’ attending tic efficacy of STI571 in breast cancer. physicians. The mean follow-up period was 68 months (range, 1–176 months). Formalin-fixed (buffered 4%), paraffin-embed- INTRODUCTION ded tumor material was available from the Institute of Pathol- KIT (CD117) is a transmembrane tyrosine kinase acting as ogy, University Hospital Basel; the Institute for Clinical Pathol- a type III receptor for mast cell growth factor. It plays an ogy in Basel; and the Triemli Hospital in Zu¨rich. The use of important role in the development of multiple cell types, includ- these specimens and data in research were approved by the ing hematopoietic cells, germ cells, and melanocytes (1). KIT Ethics Committee of the Basel University Hospital. The patho- can also be detected in various tumor entities (2–6). KIT alter- logical stage and nodal status were obtained from the primary pathology reports. All slides from all tumors were reviewed by one of two pathologists (J. T. and G. S.) to define the histolog- ical grade according to Elston and Ellis (BRE: Ref. 25) and the Received 4/8/03; revised 9/8/03; accepted 9/8/03. histological tumor type. For the TMA composition, see Table 2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked TMA construction was as described previously (26). Briefly, advertisement in accordance with 18 U.S.C. Section 1734 solely to tissue cylinders with a diameter of 0.6 mm were punched from indicate this fact. representative tumor areas of a “donor” tissue block by use of a Requests for reprints: Guido Sauter, Institute of Pathology, Division of semiautomatic robotic precision instrument and placed in six Molecular Pathology, University of Basel, Schoenbeinstrasse 40, CH- 4031 Basel, Switzerland. Phone: 41 61 265 2889; Fax: 41 61 265 2966; different recipient paraffin blocks, each containing 342–522 E-mail: [email protected]. individual samples. Four-m sections of the resulting multitu- Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 179 Table 1 Primer sequences for KIT sequence analysis Exon Direction Sequence (5Ј 3 3Ј) 2 Forward CAT CCA TCC ATC CAG GAA AA Reverse 1 ACT GCA GAA AGC CAA GCA TT Reverse 2 (nested) GCA GAA AGC CAA GCA TTT ACC 8 Forward GCT GAG GTT TTC CAG CAC TC Reverse 1 CAG TCC TTC CCC TCT GCA T Reverse 2 (nested) CCT CTG CTC AGT TCC TGG AC 9 Forward 1 TCC TAG AGT AAG CCA GGG CTT Forward 2 (nested) AGC CAG GGC TTT TGT TTT CT Reverse TGG TAG ACA GAG CCT AAA CAT CC 11 Forward CCA GAG TGC TCT AAT GAC TG Reverse 1 AGC CCC TGT TTC ATA CTG AC Reverse 2 (nested) GAG TTT CCC AGA AAC AGG CTG AGT 13 Forward 1 GCT TGA CAT CAG TTT GCC AG Forward 2 (nested) TGA CAT CAG TTT GCC AGT TG Reverse AAA GGC AGC TTG GAC ACG GCT TTA 17 Forward TCC TTA CTC ATG GTC GGA TC Reverse 1 CAG GAC TGT CAA GCA GAG AA Reverse 2 (nested) ACT GTC AAG CAG AGA ATG GG mor TMA blocks were transferred to an adhesive-coated slide ers designed for the PCR and the sequence reactions are listed in system (Instrumedics Inc., Hackensack, NJ). Table 1. Sequence products were analyzed on an ABI Prism 310 Immunohistochemistry (IHC). IHC was performed Genetic Analyzer (Applied Biosystems). with the anti-CD117 antibody from DAKO (A4502). In a com- parison of multiple antibodies, A4502 had previously yielded the highest specificity and the least background.4 A4502 was RESULTS applied at a dilution of 1:300 at room temperature for 2.5 h, after IHC. The IHC staining was interpretable in 1654 3 min of pressure cooking in 10 mM sodium citrate buffer (pH (75.3%) of the 2197 arrayed tissue spots on the TMA. KIT 6.0) for antigen retrieval. Endogenous peroxidase was blocked staining was observed in the membranes of 43 (2.6%) of these with 0.3% hydrogen peroxide diluted in methanol for 30 min. A tumors. Staining was considered weak in 29 (1.8%) and strong standard kit (Vector ABC) was used for visualization. For check in 14 (0.8%) tumors, according to our arbitrarily selected defi- for specificity, we performed preabsorption experiments with nitions. Examples of KIT-positive tumors are shown in Fig. 1. A the antibody (CD117 peptide stock solution; PP1518; Neomar- comparison with tumor phenotype revealed that KIT expression kers). This antigen matches the sequence of the epitope recog- was particularly frequent in medullary breast cancer, in which 9 nized by the DAKO polyclonal antibody. A gastrointestinal of 47 tumors (19.1%) were positive. An increased frequency stroma tumor sample was used as a positive control. For each was also seen in papillary (2 of 23 positive; 8.7%) and apocrine tissue sample, the percentage of positive cells was estimated and cancers (1 of 11 positive; 9%), but the numbers of analyzed the staining intensity was recorded semiquantitatively as 1ϩ, tumors were too low for these subtypes to allow us to draw firm 2ϩ,or3ϩ. For statistical analyses, the staining results were conclusions. There was no association between KIT expression categorized into three groups. Tumors without any staining were and pT or pN stage, but there was a significant relationship considered negative. Tumors with 1ϩ staining and tumors with between KIT positivity and BRE grade (Table 2). This was also 2ϩ staining in Ͻ50% of cells were considered weakly positive. true if medullary carcinomas were excluded from analysis (P ϭ All remaining samples (2ϩ staining in Ն50% of cells and 0.0002). There was no association between KIT expression and samples with 3ϩ staining) were considered strongly positive. patient survival (Fig. 2). Conventional “large sections” were analyzed by IHC from 36 A comparison of TMA data with large-section KIT staining KIT-positive and 28 KIT-negative tumors to further validate the of 64 tumors revealed good concordance. Detectable KIT stain- IHC results obtained on TMAs.