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Journal of Clinical Oncology, Vol 22, No 22 (November 15), 2004: pp. 4514-4522 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.10.125 Prevalence of KIT Expression in Human TumorsFrom the Institute of Pathology, University of Basel; Novartis Pharma AG, Basel, Switzerland; and Diomeda Life Sciences Inc, Rockville, MD Address reprint requests to Guido Sauter, MD, Institute of Pathology, University of Basel, Schönbeinstrasse 40, 4031 Basel, Switzerland; e-mail: guido.sauter{at}unibas.ch
PURPOSE: KIT is a target for imatinib mesylate (Gleevec; Novartis Pharma, Basel, Switzerland). Gastrointestinal stromal tumors (GISTs) express KIT and respond favorably to imatinib therapy. To determine other tumors in which such a molecular targeted therapy might be indicated, we investigated KIT expression in different human tumor types. Because recent studies in GISTs suggest that KIT-activating mutations predict response to imatinib therapy, we also sequenced a subset of positive tumors. MATERIALS AND METHODS: More than 3,000 tumors from more than 120 different tumor categories were analyzed by immunohistochemistry in a tissue microarray format. Seven commercially available anti-KIT antibodies were initially evaluated. The antibody A4502 (DAKO) was selected for analysis because of a high frequency of positivity in GIST and low staining background in other tissues. To determine the frequency of KIT mutations in various tumor types, the exons 2, 8, 9, 11, 13, and 17 (where mutations previously were reported) were sequenced in 36 tumors with strong KIT expression. RESULTS: KIT positivity was detected in 28 of 28 GISTs (100%), 42 of 50 seminomas (84%), 34 of 52 adenoid-cystic carcinomas (65%), 14 of 39 malignant melanomas (35%), and eight of 47 large-cell carcinomas of the lung (17%), as well as in 47 additional tumor types. KIT mutations were found in six of 12 analyzed GISTs, but only in one of 24 other tumors. CONCLUSION: The results suggest that KIT expression occurs infrequently in most tumor types and that, with the exception of GISTs, KIT gene mutations are rare in immunohistochemically KIT-positive tumors.
KIT (CD117) is a transmembrane tyrosine kinase that acts as a receptor for mast cell growth factor (also known as stem cell factor or kit ligand). It belongs to the type III family of receptor kinases and can be detected in several normal cell types including hematopoietic cells, germ cells, interstitial cell of Cajal, ductal breast epithelium, mast cells, and melanocytes.1-5 KIT expression has been detected in a variety of different tumor entities. In gastrointestinal stromal tumors (GISTs), the frequency of KIT positivity is so high (90% to 95%) that immunohistochemical KIT detection is considered a prerequisite for the histologic diagnosis of GISTs. A smaller fraction of KIT-positive specimens has been described in at least 46 additional tumor entities.1,3,4,6-10 KIT expression in malignant tumors is of topical interest because KIT is one of the targets of the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec). Imatinib initially was shown to be effective in the treatment of chronic myeloid leukemia, in which it targets the kinase function of the BCR/ABL fusion protein.11,12 Subsequently, significant treatment responses were also reported in patients with advanced KIT-positive GIST.13-15 It is has been suggested that the response rate to imatinib may be particularly high in KIT-expressing tumors that also harbor activating KIT mutations.16 Previous studies investigating KIT expression in human tumors have used a variety of antibodies, staining protocols, and scoring criteria,1,3,4,6-10,17,18 which reduce the comparability of these studies. Thus, identification of tumor entities that may benefit from imatinib therapy requires investigation of KIT expression in a wide variety of tumors using comparable assessment protocols. To this end, we used a multitumor tissue microarray (TMA) containing more than 3,500 paraffin-embedded tumor samples representing more than 120 tumor types and subtypes19 to evaluate the epidemiology of KIT expression across a diverse array of human tumors. In addition, selected KIT-positive tumors were also sequenced to elucidate the epidemiology of KIT gene mutations.
TMA A total of 3,911 tissue samples from the archives of the Institute of Pathology, University of Basel (Basel, Switzerland), were assessed including 3,556 primary tumors from 134 tumor types and subtypes, and 355 samples from 34 different normal tissues. Tissues were fixed in formalin for variable time periods and then embedded in paraffin. TMA construction was as described previously.19 Briefly, tissue cylinders with a diameter of 0.6 mm were punched from representative tumor areas of a donor tissue block using a semiautomated precision instrument and brought into seven different recipient paraffin blocks each containing between 400 and 612 individual samples. Four-micrometer sections of the resulting multitumor TMA blocks were transferred to an adhesive-coated slide system (Instrumedics Inc., Hackensack, NJ).
Immunohistochemistry
In normal tissues, a cell type-specific distribution of KIT expression was recorded (+, weak; ++, moderate; +++, strong). For tumor tissues, the percentage of positive cells was estimated and the staining intensity was semiquantitatively recorded as 1+, 2+, or 3+. For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered negative. Tumors with 1+ staining intensity in less than 60% of cells and 2+ intensity in less than 30% of cells were considered weakly positive. Tumors with 1+ staining intensity in
c-KIT Sequence Analysis
Antibody Evaluation The results of our comparison of seven different antibodies are listed in Table 2. The antibodies sc-1494, sc-168, and MS-271 were not pursued further because of an unacceptably high level of background staining in stromal cells (Figs 1C to 1H). No background problem was observed for the antibodies A4502, sc-13508, No. 566, and NCL-CD117. Among these, A4502 was selected because the data suggested the highest sensitivity for this antibody. All other remaining antibodies (sc-13508, No. 566, NCL-CD117) failed to stain all GISTs and yielded a lower rate of positivity in our non-GIST test tumors. Examples of GIST that could not be stained with sc-13508 and NCL-CD117 are shown in Figs 1I to 1L. Overall, our comparative data suggested that A4502 was the most sensitive and specific antibody. In addition, A4502 had the advantage that a specific peptide was available for negative control experiments, which blocked specific reactions by adding a surplus of KIT antigen. A4502 was therefore selected to analyze the large multitumor TMA. A4502 was optimally applied at a 1:300 dilution at room temperature for 2.5 hours, after 3 minutes of cooking under pressure in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval.
KIT Immunostaining in Normal Tissues In normal tissues, an unequivocal membranous KIT staining was observed in the following cell types: secretory cells of mammary glands (++), basal cells of skin (++), thymic epithelial cells (++), mast cells (++), interstitial cells of Cajal (++), and spermatogonia in testicular tubules (+). Organs found to be completely KIT-negative included cerebrum, uterine cervix, colon, endometrium, esophagus, fat tissue, gall bladder, heart, kidney, liver, lung, lymph node, myometrium, oral cavity mucosa, ovary, pancreas, parathyroid, salivary gland, prostate, skeletal muscle, small intestine, intestinal smooth muscle, stomach mucosa, thyroid, and urothelium.
KIT Immunostaining Tumors
Mutation Analysis Mutations in exon 11 were seen in six of 12 GISTs (50%) and one of two KIT-expressing melanomas. The types of mutations are shown in Table 5. No mutations in exons 2, 8, 9, 11, 13, and 17 were found in seminoma (n = 18), giant-cell tumor of the tendon sheath (n = 1), lymphoepithelial carcinoma of the pharynx (n = 1), or small cell lung cancer (n = 2).
After trastuzumab (Herceptin) for treatment of HER-2–positive breast cancer, imatinib therapy of KIT-positive GISTs represents another example of a U.S. Food and Drug Administration (FDA) -approved rationally targeted cancer therapy requiring immunohistochemical tumor analysis to identify patients most amenable to such therapy. However, the chronology of the development of drug and diagnostic tools differs between trastuzumab and imatinib. Trastuzumab was specifically designed to target the HER-2 protein, and a diagnostic kit to identify potentially responding tumors was developed early and approved by the FDA simultaneously with the drug in September 1998.20 Imatinib was initially designed to inhibit the BCR/ABL fusion protein in chronic myeloid leukemia and was approved for this application by the FDA in May 2001.21 Only later was it discovered that imatinib is also effective against KIT-positive GISTs.14 More than 25 clinical studies are now investigating the effect of imatinib on KIT-positive tumors of various origins (http://www.clinicaltrials.gov). Despite of these activities, generally accepted guidelines for identification of KIT-positive tumors are still lacking. Previous studies have therefore used a wide variety of different KIT antibodies, protocols, and scoring systems to identify KIT-positive tumors. These studies often yielded discrepant results.1-4 For example, in small-cell lung cancer22,23 the frequency of KIT positivity ranged from 36%3 to 91%1 and in malignant melanoma from 0%4 to 20%.7 Because of the potential importance of assessing KIT expression for treatment decisions, a reliable procedure to identify KIT overexpression is needed. The TMA approach is ideally suited for development and comparison of immunohistochemical assays. Hundreds of tumors can be immunostained simultaneously under highly standardized conditions on one TMA section. The use of consecutive sections of a TMA block in combination with the small diameter of each arrayed tissue sample limits each comparison to a small tissue area with a minimal likelihood of genetic or tissue processing heterogeneity. Our initial comparison of seven commercially available antibodies showed considerable variance in staining intensity and signal-to-noise ratio. The A4502 antibody was selected because it had the highest sensitivity and produced minimal background in other tissues. A4502 recognizes an intracellular component of the transmembranous protein, 963 to 976 amino acids to the C-terminal. It was therefore not surprising that our preabsorption experiments always confirmed membranous or membranous and cytoplasmic staining as specific, whereas tumors with pure cytoplasmic KIT positivity proved to be false-positive. Multitumor TMAs containing samples from a wide variety of tumor entities are optimally suited to identify those samples with frequent alterations of a specific gene.24 This holds true even though the absolute number of positive cases detected in a TMA study may be somewhat lower than the true number of tumors due to regional heterogeneity of immunostaining. This presumed weakness of the TMA approach, however, is apparently compensated for by the perfect standardization of staining. All tumors of a TMA study can be stained under absolutely identical reaction conditions. Previous TMA studies have shown that representative information is obtained despite the small size of arrayed tissue per tumor.25-28 On a multitumor TMA, the most valuable information is the relative frequency of molecular parameters across all tumor types, which results in a rank order of potentially affected tumors. The results of this study confirm that KIT expression is a consistent finding in GIST and, as reported in previous studies, relatively frequent in melanoma, seminoma, and adenoid-cystic carcinoma.1,3 An additional 48 tumor types and subtypes with varying frequencies of KIT expression were also identified by our TMA approach. The development of a KIT immunostaining protocol, which is well supported by experimental data, does not automatically solve the problem of immunohistochemical KIT detection in clinical praxis. The experience with HER-2 testing of tumors potentially suited for trastuzumab therapy has highlighted several inherent difficulties of immunohistochemical testing. Immunohistochemistry has been shown to be highly dependent on preanalytical handling (especially fixation), and significant interlaboratory variations have been reported despite the availability of high-quality FDA-approved immunohistochemical test kits.29-31 These difficulties are seen especially in low-throughput laboratories.32,33 In any case, using immunohistochemistry for predictive tumor analysis will necessitate rigorous quality-assurance steps and highly skilled certified laboratories. It is also noteworthy that immunohistochemistry testing may be even more difficult and perhaps less relevant for targets other than HER-2. For example, initial studies using anti-epidermal growth factor receptor drugs have shown little influence of the epidermal growth factor receptor expression level (as detected by immunohistochemistry) on response to therapy.34,35 Other targets such as CD20, as a target for rituximab, are expressed in all tumors of a certain type, making testing unnecessary once a definite diagnosis is established.36 Recent data have indeed suggested that response to imatinib may not be driven primarily by the KIT expression level. Studies have shown that not all KIT-expressing tumors will benefit from imatinib therapy.37 They indicate that the response rate may depend on the presence of KIT mutations in the tumor and potentially also on the location and type of mutation.38,39 These data suggest that tumors with exon 11 mutations respond better than those tumors with exon 9 mutations, whereas the response rate is minimal in tumors without mutations.16 Our sequencing analysis revealed 50% exon 11 mutations in GISTs, which is in the range of previous studies.40 The results of sequencing analyses are more controversial in seminomas. In this study, we failed to find mutations in 18 examined seminomas, despite a comprehensive analysis of exons 2, 8, 9, 11, 13, and 17. Previous studies analyzing a total of 108 pure seminomas and dysgerminomas have found 1% exon 11 and 20% exon 17 mutations.38,41-43 The only tumor type other than GIST that showed a KIT mutation in our study was melanoma. However, our finding of an exon 11 mutation in one of two melanomas analyzed seems to be an overestimate of the true mutation prevalence. In a follow-up study, we were unable to detect KIT mutations in 10 additional KIT-expressing melanomas.44 Overall, the prevalence of KIT mutations in tumors other than GISTs and perhaps seminoma seems to be low. Recent studies investigating 10 breast,45 10 ovarian,46 and 26 small-cell lung cancers22 failed to find any mutations. Our study gives a comprehensive overview of KIT expression in a diverse set of human tumors. With the exception of seminoma and melanoma, immunohistochemical KIT-positivity was below 30% in frequently occurring tumor entities. Together with the low prevalence of mutations in KIT-expressing tumors, this suggests a relatively low proportion of patients who might benefit from imatinib therapy subsequent to KIT activation. However, KIT protein is not the only target of imatinib. In addition to chronic myelogenous leukemia, responses to imatinib are known to occur in dermatofibrosarcoma protuberans,47-49 chronic myelomonocytic leukemia,50 and hypereosinophilic syndrome,51 in which other tyrosine kinases are inhibited by imatinib.
The authors indicated no potential conflicts of interest.
We thank Y. Knecht and M. Kaspar for skillful technical assistance in laboratory work, and J. Schwegler for the photographic montage.
The first two authors contributed equally to this work. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
60% of cells, 2+ intensity in 30% to 79%, or 3+ intensity in less than 30% were considered moderately positive. Tumors with 2+ intensity in 
