KIT (CD117)-Positive Breast Cancers Are Infrequent and Lack KIT Gene Mutations

178 Vol. 10, 178–183, January 1, 2004 Clinical Cancer Research

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 histological 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-

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 comparison 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. ing was not seen in large sections of all 28 tumors that were Sequence Analysis. Ten breast cancers showing intense negative on the TMA, but was detectable on 24 of 36 tumors CD117 staining were selected for KIT mutation analysis. The that were scored positive on the TMA (Table 3; P Ͻ 0.001). KIT formalin-fixed tissues were deparaffinized and the DNA was staining intensity on large sections was related to the KIT extracted according to the protocols provided by Qiagen (Basel, expression level detected on the TMA. In 5 of 11 cases, tumors Switzerland). Exons 2, 8, 9, 11, 13, and 17 of the KIT gene were that were considered strongly positive on the TMA were also amplified by semi-nested PCR. All exons were sequenced di- scored strongly positive on large sections. However, only 4 of rectly using Big Dye Terminators Cycle Sequencing Ready 25 tumors that were scored as weakly positive on TMA sections Reaction Kit (Applied Biosystems, Foster City, CA). The prim- were considered strongly positive on large sections (P Ͻ 0.0001). In 12 cases, the TMA analysis had suggested KIT positivity, which was subsequently not confirmed on large sections. A review of these cases suggested that in the majority of 4 P. Went, unpublished data. these samples, some weak cytoplasmic background staining had

impact. Numerous studies have shown that associations between molecular alterations and clinical or pathological information can be readily detected in TMA analyses (28–34). Multiple other studies have reported a high level of concordance between TMA findings and results obtained on corresponding large sections (28, 31, 32, 35–47). In this project, a TMA was used for

Table 2 Immunohistochemical KIT expression in breast cancer KIT expression On array Analyzable Weak Strong (n) (n) (%) (%) P All samples 2197 1654 1.8 0.8 Histology Ductal carcinoma 1531 1183 1.5 0.6 Lobular carcinoma 311 205 0.5 0.0 Medullary carcinoma 57 47 10.6 8.5 Ͻ0.0001a Mucinous carcinoma 69 53 1.9 0.0 Tubular carcinoma 56 42 2.4 0.0 Cribriform carcinoma 64 47 2.1 0.0 Papillary carcinoma 30 23 4.3 4.3 Other typesb 79 54 1.9 3.7 Stage pT1 804 577 0.9 0.5 pT2 1015 779 2.3 0.8 pT3 124 92 4.3 3.3 NSc pT4 242 199 0.5 1.0 Nodal stage pN0 936 681 1.9 0.7 pN1 783 590 2.4 1.0 NS pN2 121 99 1.0 1.0 BRE grade 1 539 384 0.3 0.8 2 839 629 1.1 0.0 Ͻ0.0001 3 646 515 3.9 2.1 Fig. 1 Examples of KIT immunohistochemistry results for tissue mi- a Medullary carcinoma vs. other histologies. croarray spots. A, normal breast tissue; B, invasive breast carcinoma b Other types include adenoid-cystic carcinoma (n ϭ 1), apocrine showing strong KIT expression in the membrane; C, tissue spot with carcinoma (n ϭ 15), atypical medullary carcinoma (n ϭ 9), carcinosar- KIT-positive normal breast and KIT-negative tumor cells (arrowhead). coma (n ϭ 2), clear cell carcinoma (n ϭ 14), histiocytic carcinoma (n ϭ The insets show magnifications of representative areas. 1), lipid-rich carcinoma (n ϭ 2), and histiocytoic carcinoma (n ϭ 2). c NS, not significant.

been overinterpreted on the TMA section. However, in an attempt to find as many positive cases as possible on the TMA, questionable cases had been scored positive. The IHC analysis was noninformative in 543 (24.7%) of 2197 arrayed tumors because of missing tissue or a lack of tumor cells in the arrayed tissue sample. Mutation Analysis. No alterations compared with the KIT genomic sequence (GenBank accession no. U63834; Ref. 27) were found in exons 2, 8, 9, 11, 13, and 17 in the set of 10 examined breast cancers.

DISCUSSION We used a TMA to evaluate the KIT expression frequency in a series of 2197 breast cancers. In this method, minute tissue samples (0.6-mm diameter) of hundreds of tumors are analyzed on one glass microscope slide. It is obvious that focal gene alterations are not always detected in a TMA setting. However, Fig. 2 Survival analysis (Kaplan–Meier model) of KIT-positive (weak this perceived disadvantage appears to have limited practical or strong immunostaining) versus KIT-negative cancers.

Table 3 KIT immunohistochemistry: Tissue microarray analysis may be particularly true in the case for medullary breast cancers, versus large section analysisa in which 19.1% positive cases were observed. However, be- Conventional large sectionsa cause of the rather good prognosis of medullary cancers, only a few of these patients may need to be enrolled in one of the TMAb Negative Weak Strong P clinical trials open at present. The increased frequency of KIT Negative 28 0 0 positivity detected in papillary and apocrine cancers may be less Weak 12 9 4 Ͻ0.0001 Strong 0 6 5 meaningful because only one or two positive cases were ob- a served in these rare tumor types. At present there is no evidence Comparison of tissue microarray analysis results from 64 breast suggesting that KIT-positive breast cancers can benefit from cancers with the results obtained from corresponding conventional large sections. STI571 therapy. Because the KIT protein is expressed in normal b TMA, tissue microarray. breast epithelium, it seems that KIT expression is down-regulated rather than up-regulated in most breast cancers in a small tumor subset. The lack of KIT mutations in our 10 tumors sequenced for exons 2, 8, 9, 11, 13, and 17 also does not provide “rare event detection.” Because of the low frequency of KIT evidence for KIT activation in breast cancer. It has been sug- expression in breast cancers, it was necessary to first analyze a gested that the response rate to STI571 may be dependent on the very large number of tumors by IHC to identify KIT-positive presence and type of KIT mutation in the tumor cells (18–24). cases for subsequent mutation analysis. Comparison of the re- If this is true, our mutation analysis data further argued against sults obtained with TMA and large section analysis indicated a possible response of KIT-positive breast cancers to STI571 that the TMA technology is highly suited for identification of therapy. However, other authors have reported some therapeutic rare positive cases. Surprisingly, we found no cases that were effect in KIT-positive tumors expressing wild-type protein (57). false negative in the TMA analysis, but there were 12 cases that Taken together, our data show that immunohistochemically were considered positive on the TMA only. A review of these detectable KIT protein is infrequent in breast cancer. Although cases revealed that a weak cytoplasmic background staining had this may reflect preservation of “normal level KIT expression” been overinterpreted as true positivity in the majority of these in breast epithelium and no KIT mutations were found, studies cases. However, a small number of false-positive cases was are required to investigate the possible beneficial effects of acceptable in our TMA analysis because in our screening mode imatinib mesylate in KIT-positive breast cancers. we attempted to identify as many of the true-positive cases as possible. Because all large-section-positive cases were identi- fied in our TMA setting, one could consider the possibility that REFERENCES TMAs made from tissues of living cancer patients can be used 1. Gibson, P. C., and Cooper, K. CD117 (KIT): a diverse protein with selective applications in surgical pathology. Adv. Anat. Pathol., 9: to select patients for future targeted therapy regimens (48). 65–69, 2002. Because there is no officially recommended method for 2. Arber, D. A., Tamayo, R., and Weiss, L. M. Paraffin section detec- IHC KIT testing, there is some variability of reported KIT tion of the c-kit gene product (CD117) in human tissues: value in the positivity rates in breast cancer (1–13%; Refs, 10–12) as well as diagnosis of mast cell disorders. Hum. Pathol., 29: 498–504, 1998. in other tumor entities (2, 4, 5, 49–56). We therefore made 3. Lammie, A., Drobnjak, M., Gerald, W., Saad, A., Cote, R., and significant efforts to optimize our IHC KIT detection system. Cordon-Cardo, C. Expression of c-kit and kit ligand proteins in normal Seven commercially available KIT antibodies were evaluated in human tissues. J. Histochem. Cytochem., 42: 1417–1425, 1994. a previous study, which revealed considerable variability of 4. Tsuura, Y., Hiraki, H., Watanabe, K., Igarashi, S., Shimamura, K., 4 Fukuda, T., Suzuki, T., and Seito, T. Preferential localization of c-kit their staining properties. Antibody A4502 (DAKO) was judged product in tissue mast cells, basal cells of skin, epithelial cells of breast, to be optimal in this study because it revealed the highest small cell lung carcinoma and seminoma/dysgerminoma in human: frequency of positivity in arrayed gastrointestinal stroma tumors immunohistochemical study on formalin-fixed, paraffin-embedded tis- and because reagents are available for preabsorption control sues. Virchows Arch., 424: 135–141, 1994. experiments to assure maximum specificity of the system. Using 5. Natali, P. G., Nicotra, M. R., Sures, I., Santoro, E., Bigotti, A., and Ullrich, A. Expression of c-kit receptor in normal and transformed this system, we found KIT expression in only 43 of 1654 human nonlymphoid tissues. Cancer Res., 52: 6139–6143, 1992. interpretable tumors (2.6%). The concordant lack of detectable 6. Matsuda, R., Takahashi, T., Nakamura, S., Sekido, Y., Nishida, K., KIT expression in both large sections and arrayed tissue spots of Seto, M., Seito, T., Sugiura, T., Ariyoshi, Y., et al. Expression of the 28 randomly selected KIT-negative tumors makes it unlikely c-kit protein in human solid tumors and in corresponding fetal and adult that examination of more tissue samples per tumor would yield normal tissues. Am. J. Pathol., 142: 339–346, 1993. a substantially higher number of positive tumors. Our result is 7. Kantarjian, H., Sawyers, C., Hochhaus, A., Guilhot, F., Schiffer, C., similar to the 1% positive cases recently reported by Tsuura et Gambacorti-Passerini, C., Niederwieser, D., Resta, D., Capdeville, R., Zoellner, U., Talpaz, M., Druker, B., Goldman, J., O’Brien, S. G., al. (10) in a large study involving 197 breast cancers. Another Russell, N., Fischer, T., Ottmann, O., Cony-Makhoul, P., Facon, T., study had previously reported KIT positivity in 13% of breast Stone, R., Miller, C., Tallman, M., Brown, R., Schuster, M., Loughran, cancers (12). This discrepancy can be explained by the use of T., Gratwohl, A., Mandelli, F., Saglio, G., Lazzarino, M., Russo, D., different antibodies, staining conditions, scoring methods, Baccarani, M., and Morra, E. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N. Engl. J. Med., and/or variations in preanalytical tissue processing. 346: 645–652, 2002. Although KIT expression is obviously infrequent in breast 8. Miettinen, M., Furlong, M., Sarlomo-Rikala, M., Burke, A., Sobin, cancer, it is still possible that a small fraction of breast cancer L. H., and Lasota, J. Gastrointestinal stromal tumors, intramural patients could potentially benefit from STI571 therapy. This leiomyomas, and leiomyosarcomas in the rectum and anus: a clinico-

pathologic, immunohistochemical, and molecular genetic study of 144 25. Elston, C. W., and Ellis, I. O. Pathological prognostic factors in cases. Am. J. Surg. Pathol., 25: 1121–1133, 2001. breast cancer. The value of histological grade in breast cancer: experi- 9. Sawyers, C. L. Imatinib GIST keeps finding new indications: suc- ence from a large study with long-term follow-up. Histopathology, cessful treatment of dermatofibrosarcoma protuberans by targeted inhi- 1991: 403–410, 1991. bition of the platelet-derived growth factor receptor. J. Clin. Oncol., 20: 26. Bubendorf, L., Kononen, J., Koivisto, P., Schraml, P., Moch, H., 3568–3569, 2002. Gasser, T. C., Willi, N., Mihatsch, M. J., Sauter, G., and Kallioniemi, 10. Tsuura, Y., Suzuki, T., Honma, K., and Sano, M. Expression of O. P. Survey of gene amplifications during prostate cancer progression c-kit protein in proliferative lesions of human breast: sexual difference by high-throughout fluorescence in situ hybridization on tissue microar- and close association with phosphotyrosine status. J. Cancer Res. Clin. rays. Cancer Res., 59: 803–806, 1999. Oncol., 128: 239–246, 2002. 27. Andre, C., Hampe, A., Lachaume, P., Martin, E., Wang, X. P., 11. Chui, X., Egami, H., Yamashita, J., Kurizaki, T., Ohmachi, H., Manus, V., Hu, W. X., and Galibert, F. Sequence analysis of two Yamamoto, S., and Ogawa, M. Immunohistochemical expression of the genomic regions containing the KIT and the FMS receptor tyrosine c-kit proto-oncogene product in human malignant and non-malignant kinase genes. Genomics, 39: 216–226, 1997. breast tissues. Br. J. Cancer, 73: 1233–1236, 1996. 28. Nocito, A., Bubendorf, L., Maria Tinner, E., Suess, K., Wagner, U., 12. Natali, P. G., Nicotra, M. R., Sures, I., Mottolese, M., Botti, C., and Forster, T., Kononen, J., Fijan, A., Bruderer, J., Schmid, U., Ackermann, Ullrich, A. Breast cancer is associated with loss of the c-kit oncogene D., Maurer, R., Alund, G., Knonagel, H., Rist, M., Anabitarte, M., product. Int. J. Cancer, 52: 713–717, 1992. Hering, F., Hardmeier, T., Schoenenberger, A. J., Flury, R., Jager, P., 13. Demetri, G. D., von Mehren, M., Blanke, C. D., Van den Abbeele, Luc Fehr, J., Schraml, P., Moch, H., Mihatsch, M. J., Gasser, T., and A. D., Eisenberg, B., Roberts, P. J., Heinrich, M. C., Tuveson, D. A., Sauter, G. Microarrays of bladder cancer tissue are highly representative Singer, S., Janicek, M., Fletcher, J. A., Silverman, S. G., Silberman, of proliferation index and histological grade. J. Pathol., 194: 349–357, S. L., Capdeville, R., Kiese, B., Peng, B., Dimitrijevic, S., Druker, B. J., 2001. Corless, C., Fletcher, C. D., and Joensuu, H. Efficacy and safety of 29. Hoos, A., Stojadinovic, A., Mastorides, S., Urist, M. J., Polsky, D., imatinib mesylate in advanced gastrointestinal stromal tumors. N. Engl. Di Como, C. J., Brennan, M. F., and Cordon-Cardo, C. High Ki-67 J. Med., 347: 472–480, 2002. proliferative index predicts disease specific survival in patients with 14. van Oosterom, A. T., Judson, I., Verweij, J., Stroobants, S., Donato high-risk soft tissue sarcomas. Cancer (Phila.), 92: 869–874, 2001. di Paola, E., Dimitrijevic, S., Martens, M., Webb, A., Sciot, R., Van 30. Hoos, A., Stojadinovic, A., Singh, B., Dudas, M. E., Leung, D. H., Glabbeke, M., Silberman, S., and Nielsen, O. S. Safety and efficacy of Shaha, A. R., Shah, J. P., Brennan, M. F., Cordon-Cardo, C., and imatinib (STI571) in metastatic gastrointestinal stromal tumours: a Ghossein, R. Clinical significance of molecular expression profiles of Phase I study. Lancet, 358: 1421–1423, 2001. Hurthle cell tumors of the thyroid gland analyzed via tissue microarrays. 15. Hernandez-Boluda, J. C., and Cervantes, F. Imatinib mesylate Am. J. Pathol., 160: 175–183, 2002. (Gleevec, Glivec): a new therapy for chronic myeloid leukemia and 31. Moch, H., Schraml, P., Bubendorf, L., Mirlacher, M., Kononen, J., other malignancies. Drugs Today (Barc.), 38: 601–613, 2002. Gasser, T., Mihatsch, M. J., Kallioniemi, O. P., and Sauter, G. High- 16. Mace, J., Sybil Biermann, J., Sondak, V., McGinn, C., Hayes, C., throughput tissue microarray analysis to evaluate genes uncovered by Thomas, D., and Baker, L. Response of extraabdominal desmoid tumors cDNA microarray screening in renal cell carcinoma. Am. J. Pathol., to therapy with imatinib mesylate. Cancer (Phila.), 95: 2373–2379, 154: 981–986, 1999. 2002. 32. Torhorst, J., Bucher, C., Kononen, J., Haas, P., Zuber, M., Ko¨chli, 17. Dagher, R., Cohen, M., Williams, G., Rothmann, M., Gobburu, J., O. R., Mross, F., Dieterich, H., Moch, H., Mihatsch, M., Kallioniemi, Robbie, G., Rahman, A., Chen, G., Staten, A., Griebel, D., and Pazdur, O., and Sauter, G. Tissue microarrays for rapid linking of molecular R. Approval summary: imatinib mesylate in the treatment of metastatic changes to clinical endpoints. Am. J. Pathol., 159: 2249–2256, 2001. and/or unresectable malignant gastrointestinal stromal tumors. Clin. 33. Barlund, M., Forozan, F., Kononen, J., Bubendorf, L., Chen, Y., Cancer Res., 8: 3034–3038, 2002. Bittner, M. L., Torhorst, J., Haas, P., Bucher, C., Sauter, G., Kallion- 18. Ma, Y., Zeng, S., Metcalfe, D. D., Akin, C., Dimitrijevic, S., iemi, O. P., and Kallioniemi, A. Detecting activation of ribosomal Butterfield, J. H., McMahon, G., and Longley, B. J. The c-KIT mutation protein S6 kinase by complementary DNA and tissue microarray anal- causing human mastocytosis is resistant to STI571 and other KIT kinase ysis. J. Natl. Cancer Inst. (Bethesda), 92: 1252–1259, 2000. inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory- 34. Schraml, P., Kononen, J., Bubendorf, L., Moch, H., Bissig, H., type mutations. Blood, 99: 1741–1744, 2002. Nocito, A., Mihatsch, M., Kallioniemi, O., and Sauter, G. Tissue microarrays for gene amplification surveys in many different tumor types. 19. Longley, B. J., Reguera, M. J., and Ma, Y. Classes of c-KIT Clin. Cancer Res., 5: 1966–1975, 1999. activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk. Res., 25: 571–576, 2001. 35. Hoos, A., Urist, M. J., Stojadinovic, A., Mastorides, S., Dudas, 20. Joensuu, H., Roberts, P. J., Sarlomo-Rikala, M., Andersson, L. C., M. E., Leung, D. H., Kuo, D., Brennan, M. F., Lewis, J. J., and Tervahartiala, P., Tuveson, D., Silberman, S., Capdeville, R., Dimitri- Cordon-Cardo, C. Validation of tissue microarrays for immunohisto- jevic, S., Druker, B., and Demetri, G. D. Effect of the tyrosine kinase chemical profiling of cancer specimens using the example of human inhibitor STI571 in a patient with a metastatic gastrointestinal stromal fibroblastic tumors. Am. J. Pathol., 158: 1245–1251, 2001. tumor. N. Engl. J. Med., 344: 1052–1056, 2001. 36. Mucci, N. R., Akdas, G., Manely, S., and Rubin, M. A. Neuroen- 21. Demetri, G. D. Targeting c-kit mutations in solid tumors: scientific docrine expression in metastatic prostate cancer: evaluation of high rationale and novel therapeutic options. Semin. Oncol., 28: 19–26, throughput tissue microarrays to detect heterogeneous protein expres- 2001. sion. Hum. Pathol., 31: 406–414, 2000. 22. Joensuu, H., and Dimitrijevic, S. Tyrosine kinase inhibitor imatinib 37. Rubin, M. A., Dunn, R., Strawderman, M., and Pienta, K. J. Tissue (STI571) as an anticancer agent for solid tumours. Ann. Med., 33: microarray sampling strategy for prostate cancer biomarker analysis. 451–455, 2001. Am. J. Surg. Pathol., 26: 312–319, 2002. 23. Verweij, J., Judson, I., and van Oosterom, A. STI571: a magic 38. Sallinen, S. L., Sallinen, P. K., Haapasalo, H. K., Helin, H. J., bullet? Eur. J. Cancer, 37: 1816–1819, 2001. Helen, P. T., Schraml, P., Kallioniemi, O. P., and Kononen, J. Identi- 24. Allander, S. V., Nupponen, N. N., Ringner, M., Hostetter, G., fication of differentially expressed genes in human gliomas by DNA Maher, G. W., Goldberger, N., Chen, Y., Carpten, J., Elkahloun, A. G., microarray and tissue chip techniques. Cancer Res., 60: 6617–6622, and Meltzer, P. S. Gastrointestinal stromal tumors with KIT mutations 2000. exhibit a remarkably homogeneous gene expression profile. Cancer 39. Engellau, J., Akerman, M., Anderson, H., Domanski, H. A., Ram- Res., 61: 8624–8628, 2001. bech, E., Alvegard, T. A., and Nilbert, M. Tissue microarray technique

in soft tissue sarcoma: immunohistochemical Ki-67 expression in ma- 48. Sauter, G., and Mirlacher, M. Tissue microarrays for predictive lignant fibrous histiocytoma. Appl. Immunohistochem. Mol. Morphol., molecular pathology. J. Clin. Pathol., 55: 575–576, 2002. 9: 358–363, 2001. 49. Yajima, T., Kanda, T., Yoshiike, K., and Kitamura, Y. Retroviral 40. Hedvat, C. V., Hegde, A., Chaganti, R. S., Chen, B., Qin, J., Filippa, vector targeting human cells via c-Kit-stem cell factor interaction. Hum. D. A., Nimer, S. D., and Teruya-Feldstein, J. Application of tissue Gene. Ther., 9: 779–787, 1998. microarray technology to the study of non-Hodgkin’s and Hodgkin’s 50. Yamasaki, T., Shimazaki, H., Aida, S., Tamai, S., Tamaki, K., lymphoma. Hum. Pathol., 33: 968–974, 2002. Hiraide, H., Mochizuki, H., and Matsubara, O. Primary small cell (oat 41. Camp, R. L., Charette, L. A., and Rimm, D. L. Validation of tissue cell) carcinoma of the breast: report of a case and review of the literature. Pathol. Int., 50: 914–918, 2000. microarray technology in breast carcinoma. Lab. Investig., 80: 1943– 1949, 2000. 51. Hochhaus, A., Kreil, S., Corbin, A., La Rosee, P., Lahaye, T., Berger, U., Cross, N. C., Linkesch, W., Druker, B. J., Hehlmann, R., 42. Fernebro, E., Dictor, M., Bendahl, P. O., Ferno, M., and Nilbert, M. Gambacorti- Passerini, C., Corneo, G., and D’Incalci, M. Roots of Evaluation of the tissue microarray technique for immunohistochemical clinical resistance to STI-571 cancer therapy. Science (Wash. DC), 293: analysis in rectal cancer. Arch. Pathol. Lab. Med., 126: 702–705, 2002. 2163, 2001. 43. Ginestier, C., Charafe-Jauffret, E., Bertucci, F., Eisinger, F., Ge- 52. Hornick, J. L., and Fletcher, C. D. Immunohistochemical staining neix, J., Bechlian, D., Conte, N., Adelaide, J., Toiron, Y., Nguyen, C., for KIT (CD117) in soft tissue sarcomas is very limited in distribution. Viens, P., Mozziconacci, M. J., Houlgatte, R., Birnbaum, D., and Am. J. Clin. Pathol., 117: 188–193, 2002. Jacquemier, J. Distinct and complementary information provided by use 53. Kantarjian, H. M., and Talpaz, M. Imatinib mesylate: clinical re- of tissue and DNA microarrays in the study of breast tumor markers. sults in Philadelphia chromosome-positive leukemias. Semin. Oncol., Am. J. Pathol., 161: 1223–1233, 2002. 28: 9–18, 2001. 44. Rassidakis, G. Z., Jones, D., Thomaides, A., Sen, F., Lai, R., 54. Holst, V. A., Marshall, C. E., Moskaluk, C. A., and Frierson, H. F., Cabanillas, F., McDonnell, T. J., and Medeiros, L. J. Apoptotic rate in Jr. KIT protein expression and analysis of c-kit gene mutation in peripheral T-cell lymphomas. A study using a tissue microarray with adenoid cystic carcinoma. Mod. Pathol., 12: 956–960, 1999. validation on full tissue sections. Am. J. Clin. Pathol., 118: 328–334, 2002. 55. Jeng, Y. M., Lin, C. Y., and Hsu, H. C. Expression of the c-kit protein is associated with certain subtypes of salivary gland carcinoma. 45. Yosepovich, A., and Kopolovic, J. [Tissue microarray technolo- Cancer Lett., 154: 107–111, 2000. gy—a new and powerful tool for the molecular profiling of tumors]. 56. Bokemeyer, C., Kuczyk, M. A., Dunn, T., Serth, J., Hartmann, K., Harefuah, 141: 1039–1041,1090, 2002. Jonasson, J., Pietsch, T., Jonas, U., and Schmoll, H. J. Expression of 46. Hendriks, Y., Franken, P., Dierssen, J. W., De Leeuw, W., Wijnen, stem-cell factor and its receptor c-kit protein in normal testicular tissue J., Dreef, E., Tops, C., Breuning, M., Brocker-Vriends, A., Vasen, H., and malignant germ-cell tumours. J. Cancer Res. Clin. Oncol., 122: Fodde, R., and Morreau, H. Conventional and tissue microarray immu- 301–306, 1996. nohistochemical expression analysis of mismatch repair in hereditary 57. Heinrich, M. C., Corless, C., Blanke, C. D., Demetri, G. D., Joen- colorectal tumors. Am. J. Pathol., 162: 469–477, 2003. suu, H., von Mehren, M., McGreevey, L., Wait, C., Griffith, D., Chen, 47. Tzankov, A., Zimpfer, A., Lugli, A., Krugmann, J., Went, P., C.-J., Haley, A., Kiese, B., Druker, B., Roberts, P. J., Eisenberg, B., Schraml, P., Maurer, R., Ascani, S., Pileri, S., Geley, S., and Dirnhofer, Singer, S., Silberman, S., Dimitrijevic, S., Fletcher, C. D., and Fletcher, S. High-throughput tissue microarray analysis of G1-cyclin alterations J. KIT mutational status predicts clinical response to STI571 in patients in classical Hodgkin’s lymphoma indicates overexpression of cyclin E1. with metastatic gastrointestinal stromal tumors (GISTs). Proc. Am. Soc. J. Pathol., 199: 201–207, 2003. Oncol., 21: 2a, 2002.

Ronald Simon, Soti Panussis, Robert Maurer, et al.

Clin Cancer Res 2004;10:178-183.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/10/1/178

Cited articles This article cites 57 articles, 9 of which you can access for free at: http://clincancerres.aacrjournals.org/content/10/1/178.full#ref-list-1

Citing articles This article has been cited by 10 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/10/1/178.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/10/1/178. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.