Expression of KIT and epidermal growth factor receptor in chemotherapy refractory non-seminomatous germ-cell tumors

A. Madani1,+, K. Kemmer2, C. Sweeney1, C. Corless2, T. Ulbright3, M. Heinrich4 and L. Einhorn1,5

1 Division of Hematology and Oncology, and 3 Department of Pathology, Indiana University Medical Center, Indianapolis, IN; 2 Department of Pathology, and 4 Division of Hematology and Oncology, Oregon Health & Science University, Portland, OR; 5 Walther Cancer Institute, Indianapolis, IN, USA

Received 4 November 2002; revised 3 January 2003; accepted 7 February 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

The majority of patients with germ-cell tumors (GCTs) are curable with standard therapy. The molecular differences between curable and incurable disease are unknown. We have studied the expression of KIT and the epidermal growth factor receptor (EGFR) to determine their incidence in chemorefractory disease.

Patients and methods:

We retrospectively analyzed 23 patients with chemorefractory non-seminomatous GCTs (15 late relapse and eight transformed teratomas). None of these 23 patients were cured by their initial chemotherapy and/or surgery. Immunohistochemical analysis of KIT and EGFR was performed on the most recently available specimen from a metastatic site. PCR amplimers of KIT exon 17 were screened for mutations by a combination of denaturing high-performance liquid chromatography and direct sequencing.

Results:

KIT was expressed (≥10% of the tumor displaying membranous or cytoplasmic staining) in 11 of 23 GCT patients [48%; 95% confidence interval (CI) 26% to 68%]. There were no activating KIT mutations in the phosphoryltransferase domain (exon 17) in 21 patients analyzed. EGFR was expressed (1+ to 3+) in 15 of 23 GCT patients (65%; 95% CI 41% to 82%).

Conclusions:

KIT and EGFR are expressed in a significant proportion of refractory GCTs. The significance of these findings will be determined by ongoing clinical trials.

Key words: c-Kit, EGFR, germ-cell tumor, refractory, testicular cancer


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Germ-cell tumors (GCTs) are the most common solid malignant neoplasm in young adult males. They are uniquely chemosensitive. Approximately 75% of patients with disseminated disease are cured with initial chemotherapy [1]. Second-line salvage chemotherapy with high-dose chemotherapy will cure ~50% of patients with testicular cancer [2]. However, despite these excellent results, there remains a group of patients who are currently incurable with standard chemotherapy. This includes patients who have progressed despite salvage with high-dose chemotherapy, patients with primary mediastinal non-seminomatous GCTs who were not cured with their initial chemotherapy, and patients experiencing a late relapse (>2 years from the start of their most recent chemotherapy) [35]. Another unfavorable group is represented by patients having teratoma with malignant transformation who are not resectable. Teratoma is pluripotent tissue that can differentiate along ectodermal, endodermal or mesodermal lines with transformation to other cell types such as rhabdomyosarcoma and primitive neuroectodermal tumor (PNET) [6].

Growth factors and their receptors are known to play critical roles in development, cell growth and differentiation. Many growth factor receptors possess an intrinsic tyrosine kinase activity that is activated upon interaction of the receptor with its cognate ligand, resulting in a variety of cellular responses including cell proliferation and differentiation. Abnormal activity of human receptor tyrosine kinase plays an important role in malignancy. This can be either through overexpression, gene amplification or activating mutations [7].

KIT, a 145-kDa transmembrane glycoprotein, is the product of the KIT gene (also known as c-Kit or stem cell factor receptor). KIT is a member of the type III receptor tyrosine kinase family that includes the receptors for platelet-derived growth factor, macrophage colony-stimulating factor, and monocyte colony-stimulating factor (FMS)-like receptor tyrosine kinase ligand (FLT-3) [8]. KIT is expressed by hematopoietic progenitor cells, mast cells, germ cells, and by the pacemaker cells of the gut (the interstitial cells of Cajal) [9]. KIT expression has been reported in GCTs, especially in seminomas [1019]. Activating mutations in the intracellular domain of KIT (exon 17) have also been reported in seminomas [19].

Epidermal growth factor receptor (EGFR, also known as HER1 or erb1), a 145-kDa transmembrane glycoprotein, is a member of the type I receptor tyrosine kinase family or erb-B family (that also includes HER2/neu, HER3 and HER4). EGFR is expressed primarily in cells of epithelial origin [20]. EGFR signaling has been shown to be important not only for proliferation but also for other processes that are crucial to cancer progression, including angiogenesis, metastatic spread, and the inhibition of apoptosis [21]. Many carcinomas have been found to overexpress EGFR with or without gene amplification, including those of the head and neck, esophagus, breast, colon, lung, prostate, kidney, ovary, stomach and bladder [22]. EGFR expression was also reported in GCTs [23, 24].

Recent advances using molecularly targeted therapy in the treatment of human malignant tumors, especially with agents targeting KIT [25] and EGFR [26], led us to evaluate the expression of KIT and EGFR in GCTs. In this study, we examine the expression of KIT and EGFR by immunohistochemistry in a series of 23 patients with refractory non-seminomatous GCTs not cured by chemotherapy and/or surgery (15 late relapse and eight transformed teratomas). Since activating mutations in the intracellular domain of the KIT gene (exon 17) have been previously described in seminomas [19, 27], we looked for similar mutations in this group of advanced non-seminomatous GCTs.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
This retrospective study included a total of 23 patients with chemotherapy-refractory non-seminomatous GCTs from Indiana University (15 late relapse and eight transformed teratomas). None of these patients were cured by their initial chemotherapy and/or surgery. All but one received two or more prior chemotherapy regimens including first-line platinum-based chemotherapy. Tissue specimens from these patients were available as part of a National Cancer Institute (NCI) funded project evaluating molecular differences between curable and incurable patients with GCTs. This study was approved by the Indiana University Institutional Review Board and informed consent was obtained. Pertinent clinical data were obtained from medical records. Patients were prognostically staged according to the International Germ Cell Cancer Consensus Group Criteria (IGCCCG) [28].

Immunohistochemistry studies (IHC)
Serial sections from the most recently available tumor specimen from a metastatic site (all but one were obtained from resection of metastases at the time of tumor recurrence) were cut and submitted for deparaffinization. In all patients, histological diagnosis of GCT (including the proportion of various tumoral elements) was confirmed by two experienced pathologists prior to performing IHC studies with anti-KIT and anti-EGFR.

IHC for KIT
Following deparaffinization, slides were subjected to epitope retrieval by heating in citrate buffer (Biogenex, San Ramon, CA, USA) for 20 min in a vegetable steamer (Black & Decker). A standard avidin–biotin staining protocol was carried out with a Dako polyclonal rabbit antibody to KIT (Dako A4502; Dako Corp., Carpinteria, CA, USA) used at 1:300 dilution, goat biotinylated anti-rabbit secondary (Vector Laboratories, Burlingame, CA, USA) 0.5 µg/ml and the Vectastain Elite kit (Vector Laboratories). Endogenous mast cells served as internal positive controls; the antibody did not stain other tissue elements (e.g. epithelial cells) at the selected titer. Assessment of cytoplasmic or membranous staining was performed using a semi-quantitative approach: KIT expression was considered negative if there was no membranous or cytoplasmic staining, or if staining was observed in <10% of tumor cells of any given tumor component; KIT expression was considered positive if there was membranous or cytoplasmic staining of ≥10% of tumor cells of any given tumor component.

IHC for EGFR
Following deparaffinization, slides were subjected to epitope retrieval by pretreatment with proteinase K (Dako) for 5 min. The primary antibody was a monoclonal antibody anti-EGFR (clone H11; Dako) used at 1:200 dilution with 30-min incubation at room temperature. Bound antibody was detected using a 3,3-diaminobenzidine plus substrate-chromogen system (Dako EnVision+ System). Positive and negative control stains were run in parallel. Assessment of membranous staining for EGFR was performed using the semi-quantitative method (Dako HercepTest) employed for HER2/neu scoring for invasive breast carcinoma [29]: EGFR expression was assessed based on the intensity of the observed signal and the pattern of membranous staining (focal versus complete). EGFR expression was considered negative if there was no membranous staining or staining was observed in <10% of tumor cells of any given tumor component. EGFR expression was considered 1+ positive if there was weak membranous staining in ≥10% of tumor cells of any given tumor component, and the cells were only stained in part of their membrane; 2+ positive if there was weak to moderate complete membranous staining in ≥10% of tumor cells of any given tumor component; 3+ positive if there was moderate to strong complete membranous staining in ≥10% of tumor cells of any given tumor component.

Genomic DNA analysis for KIT gene mutations
Tumor tissue on unstained slides was identified by comparison with a hematoxylin–eosin (H&E) stained slide and selected areas were scraped with a sterile scalpel blade into a 0.5 ml tube. Following deparaffinization in xylenes and ethanol, DNA was extracted from the tissue scrapings using the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA). DNA was not extracted from two patients because of the presence of intermixed normal tissue elements that prevented the collection of pure tumor. PCR amplification of genomic DNA 500 ng was performed using the High Fidelity PCR system (Roche 1732078) for the following KIT exon 17 primer pair:

KIT exon 17 forward 81318 5'-TGTATTCACAGAGACTTGGC-3'

KIT exon 17 reverse 81534 5'-GGATTTACATTATGAAAGTCACAGG-3'

DNAs were amplified in 50 µl PCR reactions of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C for 45 cycles (GeneAmp"PCR System 9700, Applied Biosystems, Foster City, CA, USA). Negative controls were included in each set of amplifications. Amplicons were analyzed by denaturing-HPLC (d-HPLC) using a transgenomic WAVE system as described previously [30]. Amplicons with an abnormal d-HPLC elution profile at 58°C were subjected to bidirectional sequence analysis on an ABI 377 sequencer using the ABI Big Dye terminator kit (Applied Biosystems).


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patient characteristics
At the initial presentation clinical information was available from the 23 patients evaluated and is summarized in Table 1: all 23 patients were male. Twenty of 23 (87%) patients had a testis primary. The majority of the patients (18 of 23, 78%) had metastatic disease at initial diagnosis, including nine patients with stage II disease and nine with stage III disease. Approximately half of the patients were in the unfavorable-risk category according to the IGCCCG, with eight patients in the intermediate-risk group and four in the poor-risk group.


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Table 1. Patient characteristics
 
Tumor samples of the 23 therapy refractory patients were obtained from the most recent metastatic site (all but one from resection of metastases at the time of tumor recurrence). Fifteen of 23 (65%) were late relapse patients, and the remaining eight patients had teratoma with malignant transformation, including five with a sarcomatous histology and two with PNET. The median time to progression was 37 months (range 6–384 months) due to the predominance of late relapse patients. None of the 23 patients were cured by their initial chemotherapy and/or surgery. All but one received at least two chemotherapy regimens including first-line platinum-based therapy. Seven of 23 (30%) died of disease; eight of 23 (35%) are alive with disease, and eight of 23 (35%) are currently alive with no evidence of disease after surgical resection.

Expression of KIT by immunohistochemistry in refractory GCTs
Using a semi-quantitative criteria approach (KIT scored positive if there was membranous or cytoplasmic staining of ≥10% of cells of any given tumoral component), positive KIT staining was observed in 11 of 23 GCT patients [48%; 95% confidence interval (CI) 26% to 68%]: the majority of teratoma with malignant transformation expressed KIT (five of eight patients, 62%; 95% CI 29% to 89%), including all five patients with a sarcomatous histology. Only six of 15 patients with late relapse demonstrated a significant KIT expression (40%; 95% CI 19% to 67%), including four of five patients with a myxoid or sarcomatoid histology. The two cases of teratoma with malignant transformation with PNET histology did not exhibit any KIT expression. In the majority of patients, the pattern of positivity was cytoplasmic (nine of 11, 82%); and the extent mostly patchy to focal (seven of 11 patients, 64%). The staining results for KIT are summarized in Tables 2 and 3 with an example in Figure 1.


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Table 2. KIT and epidermal growth factor receptor (EGFR) expression in germ-cell tumors (GCTs) by immunohistochemistry with KIT (exon 17) mutational status in GCTs
 

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Table 3. KIT and epidermal growth factor receptor (EGFR) expression in refractory germ-cell tumors (GCTs)
 


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Figure 1. Epidermal growth factor receptor (EGFR) and KIT expression in chemotherapy refractory non-seminomatous germ-cell tumors by immunohistochemistry. Left panels: anti-KIT immunostaining. Right panels: anti-EGFR immunostaining. (GCT 3) PNET (negative KIT and EGFR stain). (GCT 12) Late relapse (KIT cytoplasmic staining in scattered tumor cells; negative EGFR stain). (GCT 13) Late relapse (negative KIT stain; strong membranous EGFR staining). (GCT 15) Late relapse (strong membranous staining for KIT and EGFR).

 
Evaluation of KIT activating mutations in refractory GCTs
Screening for KIT activating mutations at the second intracellular kinase domain (exon 17) was conducted in 21 GCT patients by using a combination of PCR amplification and d-HPLC with direct sequencing. There were no activating mutations at exon 17 of the KIT gene in the 21 GCT patients tested (Table 2).

Expression of EGFR by immunohistochemistry in refractory GCTs
Positive EGFR staining was observed in 15 of 23 GCT patients (65%; 95% CI 41% to 82%) including 14 patients with 2+ to 3+ EGFR overexpression. The majority of late relapse patients expressed EGFR (11 of 15, 73%; 95% CI 45% to 90%), including nine of 10 patients with yolk sac tumor histology (90%; 95% CI 60% to 99%). A significant proportion of teratoma with malignant transformation cases stained positive for EGFR (four of eight, 50%; 95% CI 19% to 81%), including two of five with a sarcomatous histology and one of two with PNET histology. The staining results for EGFR are summarized in Tables 2 and 3 with an example in Figure 1.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The recent availability of molecularly targeted therapy with tyrosine kinase inhibitors, particularly with agents directed against KIT and EGFR, offers new hope for effective and better-tolerated therapy for human malignancies. We designed a preliminary study evaluating the expression of KIT and EGFR to explore the feasibility of this therapeutic approach in a population of chemotherapy-refractory non-seminomatous GCTs composed of late relapse and transformed teratoma patients.

Although KIT expression by IHC is detectable in a high percentage of primary seminomatous GCTs with a membranous pattern, only a minority of primary non-seminomatous GCTs displayed KIT expression with a diffuse cytoplasmic pattern [13, 14, 1719]. By using a threshold of expression likely to be of clinical relevance (≥10% of cells in any given tumoral component) we found that positive staining for KIT was present in 11 of 23 patients (48%), with mostly a cytoplasmic pattern (nine of 11, 82%). Our results in this particular population of chemotherapy-refractory non-seminomatous GCTs suggest that KIT expression is similar to that observed in primary non-seminomatous GCTs [1118]. Interestingly, KIT expression was particularly prevalent in patients with teratoma with transformation and a secondary malignant component (five of eight, 62%), with all five patients having a sarcomatous histology displaying positive KIT staining. Significant KIT expression was also seen in four of five patients (80%) with late relapse with myxoid or sarcomatoid histology. By contrast, GCT elements of the usual type (e.g. embryonal carcinoma and teratoma) did not express KIT. The expression of KIT was limited to the transformed teratoma elements, especially those with a sarcomatous histology, and late relapse with yolk sac tumor histology containing myxoid/sarcomatoid or papillary/glandular elements. This observed KIT expression in transformed teratoma with a sarcomatous histology and late relapse with myxoid or sarcomatoid histology is in line with other reports of occasional KIT staining in soft tissue sarcomas of neuroectodermal origin [3135].

Recently, Kollmannsberger et al. evaluated the expression of KIT by IHC in a series of 22 patients with platinum-resistant GCTs (platinum-refractory or failed second-line platinum-based salvage chemotherapy) [36]. They reported that the expression of KIT was restricted to the seminoma component of mixed non-seminomatous GCTs and the one case of refractory seminoma with clear membranous staining. Non-seminomatous GCTs showed neither membranous nor scattered intracellular positive staining. There are several differences between our study and that of Kollmannsberger et al.: clearly, our study population consisting of late relapse and transformed teratoma patients is different from the study population of patients with platinum-resistant GCTs studied by Kollmannsberger et al. Furthermore, the observed KIT expression in our patients was restricted to the transformed teratoma elements with a sarcomatous histology, and late relapse with exclusively a yolk sac tumor histology containing myxoid/sarcomatoid or papillary/glandular elements. None of the GCT elements of the usual type (e.g. embryonal carcinoma and teratoma) expressed KIT. Finally, since we did not analyze the expression of KIT in the primary tumors we cannot exclude a differential of expression between metastatic and primary tumors from the same patient.

Tian et al. reported an activating mutation of KIT at exon 17 (D816H) in a minority of seminomas/dygerminomas (two of 23 tumors, 8%). In one case of mixed ovarian dysgerminoma/yolk sac tumor the mutation was present in both components of the tumor [19]. A preliminary report by Kemmer et al. reported a higher frequency of exon 17 mutations in seminomas (12 of 46, 26%), probably owing to the use of more sensitive techniques [27]. Since all the mutations found to date in GCTs were in exon 17 of KIT, we looked for similar mutations in our patient population of refractory GCTs. In the 21 patients analyzed by a combination of PCR amplification and d-HPLC with direct sequencing, we did not find any activating KIT mutations in the phosphoryltransferase domain (exon 17). This suggests that either KIT is rarely if at all the target of activating mutations in refractory GCTs or that other mutations occur in non-evaluated sequences of the gene.

The clinical experience with agents targeting KIT, such as imatinib mesylate, suggests that the likelihood of clinical benefit correlates with the biological importance of the KIT-receptor pathway in the tumors treated, and this is mostly reflected through the constitutional activation of KIT by activating mutations in patients with T gastrointestinal stromal tumor (GIST) [37]. Even the presence of KIT mutations does not imply sensitivity and response to imatinib mesylate, as illustrated by Heinrich et al. in their pre-clinical studies of the mutation D816V reported in seminomas [25]. Although the significance of the observed KIT expression by IHC in our patients (especially in those with transformed teratoma and a sarcomatous histology) remains unknown, the absence of any detectable activating KIT mutations makes it unlikely that they would respond to targeted therapy with imatinib mesylate unless this agent targets other biologically important tyrosine kinases to GCTs.

The expression of EGFR has been previously evaluated in primary GCTs. Shuin et al. reported the expression of EGFR at the transcriptional level in two of three immature teratomas but no expression could be demonstrated in 15 seminomas and six embryonal carcinomas [23]. Moroni et al. evaluated the expression of EGFR by IHC in a series of 24 testicular tumors. The expression of EGFR appeared to be restricted to the ß-human chorionic gonadotrophin (HCG) positive component (choriocarcinoma) in 16 of 18 primary non-seminomatous GCTs studied. In contrast, one Leydig cell tumor, five seminomas, and ß-HCG negative components of GCTs did not express EGFR [24]. Recently, Kollmannsberger et al. evaluated the expression of EGFR by IHC in a series of 22 patients with platinum-resistant GCTs and 12 patients with chemosensitive GCTs. They reported that the presence of EGFR was restricted to trophoblastic giant cells and the syncytiotrophoblastic elements of four non-seminomas, including one pure choriocarcinoma, and to a secondary non-germ-cell malignancy arising from a transformed teratoma. There were no differences in the pattern of EGFR expression between platinum-resistant and -sensitive patients [36].

In the present study, we found the expression of EGFR to be present in the majority of patients (15 of 23, 65%) with refractory non-seminomatous GCTs, especially in late relapse patients with yolk sac tumor histology (nine of 10, 90%). A significant proportion of transformed teratoma patients (four of eight, 50%) also displayed EGFR expression. In one patient no EGFR expression was detected even in the minor choriocarcinoma component. Several factors may have contributed to the discrepancy between our results and those reported by Moroni et al. and Kollmannsberger et al. First, our study of late relapse and transformed teratoma patients clearly differs from that of patients with primary GCTs [24] and from that of patients with platinum-resistant disease (platinum-refractory or failure of second-line platinum-based salvage chemotherapy) [36]. Secondly, the tumor samples from our study were from the most recent metastatic site and not from the original primary testicular tumor, and since we did not analyze the expression of EGFR in the primary tumors we cannot exclude a differential of expression between metastatic and primary tumors from the same patient. Thirdly, the use in all three studies of different anti-EGFR antibodies recognizing distinct epitopes may have also contributed to this expression discrepancy. Fourthly, although Kollmannsberger et al. showed absence of EGFR expression in other germ-cell elements such as yolk sac or embryonal carcinoma present in various proportions in their patients with mixed non-seminomatous GCTs, they did demonstrate EGFR expression in the only case of transformed teratoma analyzed; in contrast, the tumors in our patients were more differentiated and homogeneous, consisting mainly of late relapse with exclusively a yolk sac histology and epithelial or myxoid differentiation, and transformed teratomas with a sarcomatous differentiation. This predominant EGFR expression in late relapse patients with an exclusive yolk sac tumor histology may be a reflection of a different biological behavior of these advanced tumors. Finally, since there is no consensus about the definition of a positive EGFR expression, the description of EGFR positivity differs from one study to another. Moroni et al. did not define the degree and extent of EGFR expression that they considered positive, whereas Kollmannsberger et al. defined EGFR positivity as at least a 2+ EGFR membranous staining in at least 50% of the cells. We have chosen to take a threshold of expression at 10% of any given tumor component as this semi-quantitative IHC approach was validated clinically with other molecular targets such as the steroid receptor and Her2neu.

In addition to the demonstration of EGFR expression in the ß-HCG-positive component (choriocarcinoma) of primary non-seminomatous GCTs, Moroni et al. evaluated the expression of Her2neu, TGF-{alpha} (EGFR ligand), and phosphorylated EGFR. They showed that the expression of Her2neu, TGF-{alpha}, and phosphorylated EGFR was detected in 25, 36, and 27% of EGFR-positive primary non-seminomatous GCTs [24]. These observations suggest that EGFR system activation or dysfunction involves not only EGFR overexpression but may also involve an autocrine loop with overexpression of its ligand TGF-{alpha}, as well as coexpression of other erb-B receptors such as Her2neu. These findings, together with the results of the present study, are relevant to support the possible role played by the EGFR signaling system in growth control of non-seminomatous GCTs.

Preliminary clinical experience with agents targeting EGFR did not show any correlation between clinical activity of these agents and the degree of expression of EGFR [38]. This may indicate that the expression of EGFR by IHC is not a good surrogate for the biological importance of EGFR or its signaling pathway to tumor growth and survival. It has now become clear that EGFR signaling occurs through a complex network with possible ‘cross talk’ or heterodimerization with other erb-b receptors (Her2neu, HER3 and HER4), and involvement of their respective signaling pathways. This may explain the versatility of EGFR signaling and its involvement in various cellular processes such as proliferation, inhibition of cellular death or apoptosis, metastatic spread and angiogenesis. As such, EGFR system activation and dysfunction may involve several mechanisms such as overexpression of activating ligands through an autocrine loop, presence of mutated and constitutively activated receptor, overexpression of EGFR or coexpression of Her2neu and other erb-B receptors. Until more biologically and clinically validated criteria become available, clinicians will continue to rely on the detection of EGFR expression by IHC for the selection of patients in trials with EGFR inhibitors. We contend that in this population with an inherently chemoresistant disease and in a relatively more homogenous population with transformed teratomas or late relapse of GCTs, a trial with an EGFR inhibitor is justified if there is a documented EGFR expression. If effectiveness with EGFR inhibitors is demonstrated, then the potential for sufficient downstaging of the tumor may allow a greater possibility of curative surgical resection and enhanced clinical benefit to these patients.

In summary, this preliminary study of a unique population of 23 patients with refractory non-seminomatous GCTs (late relapse and transformed teratoma) documents that the expression of KIT and EGFR occur in a significant proportion of these tumors. Although the exact significance of this has yet to be determined, the absence of activating KIT mutations argues against the potential benefit from agents targeting KIT such as imatinib mesylate. The therapeutic potential with agents targeting EGFR in this population deserves further exploration.


    Acknowledgements
 
Supported in part by NCI grant PO1 CA 74295. Presented in part at the 31st Annual Meeting of the American Society of Clinical Oncology, Orlando, FL, USA, 18–22 May 2002.


    Footnotes
 
+ Correspondence to: Dr A. Madani, Division of Hematology and Oncology, Indiana Cancer Pavilion, 535 Barnhill Drive, RT 473, Indianapolis, IN 46202, USA. Tel: +1317-274-0920. Fax: +13176-278-0079; E-mail: amadani{at}iupui.edu Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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