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Expression and Mutational Status of c-kit in Small-Cell Lung Cancer

Prognostic Relevance

Departments of ¹ Surgery, ² Oncology, Transplants and Advanced Technologies in Medicine, and ³ Cardio-Thoracic Surgery, University of Pisa, Pisa, Italy

	ABSTRACT

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Purpose: The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family. Kinase activity has been implicated in the pathophysiology of many tumors, including small-cell lung carcinoma (SCLC). Autocrine or paracrine activation of c-kit by its ligand has been postulated for lung cancer, but this receptor can also be activated by mutations of the c-kit gene. We examined c-kit expression and mutational status in SCLC to verify its putative expression and genetic alterations, as well as its eventual prognostic impact.

Experimental Design: We studied 60 SCLC samples to determine the mutations of the coding region of the gene; the exons 9 and 11 were analyzed by PCR-single-strand conformational polymorphism and automated sequencing. Moreover, c-kit expression was evaluated in 55 samples by immunohistochemical method.

Results: Expression of c-kit was demonstrated in about 40% of SCLC samples. Two mutations in exon 9 and three mutations in exon 11 were found. Kaplan-Meier analysis revealed no prognostic significance of c-kit expression for survival.

Conclusions: In our series, the expression of c-kit and its mutational status failed to appear relevant or to have a significant impact on survival; this makes the therapeutic approach with an inhibitor of tyrosine kinase more difficult in SCLC until a sure demonstration of c-kit implication is obtained for this tumor.

	INTRODUCTION

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Molecular biological studies have revealed that a series of genetic changes in dominant and recessive oncogenes is involved in the pathogenesis of lung cancer. In addition, certain growth factors may play an important role in the development of this type of cancer (1) . Kit, a 145-KD transmembrane glycoprotein, is the product of the c-kit gene, the normal cellular homologue of the viral oncogene v-kit. The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family (2) . Steel factor, which is also designated Stem Cell Factor, is the cognate ligand of this receptor. Activation of tyrosine kinase through dimerization of the c-kit receptor occurs after binding of the cognate ligand and results in the phosphorylation of a variety of substrates involved in intracellular signal transduction (3 , 4) . Kit expression has been documented in a wide variety of human malignancies, and kinase activity has been implicated in the pathophysiology of a number of these tumors, including mastocytosis/mast cell leukemia (5 , 6) , gastrointestinal stromal tumors (GIST; Refs. 7 , 8 ), acute myelogenous leukemia (9, 10, 11) , melanoma (12 , 13) , ovarian (14) , breast (15 , 16) , and small-cell lung carcinoma (SCLC; Ref. 17 ). Autocrine or paracrine activation of c-kit kinase by Stem Cell Factor has been postulated for a number of these malignancies, including SCLC, and treatment with tyrosine kinase inhibitors, such as STI571, could be a promising strategy in c-kit positive patients, especially in SCLC, in which chemotherapy has been up to now the best therapeutic approach. In this sense, a better understanding of the real clinical value of c-kit in SCLC is necessary.

The tyrosine kinase receptor can be activated by mutation of the c-kit gene (18, 19, 20, 21) . These mutations result in ligand-independent kinase activity with consequent receptor autophosphorylation and stimulation of downstream signaling pathways. GISTs, the most common mesenchymal neoplasms in the human gastrointestinal tract, are almost exclusively positive for c-kit, ranging from 89 to 100% (8 , 22) and commonly have activating mutations (approximately 70%) in the c-kit gene (8 , 23, 24, 25, 26) . SCLC, a distinct clinicopathological entity among lung cancers, has a highly aggressive clinical course and results in significant morbidity and mortality (27) . Despite years of clinical research, the prognosis for SCLC patients treated with combination chemotherapy has improved only minimally. Because the c-kit oncoprotein seems to be expressed in SCLC (28 , 29) , the mutational status of the c-kit gene could play an important role in tumor growth and in the pathogenesis of this tumor, although only two studies on the mutational status of the c-kit gene in SCLC have been reported (30 , 31) . Therefore, we examined c-kit expression and mutational status in 60 SCLC samples to clarify the prognostic value of c-kit in SCLC and to discern whether or not patients with c-kit alterations could benefit from treatment with an inhibitor of tyrosine kinase activity.

	PATIENTS AND METHODS

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Surgical Specimens.
Sixty SCLC patients, who had undergone curative surgical resection at the Department of Surgery, University of Pisa, between 1976 and 1997, were analyzed. There were 56 males and 4 females (mean age 59.01 years, median 59.5 years, range 34–75 years). All of the patients underwent a complete preoperative staging. This included a detailed history and a physical examination; the evaluation of the performance status according to Karnofsky; a complete blood count and biochemical profile; cardiac and pulmonary function tests; chest X-ray; bronchoscopy; computed tomography of chest, the upper abdomen, and brain; abdominal ultrasonography; bone scan; Gallium-67 scan; bilateral bone marrow biopsy; and aspiration. Preoperative mediastinoscopy was not routinely performed where there was an absence of Gallium up-take on the mediastinum, and no bulky mediastinal lymph-node involvement was evident. All of the patients received surgery as their first-line therapy, followed by adjuvant chemotherapy and, in the cases of hilar or mediastinal involvement, also by radiotherapy. According to tumor status, there were 11 T₁, 36 T₂, 12 T₃, and 1 T₄; 29 patients did not show nodal metastasis at the moment of diagnosis, whereas 31 did show hilar and/or mediastinal metastatic involvement. Twenty-two patients were clinically staged as stage I (S1) whereas 12 patients were staged as stage II (S2), and the other 26 had stage III (S3). Data on clinical behavior were available in all 60 cases (median follow-up 243 months, range 57–316). Eleven of the patients are alive, whereas 49 died. Tumor samples, from the primary tumor side, were formalin-fixed and paraffin-embedded for histological and immunohistochemical processing. Tumors were classified according to the WHO classification (1982; Ref. 32 ) and according to the guidelines of the American Joint Committee for Cancer Staging (1992; Ref. 33 ). Furthermore, paraffin-embedded GIST samples were used as positive control for c-kit expression and mutations.

Immunohistochemistry.
Immunohistochemical evaluation of c-kit expression was performed using two different monoclonal antibodies (Dako, dilution 1:100; Novocastra, NCL-CD117 clone, dilution 1:20; 30'), to determine which one provided the best results in terms of both sensitivity and specificity. Five-µm sections of 55 tumor samples were stained with the above anti-c-kit antibodies using a Ventana (Tucson, AZ) automated immunohistochemical stainer following the manufacturer’s guidelines. To unmask the antigens, the slides were microwave-treated in 10 mM citrate buffer (pH 6) for a total of 10 min.

The grade of positivity was evaluated semiquantitatively by counting positive cells on at least 100 tumor cells. We considered as positive tumor cells those that showed cytoplasmatic and/or membrane immunoreactivity for c-kit. We evaluated as "positive" the tumors with a number of immunoreactive cells above the threshold value of 1%.

DNA Extraction.
Genomic DNA was isolated from 60 paraffin-embedded tissues. The paraffin was removed by xylene extraction, and the samples were subsequently lysed by proteinase-K. DNA extraction was then performed using the spin column procedure (QIAamp Tissue kit, Qiagen). The purification procedure was comprised of the following three steps: loading of the entire lysate onto the spin column with adsorption of DNA to the silica membrane, removal of residual contaminants by washing in the centrifugation step, and elution in sterile water.

PCR-Single-Strand Conformational Polymorphism (PCR-SSCP) Screening for c-kit Gene Mutations.
The eluted DNA was used as template in a standard 20 µl-PCR reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂ (pH 8.3), 0.2 mM deoxynucleoside triphosphate, 8 pmoles each of sense and antisense primer, and 1 unit of Amplitaq DNA Polymerase (Perkin-Perkin-Elmer Corp.). Primers used for the amplification were as follows: for exon 9, 5'-TTTGGAAAGCTAGTGGTTCA-3'/5'-ATGGTAGACAGAGCCTAAAC-3' and for exon 11, 5'-GATCTATTTTTCCCTTTCTC-3'/5'-AGCCCCTGTTTCATACTGAC-3'. PCR product size for c-kit exons 9 and 11 were 190 and 174 bp, respectively. The fragment sizes generated by the amplimer pairs were within the optimum range for detection of sequence alterations by the bandshift in SSCP analysis. Because both the primers had similar melting temperatures, the same PCR conditions could be used to simultaneously amplify the two exons (in separated reaction tubes). Conditions of c-kit exons 9 and 11, after initial denaturation at 95°C (5 min), were 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and synthesis at 72°C for 30 s, followed by final extension for 10 min. As negative control, the DNA template was omitted in the reaction. The amplification products were separated on 2% agarose gels and visualized by ethidium-bromide staining.

The PCR products were diluted 1:1 with denaturing solution (1% xylene cyanol, 1% bromphenol blue, 0.1 mM EDTA, and 99% formamide), boiled for 5 min, and thereafter directly placed on ice to prevent reannealing of the single-stranded product. SSCP screening for both genes was carried out on the GenePhor Electrophoresis unit using GeneGel Excel 12.5/24, following the instructions supplied with the kit (Pharmacia Biotech). Electrophoresis was performed at 18°C, at 600 V, 25 mA, 15 W for 80 min. Gels were stained using PlusOne Silver Staining kit (Pharmacia Biotech), following the supplied instructions. Tumor samples demonstrating aberrantly migrating bands in two or more independent PCR-SSCP runs were considered to contain one mutation.

For the detection of mutations, PCR products showing mobility shifts were purified with QIAquick PCR Purification kit (Qiagen) and sequenced using cyclic sequencing kit (ALFexpress II, Amersham Biosciences) following the manufacturer’s recommendations.

Statistical Analysis.
All statistical analyses were carried out using STATISTICA software (Stat-soft). A ² test was used to analyze the associations between the different variables. Survival analysis was determined according to the Kaplan-Meier method, and the statistical significance of the differences in survival distribution was evaluated by the log-rank test. The a priori level of significance was set at P < 0.05.

	RESULTS

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Clinicopathological Parameters and Overall Survival.
Among the clinicopathological parameters, metastatic nodal-involvement (P = 0.0003) and advanced stage (P = 0.009) were strongly associated with a worse overall survival (Table 1) . A similar statistically significant association was observed between these characteristics and disease-free survival (data not shown). Fig. 1 shows Kaplan-Meier survival plots generated on the basis of nodal-status (Fig. 1A) and stage (Fig. 1B) .

View larger version (24K): [in this window] [in a new window]   Fig. 1. Kaplan-Meier overall survival in relation to p-nodal-status (A) and p-stage (B). N, nodal; S, stage.

View larger version (169K): [in this window] [in a new window]   Fig. 2. Immunohistochemical staining of SCLC using monoclonal antibodies against c-kit. A and B, CD117-positive SCLC samples, respectively weak and strong; C, CD117-negative SCLC sample; D, representative CD117-positive gastrointestinal stromal tumor sample for comparison. SCLC, small-cell lung carcinoma.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Electrophoretic analysis of PCR products for exon 9 (190 bp) and exon 11 (174 bp) of c-kit gene. Lanes M, molecular weight marker (100 bp ladder, Pharmacia); Lanes 1–7, small-cell lung carcinoma samples; Cn, negative control, with no DNA added.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Single-strand conformational polymorphism analysis in small-cell lung carcinoma tissues. Sample 4 for c-kit exon 9 and samples 1 and 2 for c-kit exon 11 show an aberrant electrophoretic pattern.

View larger version (63K): [in this window] [in a new window]   Fig. 5. An example of electropherogram with relative sequence for small-cell lung carcinoma samples with heterozygous alterations of c-kit exon 9 and 11.

c-kit and Clinicopathological Parameters.
When we analyzed the relationship between c-kit expression and clinicopathological parameters, such as sex and age of the patients, p-tumor, -nodal status and -stage, we were unable to find any statistical association among these variables (Table 4) .

	DISCUSSION

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Therapies targeting signaling pathways involved in the pathogenesis of different tumors have been recently developed. Various studies have shown that the tyrosine kinase inhibitor STI-571 (Gleevec) is successfully being used against tumors expressing the c-kit oncogene, such as GISTs. In addition to GISTs, c-kit is also expressed in a variety of hematological malignancies and solid tumors, including SCLC. In fact, approximately 40–70% of SCLC cell lines and tumor specimens coexpress c-kit and its natural ligand Stem Cell Factor, and the Stem Cell Factor/c-kit pathway is functional in an autocrine or a paracrine fashion in SCLC (28 , 30 , 34, 35, 36) . These observations provided the scientific rationale for considering the chemotherapeutic approach with STI571 as a valid alternative tool in treating SCLC. Because in vitro studies demonstrated an antiproliferative effect of STI-571 on SCLC cell lines (37 , 38) , clinical trials have been underway with this compound (39) . In particular, Phase III and Phase II studies using STI-571 in SCLC have been recently performed, although no evidence of antitumor activity has been reported (40 , 41) . The authors of these trials, after no objective responses were seen in patients, performed immunohistochemical analyses of the SCLC tumor blocks available, observing that c-kit expression was absent in the majority of the cases. Other recent works have reported Kit positivity in tumor samples as being far less common than that turned up in the literature published previously (42 , 43) ; Micke et al. (44) , in fact, showed a percentage (37%) of positive c-kit expression in SCLC that was very similar to that obtained in our present study. The discrepancy in these data are probably attributable to the use of cell lines in the majority of the previous studies (28 , 34, 35, 36 , 45) . The influence of c-kit expression on prognosis is an area of on-going investigation, mostly in relation to recent therapies targeting signaling pathways involved in the pathogenesis of different types of cancer. The overall survival analysis in our series of SCLC revealed no significant association between c-kit expression and outcome; moreover, no statistical association was found between c-kit status and pathological parameters such as p-tumor status, p-nodal status, and p-stage, which represent established predictors of survival in this type of tumor. Pathological parameters (i.e., nodal and stage) showed, in our present series, a significant influence on overall and disease-free survival, confirming that our cohort is, however, representative of SCLC in general. Our findings are in agreement with recent studies (4 , 46 , 47) that investigated the prognostic impact of c-kit expression on overall survival and in which no influence of this factor was found in relation to clinical outcome. As a matter of fact, in data reported by Naeem et al. (48) although there was a tendency toward lower survival for c-kit positive patients compared with negative ones, this difference failed to reach a statistical significance. On the other hand, Micke et al. (44) revised a sizeable group (102 cases) of SCLC showing a prognostic significance of c-kit expression on overall survival, although the percentage of cases positive for c-kit expression was lower than those reported by Blackhall (4) and Naeem (48) . In our opinion, some differences in our article as compared with the papers mentioned above have to be pointed out, first and most important of which regards the type of patients we analyzed. All our tumor samples arose from surgical curative resection, i.e., all our patients received surgery as their first-line therapy, in contrast to the series analyzed by Micke et al. (44) , Naeem et al. (48) , and Blackhall et al. (4) , who investigated small biopsy from bronchoscopy and/or mediastinoscopy of patients treated with chemoradiotherapy. This may considerably modify the evaluation of c-kit antigen expression, because in our experience it shows a heterogeneous distribution throughout tumor samples. Small biopsies could be insufficiently representative of the entire tumor, altering data concerning the real c-kit protein distribution and levels.

The biological mechanism for the influence of c-kit on prognosis will be the subject of future studies. With respect to GIST, constitutive c-kit tyrosine-kinase activity results from activating or gain-of-function mutations; to our knowledge, only two papers concerning the mutational status of the c-kit gene in SCLC have been published. In the first work (30) , a single c-kit mutation was identified, even if this aminoacidic substitution at the transmembrane domain was observed in only 6.6% and 7.7% of, respectively, SCLC cell lines and primary tumor samples. Moreover, the functional consequence of this mutation is yet to be clarified. In the second, more recent, study (31) mutational analysis was restricted to DNA alterations in exon 11, encoding juxtamembrane domain of the c-kit oncoprotein: no activating mutations were found, using either SSCP or sequencing technique. Up to now, DNA aberrations in exon 13, encoding the first catalytic tyrosine kinase domain, or in exon 17, encoding TK2, are rare and seem unlikely to contribute to c-kit oncogene activation. On the basis of these considerations, we restricted our mutational analysis to exon 9, encoding the extracellular domain, and exon 11 of the c-kit gene. The low percentage of mutations of c-kit gene (8.33%) we found seems to provide further evidence that c-kit oncoprotein expression in SCLC is not strongly correlated with activating mutations. Because the number of mutations in our series is constitutively low, we believe that the analysis of survival, according to the presence of mutations, does not have a real impact on the clinical behavior of these patients. Most of the c-kit-positive tumors showed no alterations of this gene; in light of these observations, we suppose that c-kit overexpression in a significant number of SCLC could be unrelated to a true activating (gain-of-function) constitutional mutation, as instead it is in GIST. Thus, other molecular mechanisms leading to kit activation are probably responsible for c-kit expression in SCLC, independent of mutations.

In conclusion, many questions remain to be answered about the prognostic role of c-kit in SCLC patients: i.e., whether this impact is attributable to an autocrine or paracrine growth stimulation or whether other biological mechanisms are involved and, moreover, whether c-kit should be taken into account only in a subtype of SCLC patients. Because only about 40% of all our SCLC patients were c-kit positive, patients enrolled in putative targeted therapies interacting with the c-kit signaling pathway have to be stratified according to c-kit expression. Taking everything into account, we strongly suggest that at least a certain number of small-cell carcinomas, mainly with extensive disease, could receive a potential benefit from therapeutic treatment with c-kit inhibitor; however, overexpression of c-kit, more than its mutational status, represents the preliminary basis for using this approach in SCLC.

	FOOTNOTES

 
Grant support: from the Associazione Italiana per la Ricerca sul Cancro.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: G. Fontanini, Department of Oncology, Division of Pathology, via Roma, 57 56126 Pisa, Italy. Phone: 39-050-992983; Fax: 39-050-992942; E-mail: g.fontanini{at}do.med.unipi.it

Received 12/ 3/03; revised 2/12/04; accepted 3/18/04.

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