Japanese Journal of Clinical Oncology Advance Access originally published online on July 14, 2006
Japanese Journal of Clinical Oncology 2006 36(8):494-498; doi:10.1093/jjco/hyl061
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© 2006 Foundation for Promotion of Cancer Research
Immunohistochemical and Mutational Analysis of c-kit in Gastrointestinal Neuroendocrine Cell Carcinoma
1 Division of Molecular Diagnosis and Cancer Prevention, 2 Division of Pathology, 3 Division of Gastroenterological Surgery, Saitama Cancer Center, Kitaadachigun, Saitama and 4 Pathology Division, Innovative Medical Research Center, National Cancer Center Hospital East, Chiba, Japan
For reprints and all correspondence: Kiwamu Akagi, Division of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, 818 Komuro Ina, Kitaadachigun, Saitama 362-0806, Japan. E-mail: Akagi{at}cancer-c.pref.saitama.jp
Received December 16, 2005; accepted May 10, 2006
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Gastrointestinal neuroendocrine cell carcinoma (NEC) is a highly aggressive tumor with poor prognosis, for which an effective therapy is highly desirable. Recently, use of a c-kit inhibitor achieved excellent results against gastrointestinal stromal tumor (GIST) that showed c-kit overexpression and mutation in most cases. According to recent studies, 17–44% of pulmonary NEC also expressed c-kit and the antitumor effect of c-kit inhibitor was demonstrated in vitro against small cell carcinoma of the lung. As gastrointestinal NECs are clinicopathologically similar to pulmonary NECs, we investigated c-kit expression and mutation in gastrointestinal NEC.
Methods: Surgically resected gastrointestinal NEC was examined for c-kit expression by immunohistochemistry and RT–PCR. Mutation of the c-kit gene was also investigated by means of single-strand conformation polymorphisms (SSCP).
Results: Twenty-six percent (6 out of 23 patients) of gastrointestinal NEC expressed c-kit protein. c-kit protein expression was demonstrated in 1 out of 4 colorectal, 1 out of 2 duodenal, 4 out of 16 gastric and no esophageal (sample size of 1) NECs. The results of immunohistochemistry for c-kit were consistent with the RT–PCR. No c-kit gene mutation was found in gastrointestinal NEC.
Conclusion: The frequency of c-kit expression in gastrointestinal NEC was similar to that previously reported for pulmonary NEC. These findings suggest that c-kit inhibitor may be effective against some gastrointestinal NECs.
Key Words: c-kit • gastrointestinal neuroendocrine cell carcinoma • c-kit inhibitor
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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The neuroendocrine cell carcinoma (NEC) of gut develops throughout the whole gastrointestinal tract (1,2) and comprises 0.1–1.0% of gastrointestinal malignancies (2–5). It has specific clinicopathological features: they include poorly differentiated carcinoma with structures associated with neuroendocrine tumors such as organoid, trabecular or rosette-like growth patterns, and expresses several antigens shared with nerve elements, a favorable but transient response to cisplatin-based chemotherapy (2), a high ability to metastasize and poor prognosis (2–7). These clinical features and histological findings are similar to those of pulmonary NEC, that is, small or large cell carcinoma (2).
Recently, it was revealed that c-kit is overexpressed at a relatively high frequency in pulmonary NEC (8–11). The c-kit gene encodes a transmembrane tyrosine kinase receptor and signaling by c-kit appears to play a central role in cellular transformation and differentiation. Therefore, aberrant activation of c-kit leads to the development (12) and progression of several human malignancies (8,9). In terms of therapeutic strategies, it has become important to evaluate the frequency of c-kit expression and mutation in malignancies since the emergence of imatinib mesylate, a potential therapeutic agent which inhibits the action of BCL-ABL in chronic myeloid leukemia and c-kit in gastrointestinal stromal tumor (GIST) and mast cell disease (12). In fact, its excellent effect against these tumors has been reported in clinical trials (13–15). Imatinib mesylate is also effective against small cell carcinoma of lung in vitro (16).
In the present study, we investigated the expression and mutation of c-kit in gastrointestinal NEC, since this could provide us with useful information concerning whether imatinib mesylate might be effective against this aggressive malignancy.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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TUMOR SPECIMENS
We collected specimens diagnosed as gastrointestinal NEC from the records of histopathology at the Saitama Cancer Center and the National Cancer Center Hospital East. All specimens were surgically resected. The diagnoses of NEC were based on the findings of hematoxylin and eosin-stained sections (sheets and nests of round to fusiform cells with minimal amounts of cytoplasm, granular nuclear chromatin and inconspicuous nucleoli) and immunohistochemical sections (chromogranin, synaptophysin, NSE and CD56). The carcinoid was excluded from this group. The definition of the WHO classification of digestive tumors published in 2000 (17) was used to differentiate NEC from carcinoid.
IMMUNOHISTOCHEMISTRY
Immunohistochemistry of c-kit was performed using the method previously reported (10). Briefly, 4 µm tissue sections were deparaffinized in xylene, dehydrated in a graded alcohol series and blocked for endogenous peroxidase with 3% H2O2 in absolute methyl alcohol. Following microwave treatment at 95°C for 20 min for antigen retrieval, sections were incubated at 4°C overnight with rabbit polyclonal anti-human c-kit (DAKO, Glostrup, Denmark) at a dilution of 1:50. Tissue sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, USA) at room temperature for 30 min and then with a streptavidin–biotin peroxidase complexed Kit (DAKO) for 30 min. Sections were exposed to 3,3'-diaminobenzidine for 3–5 min. Membrane or cytoplasmic staining of c-kit was evaluated as positive. Each tumor was scored as ‘–’ when no tumor cells were stained, ‘1+’ when 0–10% of the tumor cells were stained and ‘2+’ when over 10% of the tumor cells were stained, as well as weak or strong according to the staining intensity where ‘strong’ was defined as the intensity comparable to that observed in a mast cell. Immunohistochemically c-kit positive GIST tissue and normal colonic mucosa were used as positive and negative controls, respectively.
RT–PCR
Total RNA was isolated from the frozen tumor tissues using the acid guanidinium thiocyanate phenol–chloroform (AGPC) extraction method. One microgram of total RNA was reverse transcribed into cDNA using oligo-dT primer and an RT–PCR kit (Invitrogen) according to the manufacturer's instructions. One-tenth of the reacted aliquot was subsequently amplified by PCR using GAPDH primers (GAPDH forward primer, 5'-TGGTATCGTGGAAGGACTCATGAC-3'; GAPDH reverse primer, 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3') as a control and c-kit primers (c-kit forward primer, 5'-AGCAAATCCATCCCCACACC-3'; c-kit reverse primer, 5'-GGCTTGAGCATCTTTACAGCGAC-3'). Thermal cycling was initiated with a 10 min reaction at 95°C to activate the Taq polymerase followed by 26 three-step cycles for GAPDH at 94°C for 30 s, 56°C for 30 s and 72°C for 30 s, and 35 three-step cycles for c-kit at 94°C for 30 s, 60°C for 30 s and 72°C for 60 s. Four microliters of the PCR product was electrophoresed through a 1.5% agarose gel and the gel subsequently stained with ethidium bromide to visualize the PCR product.
EXAMINATION OF C-KIT GENE MUTATION BY PCR–SSCP
DNA was isolated from the frozen tissues or formalin-fixed paraffin-embedded tissue sections. DNA from GIST harboring a c-kit mutation at exon 11 and normal colonic mucosa was used as positive and negative controls, respectively. We performed PCR–single-strand conformation polymorphism (SSCP) in an effort to analyze mutations in the c-kit gene. PCR was carried out at a final concentration of 1x PCR buffer (10x PCR buffer; 150 mM Tris–HCl, pH 8.0, 500 mM KCl), 50 ng of genomic DNA, 2.5 mM MgCl2, 0.5 mM of each forward and reverse primer, and 1.25 U of ‘AmpliTaq Gold’ (PE Applied Biosystems, Branchburg, NJ) in a total volume of 20 µl. All PCRs were initiated at 95°C for 9 min, followed by 35 three-step cycles for exon 9–13 of the c-kit gene at 94°C for 30 s, 65°C for 30 s and 72°C for 30 s. The following primers were used: exon 9 f, 5'-ATTTATTTTCCTAGAGTAAGCCAGGG-3'; exon 9 r, 5'-ATCATGACTGATATGGTAGACAGAGC-3'; exon 10 f, 5'-TGGTAGAGATCCCATCCTGC; exon 10 r, 5'-TGGGGAGAAAGGGAAAAATAG-3'; exon 11 f, 5'-TTGTTCTCTCTCCAGAGTGCTC-3'; exon 11 r, 5'-AAGGTGACATGGAAAGCCC-3'; exon 12 f, 5'-CAGCACCATCACCACTTACC-3'; exon 12 r, 5'-AGCAAAAAGCACAACTGGC-3'; exon 13 f, 5'-AGATGCTCAAGCGTAAGTTC C-3'; and exon 13 r, 5'-CCTGACAGACAATAAAAGGCAG-3'.
Two microliters of PCR products was mixed with 20 µl of SSCP gel loading dye, heated for 5 min at 95°C and then chilled on ice. Approximately 6 µl of this mixture was loaded in each well of a 10% acrylamide gel, electrophoresed at 10, 15 and 20°C for 3 h at 300 V, and the gel subsequently stained with Syber Gold to visualize the PCR product.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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We examined 23 gastrointestinal NECs for c-kit expression. Results are summarized in Table 1. They consisted of 1 esophageal, 16 gastric, 2 duodenal and 4 colorectal NECs. The average age of the patients was 60 years, and the ratio of men to women was 19:4. Most patients had a non-NEC component, such as squamous cell carcinoma with esophageal NEC and adenocarcinoma with gastric or intestinal NEC.
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c-kit protein expression was found in 6 out of 23 gastrointestinal NECs but not in any normal epithelial cells of the gut. Mast cells localized in the stroma were used as a positive internal control. Two NECs showed a 2+ distribution score and strong immunoreactivity for c-kit, while four NECs showed a 1+ distribution score and weak immunoreactivity for c-kit (Fig. 1A and B). Cytoplasmic localization of c-kit protein was observed in three out of four weak c-kit positive NECs. With respect to organ type, 4 out of 16 gastric, 1 out of 2 duodenal and 1 out of 4 colorectal NECs showed c-kit expression (Table 1).
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None of the concordant non-NEC components showed detectable c-kit expression. As a representative case (case no. 5), hematoxylin and eosin staining and immunohistochemical staining for chromogranin A, CD56 and c-kit is shown in Fig. 1C–F.
c-kit expression was also evaluated in three frozen tumor specimens using RT–PCR and the findings were consistent with the immunohistochemistry (Fig. 2, Table 1).
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Since most mutations were observed at the juxtamembrane domain of the c-kit gene in GIST, we analyzed the exon 9–13 of the c-kit gene in six gastrointestinal NECs that were obtained as frozen samples or paraffin-embedded tissue sections by PCR–SSCP. However, no c-kit mutation was detected (Fig. 3, Table 1).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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We demonstrated that 26% of gastrointestinal NECs showed c-kit protein expression. This finding is similar to the frequency of c-kit expression of colorectal NEC reported recently (3) and also the frequency is in the range of c-kit expression in pulmonary small or large cell carcinoma (17–44%) (8–11). The similarity in clinical and pathological features between pulmonary NEC and gastrointestinal NEC has been known, and thus the present study revealed that c-kit expression is also similar. The concordant non-NEC component, i.e. squamous cell carcinoma in esophagus and adenocarcinoma in stomach and intestine, was completely negative for c-kit (3,8,9,18,19).
Although we examined c-kit mutation by PCR–SSCP, no c-kit mutation was found in gastrointestinal NECs at exon 9–13, the hot spot of mutation in GIST (3). Aberrant activation of c-kit signaling occurs as a result of gain-of-function mutation of c-kit in GIST and mast cell disease. It has been speculated that the autocrine or paracrine growth loop of c-kit and stem cell factor, the natural ligand of c-kit, causes c-kit activation in pulmonary NEC where overexpression of both was found (16). Although stem cell factor was not examined in this study, the finding that no c-kit mutation was observed in gastrointestinal NEC might suggest that the mechanism of c-kit activation is similar to that of pulmonary NEC.
It is important to investigate c-kit expression in malignancies from a therapeutic standpoint, since imatinib mesylate, a specific inhibitor of c-kit, may be effective against these malignancies. We prescribed imatinib mesylate to a 54-year-old man (case no. 5) harboring lung metastasis of gastric NEC on the approval of the ethics committee and the patient's informed consent. He underwent total gastrectomy (32 months previously) due to gastric NEC and partial hepatectomy (18 and 16 months previously) due to hepatic metastasis. The patient's gastric lesion and hepatic metastasis were strongly positive for c-kit (Figs 1F and 2 lane 2). However, imatinib mesylate therapy was discontinued after 10 days due to systemic edema classified as grade 3, according to the National Cancer Institute Common Toxicity Criteria Version 2.0, and grade 2 anorexia. Although we could not access its effect, the apparent effectiveness was not confirmed in the CT performed 1 month following initiation of the therapy.
According to recent studies, imatinib mesylate was not useful as a single agent in treating patients with small cell carcinoma of the lung (20–22), despite the positive antitumor activity of imatinib mesylate against several tumors in vitro (16). The possible reason for this resistance in vivo might be the diversification of cancer cells, since we found that concomitant non-NEC components did not express c-kit protein. This finding might suggest that the use of imatinib mesylate as a single agent does not exert sufficient antitumor activity, but its benefit could be attained by combination with other chemotherapeutic agents.
In conclusion, the present study demonstrated the expression of c-kit in gastrointestinal NEC. Further studies are necessary to clarify the mechanisms of c-kit expression in order that effective therapeutic strategies are developed.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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