Clinical Oncology (2007) 19: 204e208doi:10.1016/j.clon.2006.12.009

Original Article

CD117 (c-kit) Expression in Human HepatocellularCarcinoma

G. Becker*, A. Schmitt-Graeffy, V. Ertelty, H. E. Blum*, H.-P. Allgaier*

*Department of Medicine II, Freiburg University Hospital, Freiburg, Germany; yInstitute of Pathology,Freiburg University Hospital, Freiburg, Germany

ABSTRACT:Aims: Although various methods of treatment have been tried, treatment options for advanced hepatocellularcarcinoma (HCC) remain limited. Expression of the platelet-derived growth factor has been shown in HCC, which mayderive from hepatic stem cells that express the c-kit proto-oncogene. Because of the promising results of imatinib andthe key role played by c-kit in gastrointestinal stromal tumours and other solid tumours, the aim of this study was todetermine the prevalence of c-kit (CD117) overexpression in patients with HCC.Materials and methods: A retrospective study of 258 archival specimens of subjects with histologically confirmed HCCwas carried out. Expression of the c-kit proto-oncogene was evaluated by immunohistochemistry using rabbit anti-CD117antibody A4502.Results: The overall percentage of positive immunohistochemical staining of HCCs was 2.3% (6/258).Conclusions: Our results suggest that CD117 is not significantly overexpressed in HCC and there seems to be no role forthe use of imatinib. Becker, G. et al. (2007). Clinical Oncology 19, 204e208

ª 2006 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Key words: CD117, hepatocellular carcinoma, imatinib, tyrosine kinase

Introduction

Hepatocellular carcinoma (HCC) is one of the majormalignancies worldwide. The highest risk areas are ineastern Asia, middle Africa and some countries of westernAfrica [1]. Recent studies have also shown an increasingHCC incidence in developed countries [2]. Surgical re-section and liver transplantation are regarded as the onlypotentially curative therapies. Because of advanced ordecompensated liver cirrhosis, co-morbidity and multi-centricity of the HCC lesions, 70e80% of patients areinoperable at the time of diagnosis [3]. Therefore, severallocal ablation methods have been developed as minimallyinvasive strategies for HCC treatment, including trans-arterial chemoembolisation and percutaneous ethanol in-jection, as well as radiofrequency thermal ablation [4]. Forpatients with advanced HCC, these local ablative therapiesare not suitable. The results of systemic HCC treatmentsincluding intravenous chemotherapy or anti-oestrogenicdrugs have been disappointing [5]. The positive influence ofthe somatostatin analogue octreotide on survival reportedin pilot studies [6] could not be confirmed in randomisedcontrolled trials [7,8]. Presently, there is no standardtherapy for advanced HCC and the prognosis is generallypoor [9,10].

0936-6555/07/190204þ05 $35.00/0 ª 2006 The Royal C

The proto-oncogene c-kit belongs to the platelet-derivedgrowth factor (PDGF) receptor family. It encodes a receptortyrosine kinase and has been shown to be expressed ina wide variety of human malignancies, including gastroin-testinal stromal tumours (GIST), colon carcinoma, salivarygland carcinoma, testicular germ cell tumours and neuro-blastomas [11e17]. c-kit expression is of topical interest,because the stem cell factor receptor (c-kit, CD117) is oneof the targets of the tyrosine kinase inhibitor imatinibmesylate (STI571, Glivec�, Gleevec�). Imatinib was initiallydesigned to inhibit the BCR/ABL fusion protein in chronicmyeloid leukaemia [17e21] and was approved for thisapplication by the Food and Drug Administration in May2001. Subsequently it was discovered that imatinib is alsoeffective against kit-positive GISTs [17e25]. The promisingresults of imatinib and the key role played by c-kit in GISTsand other solid tumours motivated us to investigatewhether c-kit is also expressed in human HCC.

Materials and MethodsPatient Selection

Paraffin-embedded tissue blocks from the archive of theInstitute of Pathology, University of Freiburg were carefully

ollege of Radiologists. Published by Elsevier Ltd. All rights reserved.

205CD117 EXPRESSION IN HUMAN HCC

reviewed and assessed in the current investigation.Specimens were analysed according to the institutionalethics policy. All tissues were obtained from patients withprior histologically confirmed HCC. Patients’ clinical datawere verified through the medical records.

Immunohistochemistry

Formalin-fixed paraffin-embedded tissue sections cut at4 mm were deparaffinised, rehydrated and heat retrievedin 1 mM citrate buffer, pH 6 for antigen detection. Rabbitanti-CD117 antibody (c-kit) was used as the primaryantibody at a dilution of 1 : 400. The sections wereincubated with the primary antibody at room tempera-ture for 30 min. The bound antibodies were detected bythe alkaline phosphataseeanti-alkaline phosphatasemethod, which produces a red reaction product, aspreviously described [26]. To control for non-specificbinding, the first antibodies were omitted. Appropriatepositive and negative controls were included. Within eachspecimen the HCC lesion was semi-quantitatively evalu-ated. Immunostaining was classified as follows: 0, noimmunostaining above the cut-off level; þ, low expres-sion; 2þ, moderate expression; 3þ, strong expressionsimilar to reactive mast cells, which were used aspositive internal controls. In addition, different immuno-histochemistry patterns were recorded: (a) predominantlycytoplasmic staining, and (b) predominantly plasmalem-mal labelling. Each case was evaluated for the expres-sion of CD117 (c-kit) by two independent investigators(ASG, VE).

Results

To investigate whether the c-kit receptor was expressed onthe cell membrane of human HCC, archival paraffin-embedded tissue blocks obtained from 258 patients withhistopathologically confirmed HCC were examined. Thedemographic and clinicopathological characteristics of thepatients are summarised in Table 1.

Tissue sections were immunostained with an anti-CD117polyclonal antibody, with appropriate results for thepositive and negative controls (Fig. 1A, B). Strong CD117expression was detected in scattered mast cells within thespecimens. The overall percentage of positive immunohis-tochemical staining of HCCs was 2.3% (6/258). Five patientsshowed predominantly cytoplasmic staining of tumourcells, whereas one patient showed predominantly plasma-lemmal labelling.

The descriptive statistics show that there was no specificpattern concerning clinicopathological characteristics inthe c-kit-positive patients in contrast to the c-kit-negativepatients. In both groups, most of the patients were maleand cirrhosis was mainly caused by alcohol. Taking intoaccount the low number of only six c-kit-positive patients,we refrained from statistically testing the distributionsof the clinicopathological characteristics between thec-kit-negative and c-kit-positive patients.

Discussion

Several protein kinases are deregulated and overexpressedin human cancers and therefore are targets for selectivepharmacological inhibitors. An analysis of the gene expres-sion pattern of HCC showed a high expression of severalprotein tyrosine kinases, such as PDGF receptor-b, intumour cells [27]. c-kit (CD117) is a transmembranetyrosine kinase that acts as a receptor for mast cell growthfactor [17]. It belongs to the type III family of receptorkinases and can be detected in several normal cells,including haematopoietic stem cells, germ cells, intestinalcells of Cajal, ductal breast epithelium, mast cells andmelanocytes [28e32]. kit expression has also been de-tected in a variety of different tumour entities, such asGISTs and at least 46 additional tumour entities [17].Although the efficacy of imatinib in GISTs has beenrecognised, only a few data exist regarding the over-expression of c-kit in other fatal cancers arising from thegastrointestinal tract, especially those of the liver.

Currently, three main mechanisms leading to the de-velopment of HCC in a cirrhotic liver are discussed [33]. Onehypothesis suggests that hepatocytes of regenerative nod-ules first become dysplastic and then neoplastic [34,35]. Forhepatic viruses, a direct influence on malignant trans-formation of hepatocytes is discussed [36]. Furthermore,mainly c-kit-positive oval cells, the somatic stem cells of theliver that are supposed to replace lost hepatocytes, mayundergo malignant transformation [37,38].

Our study of 258 HCC tissue sections showed that c-kitreceptors were expressed in only 2.3%. This result confirmsthe results of previous smaller studies. Potti et al. [39]found only one c-kit-positive tumour (4%) in 25 HCCs anda study by Went et al. [17] investigating c-kit expression indifferent human tumours found none of the 40 HCC tissuesections positive for c-kit. A recently published phase II trialin 17 patients with advanced HCC showed no therapeuticeffect for imatinib [25]. Only one of 15 tumours waspositive for PDGF receptor and none of the 14 tumours waspositive for c-kit. In contrast to these results, Chung et al.[16] found 22 of 86 HCC tissue sections (26%) positive for c-kit by immunohistochemical staining and confirmed mRNAexpression by reverse transcription-polymerase chain re-action. Although even in this study most tumours werenegative for c-kit, these results suggest that the cellularpopulation of HCC is probably heterogenous and may reflectdifferent genetic expression patterns due to cellularheterogeneity or the distinct stages of cell differentiation.In the study by Chung et al. [16], over 90% of patients hadchronic hepatitis B or C, whereas in our sample cirrhosiswas caused by alcohol in about 50% of patients. On theother hand, only 1/90 patients with hepatitis was positivefor c-kit in our study. We have no valid explanation why inthe study by Chung et al. 26% of HCC tissues were positivefor c-kit, whereas in our study and in the studies by Pottiet al. [39] and Went et al. [17] only 4% or less of HCC tissueswere positive for c-kit.

Analysing their data, Chung et al. [16] suggested thatc-kit-positive patients exhibited a better survival rate than

206 CLINICAL ONCOLOGY

Table 1 e Demographic and clinicopathological characteristics of the 258 hepatocellular carcinoma patients

c-kit (�) c-kit (þ) Total

n % n % n %

Total 252 6 258Male 200 77.7 5 83.3 205 79.6Age (mean years) [range] 65.1 70.8 65.3 [11e86]

Cause of cirrhosis 241 3 (md: 3) 247Alcohol 115 47.7 2 66.6 117 48.0Hepatitis C 63 26.1 1 33.3 64 26.2Hepatitis B 27 11.2 0 0 27 11.0Haemochromatosis 14 5.8 0 0 14 5.7Other/unknown 22 9.1 0 0 22 9.0

Alpha-fetoprotein 227 5 (md: 1) 233!100 ng/ml 144 63.4 1 20 145 62.5O100 ng/ml 83 36.6 4 80 87 37.5

ChildePugh stage 236 5 (md: 1) 242Child A 134 56.8 4 80 138 57.3Child B 73 30.9 1 20 75 31.1Child C 28 11.9 0 0 28 11.6

Okuda stage 225 5 (md: 1) 231Okuda I 104 46.2 4 80 108 47.0Okuda II 98 43.6 1 20 99 43.0Okuda III 23 22.1 0 0 23 10.0

Portal vein thrombosis 175 md: 6 181Yes 54 30.9 0 0 54 30.9No 121 69.1 0 0 121 69.1

Diameter (cm) 215 3 (md: 3) 221!5 110 51.2 2 66.6 112 51.4O5 105 48.8 1 33.3 106 48.6

Type of tumour 252 5 (md: 1) 258Trabecular 140 55.6 4 80 144 56.0Glandular 9 3.6 0 0 9 3.5Solid 27 10.7 0 0 27 10.5Mixed 76 30.2 1 20 77 30.0

Grading of tumour 252 5 (md: 1) 258Grade 1 106 42.1 4 80 110 42.8Grade 2 86 34.1 1 20 87 33.9Grade 3 60 23.8 0 0 60 23.3

md, missing data.

those with negative c-kit expression. However, in theiranalysis, they only incorporated the cause of cirrhosis. Theydid not take into account the staging of cirrhosis using theChildePugh score [40], tumour staging using the Okudasystem [41] or the CLIP score [42,43]. Parameters predict-ing poor survival, such as evidence of portal vein thrombosis[44,45], were not analysed. Although Chung et al. [16]found a better survival in the c-kit-positive patients, theexpression of c-kit cannot be used as a prognostic factor forHCC. Further investigation would be needed to test thishypothesis. Discrepant results may be due to a wide varietyof different antibodies, protocols and scoring systems toidentify c-kit-positive tumours. Went et al. [17] pointed outthat, because of different antibodies and scoring systems,

the frequency of c-kit positivity ranged from 36% [30] to91% [28] in small cell lung cancer and from 0% [31] to 20%[46] in malignant melanoma. Went et al. compared sevendifferent antibodies and found that antibody A4502 was themost sensitive and specific antibody [17]. However, theiranalysis of 40 HCC tissue sections was negative in allspecimens. This is consistent with the results of our studyinvestigating the expression of c-kit in over 250 HCC tissuesections also using the most sensitive and specific antibody(A4502). These results, together with the recently pub-lished phase II trial with imatinib [25], suggest that c-kit isa questionable target in the treatment of HCC. It seemsunlikely that CD117 has a key role in the pathogenesis ofHCC.

207CD117 EXPRESSION IN HUMAN HCC

Fig. 1 e (A) Immunohistochemical staining of c-kit oncoprotein in hepatocellular carcinoma cells (�400). (B) c-kit-negative hepatocellularcarcinoma showing mast cells as the positive internal control (�400).

Acknowledgement. This study was supported by a grant fromNovartis Pharma. Dr Allgaier reports having received lecture feesfrom Novartis Pharma.

Author for correspondence: G. Becker, Department of Medicine II,University Hospital Freiburg, Hugstetter Strasse 55, D-79106Freiburg, Germany. Tel: þ49(0)7612703213; fax: þ49(0)7612703291; E-mail: becker@med1.ukl.uni-freiburg.de

Received 12 August 2006; received in revised form 5 December2006; accepted 14 December 2006

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