It is well known that hepatocellular carcinoma (HCC) is a type of hypervascular lesions (El-Assal et al., 1998). Angiogenesis is critical for tumor growth, and therefore tumors may continuously produce certain mediators that induce angiogenesis, permitting tumor cell proliferation and expansion (Park et al., 2000). Tumor cells induce adjacent blood vessels to sprout new vessels toward the tumor. Immature microvessels are not covered by pericytes, and they are irregular and leaky; thus, tumor cells can more easily penetrate immature microvessels than mature microvessels (Sun and Zhang, 2006). CD34, a differentiation stage-specific leukocyte antigen, is an approximately 115 kDa single-chain transmembrane glycoprotein present on leukemic cells, endothelial cells, and stem cells (Fina et al., 1990). In addition, it is localized on cells of the splenic marginal zone, dendritic cells around vessels, nerves, hair follicles, muscle bundles, and sweat glands in a variety of tissues and organs. Its function is still unclear. It may be related to cellular adhesion and/or migration because of its structural characteristics and its relative abundance on cellular surface mainly in the region of cell adhesion in the endothelium (Bono et al., 2002). It is used for leukemia diagnosis and subclassification and for diagnosis of vascular tumors. Antibodies to CD34 also strongly label gastrointestinal stromal tumors, and the antigen is invariably found in solitary fibrous tumor and dermatofibrosarcoma protuberans (Pusztaszeri et al., 2006). The relationship between the amount of microvessels expressing CD34 and metastases has been found in melanoma (Srivastava et al., 1986), in breast cancer (Weidner et al., 1991), cervical cancer (Sillman et al., 1981), stomach (Maeda et al., 1995), lung (Macchiarini et al., 1992), and in prostate cancer (Bettencourt et al., 1998; Bono et al., 2002).
Recently, distinctly hepatocellular nodules [referred to as dysplastic nodules (DNs), adenomatous hyperplasia, or macroregenerative nodules] have been found in chronic hepatic diseases and cirrhosis, and they have been considered to be premalignant lesions (Hytiroglou et al., 1995). DNs may be the first step of hepatocarcinogenesis, subsequently developing into a HCC through low-grade DN, high-grade DN, and early HCC (Park et al., 2000). Angiogenesis is important in hepatocarcinogenesis (Kimura et al., 1998) and also increased expression of CD34, as has been reported in HCC (Lu et al., 2004).
C-myc gene is included among the oncogenes, the amplification of which has been detected in cancers in various organs (Takahashi et al., 2007). However, there have been few reports on the amplification of this gene in HCC. Results of several studies (Watson et al., 1996; Sierra et al., 1999) indicate that genetic alterations in the c-myc oncogene play an important role in the induction and progression of human cancer. The product of the c-myc protooncogene is c-myc protein, and overexpression of c-myc protein in breast cancer to some extent correlates with gene amplification (Facchini and Penn et al., 1998). C-myc protein exerts diverse effects on cell behavior. Data gathered to date indicate that c-myc protein plays a critical role in normal progression of the cell cycle, in inhibition of terminal differentiation, and in induction of programmed cell death (Thompson et al., 1998; Yu et al., 2003). The occurrence of c-myc oncogene amplification in breast cancer has been related to a poor prognosis (Berns et al., 1996).
However, no attempt was made in any of these studies to investigate the role of CD34 and c-myc oncoproteins in hepatocytic dysplastic lesions and their progression to the subsequent HCC.
The purpose of this study was to evaluate the degree of CD34 expression (as a marker of microvessel density) and the proliferation activity of hepatocytes and HCC cells, evaluated by the level of c-myc expression in low-grade DNs, high-grade DNs, early and advanced HCCs to understand their role in hepatocarcinogenesis. Moreover, we will determine whether there is any relationship between their expression and the histologic grade and stage of HCC to elucidate the potential role of them as prognostic markers in HCC.
Materials and methods
This is a retrospective study of selected 60 hepatic lesions, 10 DNs (six low grade and four high grade), and 50 HCCs (11 GI, 23 GII, and 16 GIII), in addition to10 control cases examined from adjacent nondysplastic, non-neoplastic hepatic tissue adjacent to the dysplastic, and the malignant lesions. Low-grade DN showed mild atypia, and high-grade DN showed moderate atypia insufficient for the diagnosis of malignant neoplasm (International Working Party, 1995). Cases of HCC were surgically resected biopsies that were selected according to the availability of paraffin blocks and follow-up data. The materials included consecutive archival formalin-fixed, paraffin-embedded blocks processed in the pathology department of the National Cancer Institute and Benha Faculty of Medicine during the period between 2000 and 2006. HCCs were graded according to the criteria described by Poon et al. (2003), and were staged according to the pathological tumor node metastasis classification (Sobin and Wittekind, 1997). Seven cases (14%) were stage I, 24 cases (48%) were stage II, 14 cases (28%) were stage III, and five cases (10%) were stage IV. As regards the tumor size, all specimens were unifocal lesions larger than 2 cm in maximum diameter. Three sections of 4 μ thickness were obtained for every case. One was hematoxylin and eosin stained for diagnosis reviewing and confirmation. The other two sections were immunohistochemically stained for CD34 and c-myc proteins.
Interpretation of immunohistochemical staining
The stained slides were microscopically examined, using 200× magnification, by two independent observers. For evaluating the degree of CD34 expression we followed the criteria given by Park et al. (2000):
(1) Negative (0): staining of endothelial cells occupying less than 10% of the sinusoidal liver cell surface.
(2) Low expression (+1): staining of endothelial cells occupying approximately 10 to 30% of the sinusoidal liver cell surface.
(3) Moderate expression (+2): staining of endothelial cells occupying approximately 30 to 50% of the sinusoidal liver cell surface.
(4) High expression (+3): staining of endothelial cells occupying more than 50% of the sinusoidal liver cell surface.
Expression of c-myc
It was scored semiquantitatively by two observers analyzing 5–10 higher power fields under an optical microscope (original magnification ×40). The results were quantified as negative (−) if no staining was observed in any cell, and as positive (+) if less than 5% of cells in the section were positive; (++), when 5–50% of cells were positive; (+++), when more than 50% were stained (Yu et al., 2003).
Data analysis was conducted using the SPSS program (0.8 for windows, SPSS Inc., Chicago, Illinois, USA) software package according to Pearson's correlation coefficient. Correlation between several variables was computed using Fisher's exact test. P value of less than 0.05 was considered to be significant, and less than 0.01 was considered to be highly significant.
C-myc immunostaining results
C-myc immunostaining was observed as nuclear staining in all 10 control cases (Table 1).
(1) All control non-neoplastic cases showed positive low c-myc expression.
(2) Dysplastic hepatic nodules showed high (+++) expression of c-myc in 50% of high-grade dysplasia (Fig. 1) and moderate (++) c-myc expression in 25% of cases examined compared with low-grade dysplastic cases in which 16.7% of cases showed moderate c-myc expression and none of them showed high c-myc expression. As regards HCC cases, 50% of them showed high c-myc expression and 25% showed moderate c-myc expression (Figs 2 and 3). The relationship was statistically significant (P<0.05).
(3) There was a statistically significant correlation between c-myc expression and the grade and stage of HCC cases (P<0.05).
(1) Non-neoplastic control cases showed focal positive CD34 expression in sinusoids, restricted to the periportal area, whereas the centrolobular sinusoids were negative (Table 2).
(2) In low-grade DNs, CD34 expression was confined mainly to the outer region with periportal and peripheral patterns. CD34 expression was more diffuse in high-grade DNs and HCCs.
(3) High (+2 and+3) CD34 expression was seen in 75% of high-grade dysplastic hepatic cases examined and in 88% of HCC cases compared with low-grade dysplastic cases, which showed low (+1) CD34 expression in 66.7% of cases. This relationship was statistically significant (P<0.05) (Figs 4 and 5).
(4) Positive correlation was seen between CD34 expression and HCC as regards tumor grading and staging, which was statistically significant (P<0.05) (Figs 6 and 7).
HCC is a common type of cancer, with approximately 260 000 new cases each year. However, specific changes of gene expression in HCC remain obscure. The expression of genes for hepatocytic growth factor c-myc protooncogene was analyzed by Yongqiang et al., 2003; Kim et al., 2005. It has been suggested that the rate of tumor growth is determined by the balance between cell proliferation and cell death: only when the rate of proliferation exceeds that of death does tumor growth occur. Alteration in oncogene expression may, therefore, potentially act on cancer cells by lowering their rate of apoptosis, raising their rate of proliferation, or both (Yongqiang et al., 2003). Data published to date indicate that oncogene amplification is one of the most common genetic alterations found in human cancers. Examination of regional c-myc gene amplification within breast tumors showed that alteration of this gene can occur at an early in-situ stage of tumor progression and often does not persist in late-stage nodal metastasis. The product of the c-myc oncogene is an important nuclear DNA-binding protein that seems to play critical roles in the regulation of cell growth and division. The c-myc oncoprotein is activated to cause cell transformation by overexpression, resulting in intracellular accumulation (Kim et al., 2005).
Data presented in this study showed that all control non-neoplastic cases showed positive low (+) c-myc expression. Dysplastic hepatic nodules showed high (+++) expression of c-myc in 50% of high-grade dysplasia and moderate (++) c-myc expression in 25% of cases examined compared with low-grade dysplastic cases in which 16.7% of cases showed moderate c-myc expression and 83.3% of them showed low (+) c-myc expression and none of them showed high c-myc expression. As regard HCC cases, 50% of them showed high c-myc expression and 25% showed moderate c-myc expression. The relationship was statistically significant (P<0.05). These results may support the assumption that alterations in the c-myc protein level are involved in formation and progression of low-grade DNs into high-grade DNs and early HCC. Thus, there is c-myc overexpression at the time of tumor presentation. Sustained c-myc overexpression results in persistent proliferation of hepatocytes, and increased occurrence of HCC development may be through activation of transforming growth factor due to an unrestrained cell cycle progression of neoplastic hepatocytes by the disruption of the cyclin D-pRb-E2F pathway (Young et al., 2000; Yoshiji et al., 2002).
With regard to the relation to the tumor grade, examined cases of HCC showed high c-myc expression in 14 (87.5%) of 16 GIII cases examined compared with none of 11 (9.1%) GI cases. These results were in accordance with the data discussed by Cui et al. (2004) and Kim et al. (2005) in which they found that c-myc overexpression was related to tumor progression and differentiation. Moreover, they stated that this overexpression has no direct relation to the recurrence, which was a later biological behavior developed at an advanced stage of the tumor. The tumor differentiation in the c-myc positive group was lower than that in the c-myc negative group. A study on the biological behavior of c-myc protein indicates that besides being a transcriptional factor it is important for cell growth and differentiation (Yu et al., 2003).
As regards the relation to TNM stage, five (100%) of SIV cases showed high c-myc expression in relation to two of seven cases (28.6%) of SI cases. In consistent to these results, Berns et al. (1996) found that the occurrence of c-myc oncogene amplification in breast cancer has been related to a poor prognosis.
HCC is known to receive its blood supply principally from the hepatic arteries. Recent studies have reported differences in the vascular supply, especially arterial supply among low-grade and high-grade DNs and HCCs. Increased expression of vascular endothelial growth factor (VEGF) has been reported in HCC. In addition, VEGF may play an important role in the early phases of hepatocarcinogenesis, but little issues are discussed in the role of CD34 in early and late stages of hepatic carcinogenesis (Young et al., 2000).
Angiogenesis is very common within normal and pathologic tissues and is regulated by many factors, such as VEGF, hypoxia-inducible factor 1 α, human macrophage metalloelastase, basic fibroblast growth factor, and so on (Ravi et al., 2000; Yoshiji et al., 2002). Vessels interact with tumor cells to construct microenvironment of tumor (Gorrin-Rivas et al., 2000). CD34, which is expressed in blood stem cells and neovessel endothelial cells, is the most distinctive marker for demonstrating vessel endothelial cells (Frachon et al., 2001), especially for demonstrating sinusoid-like vessels in tumor tissues (Gottschalk-Sabag et al., 1998). In this research, non-neoplastic control cases showed focal positive CD34 expression in sinusoids restricted to the periportal area, whereas the centrolobular sinusoids were negative. All dysplastic and HCC cases were positive for CD34 antibody in sinusoidal endothelial cells. High CD34 expression was seen in 75% of high-grade dysplastic hepatic cases examined and in 88% of HCC cases compared with low-grade dysplastic cases, which showed low CD34 expression in 66.7% of cases. This relationship was statistically significant (P<0.05). This means that the degree of sinusoidal capillarization and angiogenesis increased gradually according to the stepwise development of hepatocarcinogenesis, as it was higher in high-grade DNs and HCCs than in low-grade DNs. Therefore, the microvessels play an important role not only in occurrence and development of HCC, but also may be helpful to identify malignant lesions or precancerous lesions (high-grade DNs) from benign or low-grade DNs.
These results are consistent with those of Roncalli et al. (1999) and De Boer et al. (2000) who found that microvessel density (MVD) determined by CD34 increased gradually from normal liver to DNs and finally to HCC. They found increased MVD at the early stages of focal dysplasia, which increased gradually from low to high grades of dysplasia. In addition, Sakamoto et al. (1991) and Lu et al. (2004) suggested that DNs may be the first step of multistep hepatocarcinogenesis and that they develop into HCC probably through high-grade DNs and subsequently into early HCCs. Moreover, Bettencourt et al. (1998), reported that there was increased density of capillaries in prostate cancer compared with benign prostate tissue.
With regard to the relation to the tumor grade, examined cases of HCC showed high (+3) CD34 expression in 12 (75%) of 16 GIII cases examined compared with five (45.4%) of 11 GI cases that showed low (+1) CD34 expression, which was statistically significant (P<0.05). These results may indicate that angiogenesis and sinusoidal capillarization increased gradually as hepatocarcinogenesis progressed. These results are in accordance with the data discussed by Ohmori et al. (2001), who found that high expression of CD34 positive sinusoidal endothelial cells is a risk factor for HCC in a patient with chronic liver disease. In addition, Lu et al. (2004) found that MVD was higher in high-grade HCC than low-grade HCC. Taku et al. (2001) reported that fine sinusoidal capillaries were positive for CD34 in well-differentiated HCC whereas thick and peripheral vessels were positive for CD34 in moderately differentiated and poorly differentiated HCC and suggested that CD34 may be useful marker in the diagnosis of the hepatic DNs and assessing the degree of the differentiation of HCC. Other reports also showed that poor differentiation and portal invasion are significantly related to MVD (Nakashima et al., 1999). Yamamoto et al. (2001) demonstrated that sinusoidal capillarization occurring in well-differentiated HCC is related to dedifferentiation of parenchymal tumor cells. As regards the relation to TNM stage, four (80%) of five SIV cases showed high (+3) CD34 expression compared with four (57.1%) of seven SI cases that showed low (+1) CD34 expression (P<0.05). Similar to these results, Lu et al. (2004) found that increased MVD was associated with advanced stages of HCC, indicating that the density of neovascularization is correlated with metastasis, resulting in poor prognosis.
In this study, c-myc oncoprotein expression increased gradually as hepatocarcinogenesis progressed. Thus, c-myc oncoprotein expression is considered to contribute to the growth advantage in hepatocarcinogenesis. In addition, the degree of MVD determined by the degree of CD34 expression increased gradually according to the stepwise development of hepatocarcinogenesis. Moreover, the significant coexpression of CD34 and c-myc in HCC in this study strongly suggests an important role for these genes in malignant growth and progression.
In conclusion, c-myc and CD34 upregulations are greatly integrated in multistep hepatocellular carcinogenesis and also in prognosis and progression of HCC.
1. Berns Els MJJ, Klijn JGM, Smid M, Van Staveren IL, Look MP, Van Putten WLJ, et al. TP53 and myc gene alteration independently predict poor prognosis in breast patients. Chromosomes Cancer. 1996;16:170–179
2. Bettencourt MC, Bauer JJ, Sesterhenn IA, Connelly RR, Moul JW. CD34 immunohistochemical assessment of angiogenesis as aprognostic marker for prostate cancer recurrence after radical prostatectomy. J Urol. 1998;160:459–465
3. Bono AV, Celato N, Cova V, Salvadore M, Chinetti S, Novario R, et al. Microvessl density in prostate carcinoma. Prostate Cancer Prostatic Dis. 2002;5:123
4. Kim CK, Lim JH, Park CK, Choi D, Lim HK, Lee WJ, et al. Neoangiogenesis and sinusoidal capillarization in hepatocellular carcinoma: correlation between dynamic ct and density of tumor microvessels. Radiology. 2005;237:529–534
5. Cui J, Dong BW, Liang P, Yu XL, Yu DJ. Effect of c-myc, Ki-67, MMP-2 and VEGF expression on prognosis of hepatocellular carcinoma patients undergoing tumor resection. World J Gastroenterol. 2004;10:1533–1536
6. De Boer WB, Segal A, Frost FA, Sterrett GF. Can CD34 discriminate between benign and malignant hepatocytic lesions in fine needle aspirates and thin core biopsies? Cancer. 2000;90:273–278
7. El-Assal ON, Yamanoi A, Soda Y, Yamaguchi M, Igarashi M, Ymamoto A, et al. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology. 1998;27:1554–1562
8. Facchini LM, Penn LZ. The molecular role of Myc in growth and transformation: recent discoveries lead to new insights. FASEB J. 1998;12:633–651
9. Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delai D, et al. Expression of the CD34 gene in vascular endothelial cells. Blood. 1990;75:2417–2426
10. Frachon S, Gouysse G, Dumortier J, Couvelard A, Nejjari M, Mion F, et al. Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study. J Hepatol. 2001;34:850–857
11. Gorrin-Rivas MJ, Arii S, Mori A, Takeda Y, Mizumoto M, , Furutani M, et al. Implication of human macrophage metalloelastase and vascular endothelial growth factor gene expression in angiogenesis of hepatocellular carcinoma. Ann Surg. 2000;231:67–73
12. Gottschalk-Sabag S, Ron N, Glick T. Use of CD34 and factor VIII to diagnose hepatocellular carcinoma on fine needle aspirates. Acta Cytol. 1998;42:691–699
13. Hytiroglou P, Theise ND, Schwartz M, Mor E, Miller C, Thung SN, et al. Macroregenerative nodules in a series of adult cirrhotic liver explants: issues of classification and nomenclature. Hepatology. 1995;21:703–708
14. International Working Party. . Terminology of nodular hepatocellular lesion. Hepatology. 1995;22:983–993
15. Kimura H, Nakajima T, Kagawa K, Deguchi T, Kakusui M, Katagishi T, et al. Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry. Liver. 1998;18:14–19
16. Lu JP, Wang J, Wang T, Wang Y, Wu WQ, Gao L, et al. Microvessel density of malignant and benign hepatic lesions and MRI evaluation. World J Gastroenterol. 2004;10:1730
17. Macchiarini P, Fontanini G, Hardin MJ, Squartini F, Angeletti CA. Relation of neovascularization to metastasis of non-small cell lung cancer. Lancet. 1992;340:145–148
18. Maeda K, Chung YS, Takatsuka S, Ogawa Y, Sawada T, Yamashita Y, et al. Tumor angiogenesis as a predictor of recurrence in gastric carcinoma. J Clin Oncol. 1995;13:477–481
19. Nakashima Y, Nakashima O, Hsia CC, Kojiro M, Tabor E. Vascularization of small hepatocellular carcinomas: correlation with differentiation. Liver. 1999;19:12–18
20. Ohmori S, Shiraki K, Sugimoto K, Sakai T, Fujikawa K, Wagayama H, et al. High expression of CD34 positive sinusoidal endothelial cells is a risk factor for hepatocellular carcinoma in patients with HCV-associated chronic liver diseases. Hum Pathol. 2001;32:1363–1370
21. Park YN, Kim YB, Yang KM, Park C. Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch Pathol Lab Med. 2000;124:1061–1065
22. Poon RT, Lau CP, Ho JW, Yu WC, Fan ST, Wong J, et al. Tissue factor expression correlates with tumor angiogenesis and invasiveness in human hepatocellular carcinoma. Clin Cancer Res. 2003;9:5339–5345
23. Pusztaszeri MP, Seelentag W, Bosman FT. Immunohistochemical expression of endothelial markers CD31, CD34, Von willebrand factor, and Fli-1 in normal human tissue. J Histochem Cytochem. 2006;54:385–395
24. Ravi R, Mookerjee B, Bhujwalla ZM, Sutter CH, Artemov D, Zeng Q. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor alpha. Genes Dev. 2000;14:34–44
25. Roncalli M, Roze E, Coggi G, Di Rocco MG, Bossi P, Minola E, et al. The vascular profile of regenerative and dysplastic nodules of the cirrhotic liver: implication for diagnosis and classification. Hepatology. 1999;30:1174–1178
26. Sakamoto M, Hirohashi S, Shimosato Y. Early stages of multistep hepatogenesis: adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol. 1991;22:172–178
27. Sierra A, Castellsague X, Escobedo A, Moreno A, Drudis T. Synergistic cooperation between c-myc and Bcl-2 in lymph node progression of T1 human breast carcinomas. Breast Cancer Res. 1999;54:39–45
28. Sillman F, Boyce J, Fruchter R. The significance of atypical vessels and neovascularization in cervical neoplasia. Am J Obst Gynecol. 1981;139:154–159
29. Sobin LH, Wittekind C TNM classification of malignant tumors. 1997.5th ed New York Wiley-Liss
30. Srivastava A, Laidler P, Hughes LE, Woodcock J. Neovascularization in human cutaneous melanoma: a quantitative morphological and Doppler ultrasound study. Eur J Cancer Clin Oncol. 1986;22:1205–1209
31. Sun XF, Zhang H. Clinicopathological significance of stromal variables: angiogenesis, lympangiogenesis, inflammatory infiltration, MMP and PINCH in colorectal carcinomas. Mol Cancer. 2006;5:43
32. Takahashi Y, Kawate S, Watanabe M, Fukushima J, Mori S, Fukusato T, et al. Amplification of c-myc and cyclin D1 genes in primary and metastatic carcinomas of the liver. Pathology International. 2007;57:437–442
33. Taku K, Yoko T, Takahash I, Miyuki T, Tomoko S, Toshitaka U, et al. Immunocytochemical localization of CD34 (endothelial cell marker) in liver disease: focus diagnosis of hepatocellular carcinoma. J Jap Soc Clin Cytol. 2001;40:571–574 No. 6.
34. Thompson BE. The many roles of Myc in apoptosis. Ann Rev. 1998;60:575–600
35. Watson PH, Singh R, Hole AK. Influence of c-myc on the progression of human breast cancer. Curr Top Microbiol Immunol. 1996;213:267
36. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis. Correlation in invasive breast carcinoma. New Engl J Med. 1991;324:1–8
37. Yamamoto T, Hirohashi K, Ikebe T, Kaneda K, Mikami S, Uenishi T, et al. Relationship of the microvascular type to the tumor size, arterialization and dedifferentiation of human hepatocellular carcinoma. Jap J Cancer Res. 2001;92:1207–1213
38. Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Hicklin DJ, et al. Synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in murine hepatocellular carcinoma. Hepatology. 2002;35:834–842
39. Yongqiang Yu, Weidong Dong, Xin Li, Enju Yu, Xiaorong Zhou, Shuhua Li, et al. Significance of c-Myc and Bcl-2 Protein Expression in Nasopharyngeal Carcinoma. Arch Otolaryngol Head Neck Surg. 2003;129:1322–1326
40. Yu Y, Dong W, Li X, Yu E, Zhou X, Li S, et al. Significance of c-Myc and Bcl-2 protein expression in nasopharyngeal carcinoma. Arch Otolaryngol Head Neck Surg. 2003;129:1322–1326