Cancer Genetics and Cytogenetics 203 (2010) 269e277
Correlation between clinical characteristics, survival and genetic alterations in patients with hepatocellular carcinoma from Saudi Arabia Ahmed Al-Qahtania, Tahani Al-Hazzanib, Turki Al-hussainc, Abdulmonem Al-Ghamdic, Hadeel Al-Manac, Saud Al-Arifid, Mohammed Al-Ahdala, Magdy Alye,f,* a
Biological and Medical Research (MBC 03), King Faisal Specialist Hospital and Research Centre, Box 3344 (MBC-03), Riyadh 11211, Saudi Arabia b Scientific Section, Department of Biology, University of Princess Nora Bent Abdul Rahman, P.O. Box 84428, Riyadh 11671, Saudi Arabia c Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, P.O. Box 3544 (MBC-03), Riyadh 11211, Saudi Arabia d Zoology Department, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia e Biology Department, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia f Zoology Department, Beni-Suef University, P.O. Box 169 Imbaba, Beni-Suef 62511, Egypt Received 16 June 2010; received in revised form 30 July 2010; accepted 8 August 2010
Abstract
Amplification of the two oncogenes ERBB2 and MYC and deletion of the tumor suppressor gene TP53 are frequently encountered in cancerous tissues. The purpose of this study was to use the fluorescence in situ hybridization (FISH) technique for the assessment of ERBB2 and MYC amplification and TP53 deletion, and to relate these molecular markers to clinical and pathologic factors in Saudi patients with hepatocellular carcinoma. The study was conducted on 40 paraffin-embedded tissue samples originally taken from either hepatitis C virus (HCV)- or HBV-infected patients using the FISH technique. The level of ERBB2, MYC, and TP53 in the malignant group was significantly increased as compared to the control group. Of the 40 patients, 3 (7.5%) had amplification of ERBB2 gene, 4 (10%) different patients had amplification of MYC, and 26 patients (65%) had evidence of deletion of at least one allele on chromosome 17 for the TP53 gene in a high proportion of cells. There was a significant correlation between amplification of MYC oncogene and the number of tumor masses. Moreover, significant correlation was observed between poorly differentiated tumors when compared with moderate or well-differentiated tumors when MYC was analyzed. On the other hand, MYC failed to reveal any significant association between oncogene amplification and other clinicopathologic variables examined. Univariate analysis revealed a strong association between deletion of TP53 and multiple tumor mass (P! 0.001). No statistical correlation could be detected between deletion of TP53 and tumor size, grade, stage, and tumor differentiation. No significant difference could be detected in the mean survival time of patients positive for the alteration of the genes compared to the patients who showed no alterations for the same genes. However, when the stage of the tumor was analyzed, there was a significant difference in the mean survival time between patients who showed gene alterations compared to patients with no changes in the studied genes. When overall survival was analyzed, only patients with MYC amplification had a lower median survival (20.75 months) than patients without MYC amplification (35.82, P50.009). Genetic alterations of ERBB2 and TP53 genes had no effect on survival 2 (see Results). The combination of ERBB2, MYC, and TP53 could be useful markers to stratify patients into different risk groups. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide. At present, approximately 600,000 new patients are diagnosed with HCC each year worldwide. HCC is reported to be the fifth most common cancer, and its mortality is third preceded by lung * Corresponding author. Tel.: þ20111300344; fax: þ208222334551. E-mail address:
[email protected] (M. Aly). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.08.011
and colon cancer [1]. However, regional differences in the incidence of HCC are significant. The highest prevalence is found in Southeast Asia and sub-saharan Africa, mostly due to the high rates of chronic viral hepatitis, a high-risk factor for HCC [2]. However, little is known about the molecular pathogenesis of HCC. In fact, the majority of HCC are associated with a background of chronic liver diseases. Therefore, development of HCC is hypothesized to involve multiple genetic changes over a long period of time. Numerous alterations of the genome, including either
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amplification of oncogenes such as ERBB2 and MYC or deletion of tumor suppressor genes such as TP53, could result in the development of malignant tumors. Infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) contributes significantly to the epidemiology and the incidence of HCC. The great majority (nearly 80%) of HCC cases worldwide are associated with chronic infections with these two viruses [3]. Evidence was first established through epidemiologic studies, then substantiated through genetic techniques, where a large number of studies linked HBV and HCV to the development of HCC [4]. For example, transfection of cells with the X gene of HBV has been shown to disrupt the control of several cellular and molecular processes, such as proliferation, apoptosis, or cell cycle. The disruption of these important pathways might be through modulation of the function of several genes, including MYC, TP53, FOS, NFKB, and many others. Therefore, the X gene is hypothesized to play an important role in the generation of genetic and cellular events that lead to the development of HCC associated with HBV [5]. Also, several proteins encoded by HCV, such as the core proteins NS5A and NS5B, are hypothesized to cause impairments of cell signaling pathways involved in transformation of hepatocytes [6]. Several studies on tissue samples taken from virally infected HCC patients have shown allelic losses on several chromosomes, including 1, 2q, 4, 5q, 6q, 8, 9, 10q, 11p, 13q, 14q, 16, 17, and 22q [7]. On the other hand, chromosomal gains were observed in 1q21, 11q13, 1q, 2q21, 5, 7q31, 8q, and many others [8]. Such chromosomal aberrations could contribute to the carcinogenesis of liver cells. The MYC oncogene is located on chromosome 8q and encodes a 67-kD transcription factor protein that plays an important role in different cellular activities including growth, differentiation, and apoptosis. Genetic deregulation of the MYC gene and overexpression of MYC protein are evident in different types of tumors [9]. In HCC, amplification of MYC was shown to correlate well with larger size, metastasized, and recurring type more significantly than primary tumors [10]. The ERBB2 oncogene is a member of the human epidermal growth receptor family. The gene encodes a protein with extracellular and intracellular domains, and the latter possesses a tyrosine kinase activity involved in signal transduction of cell growth and development. Amplification of the ERBB2 gene has been implicated in the pathogenesis and aggressive progression of several types of tumors [11]. The human TP53 gene encodes a 53-kD nuclear phosphoprotein consisting of 393 amino acids. The wild-type TP53 protein is largely responsible for blocking the entry of cells into S phases, allowing cells to repair any damage in DNA before duplicating the genome during the cell cycle. TP53 can also induce apoptosis when DNA repair fails. Mutations in TP53 have been found in nearly all types of cancer. Some of these mutations not only allow
propagation of genetically damaged cells, but also could result in inhibition of apoptosis [12]. Fluorescence in situ hybridization (FISH) is a powerful technology that utilizes centromeric, whole-chromosome, and unique sequence DNA probes to cytogenetically characterize cell samples [13]. By combining a fluorescence-labeled centromeric probe with a different color fluorescence-labeled DNA probe specifically directed against a known gene on the chromosome, these characteristics of a sample can be quantified. Normal gene copy number, gene amplification, or gene deletion is determined by the number of DNA probe signals identified adjacent to the centromere. Therefore, the purpose of this study was to determine the cellular copy number of three recognized tumor-related genes (ERBB2, MYC, and TP53) by FISH in a group of Saudi patients with viral hepatitiseassociated hepatocellular carcinoma. Furthermore, the results were compared with clinicopathologic factors, and the correlation between disease-free and overall survival was examined. To our knowledge, this is the first report to demonstrate the existence of genetic alterations in Saudi patients with HCC using the FISH technique.
2. Materials and methods 2.1. Patients Parafinized HCC tissue samples from patients (n5 40) infected with either HCV or HBV were obtained from the archived collection at the Pathology Department at King Faisal Specialist Hospital & Research Center (KFSHRC, Riyadh, Saudi Arabia). No preoperative radiation therapy or chemotherapy was administered to patients before liver biopsy. The age of the patients ranged from 36 to 83 years, and the group consisted of 25 males and 15 females. Tenmicron thick, formalin-fixed, paraffin-embedded sections of the tumor were examined in all 40 cases. As controls, paraffin-embedded liver tissue samples (n55) were obtained from the same collection described above. 2.2. FISH analysis Blocks of paraffin-embedded HCC tissue were cut at a thickness of 10 microns and placed on glass slides. One slide for each case was stained with hematoxylin & eosin stain, and the area containing the cancerous tissue was marked. Three slides were prepared for each case, and each slide was used for hybridization with a probe cocktail, one probe specific for the gene under investigation and the other is specific for the chromosome where the gene is located. For example, the ERBB2 probe cocktail consists of ERBB2 probe (SpectrumOrange) and chromosome 17 centromere-specific probe (SpectrumGreen). All probe cocktails for ERBB2, MYC, and TP53 genes were purchased from Abbott Molecular (Des Plaines, IL). Unstained slides from the same block were deparaffinized in xylene twice for 10 minutes, denatured in
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70% formamide at 70 C for 3 minutes, and dehydrated in a chilled alcohol series of 70, 80, 90, and 100% for 2 minutes each. Ten microliters of the denatured probe was placed on each slide, covered with a glass coverslip, and sealed with rubber cement. Hybridization was performed overnight at 37 C. Slides were washed twice in 50% formamide at 47 C for 2 minutes and then twice in 2 standard saline citrate at room temperature for 2 minutes. The slides were scanned using a 90i Nikon fluorescent microscope equipped with a 100-watt mercury lamp and triple bandpass filter unit (Chroma Technology, Brattleboro, VT) with DAPI as a counterstain at a magnification of 1,000. Only intact, nonoverlapping nuclei were evaluated; positive signals were required to be bright and of approximately equal intensity among the nuclei. Genes were considered amplified if they showed a gene/centromere ratio of more than 2.2 after counting at least 100 nuclei. Ratio under 2.0 is considered unamplified [14,15]. For TP53, we also defined the FISH score as the percentage of cells for which the nuclei had lost at least one signal. The specificity of the probes and the validity of this method were checked by dual-color FISH using normal human male peripheral lymphocytes. 2.3. Statistical analysis
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Table 1 Clinicopathologic data of patients with hepatocellular carcinoma (HCC) Parameters
Number (%)
No. of cases Age (yr) Range Mean SD Median Sex Males Females Differentiation Poorly differentiated Moderately differentiated Well differentiated Tumor size O5 cm !5 cm Number of masses Single mass Multiple masses Tumor grades I II III IV Tumor stages II III IV
40 (100%) 36e83 60.22 9.88 60 25 (62.5 %) 15 (37.5 %) 4 (10 %) 15 (37.5 %) 21 (52.5 %) 26 (65 %) 14 (35 %) 19 (47.5 %) 21 (52.5 %) 8 25 4 3
(20 %) (62.5 %) (10 %) (7.5 %)
2 (5 %) 7 (17.5 %) 31 (77.5 %)
The data were expressed as mean G SD. They were analyzed statistically by the unpaired t-test or one-way analysis of variance followed by Duncan’s test to estimate the effect of the tumor grades, stages, and differentiation on the expression of the studied genes. The correlations among the studied factors and parameters were computed by Pearson’s product moment correlation coefficient. Survival analysis was performed by the Kaplan-Meier method. The full statistical analyses were executed by SPSS 15.0 software.
patients recurred in this time period, and three patients died. Estimated 2-year overall survival was analyzed by 94%. Estimated 2-year disease-free survival was 84%.
3. Results
FISH studies were successful in all cases studied. Genetic alterations in ERBB2, MYC, and TP53 were statistically significant over that of the control, as shown in Table 2. FISH analysis found 35/40 patients (87.5%) to be disomic, 4 (10%) monosomic, and 1 (2.5%) polysomic
To determine the efficiency of in situ hybridization, normal liver sections from five individuals were hybridized with the three probe cocktails. In most of the cells, two orange signals for the single-copy probe (MYC, ERBB2, and TP53), and two green signals for chromosome 17 or 8 were observed. The major characteristics and clinicopathologic data of the patients are summarized in Table 1. The median age was 60 years (range: 36e83). Twenty cases had virus B and 20 had virus C. There were 4 patients with poorly differentiated tumors and 15 with moderately differentiated tumors, whereas 21 cases were well differentiated. A total of 26 cases had tumor size more than 5 cm, and 14 had tumor size 5 cm or greater. Stage II disease represented 5% of the total, 7% were stage III, and 77.5% were stage IV. Grade I tumors represented 20%, grade 2 represented 62.5%, grade 3 were 4%, and grade 4 were 7.5%. The median follow-up time was 23 months (range: 14e32). Five
3.1. Genetic alterations of ERBB2, MYC, and TP53 in control and malignant group
Table 2 ERBB2, MYC, and TP53 alterations in patients with HCC and control
ERBB2 Mean SD Median Range MYC Mean SD Median Range TP53 alterations Positive Negative
HCC (n 5 40)
Control (n 5 5)
P
1.91 0.83 1.43 0.90e4.20
1.17 0.23 1.11 0.96e1.50
!0.001
1.80 0.77 1.35 0.95e3.53
1.28 0.30 1.22 0.98e1.66
!0.05
25 (62.5%) 15 (37.5%)
1 (20%) 4 (80%)
!0.01 !0.01
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Table 3 Chromosome 17 and 8 centromere copy number and ERBB2, MYC, and TP53 signals in HCC sections analyzed Case no.
Chromosome 17 nos. (O80%)
ERBB2 signal nos. (O80%)
TP53 signal nos. I
Chromosome 8 nos. (80%)
MYC signal nos. (O80%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1 2 1 2 2 2 1 3e8 2 1 or 0 2 2 2 2 2 2 2 2 2 2 2 2 2 4e8
2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1 2 1 2 2 2 4e8 2e8 2 1 or 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2e20
2 1 2 2 1 0 1 2 1 or 0 1 or 0 1 1 2 2 2 1 1 1 or 0 1 1 0 1 0 0e4 2 1 or 0 1 2 2 1 1 1 1 2 1 1 2 1 or 0 2 0
2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 1 2 1 2 2 2 2e6 2 2 2 2 2 2 2 2
4e15 2 2 2 2 2 2 2 2 8e15 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 4e15 2 1 2 2 2 4e15 2 2 2 2 2 2 2
for chromosome 8 in at least 80% of their cells (Table 3). A total of 33/40 cases (82.5%) were disomic, 5 (12.5%) monosomic, and 2 cells (5%) were polysomic for chromosome 17 in at least 80% of their cells. Of the 40 patients, 3 (7.5%) had amplification of the ERBB2 gene (Fig. 1), 4 (10%) were lacking one ERBB2 gene, and 33 (82.5%) had the normal two copies of the gene present in at least 80% of cells. Also, of the 40 patients, 4 (10%) different patients had amplification of the MYC probe signal, 2 (5%) had one MYC gene deleted, and 34 (85%) had two copies of the MYC gene in at least 80% of cells (Table 3). For the TP53 gene, 17 cases (42.5%) showed a single copy of the gene (Fig. 2), 9 cases (22.5) lacked both signals of the gene, while 14 cases (35%) had 2 normal copies of TP53 gene in at least 80% of cells (Table 3).
3.2. Correlation between the alterations of genes and the clinicopathologic characteristics Univariate analysis was used to correlate clinicopathologic characteristics of patients and genetic alterations. There was a significant correlation between amplification of the MYC oncogene and the number of tumor masses (Table 4). Moreover, significant correlation was observed between poorly differentiated tumors when compared with moderate or well-differentiated tumors when MYC was analyzed (Table 5). On the other hand, MYC failed to reveal any significant association between oncogene amplification and other clinicopathologic variables examined. With regard to the TP53 tumor suppressor gene, 26 patients (65%) had evidence of deletion of at least one allele on chromosome 17 in more than 80% of cells
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Table 4 Effect of the tumor size on the alterations of ERBB2 and MYC in patients with HCC
Fig. 1. Hybridization of HCC sections with probes specific to ERBB2 gene (orange) and chromosome 17 centromere. Two signals for chromosome 17 and three signals for the ERBB2 gene are evident.
examined. Univariate analysis revealed a strong association between deletion of TP53 and multiple tumor mass (P! 0.001; Fig. 3). No statistical correlation could be detected between deletion of TP53 and tumor size, grade, stage, and tumor differentiation. No significant difference in FISH scores was observed among gender of the patients or between HCC due to HBV and HCV infection (data not shown). 3.3. Survival analysis In the present study and according to the Kaplan-Meier method, we have divided the analysis of patients’ survival into the following two types: (1) survival of patients with the tumor (survival 1), and (2) the overall survival, which
Tumor size
ERBB2
O5cm (n 5 14) !5cm (n 5 26) P Single mass (n 5 19) Multiple masses (n 5 21) P
1.89 1.91 NS 1.70 2.09 NS
0.99 0.78 0.76 0.91
MYC 1.97 1.71 NS 1.51 2.07 0.019
0.90 0.69 0.61 0.81
includes the survival of patients with the tumor and after its removal (survival 2). As shown in Table 6, all the studied genes were compared with respect to their positivity or negativity for genetic alterations, no significant difference could be detected in the mean survival time (survival 1) of patients positive for the alteration of the genes compared to the patients who showed no alterations for the same genes. However, when the stage of the tumor was analyzed, there was a significant difference in the mean survival time (survival 1) between patients who showed gene alterations compared to patients with no changes in the studied genes (Table 6; Fig. 4) No significant differences were detected with respect to the gender, presence of viruses, tumor size, number of masses, tumor grade, and differentiation. When survival 2 was analyzed, only patients with MYC amplification had a lower median survival (20.75 months) than patients without MYC amplification (35.82; P5 0.009; Table 7). Genetic alterations of the ERBB2 and TP53 genes had no effect on survival 2.
4. Discussion HCC is one of the most malignant neoplasms of all tumors, and it shows a trend toward recurrence and metastasis after resection, and therefore the postoperative patients with HCC have a poorer prognosis [16,17]. FISH was used in this study to examine some of the molecular aberrations that take place and could contribute to the development of HCC in Saudi patients. Oncogene amplification forms the molecular genetic basis of oncoprotein over-expression in cancerous tissues. Table 5 Effect of differentiation on the alterations of ERBB2 and MYC in patients with HCC
Fig. 2. Fluorescence in situ hybridization (FISH) results (the signal for the TP53 gene is orange and those for the centromere of chromosome 17 are green).
Differentiation
ERBB2
MYC
Poorly differentiated (n 5 4) Moderately differentiated (n 5 15) Well differentiated (n 5 21) F ratio P
2.95 1.21 1.84 0.86 1.82 0.75 1.47 NS
3.18 0.56b 1.78 0.71a 1.55 0.56a 11.42 !0.001
a,b
Statistically significant different means.
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20
TP53 positive
18
Number of patients
TP53 negative 16
12
12 7 8
3 4 0 single mass
multiple masses
Tumor masses Fig. 3. Correlation between number of tumor masses and the alterations of TP53 in patients with HCC.
ERBB2, an oncogene located at chromosome 17q11.2~q12, was demonstrated to be amplified in various kinds of tumors, such as mammary carcinoma, ovarian carcinoma, and lung cancer. It is hypothesized that amplification of this gene is directly related to metastasis, recurrence, and prognosis of such tumors [18e21]. In this study and with fluorescencelabeled ERBB2 genomic DNA probes, we observed amplification of ERBB2 gene (17q11.2~q12) at a single nucleus level in HCC sections from Saudi patients. ERBB2 amplification exists in only 7.5% of HCC patients in this study. This is in contrast to the findings of Huang et al. [22], which detected ERBB2 amplification in 21.4% of HCC patients. Our results
Fig. 4. Correlation between survival with the tumor (survival 1) and the different stages of the tumor (P ! 0.05).
are similar to those observed in patients with cervical carcinoma and melanoma (5e20%). Data related to ERBB2 and HCC is currently limited to serology, immunohistochemistry, and CGH. Molina et al. [23] reported abnormal concentrations of ERBB2 protein antigen in 26.7% of HCC patients. Immunohistochemical analysis revealed that ERBB2 overexpression is as high as 92.3% in HCC [24] but no obvious
Table 6 Correlation between survival 1 and the clinicopathologic data 95% Confidence interval
ERBB2 MYC TP53 Gender Viruses Tumor size Tumor masses Stages
Grades
Differentiation
Negative Positive Negative Positive Negative Positive Males Females HCV HBV !5 cm O5 cm Single Multiple I II III I II III IV Poor Moderate Well
* P % 0.05 is considered significant.
Mean survival (mo)
SE
Lower bound
Upper bound
P
6.25 7.81 7.30 6.29 8 6.2 6.24 7.93 7.6 6.15 7 6.64 5 8.57 7.54 5.28 2 6.37 7.56 5 5 10.75 5.26 7.28
1.11 2.19 1.50 1.62 1.84 1.36 1.29 1.99 1.76 1.31 1.43 1.70 0.98 1.83 1.36 1.22 0 2.21 1.59 0.70 1.52 5.15 1.22 1.63
4.06 3.51 4.36 3.11 4.39 3.51 3.71 4.01 4.13 3.57 4.19 3.30 3.07 4.98 4.88 2.87 2 2.04 4.42 3.61 2.00 0.64 2.86 4.07
8.43 12.10 10.24 9.47 11.61 8.88 8.76 11.85 11.06 8.72 9.80 9.98 6.92 12.15 10.21 7.69 2 10.71 10.69 6.38 7.99 20.85 7.66 10.49
0.582 NS 0.515 NS 0.238 NS 0.243 NS 0.615 NS 0.714 NS 0.206 NS 0.023*
0.997 NS
0.703 NS
A. Al-Qahtani et al. / Cancer Genetics and Cytogenetics 203 (2010) 269e277 Table 7 Overall period of survival (survival 2) and the clinicopathologic data
ERBB2
Negative Positive MYC Negative Positive TP53 Negative Positive Gender Males Females Viruses HCV HBV Tumor size !5 cm O5 cm Tumor masses Single Multiple Stages I II III Grades I II III IV Differentiation Poor Moderate Well
95% confidence interval
Mean survival (mo)
SE
Lower bound
Upper bound
24.41 30.93 35.82 20.52 28.26 26.28 27 27.06 30.1 23.95 29.03 23.28 23.10 30.57 31 30.42 26 26.87 24.04 31.75 46 44 24 25.95
4.31 3.61 4.84 3.18 4.18 4.10 3.71 5.12 3.52 4.78 3.88 4.51 4.54 3.84 1 9.86 3.20 5.77 3.33 6.86 23.43 9.92 3.34 4.61
15.96 23.85 26.33 14.27 20.06 18.24 19.71 17.02 23.18 14.57 21.42 14.43 14.20 23.04 29.04 11.08 19.71 15.56 17.49 18.30 0.07 24.54 17.43 16.90
32.86 38.02 45.31 26.77 36.47 34.31 34.28 37.11 37.01 33.32 36.64 32.13 32.00 38.09 32.96 49.76 32.28 38.18 30.58 45.19 91.92 63.45 30.56 35.00
P 0.225 NS 0.009* 0.650 NS 0.678 NS 0.197 NS 0.464 NS 0.144 NS 0.870 NS 0.483 NS
0.221 NS
* Significant at P ! 0.01.
relationship was observed between ERBB2 expression and HCC recurrence or migration. As for comparative genomic hybridization (CGH) results, chromosome 17q amplification was observed in 30% of HCC samples [25]. However, no evidence could verify ERBB2 gene amplification at 17q11.2~q12. Heinze et al. [26] determined ERBB2 protein level in advanced HCC patients with the ELISA method. The 1- and 2-year survival rates were 20 and 10% in highprotein level patients, but 56 and 22% in low-protein level patients, respectively (P ! 0.05). Thus, ERBB2 protein expression was considered a valuable predictor for prognosis. Zekri et al. [27] found that HCC patients infected with HCV1a or HCV-4a have high ERBB2 expression, and suggested that this could contribute to HCC occurrence. ERBB2 was not associated with special clinical characteristics or survival time of patients. This is interesting to note, since the proliferative capacities of tumor cells with increased expression of ERBB2 are thought to determine aggressiveness of the tumor and to influence the patient’s survival [28]. However, our findings are in agreement with other reports that show lack of association among proliferation markers, clinical characteristics of the tumor, and survival of the patient [29]. The MYC oncogene was amplified in 10% of samples in the current study. Univariate analysis failed to reveal any significant association between oncogene amplification and the clinicopathologic variables examined, except with the number of masses and differentiation of the tumors.
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Amplification of MYC in patients with multiple tumor masses recorded significantly increased values compared to patients with a single mass. Poorly differentiated tumors were statistically significant over moderate or welldifferentiated tumors. The incidence of MYC amplification in HCC (33%) in the present study was lower than that found in previous reports (27e50%), as indicated by Southern blotting, polymerase chain reactionebased analysis, or FISH [30e32]. Amplification and overexpression of the MYC oncogene has been reported in various solid tumors, including HCC. Regarding tumor size, Aulmann et al. [33] examined MYC oncogene amplification using FISH in a series of 96 pure ductal carcinoma in situ (DCIS), and observed that MYC oncogene amplification was significantly associated with larger tumor size. They concluded that the MYC oncogene appears to be involved in the development of a more aggressive phenotype of DCIS. Shanmugham et al. [34] reported that MYC expression, assessed by immunohistochemical staining, showed a positive association with increasing grade. With regard to disease staging, Yang et al. [35] and Aulmann et al. [33] showed a significant association between MYC expression and amplification and advanced clinical stage of the disease. They implicated the role of MYC in tumor development and, consequently, disease progression. Furthermore, it has been shown that MYC amplification is a late event in the progression of HCC and it could be used as a prognostic indicator [36]. TP53 is a tumor suppressor gene that is located on chromosome 17p and has been widely studied in various types of cancers [37]. Deletion of 17p is common in various cancers, including breast [38] and colon cancer [39]. The frequent deletion of 17p may affect the tumor suppressor gene TP53 on 17p13.1. TP53 is frequently inactivated in various types of malignant tumors, including HCC [40]. However, Yumoto et al. [41] showed that loss of 17p occurred in 18/31 (58%) of HCC cases when analyzed by LOH, while the TP53 mutation was observed in only 8/31 (26%) of HCC cases. In another study, loss of one allele at 17p13.3 distal to the TP53 gene was observed in 48/94 (51%) HCC, whereas LOH at 17p13.1, near the TP53 gene, was detected in 30/94 (32%) cases, and TP53 mutation was detected in only 22/94 HCC (23%) [42]. In this study, deletion of the TP53 gene on chromosome 17 was the most significant finding. Twenty-six patients (65%) had evidence of deletion of at least one allele on chromosome 17 for the TP53 gene in a high proportion of cells. Univariate analysis revealed a strong association between deletion of TP53 and multiple tumor mass (P 5 0.028), tumor grade (P 5 0.001), tumor stage (P 5 0.003), and poorly differentiated tumors (P 5 0.048). Deletion of TP53 was negatively associated with survival and with degree of differentiation. Our findings are in consistence with several reports in other populations showing that positive or negative TP53 expression did not correlate with the survival of the patients with HCC [40,43,44].
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In conclusion, more studies should be done with larger databases of patients that would integrate clinical, pathologic, and molecular oncogene parameters. Availability of such data may provide clinicians prognostic information that could accurately predict the behavior and aggressiveness HCC. As FISH has been demonstrated to be an extremely helpful methodology, further FISH studies using more archival tissue samples to increase the sample size with more genes are underway.
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