PCNA-associated factor (KIAA0101/PCLAF) overexpression and gene copy number alterations in hepatocellular carcinoma tissues.

BACKGROUND: PCNA-associated factor, the protein encoded by the KIAA0101/PCLAF gene, is a cell-cycle regulated oncoprotein that regulates DNA synthesis, maintenance of DNA methylation, and DNA-damage bypass, through the interaction with the human sliding clamp PCNA. KIAA0101/PCLAF is overexpressed in various cancers, including hepatocellular carcinoma (HCC). However, it remains unknown whether KIAA0101/PCLAF overexpression is coupled to gene amplification in HCC. METHODS: KIAA0101/PCLAF mRNA expression levels were assessed by quantitative real-time PCR (qRT-PCR) in 40 pairs of snap-frozen HCC and matched-non-cancerous tissues. KIAA0101/PCLAF gene copy numbers were evaluated by droplet digital PCR (ddPCR) in 36 pairs of the tissues, and protein expression was detected by immunohistochemistry (IHC) in 81 pairs of formalin-fixed paraffin-embedded (FFPE) tissues. The KIAA0101/PCLAF gene copy number alteration and RNA expression was compared by Spearman correlation. The relationships between KIAA0101 protein expression and other clinicopathological parameters, including Ki-67, p53, and HBsAg protein expression in HCC tissues, were evaluated using Chi-square test. RESULTS: Our results demonstrated that KIAA0101/PCLAF mRNA levels were significantly higher in HCC than in the matched-non-cancerous tissues (p < 0.0001). The high KIAA0101/PCLAF mRNA levels in HCC were associated with poor patient survival. The KIAA0101/PCLAF gene was not amplified in HCC, and KIAA0101/PCLAF gene copy numbers were not associated with KIAA0101/PCLAF transcript levels. KIAA0101 protein was overexpressed in the majority of HCC tissues (77.8%) but was not detectable in matched-non-cancerous tissues. Significant correlations between the expression of KIAA0101 protein in HCC tissues and p53 tumor suppressor protein (p = 0.002) and Ki-67 proliferation marker protein (p = 0.017) were found. However, KIAA0101 protein levels in HCC tissues were not correlated with patient age, tumor size, serum AFP level, or the HBsAg expression. CONCLUSIONS: KIAA0101/PCLAF mRNA and protein overexpression is frequently observed in HCC but without concurrent KIAA0101/PCLAF gene amplification. Significant correlations between the expression of KIAA0101 protein and p53 and Ki-67 proteins were observed in this study. Thus, detection of KIAA0101/PCLAF mRNA/protein might be used, along with the detection of p53 and Ki-67 proteins, as potential biomarkers to select candidate patients for further studies of novel HCC treatment related to these targets.

HCC-derived EGFR mutants are functioning, EGF-dependent, and erlotinib-resistant.

BACKGROUND: Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target. Overexpression of EGFR is frequently observed in hepatocellular carcinoma (HCC) and EGFR activation has been proven to be a potential determinant of primary resistance of HCC cells to sorafenib. In our previous study, we found 13 missense mutations in EGFR exon 19-23 from hepatocellular carcinoma (HCC) tissues, but the functions of these mutations have not been determined. This study aims to determine the kinase activity and sensitivity to erlotinib, a 1st-generation EGFR-tyrosine kinase inhibitor (TKI), of seven HCC-derived mutants (K757E, N808S, R831C, V897A, P937L, T940A, and M947T). RESULTS: Using transduction of pBabe-puro retroviral vector with or without EGFR, we constructed and determined the function of EGFRs in NIH-3T3 cells stably harboring each of the seven mutants, as well as the erlotinib-sensitive L858R-mutant, the erlotinib-resistant T790M-mutant, and EGFR wild type (WT). Our results indicate that the seven mutants are functioning, EGF-dependent, EGFRs. Cells harboring six of the seven mutants could generate some level of EGFR phosphorylation in the absence of EGF, indicating some constitutive kinase activity, but all of the seven mutants remain primarily EGF-dependent. Our results demonstrate that erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs: complete apoptosis and autophagy (cleavage of both caspase-3 and PARP, and marked LC3-II increment) in L858R-mutant; partial apoptosis and autophagy (only cleavage of caspase-3, and moderate LC3-II increment) in WT and HCC-derived mutants; and no apoptosis and minimal autophagy (no cleavage of caspase-3 and PARP, and minimal LC3-II increment) in T790M-mutant. The seven HCC-derived mutants are erlotinib-resistant, as treatment with erlotinib up to high concentration could only induce partial inhibition of EGFR phosphorylation, partial or no inhibition of AKT and ERK phosphorylation, and partial apoptosis and autophagy. CONCLUSION: The seven HCC-derived EGFR mutants in this study are functioning, EGF-dependent, and erlotinib-resistant. Erlotinib induces differential degree of apoptosis and autophagy among cells harboring different EGFRs. The degree of inhibition of EGFR phosphorylation by erlotinib is the determining factor for the degree of apoptosis and autophagy amongst cells harboring EGFR mutants. This study paves the way for further investigation into the sensitivity of these HCC-derived mutants to the 3rd-generation irreversible EGFR-TKI, osimertinib.

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues.

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa = 0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3-17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6-3.6, p < 0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.

Reduced HBV cccDNA and HBsAg in HBV-associated hepatocellular carcinoma tissues.

Approximately 50% of hepatocellular carcinoma (HCC) is attributable to chronic infection with hepatitis B virus (HBV). Serum hepatitis B surface antigen (HBsAg) is an important diagnostic marker of HBV infection, whereas intrahepatic HBV covalently closed circular DNA (cccDNA) is a surrogate marker of HBV persistence. This study aimed to investigate relationships between serum HBsAg, intrahepatic HBsAg, and intrahepatic cccDNA in HBV-associated HCC. Intrahepatic HBsAg was determined by immunohistochemistry in matched non-cancerous and HCC tissues from 88 patients; 56 patients (63.64%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic HBsAg was positive staining in 73.2% of non-cancerous tissues, but only in 10.7% of HCC tissues. Significant correlation between serum HBsAg and intrahepatic HBsAg was observed in non-cancerous tissues (p < 0.001), but not in HCC tissues (p = 0.415). Absolute quantification of intrahepatic cccDNA was performed by droplet digital PCR in tissues from 30 patients; 18 patients (60%) were serum HBsAg positive. In serum HBsAg-positive group, intrahepatic cccDNA was detected in 66.66% of non-cancerous tissues, but only in 5.55% of HCC tissue; intrahepatic cccDNA levels in non-cancerous tissues were significantly higher than those in HCC tissues (p < 0.001), and correlated with serum HBsAg (p < 0.01). Significant correlations between intrahepatic HBsAg and intrahepatic cccDNA were found in both non-cancerous tissues (p < 0.01) and HCC tissues (p < 0.05). We concluded that HBV cccDNA and intrahepatic HBsAg in HBV-associated HCC tissues were significantly reduced, as compared with matched non-cancerous tissues. This warrants further investigation into the impacts and the cause(s) of cccDNA reduction in HBV-associated HCC tissues, which might yield novel immune-related therapy for HBV-associated HCC.

Missense Mutations in Exons 18-24 of EGFR in Hepatocellular Carcinoma Tissues.

Epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase receptor, plays important roles in various cancers. In nonsmall cell lung cancer (NSCLC), EGFR mutations cluster around the ATP-binding pocket (exons 18-21) and some of these mutations activate the kinase and induce an increased sensitivity to EGFR-tyrosine kinase inhibitors. Nevertheless, data of EGFR mutations in HCC are limited. In this study, we investigated EGFR expression by immunohistochemistry and EGFR mutations (exons 18-24) by PCR cloning and sequencing. EGFR overexpression in HCC and matched nontumor tissues were detected in 13/40 (32.5%) and 10/35 (28.6%), respectively. Moreover, missense and silent mutations were detected in 13/33 (39.4%) and 11/33 (33.3%) of HCC tissues, respectively. The thirteen different missense mutations were p.L730P, p.V742I, p.K757E, p.I780T, p.N808S, p.R831C, p.V851A, p.V897A, p.S912P, p.P937L, p.T940A, p.M947V, and p.M947T. We also found already known SNP, p.Q787Q (CAG>CAA), in 13/33 (39.4%) of HCC tissues. However, no significant association was detected between EGFR mutations and EGFR overexpression, tissue, age, sex, tumor size, AFP, HBsAg, TP53, and Ki-67. Further investigation is warranted to validate the frequency and activity of these missense mutations, as well as their roles in HCC tumorigenesis and in EGFR-targeted therapy.

TOP2A amplification and overexpression in hepatocellular carcinoma tissues.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death in men worldwide owing to limited insights into pathogenesis and unsatisfactory efficacy of current therapies. HER2 and TOP2A genes are coamplified in breast and some other cancers. In this study, we investigated gene aberrations of HER2 and TOP2A and protein expressions of HER2, TOP2A, Ki-67, and p53 in tumor and matched nontumor tissues, as well as their associations with clinicopathological features. Gene aberrations were evaluated by FISH and protein expressions by IHC. Neither HER2 overexpression nor HER2 gene amplification was observed in both tumor tissues and matched nontumor tissues. By contrast, TOP2A overexpression was detected in 72.5% of tumor tissues but not detected in matched nontumor tissues. However, TOP2A gene amplification was not observed in both tumor and matched nontumor tissues. TOP2A overexpression was significantly associated with HCC tumor tissues (P < 0.001), hepatitis B surface antigen (HBsAg) in the serum (P = 0.004), and Ki-67 (P = 0.038) but not with age, tumor size, alpha-fetoprotein, TP53, and copy number of TOP2A gene and chromosome 17 centromere. In conclusion, TOP2A overexpression in HCC was not secondary to gene amplification. In addition, neither HER2 amplification nor overexpression could be used as prognostic and predictive marker in HCC.

HER2 expression in breast cancer with nonamplified HER2 and gains of chromosome 17 centromere.

Gains of chromosome 17 centromere (CEP17) may be accompanied by gains of chromosome 17q. To evaluate the effect of CEP17 gains (CEP17>3 copies per tumor nucleus) on the expression of the HER2 gene, which is located on chromosome 17q12-21.32, we analyzed HER2 amplification and expression in breast carcinomas with and without CEP17 gains. We isolated tumor nuclei from frozen tissues of 37 breast carcinomas for analysis of the HER2 gene and CEP17 by fluorescence in situ hybridization. HER2 expression was detected by immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded sections of the corresponding tumors. Tumors with amplified HER2 as determined by both HER2 copy number and HER2/CEP17 ratio were detected in 29.7% (11/37). CEP17 gains were significantly associated with HER2 amplification (P=0.005) but not associated with estrogen receptor status, tumor grade, and lymph node status (P>0.05). In contrast, HER2 amplification was significantly associated with estrogen receptor negativity (P=0.020) but not with tumor grade and lymph node status (P>0.05). IHC analysis was performed in 7 HER2-amplified tumors and all of these were IHC 3+, which were used as positive controls. Among HER2-non-amplified tumors with CEP17 gains, only 1 tumor (1/8, 12.5%) was IHC 3+. However, none of the HER2-non-amplified tumors without CEP17 gains was IHC 3+. In HER2-non-amplified tumors, there was no significant association between HER2 protein expression as detected by IHC and CEP17 or HER2 copy number (P=0.999, P=0.785, respectively). These findings indicate that in the absence of HER2 amplification, CEP17 gains do not have a significant effect on HER2 protein expression.

Fluorescence in situ hybridization method in isolated single nuclei extracted from paraffin-embedded hepatocellular carcinoma tissues.

BACKGROUND: Genetic analysis using the fluorescence in situ hybridization (FISH) method applied to intact tissue sections of formalin-fixed paraffin embedded (FFPE) tissue is well known to be relatively difficult. The frequent technical problems include unsuccessful hybridization as a result of poor probe penetration, excessive probe requirement, excessive background, auto-fluorescence, and overlapping or incomplete nuclei. These problems lead to absence or insufficiency of fluorescent signals, resulting in an inaccurate analysis. Formalin-fixed paraffin embedded tissue can be analyzed either as intact tissue sections or as a suspension of disaggregated, but intact, nuclei. Intact tissue sections have the advantage of preserved tissue architecture and morphology but have the intrinsic disadvantage of poor probe penetration, overlapping or incomplete nuclei and auto-fluorescence, accordingly reducing the accuracy of fluorescent signals evaluation. OBJECTIVE: To present the effective FISH method applied to isolated of single nuclei and the procedures for isolation of a single nuclei from formalin-fixed paraffin embedded tissues of hepatocellular carcinoma. MATERIAL AND METHOD: Ten paraffin-embedded blocks of hepatocellular carcinoma tissues from the department of pathology, Ramathibodi hospital, Thailand were studied. Isolated single nuclei were extracted from 10-microm sections of paraffin-embedded blocks of hepatocellular carcinoma tissue and hybridized with alpha-satellite centromeric DNA enumeration probes for chromosomes X (CEP X, spectrum green) and satellite III for chromosomes Y (CEP Y spectrum orange). The signal of at least, 200 interphase nuclei were counted from each specimen. RESULTS: The efficacy of this method has been evaluated in 10 formalin-fixed paraffin embedded tissue of hepatocellular carcinoma. The results showed bright, planar and an easy to score signal. CONCLUSION: FISH procedure described here is particularly suitable for retrospective studies of genetic aberration applied to formalin-fixed paraffin embedded tissues.