Comparing the clinicopathological characteristics of combined hepatocellular-cholangiocarcinoma with those of other primary liver cancers by use of the updated World Health Organization classification.

AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is an uncommon hepatic malignancy with a poor outcome. The 2019 World Health Organization (WHO) classification modified the definition and discarded the subtypes with stem cell features. However, the differences among cHCC-CCA, hepatocellular carcinoma (HCC), HCC with stem cell/progenitor features (HCCscf) and intrahepatic cholangiocarcinoma (iCCA) remain undetermined. The aim of this study was to investigate the characteristics of cHCC-CCA in comparison with those of other primary liver cancers by utilising the updated WHO classification. METHODS AND RESULTS: We retrospectively analysed 64 cHCC-CCA patients and 55 HCCscf patients from December 2007 to May 2018. Propensity score matching was conducted to compare these with HCC and iCCA patients. Clinicopathological characteristics, event-free survival and overall survival were evaluated with multivariate Cox proportional hazard regression. During a median follow-up of 55.9 months, cHCC-CCA patients had significantly poorer survival than HCCscf patients, and survival intermediate between that of HCC patients and that of iCCA patients. Hepatitis B virus (HBV) infection and high levels of tumour-infiltrating lymphocytes (TILs) were associated with favourable survival in cHCC-CCA patients. In the multivariate analysis, poor hepatic reserve, absence of HBV infection, stage IV disease and low levels of TILs were significant negative prognostic factors in cHCC-CCA patients. After being pooled with other primary liver cancers, cHCC-CCA and iCCA resulted in the worse survival. CONCLUSIONS: cHCC-CCA patients have survival intermediate between that of HCC patients and iCCA patients, and HBV infection and high levels of TILs predict favourable survival. Our study provides clinical correlations for the new 2019 WHO classification.

Hepatitis B virus surface gene pre-S2 mutant as a high-risk serum marker for hepatoma recurrence after curative hepatic resection.

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).

NEK2 Promotes Hepatoma Metastasis and Serves as Biomarker for High Recurrence Risk after Hepatic Resection.

INTRODUCTION AND AIM: Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients. MATERIAL AND METHODS: The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence. RESULTS: NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2 level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients. CONCLUSIONS: NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.

A clustered ground-glass hepatocyte pattern represents a new prognostic marker for the recurrence of hepatocellular carcinoma after surgery.

BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after hepatectomy is a serious event. It has been demonstrated that different ground-glass hepatocyte (GGH) patterns harbor specific hepatitis B virus (HBV) pre-S deletion mutants and represent preneoplastic lesions in chronic HBV infection. In the current study, the authors investigated whether a specific GGH pattern in nontumorous liver tissues was associated with the recurrence of HBV-related HCC after surgery. METHODS: Clinicopathologic data from 82 patients with HBV-related HCC were reviewed. GGH patterns were assessed on hematoxylin and eosin-stained sections. Tissue hepatitis B surface antigen (HBsAg) expression was evaluated by immunohistochemical staining. Serum profiles of pre-S status, viral load, and HBV genotype were determined and correlated with clinical recurrence and survival after surgery. RESULTS: The results indicated that the clustered pattern of GGHs or HBsAg expression was associated significantly with decreased local recurrence-free survival (LRFS) during a mean follow-up of 46.4 months (P<.001). This biomarker was comparable to or better than the prognostic value of other parameters, such as multifocal tumors (P = .022), satellite nodules (P = .005), small cell dysplasia (P = .045), or elevated viral load (P = .027), to predict recurrent HCC. Multivariate analysis also revealed that type II GGHs, which expressed marginal HBsAg and consistently clustered in nodules, were independent variables associated with LRFS (P<.001) and overall survival (P = .003). CONCLUSIONS: The current results indicated that the assessment of GGH patterns or HBsAg expression in nontumorous liver tissues provides an easily recognized, new risk marker for the recurrence of HBV-related HCC after hepatic resection.

Progesterone receptor membrane component 1 as a potential prognostic biomarker for hepatocellular carcinoma.

AIM: To investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC). METHODS: We performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion. RESULTS: We found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects. CONCLUSION: PGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.

Prohibitin in the pathogenesis of transitional cell bladder cancer.

Our profiling experiment demonstrated that prohibitin 1 (PHB) was ubiquitously expressed in uroepithelial and urothelial carcinoma cell lines and exhibited a trend toward a positive relationship with tumor progression. The aim of this study was, therefore, to examine the potential role of PHB in multistage bladder carcinogenesis and predicting patient outcome. Immunohistochemical staining showed that PHB was overexpressed in 141 out of 167 cases (84.4%) of bladder cancer. This expression was positively related to met receptor overexpression (p = 0.04) and to multiple tumors (p = 0.05). Independent factors in predicting patient survival were multiple tumors (p = 0.002), muscle invasion (p = 0.003), and met overexpression (p = 0.05) in a multivariate analysis. Interestingly, patients with superficial bladder cancer overexpressing both PHB and met had a significantly lower recurrence-free survival rate than those not expressing PHB (p = 0.04). Taken together, our findings showed that PHB was activated at an early stage of carcinogenesis and that it may play a synergistic role with met in the progression of human bladder cancer. In addition, we demonstrated that genistein and justicidin A, natural chemoprevention agents, could suppress the expression of PHB in vitro. Thus, targeting PHB would be a useful approach for treating and preventing human bladder cancer.

The significance of prohibitin and c-Met/hepatocyte growth factor receptor in the progression of cervical adenocarcinoma.

To examine the importance of prohibitin 1 and c-Met/hepatocyte growth factor receptor (HGFR) expression in human cervical adenocarcinomas, 85 patients (69 with invasive adenocarcinoma [ACA] and 16 with adenocarcinoma in situ [AIS]) were studied using immunohistochemistry. High prohibitin 1 expression was found in 51 (73.9%) of the 69 ACAs and 11 (68.7%) of the 16 AIS lesions. Prohibitin 1 overexpression was significantly higher in ACA and AIS than in adjacent nonneoplastic glandular epithelium (P < .001 for both comparisons). Prohibitin 1 expression was also positively related to tumor size (P = .019) or parametrial involvement (P = .027) in ACA. c-Met was expressed in 21 ACAs (30.4%) and was positively correlated with the Fédération Internationale de Gynécologie et d'Obstétrique (International Federation of Gynecology and Obstetrics) stage classification (P = .007) or nodal metastasis (P = .047). Nodal metastasis (P = .028) and c-Met expression (P = .022) were independent predictors for the overall survival of patients in multivariate analysis using the Cox regression method. Prohibitin 1 activation seems to be an early event, whereas c-Met overexpression may be important for the progression of cervical adenocarcinomas. Evaluation of c-Met expression status may identify a subset of patients with cervical adenocarcinoma who require more intensive treatment.

Genetic polymorphisms in CYP1A1 and GSTM1 predispose humans to PCBs/PCDFs-induced skin lesions.

INTRODUCTION: Polychlorinated biphenyls (PCBs) and dibenzofurans (PCDFs) are ubiquitous persistent pollutants in humans. Whether people with different genotypes are with different susceptibility to these chemicals are unknown. In a group of people highly exposed to PCBs/PCDFs, we tested the hypothesis that genotypic polymorphisms affected susceptibility for development of skin manifestations. METHODS: In 1979, approximately 2000 people in central Taiwan ingested cooking oil contaminated with PCBs/PCDFs. Skin disorder such as chloracne, abnormal nail, hyperkeratosis and skin allergy were found in PCBs/PCDFs exposed group. We recruited exposed and community background exposure subjects for blood testing and telephone-interview. Single nucleotide polymorphisms, AhR Arg554Lys, CYP1A1 Ile462Val, CYP1A1 T6235C, and GSTM1/T1 deletion, were determined. Occurrence of skin manifestations was compared among people with different genotypes while stratified by PCB exposure levels by logistic regression. RESULTS: Data on exposure, medical history, and genotypes were obtained from 393 exposed and 181 background exposure groups. Skin manifestations including chloracne, allergy, abnormal nail, and hyperkeratosis were more prevalent in exposed people in a dose-related manner. Among highly exposed individuals, combined CYP1A1-MspI mutant genotype and GSTM1-null genotype were associated with increased risk of chloracne (odds ratio 2.8, 95% confidence interval 1.1-7.6). Among intermediately exposed individuals, GSTM1 null genotype was associated with skin allergy. AhR Arg554Lys genotype and GSTT1 null genotype were not related to susceptibility to skin manifestations in PCB/PCDF-exposed population. CONCLUSION: CYP1A1 and GSTM1 genotypic polymorphisms might be related to the susceptibility to PCB/PCDF-induced skin manifestations.

Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma.

Ground glass hepatocytes (GGHs) have been shown to predict the development of hepatocellular carcinoma (HCC). Type I GGH and type II GGH harbor hepatitis B virus (HBV) pre-S1 and pre-S2 deletion mutants, respectively. Whether anti-HBV therapy can inhibit the expression of GGHs and potentially reduce HCC development is explored in this study. Two sets of liver specimens were included: the first contained 31 paired biopsy specimens obtained from chronic HBV patients receiving oral nucleos(t)ide analogue (NA) treatment; the second contained 186 resected liver tissues obtained from HBV-related HCC patients receiving surgery: 82 received NA before surgery and 104 did not. Compared with the baseline biopsy specimens, type I (P=0.527) and type II GGH (P=0.077) were not significantly decreased after 48 weeks of NA treatment in the first set of patients. In the second set, despite suppression of viral load (P<0.001) and periportal necrosis (P=0.006) in treated patients, GGH (P=0.594), cccDNA (P=0.172) and serum pre-S mutants (p=0.401) were not significantly suppressed. A significant decrease of type I (P=0.049) and type II GGH (P=0.029) could only be observed in patients after long duration of treatment (median duration: 4.3 years). In the treated patients, the persisted type II GGH remained an independent variable associated with decreased local recurrence-free survival of HCC (P=0.019) as in non-treated patients (P=0.001). In conclusion, the persistence of GGHs could explain the residual risk of HCC development under anti-HBV treatment. Therefore, intrahepatic GGHs and pre-S mutant are potential additional targets for HCC prevention in patients already receiving anti-HBV treatment.

Overexpression of c-met as a prognostic indicator for transitional cell carcinoma of the urinary bladder: a comparison with p53 nuclear accumulation.

PURPOSE: The c-met proto-oncogene encodes a receptor tyrosine kinase (Met) and has been shown to play a role in oncogenesis. Given that high titers of hepatocyte growth factor, the specific ligand for Met, are excreted in the urine and tend to reflect disease activity of bladder cancer, we performed this study to examine the clinical significance of Met in human bladder cancer. MATERIALS AND METHODS: We studied the mRNA expression and genomic alteration of c-met in five bladder cancer cell lines. Significance of Met overexpression was then compared with p53 nuclear accumulation (TP53) in primary bladder cancer (n = 142 patients). RESULTS: Expression of c-met mRNA tended to positively correlate with differentiation of cancer cell lines in the absence of point mutation. High expression of Met was found in seven cases (4.9%), low expression in 32 cases (22.5%), and negative expression in 103 cases (72.5%). Expression of Met was positively associated with histologic grade, stage classification, tumor size, and nodular tumor growth (P <.05, respectively); however, it was not related to TP53 status. Factors that predicted disease progression were tumor stage, Met status, and TP53 accumulation (P <.05, respectively). Indicators for poor long-term survival were invasive cancer, multiple tumors, and Met overexpression (P =.0006,.01, and.04, respectively). CONCLUSION: The c-met proto-oncogene plays a more important role in the progression of bladder carcinogenesis than p53. Evaluation of Met expression could identify a subset of bladder cancer patients who may require a more intensive treatment strategy.

An ancova approach to normalize microarray data, and its performance to existing methods.

A microarray experiment includes many steps, and each one of them may include systematic variations. To have a sound analysis, the systematic bias must be identified and removed prior to the data being analyzed. Based on the M-A dependency observed by Dudoit et al. (2002), we suggest that, instead of using the lowess normalization, a new normalization method called ANCOVA be used for dealing with genes with replicates. Simulation studies have shown that the performance of the suggested ANCOVA method is superior to any of the available approaches with regards to the Fisher's Z score and concordance rate. We used a microarray data from bladder cancer to illustrate the application of our approach. The edge the ANCOVA method has over the existing normalization approaches is further confirmed through real-time PCR.

Lin28B is an oncofetal circulating cancer stem cell-like marker associated with recurrence of hepatocellular carcinoma.

By using an expressed sequence tag bioinformatic algorithm, we identified that Lin28 homolog B (Lin28B) may have an oncofetal expression pattern which may facilitate detecting cancer cells in adults. It is also reported to be a potential marker for cancer stem cells. Therefore, we sought to verify oncofetal-stemness characters of Lin28B and test its potential as a circulating cancer stem cell-like marker in adult HCC patients. Lin28B mRNA was examined in a panel of fetal tissue, adult tissue and tumors. Lin28B was over-expressed or knocked down in HepG2 cells to evaluate its potential as a stem cell-like marker. RT-qPCR for Lin28B was performed in the peripheral blood mononuclear cells from patients with HCC receiving surgery (n=96) and non-HCC controls (n=60) and analyzed its clinical significance. Lin28B showed an oncofetal expression pattern. Its overexpression could upregulate stemness markers (OCT4, Nanog and SOX2) and enhance tumorsphere formation in vitro. Lin28B knockdown had opposite effects. Circulating Lin28B was detected in peripheral blood mononuclear cells in 3 cases (5%) of non-HCC controls and 32 cases (33.3%) of HCC patients. In HCC patients, circulating Lin28B was associated with high tumor grade (P=0.046), large size (P=0.005), high AJCC stage (P=0.044) and BCLC stage (P=0.017). Circulating Lin28B was significantly associated with decreased recurrence-free survival (P<0.001). Circulating Lin28B separated early stage HCC into 2 recurrence-free survival curves (P=0.003). In multivariate analysis, circulating Lin28B was an independent variable associated with early recurrence (P=0.045) and recurrence in early stage HCC (P=0.006). In conclusion, the oncofetal gene Lin28B is a potential oncofetal cancer-stem-cell-like circulating tumor cell marker that correlates with HCC recurrence after hepatectomy. Circulating Lin28B could refine early AJCC stages. Our finding supports the possible use of a TNMC (C for circulating tumor cells) staging system in HCC.

Nasopharyngeal carcinoma-susceptibility locus is localized to a 132 kb segment containing HLA-A using high-resolution microsatellite mapping.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor uniquely prevalent in southern Chinese. HLA-A2 is associated with NPC. In a previous study, we showed that the genes associated with susceptibility to NPC are primarily located within the HLA-A locus in Taiwanese NPC patients. However, the pathogenic genes causing NPC susceptibility remain unknown. Here, 8 polymorphic microsatellite markers distributed over a 1 megabase region surrounding the HLA-A locus were subjected to genetic analysis for the NPC-susceptibility locus. Statistical studies of associated alleles detected on each microsatellite locus showed that the NPC- susceptibility genes are most likely located between the D6S510 and D6S211 markers within a 132 kb segment containing the HLA-A locus. These results undoubtedly would facilitate the further positional cloning of the NPC-susceptibility locus, which has been elusive for the past 30 years.

Genetic susceptibility to nasopharyngeal carcinoma within the HLA-A locus in Taiwanese.

NPC is an epithelial tumor that is highly prevalent among the southern Chinese. Numerous studies have indicated that specific HLA haplotypes and genes within the HLA complex are associated with NPC. As a first effort to localize the gene responsible for susceptibility, the HLA-A, -B, and -A2 subtypes were examined for their association to NPC. Consistent with previous reports, frequencies of HLA-A2 [OR = 2.50, pc = 0.020 (study population); OR = 3.73, pc = 0.0030 (> or =40 years old)] were significantly higher in patients with NPC than in healthy controls. Two-locus analysis indicated that A2(+)B46(+) individuals are at greater risk for NPC than A2(-)B46(-) individuals in both the population studied and the > or =40-year-old group. This, however, may be due to the close linkage of these 2 genes. Moreover, A2(+)B38(+) individuals were at higher risk than A2(-)B38(-) individuals in both the population studied and the > or =40-year-old group; A2 and B38 are not genetically linked. These findings suggest that B38 or B46 alone cannot confer a high risk of NPC but that, in conjunction with A2, B38 or B46 positivity greatly increases risk. None of 5 A2 subtypes identified from studied populations was significantly associated with NPC. Microsatellite marker D6S211, located 97 kb telomeric to HLA-A, was analyzed for its association with NPC. Allele 4 of D6S211 was significantly associated with NPC (OR = 3.97, pc = 0.0042). These results strongly support the hypothesis that genes associated with susceptibility to NPC in the HLA region are within the HLA-A locus.

Clinical significance of allelotype profiling for urothelial carcinoma.

OBJECTIVES: To perform a global loss of heterozygosity (LOH) analysis on a cohort of urothelial carcinoma to investigate the clinical implication of specific chromosomal loss. Allelic deletions detected as LOH have been used to study the markers for carcinogenesis. METHODS: We examined the allelic loss on 14 chromosomal regions in a total of 71 cases of urothelial carcinoma. The results were analyzed in relation to biologic indicators of urothelial carcinoma and the clinical outcome with a mean follow-up of 101 months. RESULTS: The incidence of LOH in order of frequency was 9p (54.9%), 9q (49.3%), 13q (40.8%), 14q (40.8%), 10q (39.4%), 17p (39.4%), 8p (38.0%), 21q (36.6%), 11p (31.0%), 18q (23.9%), 4q (21.1%), 3p (16.9%), 6q (14.1%), and 1q (8.5%). Positive association with one of the indicators was observed in 3p, 9p, 9q, 10q, 14q, and 18q. The chromosomes that correlated with two biologic indicators were 4q, 6q, 11p, 17p, and 21q. Univariate analysis found that patients having combined 9p and 14q deleted tumors had particularly poor long-term survival compared with those with other patterns of chromosomal alterations (P = 0.01). In the multivariate model, nonpapillary tumors had a greater risk of recurrence, and stage classification was the only important indicator in predicting patient survival (P = 0.04). CONCLUSIONS: LOH assessment does not provide independent prognostic value compared with stage classification. However, chromosomes 4q, 6q, 9p, 11p, 14q, 17p, and 21q may harbor important tumor suppressor genes involved in the progression of urothelial carcinogenesis.