Transcriptomic profiling of intermediate cell carcinoma of the liver.

BACKGROUND: Intermediate cell carcinoma (Int-CA) is a rare and enigmatic primary liver cancer characterized by uniform tumor cells exhibiting mixed features of both HCC and intrahepatic cholangiocarcinoma. Despite the unique pathological features of int-CA, its molecular characteristics remain unclear yet. METHODS: RNA sequencing and whole genome sequencing profiling were performed on int-CA tumors and compared with those of HCC and intrahepatic cholangiocarcinoma. RESULTS: Int-CAs unveiled a distinct and intermediate transcriptomic feature that is strikingly different from both HCC and intrahepatic cholangiocarcinoma. The marked abundance of splicing events leading to intron retention emerged as a signature feature of int-CA, along with a prominent expression of Notch signaling. Further exploration revealed that METTL16 was suppressed within int-CA, showing a DNA copy number-dependent transcriptional deregulation. Notably, experimental investigations confirmed that METTL16 suppression facilitated invasive tumor characteristics through the activation of the Notch signaling cascade. CONCLUSIONS: Our results provide a molecular landscape of int-CA featured by METTL16 suppression and frequent intron retention events, which may play pivotal roles in the acquisition of the aggressive phenotype of Int-CA.

Utilizing stem cell-secreted molecules as a versatile toolbox for skin regenerative medicine.

Stem cells are recognized as an important target and tool in regenerative engineering. In this study, we explored the feasibility of engineering amniotic fluid-derived mesenchymal stem cell-secreted molecules (afMSC-SMs) as a versatile bioactive material for skin regenerative medicine applications in a time- and cost-efficient and straightforward manner. afMSC-SMs, obtained in powder form through ethanol precipitation, effectively contributed to preserving the self-renewal capacity and differentiation potential of primary human keratinocytes (pKCs) in a xeno-free environment, offering a potential alternative to traditional culture methods for their long-term in vitro expansion, and allowed them to reconstitute a fully stratified epithelium sheet on human dermal fibroblasts. Furthermore, we demonstrated the flexibility of afMSC-SMs in wound healing and hair regrowth through injectable hydrogel and nanogel-mediated transdermal delivery systems, respectively, expanding the pool of regenerative applications. This cell-free approach may offer several potential advantages, including streamlined manufacturing processes, scalability, controlled formulation, longer shelf lives, and mitigation of risks associated with living cell transplantation. Accordingly, afMSC-SMs could serve as a promising therapeutic toolbox for advancing cell-free regenerative medicine, simplifying their broad applicability in various clinical settings.

Human induced neural stem cells support functional recovery in spinal cord injury models.

Spinal cord injury (SCI) is a clinical condition that leads to permanent and/or progressive disabilities of sensory, motor, and autonomic functions. Unfortunately, no medical standard of care for SCI exists to reverse the damage. Here, we assessed the effects of induced neural stem cells (iNSCs) directly converted from human urine cells (UCs) in SCI rat models. We successfully generated iNSCs from human UCs, commercial fibroblasts, and patient-derived fibroblasts. These iNSCs expressed various neural stem cell markers and differentiated into diverse neuronal and glial cell types. When transplanted into injured spinal cords, UC-derived iNSCs survived, engrafted, and expressed neuronal and glial markers. Large numbers of axons extended from grafts over long distances, leading to connections between host and graft neurons at 8 weeks post-transplantation with significant improvement of locomotor function. This study suggests that iNSCs have biomedical applications for disease modeling and constitute an alternative transplantation strategy as a personalized cell source for neural regeneration in several spinal cord diseases.

Albumin, filamin-A and cytokeratin 19 help distinguish intrahepatic cholangiocarcinoma from extrahepatic adenocarcinoma.

BACKGROUND: The differential diagnosis of intrahepatic cholangiocarcinomas (iCCAs) from metastatic adenocarcinomas from organs adjacent to the liver (gallbladder, pancreas, and stomach) is difficult due to histopathological similarity and a lack of specific markers. This study aimed to develop a method to differentiate iCCA and adenocarcinomas originated from extrahepatic organs adjacent to the liver. METHODS: We retrospectively enrolled surgically resected iCCA (n = 181) and adenocarcinomas from extrahepatic organs (n = 30, n = 28, and n = 38 from gallbladder, pancreas, and stomach, respectively) between 2007 and 2013. The albumin mRNA in situ hybridization (ISH) and immunohistochemistry (IHC) of filamin-A and cytokeratin 19 (CK19) were performed using tissue microarray. Using logistic regression analysis of three markers, iCCA-score was developed, and its diagnostic performance was evaluated. RESULTS: The iCCAs were more frequently positive for albumin ISH (23.2% vs. 0%), filamin-A IHC (47.5% vs. 12.5%) and CK19 (68.5% vs. 40.6%) than extrahepatic adenocarcinomas (p < 0.001 for all). The iCCA-score consisting of these three markers was developed, and it showed higher diagnostic performance (area under the curve [AUC], 0.798 vs. 0.616, p < 0.001). Taking an iCCA-score of 2 or higher as the threshold for iCCA, the sensitivity was substantially higher than albumin ISH alone (45.9% and 23.2%, respectively; p < 0.001), but maintained high specificity (94.8% and 100%, respectively). CONCLUSION: Albumin ISH and IHC staining for filamin-A and CK19 showed distinct expression patterns between iCCA and extrahepatic adenocarcinomas from gallbladder, pancreas, and stomach. We developed iCCA-score that consisted of those three markers, and it showed better diagnostic performance than albumin ISH alone.

Molecular and radiopathologic spectrum between HCC and intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Primary liver cancers (LCs), including HCC and intrahepatic cholangiocarcinoma (iCCA), are derived from a common developmental lineage, conferring a molecular spectrum between them. To elucidate the molecular spectrum, we performed an integrative analysis of transcriptome profiles associated with patients' radiopathologic features. APPROACH AND RESULTS: We identified four LC subtypes (LC1-LC4) from RNA-sequencing profiles, revealing intermediate subtypes between HCC and iCCA. LC1 is a typical HCC characterized by active bile acid metabolism, telomerase reverse transcriptase promoter mutations, and high uptake of gadoxetic acid in MRI. LC2 is an iCCA-like HCC characterized by expression of the progenitor cell-like trait, tumor protein p53 mutations, and rim arterial-phase hyperenhancement in MRI. LC3 is an HCC-like iCCA, mainly small duct (SD) type, associated with HCC-related etiologic factors. LC4 is further subclassified into LC4-SD and LC4-large duct iCCAs according to the pathological features, which exhibited distinct genetic variations (e.g., KRAS , isocitrate dehydrogenase 1/2 mutation, and FGF receptor 2 fusion), stromal type, and prognostic outcomes. CONCLUSIONS: Our integrated view of the molecular spectrum of LCs can identify subtypes associated with transcriptomic, genomic, and radiopathologic features, providing mechanistic insights into heterogeneous LC progression.

Noninvasive surrogates are poor predictors of liver fibrosis in patients with Fontan circulation.

OBJECTIVES: Patients with Fontan circulation exhibit a high incidence of liver fibrosis and cirrhosis. Transient elastography (TE) and the enhanced liver fibrosis (ELF) test have proven useful as noninvasive surrogate markers of liver fibrosis for other chronic liver diseases. We evaluated whether TE and the ELF score can predict the degree of liver fibrosis in patients with Fontan circulation. METHODS: We retrospectively reviewed the medical records of 45 adult patients with at least 10 years of Fontan duration who had undergone liver biopsy and investigated the relation between the fibrosis stage and TE and the ELF test results. Additionally, the association of these variables and other biochemical and hemodynamic parameters was assessed. RESULTS: The mean age was 25.9 years and the mean Fontan duration was 20.8 years. Advanced liver fibrosis was present in 36 (80.0%) patients. TE or ELF score are comparable for patients with and without advanced liver fibrosis (mean 23.3 vs 24.8 kPa [P = .85] for TE; mean 8.94 vs 9.25 [P = .44] for the ELF score). However, N-terminal pro-brain natriuretic peptide level and ventricular end-diastolic pressure were higher in patients with advanced liver fibrosis (mean 224 vs 80 pg/mL [P < .01]; and mean 12 vs 9 mm Hg [P = .04], respectively). No independent predictor of advanced liver fibrosis was found in multivariate analysis. CONCLUSIONS: TE and the ELF score were unable to predict the degree of liver fibrosis in Fontan patients. Liver biopsy remains as the only valid method to assess fibrotic burden in this population.

Circulating Cancer Stem Cells Expressing EpCAM/CD90 in Hepatocellular Carcinoma: A Pilot Study for Predicting Tumor Recurrence after Living Donor Liver Transplantation.

Background/Aims: Circulating tumor cells (CTCs) with cancer stemness have been demonstrated to be a direct cause of tumor recurrence, and only few studies have reported the role of CTCs in liver transplantation (LT) for hepatocellular carcinoma (HCC). Methods: Epithelial cell adhesion molecule+ (EpCAM+), cluster of differentiation 90+ (CD90+) and EpCAM+/CD90+ CTCs were sorted via fluorescence-activated cell sorting, and transcripts level of EpCAM, K19 and CD90 in the peripheral blood were analyzed via real-time polymerase chain reaction preoperatively and on postoperative days 1 and 7 in 25 patients who underwent living donor liver transplantation (LDLT) for HCC. EpCAM protein was assessed in HCC tissue using immunohistochemical staining. The median follow-up duration was 40 months. Results: HCC after LDLT recurred in four out of 25 patients. Detection of EpCAM+ or CD90+ CTCs correlated well with their messenger RNA levels (p<0.05). EpCAM+ CTCs were readily detected in HCC tissue expressing EpCAM protein. The detection of EpCAM+ CTCs or EpCAM+/CD90+ CTCs before surgery and on postoperative day 1 was significantly associated with HCC recurrence after LT (all p<0.05). Pretransplant serum PIVKA-II >100 mAU/mL and postoperative day 1 EpCAM+/CD90+ CTCs were independent risk factors for HCC recurrence (hazard ratio, 14.64; 95% confidence interval, 1.08 to 198.20; p=0.043 and hazard ratio, 26.88; 95% confidence interval, 1.86 to 387.51; p=0.016, respectively). Conclusions: EpCAM+/CD90+ CTCs can be used preoperatively and 1 day after LDLT as key biological markers in LT candidate selection and post-LDLT management.

Genetic, Clinicopathological, and Radiological Features of Intrahepatic Cholangiocarcinoma with Ductal Plate Malformation Pattern.

Background/Aims: Intrahepatic cholangiocarcinoma (iCCA) with a ductal plate malformation (DPM) pattern is a recently recognized rare variant. The genomic profile of iCCA with DPM pattern needs to be elucidated. Methods: Cases of iCCA with DPM pattern were retrospectively reviewed based on the medical records, pathology slides, and magnetic resonance imaging (MRI) reports collected between 2010 to 2019 at a single center. Massive parallel sequencing was performed for >500 cancer-related genes. Results: From a total of 175 iCCAs, five (2.9%) cases of iCCA with DPM pattern were identified. All cases were of the small duct type, and background liver revealed chronic B viral or alcoholic hepatitis. Three iCCAs with DPM pattern harbored MRI features favoring the diagnosis of hepatocellular carcinoma, whereas nonspecific imaging features were observed in two cases. All patients were alive without recurrence during an average follow-up period of 57 months. Sequencing data revealed 64 mutated genes in the five cases, among which FGFR and PTPRT were most frequently mutated (three cases each) including an FGFR-TNC fusion in one case. Mutations in ARID1A and CDKN2A were found in two cases, and mutations in TP53, BAP1, ATM, NF1, and STK11 were observed in one case each. No IDH1, KRAS, or PBRM1 mutations were found. Conclusions: iCCAs with DPM pattern have different clinico-radio-pathologic and genetic characteristics compared to conventional iCCAs. Moreover, FGFR and ARID1A variants were identified. Altogether, these findings further suggest that iCCA with DPM pattern represents a specific subtype of small duct type iCCA.

Combined tumor epithelial and stromal histopathology with keratin 81 expression predicts prognosis for pancreatic ductal adenocarcinoma.

BACKGROUND: Development of a pragmatic pathologic classifier of pancreatic ductal adenocarcinoma (PDAC) that reflects biological behavior is needed. METHODS: The tumor epithelial and stromal features of PDAC and molecular subtype-related markers were evaluated in three independent cohorts. RESULTS: In the non-neoadjuvant therapy cohort (n = 108), regarding tumor-epithelial feature, non-gland-forming type showed worse prognosis compared to gland-forming type (P < .001). For tumor-stromal feature, in gland-forming type, the prognosis was good in order of inactivated stroma-rich, stroma-poor, and activated stroma-rich (P = .027). Whereas, non-gland-forming type revealed no difference of prognosis according to tumor stroma. Of molecular subtype-related markers, keratin 81 expression was correlated with non-gland-forming type and poor prognosis (P = .005 and P = .021, respectively). Other markers (HNF1A, c-MET, and p53) showed no significant differences in prognosis. In the neoadjuvant therapy cohort (n = 68), non-gland-forming type was correlated with high residual tumor volume (≥20%) (P < .001) and gland-forming/stroma-poor type was not present. In the next-generation sequencing cohort (n = 55), non-gland-forming type was correlated with a higher number of the KRAS, TP53, CDKN2A, and SMAD4 mutations (P = .038). CONCLUSION: Combined tumor epithelial and stromal histopathology with keratin 81 expression is suggested to be useful for predicting prognosis of PDAC.

Lung and lymph node metastases from hepatocellular carcinoma: Comparison of pathological aspects.

BACKGROUND & AIMS: Extrahepatic metastasis from hepatocellular carcinoma (HCC) is a catastrophic event, yet organ-specific pathological characteristics of metastatic HCC remain unclear. We aimed to characterize the pathological aspects of HCC metastases to various organs. METHODS: We collected intrahepatic HCC (cohort 1, n = 322) and extrahepatic metastatic HCC (cohort 2, n = 130) samples. Clinicopathological evaluation and immunostaining for K19, CD34, αSMA, fibroblast-associated protein (FAP), CAIX, VEGF, PD-L1, CD3, CD8, Foxp3, CD163 and epithelial-mesenchymal transition (EMT)-related markers were performed. RESULTS: Independent factors for extrahepatic metastasis included BCLC stage B-C, microvascular invasion (MVI), vessels encapsulating tumour clusters (VETC)-HCC, K19 and FAP expression, and CD163+ macrophage infiltration (cohort 1, P < .05 for all). Lung metastases (n = 63) had the highest proportion of VETC-HCC and macrotrabecular-massive (MTM)-HCC. Lymph node metastases (n = 19) showed significantly high rates of EMT-high features, K19 expression, fibrous tumour stroma with αSMA and FAP expression, high immune cell infiltration, PD-L1 expression (combined positive score), CD3+, CD8+, Foxp3+ T cell and CD163+ macrophage infiltration (adjusted P < .05 for all). In both cohorts, EMT-high HCCs showed higher rates of K19 expression, fibrous tumour stroma, high immune cell infiltration, PD-L1 expression and CD3+ T cell infiltration, whereas EMT-low HCCs were more frequent among VETC-HCCs (P < .05 for all). Overall phenotypic features were not significantly different between paired primary-metastatic HCCs (n = 32). CONCLUSIONS: Metastatic HCCs to various organs showed different pathological features. VETC and MTM subtypes were related to lung metastasis, whereas K19 expression, EMT-high features with fibrous tumour stroma and high immune cell infiltration were related to lymph node metastasis.

The dual role of transforming growth factor-beta signatures in human B viral multistep hepatocarcinogenesis: early and late responsive genes.

Background/Aim: Transforming growth factor-beta (TGF-ß) has a dichotomous role, functioning as a tumor suppressor and tumor promoter. TGF-ß signatures, explored in mouse hepatocytes, have been reported to predict the clinical outcomes of hepatocellular carcinoma (HCC) patients; HCCs exhibiting early TGF-ß signatures showed a better prognosis than those with late TGF-ß signatures. The expression status of early and late TGF-ß signatures remains unclear in defined lesions of human B-viral multistep hepatocarcinogenesis. Methods: The expression of TGF-ß signatures, early and late responsive signatures of TGF-ß were investigated and analyzed for their correlation in cirrhosis, low-grade dysplastic nodules (DNs), high-grade DNs, early HCCs and progressed HCCs (pHCCs) by real-time PCR and immunohistochemistry. Results: The expression levels of TGF-ß signaling genes (TGFB1, TGFBR1, TGFBR2 and SMAD4) gradually increased with the progression of hepatocarcinogenesis, peaking in pHCCs. The expression of early responsive genes of TGF-ß (GADD45B, FBP1, CYP1A2 and CYP3A4) gradually decreased, and that of the late TGF-ß signatures (TWIST and SNAI1) significantly increased according to the progression of multistep hepatocarcinogenesis. Furthermore, mRNA levels of TWIST and SNAI1 were well correlated with those of stemness markers, with upregulation of TGF-ß signaling, whereas FBP1 expression was inversely correlated with that of stemness markers. Conclusions: The enrichment of the late responsive signatures of TGF-ß with induction of stemness is considered to be involved in the progression of the late stage of multistep hepatocarcinogenesis, whereas the early responsive signatures of TGF-ß are suggested to have tumor-suppressive roles in precancerous lesions of the early stage of multistep hepatocarcinogenesis.

The Clinicopathological Significance of YAP/TAZ Expression in Hepatocellular Carcinoma with Relation to Hypoxia and Stemness.

Background/Aims: Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) activation has been implicated in hepatocarcinogenesis and hepatic progenitor cell differentiation, and hypoxia has been shown to induce nuclear translocation of YAP in cancer cells. Here, we aimed to investigate the relationship between hypoxia, YAP and TAZ expression and stemness-related marker expression in human hepatocellular carcinomas (HCCs) and its clinical implications. Methods: Immunohistochemical stains were performed on tissue microarrays from 305 surgically resected HCCs, and the expression status of YAP and TAZ were correlated with CAIX, stemness markers (K19, EpCAM) and epithelial-mesenchymal transition (EMT)-related markers (uPAR, ezrin). The clinicopathological significance of YAP/TAZ expression was analyzed with relation to CAIX expression status. Results: YAP and TAZ expression were seen in 13.4 and 4.3% of HCCs, respectively. YAP/TAZ-positive HCCs frequently demonstrated higher serum AFP levels, microvascular invasion, advanced tumor stage, increased proliferative activity and expression of stemness- and EMT-related markers, CAIX, p53 and Smad2/3 (p < 0.05, all). Interestingly, YAP/TAZ-positivity was associated with microvascular invasion, higher serum AFP levels, stemness and EMT-related marker expression only in tumors expressing CAIX (p < 0.05, all), while these associations were not seen in CAIX-negative HCCs. Conclusions: YAP/TAZ expression is associated with vascular invasion, stemness and EMT in HCCs with hypoxia marker expression. The effect of Hippo signaling pathway deregulation in HCC may depend on the presence or absence of a hypoxic microenvironment, and hypoxia marker expression status should be taken into account when considering the use of YAP/TAZ as markers of aggressive biologic behavior in HCC.

YAP inactivation in estrogen receptor alpha-positive hepatocellular carcinoma with less aggressive behavior.

The expression of estrogen receptor alpha (ERα, encoded by ESR1) has been shown to be associated with the prognostic outcomes of patients in various cancers; however, its prognostic and mechanistic significance in hepatocellular carcinoma (HCC) remain unclear. Here, we evaluated the expression of ERα and its association with clinicopathological features in 339 HCC patients. ERα was expressed in 9.4% (32/339) of HCCs and was related to better overall survival (OS; hazard ratio [HR] = 0.11, p = 0.009, 95% C.I. = 0.016-0.82) and disease-free survival (DFS, HR = 0.4, p = 0.013, 95% C.I. = 0.18-0.85). ERα expression was also associated with features related to more favorable prognosis, such as older age, lower serum alpha-fetoprotein level, and less microvascular invasion (p < 0.05). In addition, to obtain mechanistic insights into the role of ERα in HCC progression, we performed integrative transcriptome data analyses, which revealed that yes-associated protein (YAP) pathway was significantly suppressed in ESR1-expressing HCCs. By performing cell culture experiments, we validated that ERα expression enhanced YAP phosphorylation, attenuating its nuclear translocation, which in turn suppressed the downstream signaling pathways and cancer cell growth. In conclusion, we suggest that ERα expression is an indicator of more favorable prognosis in HCC and that this effect is mediated by inactivation of YAP signaling. Our results provide new clinical and pathobiological insights into ERα and YAP signaling in HCC.

Pathological predictive factors for late recurrence of hepatocellular carcinoma in chronic liver disease.

BACKGROUND & AIMS: Late recurrence of hepatocellular carcinoma (HCC) is regarded as de novo HCC from chronic hepatitis. This study investigated clinicopathological and molecular factors to develop a nomogram for predicting late HCC recurrence (>2 years after curative resection). METHODS: The training and validation cohorts included HCC patients with a major aetiology of hepatitis B who underwent curative resection. Clinicopathological features including lobular and porto-periportal inflammatory activity, fibrosis and liver cell change were evaluated. Proteins encoded by genes related to late recurrence were identified using a reverse phase protein array of 95 non-tumourous liver tissues. Immunoexpression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), plasminogen activator inhibitor-1, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and spleen tyrosine kinase (SYK) was measured. RESULTS: Late recurrence occurred in 74/402 (18%) and 47/243 (19%) in the training and validation cohorts respectively. Cirrhosis, moderate/severe lobular inflammatory activity, and expression of pSTAT3, pERK1/2, and SYK proteins correlated to the gene signature of hepatocyte injury and regeneration were independently associated with late recurrence, with odds ratios (95% confidence intervals) of 2.0 (1.2-3.3), 21.1 (4.3-102.7) and 6.0 (2.1-17.7) respectively (P < .05 for all). A nomogram based on these variables (histological parameters and immunohistochemical marker combinations) showed high reliability in both the training and validation cohorts (Harrell's C index: 0.701 and 0.716; 95% confidence intervals: 0.64-0.76 and 0.64-0.79 respectively). CONCLUSIONS: The combination of pSTAT3, pERK1/2 and SYK immunoexpression with high lobular inflammatory activity and cirrhosis (fibrosis) predicts late HCC recurrence.

Clinical and survival outcomes after hepatectomy in patients with non-alcoholic fatty liver and hepatitis B-related hepatocellular carcinoma.

BACKGROUND: The prevalence of non-alcoholic fatty liver disease-related hepatocellular carcinoma (NAFLD-HCC) has increased parallelly with that of metabolic syndrome. This study aimed to compare the clinical and survival outcomes of NAFLD-HCC and HBV-related HCC(HBV-HCC). METHODS: The medical records of patients who underwent hepatectomy for HCC at Severance Hospital between 2005 and 2015 were retrospectively reviewed. Occult HBV infection was identified by nested PCR. Propensity score matching (PSM) was conducted to minimize lead-time bias caused by the lack of surveillance in NAFLD patients. Surgical and oncologic outcomes were compared between the two groups. RESULTS: There were 32 patients (7%) with NAFLD-HCC, 200 (46%) with HBV-HCC, and 194 (44%) with HBV/NAFLD-HCC (HBV and NAFLD). Before PSM, cirrhosis was more frequently detected in HBV-HCC patients (55% vs 15%, p < 0.001) and the average tumor size was larger in the NAFLD-HCC group than in the HBV-HCC group (4.4 ± 3.3 cm vs 3.4 ± 1.8 cm, p = 0.014). After a median follow-up of 74 months (range 0-157 months), survival analyses before PSM showed better 5-year overall survival (OS) in HBV-HCC patients than in NAFLD-HCC patients (80% vs 63%, p = 0.041). After PSM, 5-year OS rates were similar (60% vs 63%, p = 0.978). There were no differences between the groups in recurrence-free or disease-specific survival before and after PSM. CONCLUSION: Patients with NAFLD-HCC were less likely to have underlying cirrhosis but more likely to have larger tumors at the time of diagnosis than patients with HBV-HCC. The OS of patients with NAFLD-HCC appeared to be worse than that of patients with HBV-HCC. Therefore, active HCC surveillance is recommended in patients with metabolic syndrome for the early detection of HCC.

Gross type of hepatocellular carcinoma reflects the tumor hypoxia, fibrosis, and stemness-related marker expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is subclassified into five gross types, namely, vaguely nodular (VN), single nodular (SN), single nodular with extranodular growth (SNEG), confluent multinodular (CM), and infiltrative (INF) type. However, the pathological background underlying differences in biological behavior of different gross types of HCC remains unclear. METHODS: The histopathological features, clinical outcomes of HCC gross types, and their relationships with stemness-related marker status and fibrotic/hypoxic tumor microenvironment (TME) were evaluated in 266 resected HCCs. The stemness-related markers (CD24, CD44, CD133, SALL4, YAP1, K19 and EpCAM), fibrous tumor stroma (αSMA), and hypoxia (CAIX) were evaluated with immunohistochemistry. RESULTS: Poorer differentiation, reduced capsule formation, higher microvascular invasion, larger tumor size and larger area of necrosis were observed in order of VN-SN-SNEG-CM-INF type (p = 0.005 for all, linear-by-linear association). The expression of summed stemness-related markers and hypoxic/fibrotic TME showed an increasing trend in order of VN-SN-SNEG-CM-INF type (p < 0.005), and their expression well correlated with each other. INF type was found only in HCCs with hypoxic/fibrotic TME or high expression of stemness-related markers. CAIX expression and tumor necrosis ≥ 30% were independent prognostic markers for disease-specific survival. Early recurrence-free survival showed a significant difference based on gross types, revealing best outcome with VN type and worst outcome with INF type. CONCLUSION: The marker expression of stemness-related and hypoxic/fibrotic TME of HCC showed an increasing trend in order of VN-SN-SNEG-CM-INF gross types, and their cross-talk may be involved in the determination of various gross-morphological features and their distinct biological behavior.

Clinicopathological characteristics of intrahepatic cholangiocarcinoma according to gross morphologic type: cholangiolocellular differentiation traits and inflammation- and proliferation-phenotypes.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is subclassified into mass-forming (MF), periductal-infiltrative (PI), and mixed types grossly; however, their clinicopathological significance remains controversial. METHODS: Clinicopathological characteristics of iCCA gross types were analysed according to histopathological type (small-duct, large-duct, indeterminate) or cholangiolocellular differentiation trait (CDT) in 108 iCCAs. The expression levels of inflammation-marker (CRP, FGB) and proliferation-marker (phospho-ERK1/2, Ki-67) were evaluated by immunohistochemistry. RESULTS: There were 87 MF, 8 PI, and 13 mixed-gross type. Small-duct-type (39, 44.8%) and CDT (19, 21.8%) were found only in MF-gross type. The inflammation-marker expression was higher in MF-type than in PI- and mixed-gross types (P = 0.023). It was high in small-duct-type, middle in indeterminate-type, and low in large-duct-type (P = 0.015), and iCCAs with CDT showed higher inflammation-marker expression compared to those without (P < 0.001). Proliferation-marker expression did not differ according to gross type; however it was lower in iCCA with CDT compared to those without (P = 0.004). Subgrouping of the gross type according to histopathological type or CDT revealed that MF-type with small-duct-type or CDT had better overall survival compared to the others (P < 0.05). CONCLUSION: MF-type iCCA is more heterogeneous than other gross types. High inflammation-marker/low proliferation-marker expression in MF-type with CDT or small-duct-type may be related to a good outcome.

Investigating the Systems-Level Effect of Pueraria lobata for Menopause-Related Metabolic Diseases Using an Ovariectomized Rat Model and Network Pharmacological Analysis.

This study was conducted to evaluate the biological activities of Pueraria lobata (PL) on menopause-related metabolic diseases and to explore the underlying mechanism of PL by network pharmacological analyses. We used ovariectomized (OVX) rats as a postmenopausal model and administered PL at different doses (50, 100, and 200 mg/kg). In OVX rats, decreased uterine weights and PPAR-γ (peroxisome proliferator-activated receptor-gamma) mRNA expression in the thigh muscle were significantly recovered after PL administration. PL also significantly alleviated OVX-induced increases in total cholesterol, triglyceride, alanine aminotransferase (ALT/GPT), and aspartate aminotransferase (AST/GOT) levels. To identify the systems-level mechanism of PL, we performed network pharmacological analyses by predicting the targets of the potential bioactive compounds and their associated pathways. We identified 61 targets from four potential active compounds of PL: formononetin, beta-sitosterol, 3'-methoxydaidzein, and daidzein-4,7-diglucoside. Pathway enrichment analysis revealed that among female sex hormone-related pathways, the estrogen signaling pathways, progesterone-mediated oocyte maturation, oxytocin signaling pathways, and prolactin signaling pathways were associated with multiple targets of PL. In conclusion, we found that PL improved various indicators associated with lipid metabolism in the postmenopausal animal model, and we also identified that its therapeutic effects are exerted via multiple female sex hormone-related pathways.

Dynamics of Genomic, Epigenomic, and Transcriptomic Aberrations during Stepwise Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis to low-grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multilayered genomic, epigenomic, and transcriptomic aberrations in the stepwise hepatocarcinogenesis. Initial DNA methylation was observed in eHCC (e.g., DKK3, SALL3, and SOX1) while more extensive methylation was observed in pHCC. In addition, eHCCs showed an initial loss of DNA copy numbers of tumor suppressor genes in the 4q and 13q regions, thereby conferring survival benefits to cancer cells. Transcriptome analysis revealed that HGDNs expressed endoplasmic reticulum (ER) stress-related genes, while eHCC started to express oncogenes. Furthermore, integrative analysis indicated that expression of the serine peptidase inhibitor, Kazal type 1 (SPINK1), played a pivotal role in eHCC development. Significant demethylation of SPINK1 was observed in eHCC compared to HGDN. The study also demonstrated that ER stress may induce SPINK1 demethylation and expression in liver cancer cells. In conclusion, these results reveal the dynamics of multiomic aberrations during malignant conversion of liver cancer, thus providing novel pathobiological insights into hepatocarcinogenesis. SIGNIFICANCE: Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.

Analysis and Identification of Active Compounds from Gami-Soyosan Toxic to MCF-7 Human Breast Adenocarcinoma Cells.

Gami-soyosan is a medicinal herbal formulation prescribed for the treatment of menopausal symptoms, including hot flashes and osteoporosis. Gami-soyosan is also used to treat similar symptoms experienced by patients with breast cancer. The incidence of breast cancer in women receiving hormone replacement therapy is a big burden. However, little is known about the components and their mechanism of action that exhibit these beneficial effects of Gami-soyosan. The aim of this study was to simultaneously analyze compounds of Gami-soyosan, and determine their cytotoxic effects on estrogen receptor (ER)-positive MCF-7 human breast adenocarcinoma cells. We established a simultaneous analysis method of 18 compounds contained in Gami-soyosan and found that, among the various compounds in Gami-soyosan, gallic acid (1), decursin (17), and decursinol angelate (18) suppressed the viability of MCF-7 cells. Gallic acid (1), decursin (17), and decursinol angelate (18) induced apoptotic cell death and significantly increased poly (ADP-ribose) polymerase (PARP) cleavage and the Bcl-2-associated X protein/ B-cell lymphoma 2 (Bax/Bcl-2) ratio. Decursin (17) increased the expression of cleaved caspases-8, -9, -7, and -3. Decursinol angelate (18) increased the expression of cleaved caspase-8 and -7. These three components altered the different apoptosis signal pathways. Collectively, gallic acid (1), decursin (17), and decursinol angelate (18) may be used to inhibit cell proliferation synergistically in patients with ER-positive breast cancer.