Microscopic Intrahepatic Metastasis Research Articles

Impact of albumin-lymphocyte-platelet-C-reactive protein index as a prognostic indicator of hepatocellular carcinoma after resection: Associated with nuclear factor erythroid 2-related factor 2.

To investigate the prognostic value of the preoperative albumin-lymphocyte-platelet-C-reactive protein (ALPC) index in patients with hepatocellular carcinoma (HCC) undergoing curative hepatectomy. We also evaluated the relationship between the ALPC index and the phosphorylated nuclear factor erythroid 2-related factor 2 (p-Nrf2) levels. Data were analyzed retrospectively from 256 patients who underwent resection for HCC. For cross-validation, patients were divided into the training and testing cohort. We assessed eight combinations of inflammatory markers for predictive value for recurrence. We examined the associations of the ALPC index with recurrence-free survival and overall survival in univariate and multivariate analyses (Cox proportional hazards model). Immunohistochemical staining of p-Nrf2 was performed on tumor samples of 317 patients who underwent hepatic resection forHCC. A high preoperative ALPC index correlated with a high serum albumin concentration, small tumor size, low rate of poor differentiation, solitary tumor, early Barcelona Clinic Liver Cancer stage, and low rate of microscopic intrahepatic metastasis in the training dataset. A high preoperative ALPC index correlated with a high serum albumin concentration, high serum alpha-fetoprotein concentration, small tumor size, a low rate of poor differentiation and a low rate of microscopic intrahepatic metastasis in the testing dataset. A higher preoperative ALPC index was an independent predictor of longer recurrence-free survival and overall survival in the training and testing datasets. A high ALPC index was associated with negative p-Nrf2 expression in HCC tumor cells. We showed that a high ALPC index was an independent prognostic factor for patients with HCC undergoing curative hepatic resection.

Transferrin Receptor is Associated with Sensitivity to Ferroptosis Inducers in Hepatocellular Carcinoma.

Transferrin receptor (TFR), a membrane protein that has a critical role in the transport of iron into cells, is known to be a ferroptosis-related marker. Although TFR is reported to be abundantly expressed in tumor cells, its relationship with ferroptosis inducers in hepatocellular carcinoma (HCC) remains unclear. The authors performed immunohistochemical staining of TFR and divided 350 HCC patients into two groups according to its expression. They analyzed the association between TFR expression and prognosis or clinicopathologic factors. In addition, the regulation of malignant activity and its effect on the efficacy of ferroptosis inducers were investigated in vitro. For this study, 350 patients were divided into TFR-positive (n =180, 51.4%) and TFR-negative (n = 170, 48.6%) groups. The TFR-positive group had more hepatitis B surface antigen (HBs-Ag) (p = 0.0230), higher α-fetoprotein (AFP) levels (p = 0.0023), higher des-gamma-carboxyprothrombin (DCP) levels (p = 0.0327), a larger tumor size (p = 0.0090), greater proportions of Barcelona Clinic Liver Cancer (BCLC) stage B or C (p = 0.0005), poor differentiation (p < 0.0001), and microscopic intrahepatic metastasis (p = 0.0066). In the multivariate analyses, TFR expression was an independent prognostic factor in disease-free survival (p = 0.0315). In vitro, TFRC knockdown decreased cell motility. In addition, TFRC knockdown abolished artesunate (AS)-, lenvatinib-, and sorafenib-induced ferroptosis in HCC cell lines. The study demonstrated that simultaneous treatment of AS with multi-kinase inhibitor augmented the ferroptosis-inducing effects of AS in HCC cell lines. TFR expression is a poor prognostic factor in HCC, but its expression increases sensitivity to ferroptosis-inducing agents.

Impact of Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status.

We examined phosphorylated nuclear factor erythroid 2–related factor 2 (P‐NRF2) expression in surgically resected primary hepatocellular carcinoma (HCC) and investigated the association of P‐NRF2 expression with clinicopathological features and patient outcome. We also evaluated the relationship among NRF2, cancer metabolism, and programmed death ligand 1 (PD‐L1) expression. In this retrospective study, immunohistochemical staining of P‐NRF2 was performed on the samples of 335 patients who underwent hepatic resection for HCC. Tomography/computed tomography using fluorine‐18 fluorodeoxyglucose was performed, and HCC cell lines after NRF2 knockdown were analyzed by array. We also analyzed the expression of PD‐L1 after hypoxia inducible factor 1α (HIF1A) knockdown in NRF2‐overexpressing HCC cell lines. Samples from 121 patients (36.1%) were positive for P‐NRF2. Positive P‐NRF2 expression was significantly associated with high alpha‐fetoprotein (AFP) expression, a high rate of poor differentiation, and microscopic intrahepatic metastasis. In addition, positive P‐NRF2 expression was an independent predictor for recurrence‐free survival and overall survival. NRF2 regulated glucose transporter 1, hexokinase 2, pyruvate kinase isoenzymes L/R, and phosphoglycerate kinase 1 expression and was related to the maximum standardized uptake value. PD‐L1 protein expression levels were increased through hypoxia‐inducible factor 1α after NRF2 overexpression in HCC cells. Conclusions: Our large cohort study revealed that P‐NRF2 expression in cancer cells was associated with clinical outcome in HCC. Additionally, we found that NRF2 was located upstream of cancer metabolism and tumor immunity.

Lymphocyte-to-C-reactive protein ratio as a prognostic factor for hepatocellular carcinoma.

Systemic inflammation has been correlated with worse survival for some cancers. We evaluated prognostic values of various inflammatory factor combinations in patients who underwent resections for hepatocellular carcinoma (HCC). We retrospectively analysed 306 consecutive patients with HCC who underwent curative liver resections. After assessing eight combinations of inflammatory markers for predictive value for recurrence, we focused on lymphocyte-to-C-reactive protein ratio (LCR) to elucidate its associations with recurrence-free survival (RFS) and overall survival (OS) in univariate and multivariate analyses (Cox proportional hazards model). We also used immunohistochemical CD34 and CD8 staining to investigate the mechanism of LCR elevation. LCR showed the highest association with RFS in HCC patients among the compared indices. High preoperative LCR correlated with a high serum albumin concentration, small tumour size, early Barcelona Clinic Liver Cancer stage and low rates of microscopic vascular invasion and microscopic intrahepatic metastasis. Higher preoperative LCR was an independent predictor of longer RFS and OS in this cohort. High LCR patients had fewer vessels encapsulating tumour clusters, and higher intratumoural CD8+ T-cell counts than low LCR patients. Preoperative LCR is a novel and convenient prognostic marker for patients with HCC, and is associated with the tumour microenvironment immune status.

Anatomical resection is useful for the treatment of primary solitary hepatocellular carcinoma with predicted microscopic vessel invasion and/or intrahepatic metastasis.

The aim of this study was to evaluate anatomical resection (AR) versus non-AR for primary solitary hepatocellular carcinoma (HCC) with predicted microscopic vessel invasion (MVI) and/or microscopic intrahepatic metastasis (MIM). This retrospective study included 358 patients who underwent hepatectomy and had no evidence of MVI and/or MIM on preoperative imaging. The predictors of MVI and/or MIM were identified. The AR group (n = 222) and the non-AR group (n = 136) were classified by number of risk factor, and the survival rates were compared. Microscopic vessel invasion and/or MIM were identified in 81 (22.6%) patients. A multivariate analysis showed that high des-gamma-carboxy prothrombin concentration [odds ratio (OR) 3.35], large tumor size (OR 3.16), and high aspartate aminotransferase concentration (OR 2.13) were significant predictors. The 5-year overall survival (OS) in the patients with zero, one, two, and three risk factors were 97.4%, 73.5%, 71.5%, and 65.5%, respectively. The OS of AR is superior to that of non-AR only in patients with one or two risk factors. The present findings suggest that AR should be performed for patients with one or two risk factors, and that AR may prevent recurrence, as these patients are at risk of having MVI and/or MIM.

ARID1A Deficiency Is Associated With High Programmed Death Ligand 1 Expression in Hepatocellular Carcinoma.

The clinicopathological features of carcinomas expressing AT‐rich interaction domain 1a (ARID1A) and programmed death ligand 1 (PD‐L1) in HCC are poorly understood. Here, we examined ARID1A and PD‐L1 expression in surgically resected primary hepatocellular carcinoma (HCC) and the association of ARID1A and PD‐L1 expression with clinicopathological features and patient outcomes. Their association with ARID1A expression and tumor‐associated CD68‐positive macrophage was further explored. Using a database of 255 patients who underwent hepatic resection for HCC, immunohistochemical staining of ARID1A, PD‐L1, and CD68 was performed. We also analyzed the expression PD‐L1 after ARID1A knockdown in HCC cell lines. Samples from 81 patients (31.7%) were negative for ARID1A. Negative ARID1A expression was significantly associated with male sex, high alpha‐fetoprotein, high des‐gamma‐carboxyprothrombin, large tumor size, high rate of poor differentiation, microscopic intrahepatic metastasis, and PD‐L1 expression. In addition, negative ARID1A expression was an independent predictor for recurrence‐free survival, overall survival, and positive PD‐L1 expression. Stratification based on ARID1A and PD‐L1 expression in cancer cells was also significantly associated with unfavorable outcomes. PD‐L1 protein expression levels were increased through phosphoinositide 3‐kinase/AKT signaling after ARID1A knockdown in HCC cells. HCC with ARID1A‐low expression was significantly correlated with high levels of tumor‐associated CD68‐positive macrophage. Conclusion: Our large cohort study showed that ARID1A expression in cancer cells was associated with a poor clinical outcome in patients with HCC, PD‐L1 expression in cancer cells, and tumor microenvironment. Therefore, ARID1A may be a potential molecular biomarker for the selection of patients with HCC for anti‐programmed death 1/PD‐L1 antibody therapy.

CMTM6 Stabilizes PD-L1 Expression and Is a New Prognostic Impact Factor in Hepatocellular Carcinoma.

CKLF‐like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a regulator of programmed death ligand 1 (PD‐L1), which induces antitumor immunity in several cancers. This study aimed to clarify the relationship between CMTM6 and PD‐L1 expression and clinical outcomes in patients with hepatocellular carcinoma (HCC). In total, 259 patients with HCC who had undergone hepatic resection were enrolled. Immunohistochemical staining for CMTM6 and PD‐L1 was performed. The relationships between CMTM6 expression and the clinicopathological characteristics and outcomes were analyzed. Additionally, the stabilization of PD‐L1 expression and regulation of malignant activities by CMTM6 were examined in vitro. Our patients were divided into high (n = 65, 25.1%) and low (n = 194, 74.9%) CMTM6 expression groups. High CMTM6 expression was significantly associated with malignant aggregates, including poor differentiation (P < 0.0001), microscopic intrahepatic metastasis (P = 0.0369), and multiple intrahepatic recurrences (P = 0.0211). CMTM6 expression was significantly correlated with PD‐L1 expression in HCC tissues (P < 0.0001). The patients were classified into three groups: high CMTM6/PD‐L1 positive (n = 21), high CMTM6/ PD‐L1 negative (n = 44), and low CMTM6 (n = 194) expression pattern groups. Overall survival was significantly different among the three groups (P < 0.0001). Additionally, immunohistochemical double staining revealed that CMTM6 and PD‐L1 were co‐expressed on HCC cells. In vitro, PD‐L1 expression was enhanced at late time points in the presence of CMTM6 expression. CMTM6 also regulated epithelial‐to‐mesenchymal transition and stemness phenotypes in HCC cells. Conclusion: Our large cohort study found that CMTM6 co‐expressed with PD‐L1 was strongly associated with the clinical outcome in patients with HCC. The evaluation of CMTM6 combined with PD‐L1 in HCC might be useful for patient selection in immune checkpoint therapy.

The tumor diameter cut-off for predicting microscopic intrahepatic metastasis of hepatocellular carcinoma patients without treatment history differs from that of hepatocellular carcinoma patients with a treatment history.

Intrahepatic metastasis (IM) of hepatocellular carcinoma (HCC) occurs via vascular invasion; the tumor diameter that affects the risk of micro intra-hepatic metastasis (MIM) should be larger than that which affects the risk of micro vessel invasion (MVI). The aim of the present study was to determine the optimum tumor diameter cut-off value for predicting the presence of MIM in HCC patients without treatment history and HCC patients with a treatment history and to compare these diameters between cases of MVI and MIM. This retrospective study included 621 patients without macroscopic vessel invasion or intrahepatic metastasis on preoperative imaging who underwent hepatectomy. The cut-off tumor diameter for predicting the presence of MIM was determined by a receiver operating characteristic curves analysis. The optimum cut-off value for predicting the presence of MIM in HCC patients without treatment history was 43mm. In contrast, the optimum cut-off value for predicting the presence of MIM in HCC patients with a treatment history was 20mm. Among 46 HCC patients with MIM without treatment history, there were 20 patients with MIM without MVI who were considered to have potential multi-centric (MC) tumors rather than IM. The cumulative overall survival rates in patients with MIM without MVI (potential MC) was significantly better than that in patients with both MIM and MVI (P = 0.022). The tumor diameter cut-off value for predicting MIM differed between HCC patients without treatment history and with a treatment history and slightly smaller than those for predicting MVI beyond our expectation.

Reappraisal of evidence of microscopic portal vein involvement by hepatocellular carcinoma cells with stratification of tumor size.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death internationally, it is necessary to reappraise evidences of HCC cells involving the portal vein, especially considering tumor size. Histopathological evidence and dynamic evidences of radiology and cytology from publication were collected and analyzed. Frequencies of microscopic portal vein involvement (MPVI) and microscopic intrahepatic metastasis (MIM) in resected specimens with single nodule HCC were lower than that of multi nodule HCC, although not significantly. Early HCC (≤1.5 cm) was with extremely low to 0 frequencies of MPVI and MIM. HCC >5 cm showed a tendency of flowing HCC cells into portal vein, which was coincident with significantly high frequency (64.1 %) of MPVI for HCC >5 cm. There were no significant difference of frequencies of MPVI and MIM between groups of tumor ≤2, ≤3, and ≤5 cm. Single nodule HCC >5 cm needs anatomic resection and the root of portal vein should be firstly ligated because of tendency of flowing HCC cells into portal vein. For single nodule HCC ≤2 cm, there was a risk of about 16.2 % of MPVI, and a risk of about 16.2-26.4 % of MPVI for those single nodule HCC ≤5 cm, however, there was a risk of extremely low to 0 of MPVI for early HCC (≤1.5 cm). Surgeons have to balance liver reserve and risk of MPVI for HCC ≤5 cm before deciding anatomic or nonanatomic resection.

PLOD2 induced under hypoxia is a novel prognostic factor for hepatocellular carcinoma after curative resection

Under hypoxia, tumour cells undergo genetic and adaptive changes that allow their survival. Previously, we reported that high expression of hypoxia-inducible factor (HIF)-1 was a significant predictive factor for recurrence in hepatocellular carcinoma (HCC). Hypoxia also stimulates expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) genes via the HIF-1 pathway. The aim was to evaluate the relationship between hypoxia stress and expression of PLOD genes in HCC in vitro and to identify a new prognostic marker in HCC patients. The PLOD2 expression was assessed under hypoxia in hepatoma cell lines and characterized in 139 HCC samples following hepatic resection using microarray experiments, quantitative RT-PCR and immunohistochemistry. Prognostic factors in HCC patients were assessed using univariate and multivariate analyses. The PLOD2 expression was induced under the hypoxia in vitro. Disease-free survival in the high PLOD2 expression group of HCC patients was significantly shorter when compared with the low-expression group (P=0.002). In a subset of HCCs, we found that the PLOD2 expression of microarray was correlated with data of quantitative RT-PCR and immunohistochemistry. Of clinicopathological factors, PLOD2 expression was significantly correlated with tumour size (P=0.022) and macroscopic intrahepatic metastasis (P=0.049). In univariate analysis, six prognostic factors (tumour multiplicity, macroscopic intrahepatic metastasis, histological grade, microscopic portal invasion, microscopic intrahepatic metastasis and PLOD2 expression) were significant for disease-free survival. PLOD2 expression was identified as a significant, independent factor of poor prognosis (P=0.013). PLOD2 is a potential novel prognostic factor for HCC patients following surgery.

Significance of dihydropyrimidine dehydrogenase and thymidylate synthase mRNA expressions in hepatocellular carcinoma

The significance of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) gene expressions for the post-surgical prognosis of hepatocellular carcinoma (HCC) has not yet been determined. In the present study, we clarified the significance of DPD and TS gene expressions for the prognosis of HCC. Seventy-four patients, who underwent curative hepatic resection for primary HCC, were evaluated. The DPD and TS mRNA levels of the resected HCC specimens were evaluated using a microdissection technique and quantative real-time RT-PCR. The patients were categorized into high and low groups for each mRNA based on the median value. Various clinicopathological factors, including prognosis, and proliferation index using Ki-67 staining were evaluated in association with the DPD and TS mRNA expression levels. The low DPD mRNA expression was related to younger age, advanced clinical stage, undifferentiated histology, and microscopic intrahepatic metastasis. The overall and recurrence-free survival were significantly lower in the low DPD group than in the high DPD group (P < 0.05). Furthermore, the proliferation index in the low DPD group was significantly higher than that in the high DPD group (P < 0.01). On the other hand, the high TS group showed a tendency of better prognosis than the low TS group, although it was not statistically significant. The low DPD mRNA expression is a significant poor prognostic factor. after curative resection of HCC.

Akt phosphorylation is a risk factor for early disease recurrence and poor prognosis in hepatocellular carcinoma

Patients with hepatocellular carcinoma (HCC) who showed early massive disease recurrence due to hematogenous intrahepatic metastasis after curative resection had a poor prognosis. The authors previously reported that Akt phosphorylation was correlated with hematogenous intrahepatic metastasis, using HCC cell lines. The authors analyzed clinicopathologic features and the status of selected biologic markers, including phosphorylated Akt, to identify risk factors for early disease recurrence and poor prognosis in HCC. In the current series, 49 postoperative patients developed intrahepatic disease recurrence within 6 months (Group 1) and 86 patients remained disease recurrence free > 3 years after resection (Group 2). Group 1 was further divided into 2 subgroups: 19 patients who died of disease recurrence within a year after resection (Group 1A) and 27 patients who survived > 1 year (Group 1B). Using univariate analysis, the risk factors for early disease recurrence were tumor size, macroscopic classification, tumor differentiation, microscopic capsule infiltration, microscopic portal vein (MPV) invasion, microscopic intrahepatic metastasis (MIM), and positive immunostaining for phosphorylated Akt, Ki-67, and p53 (P < 0.05). The risk factors for poor prognosis were the number of intrahepatic metastases, tumor differentiation, and positive immunostaining for phosphorylated Akt and Ki-67 (P < 0.05). Multivariate analysis revealed that the risk factors for early disease recurrence were MPV invasion, MIM, and positive immunostaining for phosphorylated Akt, and that the risk factors for poor prognosis were positive immunostaining for phosphorylated Akt and Ki-67 (P < 0.05). The current clinical study showed the critical involvement of Akt phosphorylation in the aggressiveness of HCC. The potential benefits of surgery should be assessed carefully in patients with any of these risk factors.