Deep Learning-Based Classification of Hepatocellular Nodular Lesions on Whole-Slide Histopathologic Images.

BACKGROUND & AIMS: Hepatocellular nodular lesions (HNLs) constitute a heterogeneous group of disorders. Differential diagnosis among these lesions, especially high-grade dysplastic nodules (HGDNs) and well-differentiated hepatocellular carcinoma (WD-HCC), can be challenging, let alone biopsy specimens. We aimed to develop a deep learning system to solve these puzzles, improving the histopathologic diagnosis of HNLs (WD-HCC, HGDN, low-grade DN, focal nodular hyperplasia, hepatocellular adenoma), and background tissues (nodular cirrhosis, normal liver tissue). METHODS: The samples consisting of surgical and biopsy specimens were collected from 6 hospitals. Each specimen was reviewed by 2 to 3 subspecialists. Four deep neural networks (ResNet50, InceptionV3, Xception, and the Ensemble) were used. Their performances were evaluated by confusion matrix, receiver operating characteristic curve, classification map, and heat map. The predictive efficiency of the optimal model was further verified by comparing with that of 9 pathologists. RESULTS: We obtained 213,280 patches from 1115 whole-slide images of 738 patients. An optimal model was finally chosen based on F1 score and area under the curve value, named hepatocellular-nodular artificial intelligence model (HnAIM), with the overall 7-category area under the curve of 0.935 in the independent external validation cohort. For biopsy specimens, the agreement rate with subspecialists' majority opinion was higher for HnAIM than 9 pathologists on both patch level and whole-slide images level. CONCLUSIONS: We first developed a deep learning diagnostic model for HNLs, which performed well and contributed to enhancing the diagnosis rate of early HCC and risk stratification of patients with HNLs. Furthermore, HnAIM had significant advantages in patch-level recognition, with important diagnostic implications for fragmentary or scarce biopsy specimens.

CD45- erythroid progenitor cells promote lymph node metastasis in gastric cancer by inducing a hybrid epithelial/mesenchymal state in lymphatic endothelial cells.

BACKGROUND AND AIMS: Specific mechanisms of lymph node (LN) metastasis in early-stage gastric cancer (GC) have not been elucidated. The role of anemia, a vital clinical feature of GC, in LN metastasis is also unclear. Since the number of erythroid progenitor cells (EPCs) is increased in chronic anemia, we investigated its association with LN metastasis in GC. METHODS: Flow cytometry and immunofluorescence analyses were performed to sort and study EPCs from the circulation and tumors of patients with stage I-III GC. The effect of these EPCs on the activation of T and B cells and on the functions of lymphatic endothelial cells (LECs) was determined, and their ability to promote LN metastasis was evaluated using a footpad-popliteal LN metastasis model based on two human adenocarcinoma GC cell lines in nude mice. The prognostic value of EPCs was also analyzed. RESULTS: The proportion of CD45- EPCs was higher in the mononuclear cells in the circulation, tumors, and LNs of GC patients with LN metastasis (N+) than in those of GC patients without LN metastasis (N0). In N+ patients, CD45- EPCs were more abundant in metastatic LNs than in non-metastatic LNs. Lymphatic vessel endothelial hyaluronan receptor 1 immunoreactivity in tumors revealed that CD45- EPCs were positively associated with nodal stages and lymph vessel density. Furthermore, CD45- EPCs increased LEC proliferation and migration through their S100A8/A9 heterodimer-induced hybrid epithelial/mesenchymal (E/M) state; however, they did not influence the invasion and tubulogenesis of LECs or T and B cell proliferation. CD45- EPCs promoted LN metastasis in vivo; the S100A8/A9 heterodimer mimicked this phenomenon. Finally, CD45- EPCs predicted the overall and disease-free survival of stage I-III GC patients after radical resection. CONCLUSIONS: The CD45- EPCs accumulated in GC tissues and metastatic LNs and promoted LN metastasis via the S100A8/9-induced hybrid E/M state of LECs, which was the specific mechanism of LN metastasis in the early stages of GC.

Epstein-Barr virus-derived circular RNA LMP2A induces stemness in EBV-associated gastric cancer.

Cancer stem cells (CSCs) are cancer-initiating cells that are not only a source of tumorigenesis but also the cause of tumour progression, metastasis and therapy resistance. EBV-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer with unique clinicopathological and molecular features. However, whether CSCs exist in EBVaGC, and the tumorigenic mechanism of EBV, remains unclear. Here, NOD/SCID mice were injected subcutaneously with the EBVaGC cell line SNU719 and treated with 5-fluorouracil weekly. Successive generations of xenografts yielded a highly malignant EBVaGC cell line, SNU-4th, which displays properties of CSCs and mainly consists of CD44+ CD24- cells. In SNU-4th cells, an EBV-encoded circRNA, ebv-circLMP2A, expression increased and plays crucial roles in inducing and maintaining stemness phenotypes through targeting miR-3908/TRIM59/p53 axis. Additionally, high expression of ebv-circLMP2A is significantly associated with metastasis and poor prognosis in patients with EBVaGC. These findings not only provide evidence for the existence of CSCs in EBVaGC and elucidate the pathogenic mechanism of ebv-circLMP2A in EBVaGC, but also provide a promising therapeutic target for EBVaGC.

Liver involvement in patients with erythropoietic protoporphyria: retrospective analysis of clinicopathological features of 5 cases.

Erythropoietic protoporphyria (EPP) is a rare inherited disease whose morbidity is about 1:75,000 to 1:200,000. It is caused by the deficiency of porphyrin ferrochelatase (FECH). Liver involvement in EPP is even rarer. The diagnosis of EPP with liver involvement mainly relies on clinical manifestations, laboratory examinations, histopathological examinations and genetic testing, which is still a huge challenge for both clinicians and pathologists. Here, 5 cases of EPP with liver injury were collected, and the clinicopathological features of these patients were analyzed. The clinical manifestations and laboratory examinations varied from person to person, whereas the liver biopsies showed that there were dark brown deposits within the hepatocytes, Kupffer cells, bile canaliculi and the lumen of bile ducts, which was a constant finding by histopathological examination. Gene tests were conducted in two of the five cases, and the results confirmed the diagnosis. Fully understanding of the diseases can help us reduce the rate of missed diagnosis and provide proper treatment as early as possible.

A urine extracellular vesicle circRNA classifier for detection of high-grade prostate cancer in patients with prostate-specific antigen 2-10 ng/mL at initial biopsy.

The aim of this study was to identify a urine extracellular vesicle circular RNA (circRNA) classifier that could detect high-grade prostate cancer (PCa) of Grade Group (GG) 2 or greater. For this purpose, we used RNA sequencing to identify candidate circRNAs from urinary extracellular vesicles from 11 patients with high-grade PCa and 11 case-matched patients with benign prostatic hyperplasia. Using ddPCR in a training cohort (n = 263), we built a urine extracellular vesicle circRNA classifier (Ccirc, containing circPDLIM5, circSCAF8, circPLXDC2, circSCAMP1, and circCCNT2), which was evaluated in two independent cohorts (n = 497, n = 505). Ccirc showed higher accuracy than two standard of care risk calculators (RCs) (PCPT-RC 2.0 and ERSPC-RC) in both the training cohort and the validation cohorts. In all three cohorts, this novel urine extracellular vesicle circRNA classifier plus RCs was statistically more predictive than RCs alone for predicting ≥ GG2 PCa. This assay, which does not require precollection digital rectal examination nor special handling, is repeatable, noninvasive, and can be easily implemented as part of the basic clinical workflow.

α-Fetoprotein mRNA in situ hybridisation is a highly specific marker of hepatocellular carcinoma: a multi-centre study.

BACKGROUND: Pathologic diagnosis of hepatocellular carcinoma (HCC) can be challenging in differentiating from benign and non-hepatocytic malignancy lesions. The aim of this study was to investigate the potential utility of α-fetoprotein (AFP) mRNA RNAscope, a sensitive and specific method, in the diagnosis of HCC. METHODS: Three independent retrospective cohorts containing 2216 patients with HCC, benign liver lesions, and non-hepatocytic tumours were examined. AFP was detected using ELISA, IHC (Immunohistochemistry), and RNAscope. Glypican3 (GPC3), hepatocyte paraffin-1 (HepPar-1), and arginase-1 (Arg-1) proteins were detected using IHC. RESULTS: AFP RNAscope improved the HCC detection sensitivity by 24.7-32.7% compared with IHC. In two surgical cohorts, a panel of AFP RNAscope and GPC3 provided the best diagnostic value in differentiating HCC from benign hepatocytic lesions (AUC = 0.905 and 0.811), and a panel including AFP RNAscope, GPC3, HepPar-1, and Arg-1 yielded the best AUC (0.971 and 0.977) when distinguishing HCC from non-hepatocytic malignancies. The results from the liver biopsy cohort were similar, and additional application of AFP RNAscope improved the sensitivity by 18% when distinguishing HCC from benign hepatocytic lesions. CONCLUSIONS: AFP mRNA detected by RNAscope is highly specific for hepatocytic malignancy and may serve as a novel diagnostic biomarker for HCC.

CD44+CD24-/low sphere-forming cells of EBV-associated gastric carcinomas show immunosuppressive effects and induce Tregs partially through production of PGE2.

EBV-associated gastric carcinoma (EBVaGC) is accompanied by massive lymphocyte infiltration, but therapy resistance and tumor progression still occur in patients with EBVaGC. Cancer stem cells (CSCs) are reported to possess immunomodulatory ability that allows them to resist immune-mediated rejection for many tumor types. However, whether and how CSCs in EBVaGC exhibit immunosuppression has not yet been elucidated. We isolated CSC-like sphere-forming cells (SFCs) from EBVaGC cell line SNU-719 using the cancer sphere method. We validated their CSC-associated properties in the expression of the epithelial-mesenchymal transition (EMT)-related genes, the ability to form colonies, and resistance to chemotherapy drug-induced apoptosis and explored their immunomodulatory ability using the coculture system with PBMC (peripheral blood mononuclear cell). These CSC-like SFCs were CD44+CD24-/low and were more tumorigenic than the parental SNU-719 cells in the xenograft mouse model. Remarkably, in the tumor-PBMC co-culturing experiments, these EBVaGC SFCs demonstrated profound immunosuppression by inhibiting the proliferation of PBMCs and T cell activation as well as inducing the generation of regulatory T cells (Tregs). Furthermore, the induction of Tregs was partially dependent on prostaglandin E2 (PGE2) produced from SFCs. Moreover, the presence of high CD44+CD24-/low cells in tumor tissues predicted a decreased disease-free survival in patients with EBVaGC. Our study collectively confirmed the existence and immune resistance of CSCs in EBVaGC and offers new insights into the development of novel anti-EBVaGC strategies by targeting CSCs.

LncRNA PVT1 triggers Cyto-protective autophagy and promotes pancreatic ductal adenocarcinoma development via the miR-20a-5p/ULK1 Axis.

BACKGROUND: Defective autophagy is thought to contribute to the pathogenesis of many diseases, including cancer. Human plasmacytoma variant translocation 1 (PVT1) is an oncogenic long non-coding RNA that has been identified as a prognostic biomarker in pancreatic ductal adenocarcinoma, but how PVT1 operates in the regulation of autophagy in pancreatic ductal adenocarcinoma (PDA) is unclear. METHODS: PVT1 expression level was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and hybridization in situ (ISH). Western blot or qRT-PCR was performed to assess the ULK1 protein or mRNA level. Autophagy was explored via autophagic flux detection under a confocal microscope and autophagic vacuoles investigation under a transmission electron microscopy (TEM). The biological role of PVT1 in autophagy and PDA development was determined by gain-of-function and loss-of-function assays. RESULTS: We found that PVT1 levels paralleled those of ULK1 protein in PDA cancer tissues. PVT1 promoted cyto-protective autophagy and cell growth by targeting ULK1 both in vitro and in vivo. Moreover, high PVT1 expression was associated with poor prognosis. Furthermore, we found that PVT1 acted as sponge to regulate miR-20a-5p and thus affected ULK1 expression and the development of pancreatic ductal adenocarcinoma. CONCLUSIONS: The present study demonstrates that the "PVT1/miR-20a-5p/ULK1/autophagy" pathway modulates the development of pancreatic ductal adenocarcinoma and may be a novel target for developing therapeutic strategies for pancreatic ductal adenocarcinoma.

Collapsin response mediator protein 4 promotor methylation level as a potential predictor for diagnosing primary malignant lymphoma of the prostate.

BACKGROUND: Primary malignant lymphoma of the prostate (PMLP) is prone to occur in the elderly, and it has no significant correlation with lactate dehydrogenase (LDH) and prostate specific antigen (PSA). Clinical symptoms and imaging data of PMLP remain unspecific, and its prognosis is poor. A previous result showed that collapsin response mediator protein 4 (CRMP4) promotor methylation can be used as a predictor for lymph node metastases in prostate biopsies. However, the relationship between CRMP4 promotor methylation and PMLP has not been studied. METHODS: We investigated the clinicopathological features of PMLP and the significance of CRMP4 methylation in PMLP. The clinical data and diagnosis information of 10 patients with PMLP were retrospectively analyzed. The CRMP4 promotor methylation level in paraffin-embedded tissues of the 10 patients with PMLP were determined and then compared to limited prostate cancer (LPCa) and its negative lymph node tissue [LPCa-LN (-) (10 cases)] and also to metastatic prostate adenocarcinoma (mPCa) and its positive lymph node tissue [mPCa-LN (+) (10 cases)]. Methylation of the CRMP4 promotor in each group was analyzed statistically. A receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of CRMP4 methylation in PMLP. RESULTS: The average methylation value of CRMP4 in 10 PMLP patients, 20 cases of prostate adenocarcinoma tissue, 10 cases LPCa-LN (-) and 10 cases mPCa-LN (+) were 42.3, 30.6, 6.7 and 20.3%, respectively. A Kruskal-Wallis test showed that the difference of CRMP4 methylation was significant (X2 = 38.0, P < 0.001). An ROC curve analysis found that CRMP4 methylation > 40.9% could diagnose PMLP. This method had 90% sensitivity and 95% specificity under conditions of CRMP4 methylation > 40.9%. The area under the curve (AUC) was 0.957. CONCLUSIONS: Methylation of the CRMP4 gene was significantly increased in PMLP, and it is expected to become a new predictor for PMLP.

Comparative analysis of 22 Epstein-Barr virus genomes from diseased and healthy individuals.

Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variations might influence EBV-associated diseases and their geographical patterns. In the present study, 22 EBV whole-genome sequences from diseased and healthy individuals were analysed to explore EBV sequence variations at the whole-genome level. We found that the 22 EBV genomes were generally highly similar to each other at the genome level. However, varying degrees of genetic diversity were detected across the entire genome, especially in the latent genes. In contrast, the sequences of promoters and non-coding RNAs were strictly conserved. These findings suggested that both latent genes and non-coding RNAs play important roles in the EBV life cycle. When we investigated changes in known T-cell epitopes in some latent and lytic proteins, we observed that some T-cell epitopes were changed, while others were conserved. These findings indicate that the effect of EBV variations in protein sequences that seem to have been selected by the host immune system should be considered when conducting EBV-targeted immunotherapy. Taken together, our results provide a global view of EBV genome sequence variation, which not only is important for designing vaccines and immunotherapy for EBV but also adds to the understanding of EBV biology and the relationships between viral sequence variation and EBV-associated diseases.

Prospective validation of DACH2 as a novel biomarker for prediction of metastasis and prognosis in muscle-invasive urothelial carcinoma of the bladder.

Metastasis is the main cause of death from muscle-invasive urothelial carcinoma of the bladder (UCB), and the metastatic potential of tumors is often unpredictable. The role of Dachshund homolog 2 gene (DACH2) in tumorigenesis remains unexplored. We aimed to investigate whether DACH2 can be used as a biomarker to predict metastasis and prognosis of muscle-invasive UCB in a sequential training and validation fashion. For the training set (n = 40), compared with UCB patients without lymph node (LN) metastasis, both DACH2 protein and mRNA expression were greatly increased in case-matched patients with LN metastasis. For the independent validation set (n = 243), patients with primary UCB that did not express DACH2 had a longer metastasis-free survival (MFS) and overall survival (OS) than did those with tumors expressing DACH2 (5-year MFS: 88% [95% CI 80-96] versus 19% [95% CI 7-31], p < 0.001; 5-year OS: 93% [95% CI 87-99] versus 37% [95% CI 23-51], p < 0.001). Multivariable analysis of DACH2 status showed hazard ratios of 7.34 (95% CI 3.15-11.87, p < 0.001) for MFS and 3.96 (95% CI 2.04-7.16, p < 0.001) for OS which were much higher than hazard ratios associated with other independent risk factors. Collectively, DACH2 is an independent prognostic marker that can be used at initial diagnosis of UCB to identify patients who have a high potential to develop metastasis.

Is gastric lymphoepithelioma-like carcinoma a special subtype of EBV-associated gastric carcinoma? New insight based on clinicopathological features and EBV genome polymorphisms.

BACKGROUND: Gastric lymphoepithelioma-like carcinoma (LELC) is a rare entity that is closely associated with Epstein-Barr virus (EBV). However, the EBV latency pattern and genome polymorphisms in gastric LELC have not been systematically explored. METHODS: The clinicopathological features, EBV latency pattern and genome polymorphisms of EBV-positive gastric LELC in Guangzhou, southern China were investigated and compared with those of ordinary EBV-associated gastric carcinoma (EBVaGC) in the same area. RESULTS: Ten (1.42%) of 702 gastric carcinoma cases were identified as gastric LELC, in which eight (80%) cases were EBV-positive. The clinicopathological characteristics and EBV latency pattern of EBV-positive gastric LELC were similar to those of ordinary EBVaGC. In EBV genotype analysis, type A strain, type F, I, mut-W1/I, XhoI- and del-LMP1 variants were predominant among EBV-positive gastric LELCs, accounting for eight (100%), six (75%), eight (100%), seven (87.5%), five (62.5%) and six (75%) cases, respectively, which are similar to those in ordinary EBVaGC. For EBNA1 polymorphisms, the V-leu and P-ala subtypes were predominant in EBV-positive gastric LELC, which is different from the predominant V-val subtype in ordinary EBVaGC. EBV-positive gastric LELC has a favorable prognosis when compared to ordinary EBVaGC (median survival time 43.0 vs. 18.0 months). CONCLUSIONS: Gastric LELC is strongly associated with EBV and EBV-positive gastric LELC should be regarded as a special subtype of EBVaGC. This, to our best knowledge, is the first time in the world that the EBV latency pattern and genome polymorphisms of EBV-positive gastric LELC are systematically revealed.

Expression and clinical significance of hypoxia-induced long non-coding RNA TCONS_I2_00001955 in breast cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to play important roles in occurrence, development, and metastasis of various tumors. We aimed to screen long non-coding RNAs (lncRNAs) that promote invasion and metastasis of breast cancer cells under hypoxia, and investigate the relationship between lncRNA expression and clinicopathological features and prognosis in invasive breast cancer. METHODS: LncRNA microarray was used to screen the differentially expressed lncRNAs in MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines cultured under normoxia and hypoxia, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the microarray results. CCK8 and Transwell experiments were performed to identify the lncRNA that promote proliferation, migration, and invasion of breast cancer cells. Expression of the lncRNA and HIF-1α in invasive breast cancer was detected by RNAscope and immunohistochemistry, respectively. Correlation between the lncRNA expression and baseline characteristics was analyzed. Prognostic value of the lncRNA was evaluated using univariate and multivariate Cox regression. RESULTS: Expression of lncRNA TCONS_I2_00001955 in all the three breast cancer cells was increased under hypoxia. Overexpression of TCONS_I2_00001955 significantly enhanced proliferation, migration, and invasion of SKBR3 cells. Positive expression of TCONS_I2_00001955 was associated with recurrence, metastasis, and high expression of HIF-1α (P < 0.05), and it was an independent risk factor for poor disease-free survival of breast cancer. CONCLUSION: Hypoxia-induced lncRNA TCONS_I2_00001955 was associated with aggressive feature and poor prognosis of breast cancer.

Morphological changes in perisynaptic astrocytes induced by dopamine neuronal degeneration in the striatum of rats.

Introduction: The typical functionality of astrocytes was previously shown to be disrupted by Parkinson's disease (PD), which actively regulates synaptic neurotransmission. However, the morphological changes in astrocytes wrapping glutamatergic synapses in the striatum after dopamine (DA) neuronal degeneration is unclear. Methods: We utilized a range of methodologies, encompassing the 6-hydroxydopamine (6OHDA)-induced PD model, as well as techniques such as immunohistochemistry, Western blotting, immunofluorescence and immunoelectron microscopy (IEM) to delve into the consequences of DA neuronal degeneration on the morphological attributes of perisynaptic astrocytes. Results: Our findings demonstrated a notable rise in glial fibrillary acidic protein (GFAP) + astrocyte density and an upregulation in GFAP protein expression within the striatum due to DA neuronal degeneration, coincided with the enlargement, elongation, and thickening of astrocyte protuberances. However, the expression levels of glutamate transporter 1 (GLT1) and glutamine synthetase (GS), which are related to glutamate-glutamine cycle, were significantly reduced. Double immunofluorescence and IEM results indicated that different proportions of vesicular glutamate transporter 1 (VGlut1)+ and vesicular glutamate transporter 2 (VGlut2) + terminals were wrapped by astrocytes. Additionally, DA neuronal degeneration increased the percentage and area of VGlut1+ and VGlut2+ terminals wrapped by GFAP + astrocytes in the striatum. Furthermore, we noted that DA neuronal degeneration increased the percentage of VGlut1+ and VGlut2+ axo-spinous synapses wrapped by astrocytes but had no effect on axo-dendritic synapses. Conclusion: Hence, perisynaptic astrocytes wrapping striatal glutamatergic synapses exhibit substantial morphological and functional alterations following DA neuronal degeneration making them a potential target for therapeutic interventions in PD.

PPM1G promotes cell proliferation via modulating mutant GOF p53 protein expression in hepatocellular carcinoma.

The serine/threonine protein phosphatase family involves series of cellular processes, such as pre-mRNA splicing. The function of one of its members, protein phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G), remains unclear in hepatocellular carcinoma (HCC). Our results demonstrated that PPM1G was significantly overexpressed in HCC cells and tumor tissues compared with the normal liver tissues at both protein and RNA levels. High PPM1G expression is associated with shorter overall survival (p < 0.0001) and disease-free survival (p = 0.004) in HCC patients. Enhanced expression of PPM1G increases the cell proliferation rate, and knockdown of PPM1G led to a significant reduction in tumor volume in vivo. Further experiments illustrated that upregulated-PPM1G expression increased the protein expression of gain-of-function (GOF) mutant p53. Besides, the immunoprecipitation analysis revealed a direct interaction between PPM1G and GOF mutant p53. Collectively, PPM1G can be a powerful prognostic predictor and potential drug-target molecule.

Expert Consensus on Pathological Diagnosis of Intrahepatic Cholangiocarcinoma (2022 version).

Intrahepatic cholangiocarcinoma (iCCA) can originate from the large bile duct group (segment bile ducts and area bile ducts), small bile duct group (septal bile ducts and interlobular bile ducts), and terminal bile duct group (bile ductules and canals of Hering) of the intrahepatic biliary tree, which can be histopathological corresponding to large duct type iCCA, small duct type iCCA and iCCA with ductal plate malformation pattern, and cholangiolocarcinoma, respectively. The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies, tissue structures, growth patterns, invasive behaviors, immunophenotypes, molecular mutations, and surgical prognoses. For these reasons, this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA, mainly based on the 5th edition of the World Health Organization Classification of Tumours of the Digestive System.

An in situ molecular signature to predict early recurrence in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: To develop an in situ molecular signature to predict postsurgical recurrence in hepatocellular carcinoma (HCC) patients. METHODS: Immunohistochemistry was performed using tissue microarrays containing both tumoral and peri-tumoral regions of the advancing tumor edge from 336 HCC patients (289 were positive for hepatitis B virus) who underwent curative resection. Forty-nine variables were analyzed in the training set (n=151) using support vector machine and stepwise algorithms to develop a classifier to predict recurrence within 1 year, which was mainly caused by invasion or metastasis from the primary tumors. The classifier was further validated in an independent cohort of 185 patients (71 internal and 114 external). RESULTS: The final signature was composed of eight IHC features: CD80(T), B7-DC(T), HLA-DR(P), FasL(P), Bcl-2(T), Ki-67(T), cyclin D1(T), and CK19(T). In the independent test set, this classifier reliably predicted recurrence within 1 year (sensitivity, 69.1%; specificity, 65.0%) with an odds ratio of 4.149 (95% CI, 2.189-7.864). Based on a multivariate logistic model, the in situ molecular signature provided significant predictive power independent of tumor number, tumor size, vascular invasion and BCLC classification (p=0.001). The highest potential clinical impact of the classifier was observed in early-stage (BCLC classification 0-A) patients (p<0.0001), and the classifier was also predictive of the time-to-recurrence and overall survival (both p<0.0001). CONCLUSIONS: This in situ molecular classifier could provide a novel approach to identify patients who are at greatest risk for postsurgical recurrence of HCC and may benefit from intensive clinical follow-up or chemopreventive strategies.

Decreased K5 receptor expression in the retina, a potential pathogenic mechanism for diabetic retinopathy.

PURPOSE: Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor and specifically binds to the voltage-dependent anion channel believed to function as the K5 receptor (K5R). To investigate the role of K5R in diabetic retinopathy, the present study measured the expression levels of K5R in the retina of diabetic retinopathy models. In cultured retinal Müller cells, K5 inhibited vascular endothelial growth factor (VEGF) expression as shown with enzyme-linked immunosorbent assay and western blot analysis, suggesting that K5 has a direct effect on Müller cells. METHODS: To identify K5R in retinal Müller cells, ligand binding and competition assays as well as real-time reverse transcriptional polymerase chain reaction were performed in Müller cells. (125)I-K5 showed saturable binding to cultured Müller cells. The binding can be competed off by an excess amount of unlabeled K5 but not by angiostatin, demonstrating the specificity of the K5 binding to Müller cells. Consistent with the binding assay, reverse transcriptional polymerase chain reaction using voltage-dependent anion channel-specific primers detected the K5R mRNA in the Müller cells. RESULTS: Interestingly, K5R mRNA expression in Müller cells was downregulated by diabetic conditions including hypoxia and high glucose medium. Incubation with K5 ligand prevented hypoxia-induced downregulation of K5R. Furthermore, K5R expression was also downregulated in the retina of the oxygen-induced retinopathy model, a model of ischemia-induced retinal neovascularization. In a type 1 diabetic rat model, K5R expression in the retina was significantly suppressed in rats that had diabetes for 5 and 8 weeks. CONCLUSIONS: These results suggest that K5R is expressed in retinal Müller cells, which may mediate the inhibitory effect of K5 on VEGF expression. In diabetes conditions, K5R expression levels are decreased in the retina, which could contribute to the VEGF overexpression in diabetic retinopathy. These findings suggest that the decreased levels of K5R may also play a pathogenic role in diabetic retinopathy.