Quantitative Assessment of Hepatic Steatosis Using Label-Free Multiphoton Imaging and Customized Image Processing Program.

Nonalcoholic fatty liver disease is rapidly becoming one of the most common causes of chronic liver disease worldwide and is the leading cause of liver-related morbidity and mortality. A quantitative assessment of the degree of steatosis would be more advantageous for diagnostic evaluation and exploring the patterns of disease progression. Here, multiphoton microscopy, based on the second harmonic generation and 2-photon excited fluorescence, was used to label-free image the samples of nonalcoholic fatty liver. Imaging results confirm that multiphoton microscopy is capable of directly visualizing important pathologic features such as normal hepatocytes, hepatic steatosis, Mallory bodies, necrosis, inflammation, collagen deposition, microvessel, and so on and is a reliable auxiliary tool for the diagnosis of nonalcoholic fatty liver disease. Furthermore, we developed an image segmentation algorithm to simultaneously assess hepatic steatosis and fibrotic changes, and quantitative results reveal that there is a correlation between the degree of steatosis and collagen content. We also developed a feature extraction program to precisely display the spatial distribution of hepatocyte steatosis in tissues. These studies may be beneficial for a better clinical understanding of the process of steatosis as well as for exploring possible therapeutic targets.

Detection of fibrotic changes in the progression of liver diseases by label-free multiphoton imaging.

Collagen fibers play an important role in the progression of liver diseases. The formation and progression of liver fibrosis is a dynamic pathological process accompanied by morphological changes in collagen fibers. In this study, we used multiphoton microscopy for label-free imaging of liver tissues, allowing direct detection of various components including collagen fibers, tumors, blood vessels, and lymphocytes. Then, we developed a deep learning classification model to automatically identify tumor regions, and the accuracy reaches 0.998. We introduced an automated image processing method to extract eight collagen morphological features from various stages of liver diseases. Statistical analysis showed significant differences between them, indicating the potential use of these quantitative features for monitoring fibrotic changes during the progression of liver diseases. Therefore, multiphoton imaging combined with automatic image processing method would hold a promising future in rapid and label-free diagnosis of liver diseases.

Aberrant regulation of autophagy disturbs fibrotic liver regeneration after partial hepatectomy.

Reports indicate that autophagy is essential for maintaining hepatocyte proliferative capacity during liver regeneration. However, the role of autophagy in fibrotic liver regeneration is incompletely elucidated. We investigated the deregulation of autophagic activities in liver regeneration after partial hepatectomy using a CCl4-induced fibrosis mouse model. The baseline autophagic activity was significantly increased in the fibrotic liver. After 50% partial hepatectomy (PHx), liver regeneration was remarkably decreased, accompanied by increased hepatocyte size and binuclearity ratio. Moreover, the expression of autophagy-related proteins was functionally deregulated and resulted in a reduction in the number of autophagosome and autophagosome-lysosome fusions. We further showed upregulation of autophagy activities through verapamil administration, improved hepatocyte proliferation capacity, and restricted cellular hypertrophy and binuclearity ratio. In conclusion, we demonstrated that the impairment of liver regeneration is associated with aberrant autophagy in fibrotic liver and that enhancing autophagy with verapamil may partially restore the impaired liver regeneration following PHx.

Verapamil induces autophagy to improve liver regeneration in non-alcoholic fatty liver mice.

Verapamil can restore intracellular calcium homeostasis, increase the fusion of autophagosomes and lysosomes, reduce lipid droplet accumulation and inhibit inflammation and insulin resistance in high-fat-fed mice. The present study aimed to investigate verapamil's effect and its underlying liver regeneration mechanism in mice with non-alcoholic fatty liver. After 50% hepatectomy was performed, the changes of autophagy and liver regeneration were evaluated by detecting cell proliferation and autophagy at each time point. Then, 25mg/kg verapamil was injected intraperitoneally for 10 d before an operation in the mild to moderate fatty liver and severe fatty liver groups. The control group and mild to moderate fatty liver group reached the peak of proliferation at 24-48h after operation, and the mice with severe fatty liver and steatohepatitis reached the peak at 48-72h. Autophagy in the normal group and mild to moderate fatty liver group reached the peak 48 hours after operation. Verapamil injection can enhance autophagy, reduce the weight of fatty liver mice, improve liver function and liver regeneration. Verapamil can induce autophagy, improve hepatocyte function and promote hepatocyte regeneration through the mTOR independent signaling pathway, thus improving the process of liver regeneration after partial hepatectomy.

A Prognostic Scoring System for Predicting Overall Survival of Patients with the TNM 8th Edition Stage I and II Hepatocellular Carcinoma After Surgery: A Population-Based Study.

PURPOSE: Postoperative prognosis prediction models for patients with stage Ⅰ and Ⅱ hepatocellular carcinoma (HCC) according to the 8th edition of the Tumor-Node-Metastasis staging system after surgery are rare. This study aimed to build a prognostic score to predict survival outcomes and stratify these patients into different prognostic strata. PATIENTS AND METHODS: We developed a web-based nomogram that incorporated four selected risk factors based on the multivariate Cox regression, using a training set (n=3567) from the Surveillance, Epidemiology, and End Results (SEER) database. It was validated with an independent internal set from the SEER database (n=1783) and an external validation set of 516 Chinese patients. The predictive performance and discrimination ability of our model were further evaluated and compared with those of the conventional HCC staging systems. RESULTS: Our nomogram consistently outperformed the conventional staging systems in the training, internal validation set, and external validation set. We quantified the nomogram model into a numerical SNIG (an abbreviation of the incorporated variables - size, number, MVI, and grade) score by summing the points assigned to each incorporated variable, leading to the optimal cut-off values of 6 and 10, which could stratify patients into 3 categories (SNIG score <6, 6-10, ≥10). This yielded significantly different median overall survivals (interquartile ranges) of 42.0 (20.0-72.0) and 37.0 (17.0-67.0); 28.0 (12.0-60.0) and 42.0 (21.75-82.0); 40.0 (18.0-70.0) and 29.0 (11.5-61.0) months for the 3 categories in the entire SEER and external validation sets, respectively. CONCLUSION: We developed a web-based SNIG model to graphically and numerically predict the overall survival of stage Ⅰ and Ⅱ HCC. This scoring system may shed light on risk stratification for these patients in clinical practice and clinical trials.

Inhibition of lncRNA KCNQ1OT1 Improves Apoptosis and Chemotherapy Drug Response in Small Cell Lung Cancer by TGF-ß1 Mediated Epithelial-to-Mesenchymal Transition.

PURPOSE: Drug resistance is one of the main causes of chemotherapy failure in patients with small cell lung cancer (SCLC), and extensive biological studies into chemotherapy drug resistance are required. MATERIALS AND METHODS: In this study, we performed lncRNA microarray, in vitro functional assays, in vivo models and cDNA microarray to evaluate the impact of lncRNA in SCLC chemoresistance. RESULTS: The results showed that KCNQ1OT1 expression was upregulated in SCLC tissues and was a poor prognostic factor for patients with SCLC. Knockdown of KCNQ1OT1 inhibited cell proliferation, migration, chemoresistance and promoted apoptosis of SCLC cells. Mechanistic investigation showed that KCNQ1OT1 can activate transforming growth factor-ß1 mediated epithelial-to-mesenchymal transition in SCLC cells. CONCLUSION: Taken together, our study revealed the role of KCNQ1OT1 in the progression and chemoresistance of SCLC, and suggested KCNQ1OT1 as a potential diagnostic and prognostic biomarker in SCLC clinical management.

Label-Free Identification of Early Stages of Breast Ductal Carcinoma via Multiphoton Microscopy.

Breast cancer can be cured by early diagnosis. Appropriate and effective clinical treatment benefits from accurate pathological diagnosis. However, due to the lack of effective screening and diagnostic imaging methods, early stages of breast cancer often progress to malignant breast cancer. In this study, multiphoton microscopy (MPM) via two-photon excited fluorescence combined with second-harmonic generation was used for identifying the early stages of breast ductal carcinoma. The results showed differences in both cytological features and collagen distribution among normal breast tissue, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, and high-grade ductal carcinoma in situ with microinvasion. Furthermore, three features extracted from the MPM images were used to describe differences in cytological features, collagen density, and basement membrane circumference in the early stages of breast ductal carcinoma. They revealed that MPM has the ability to identify early stages of breast ductal carcinoma label-free, which would contribute to the early diagnosis and treatment of breast cancer. This study may provide the groundwork for the further application of MPM in the clinic.

Immunohistochemical Signature Add Prognostic Value in Patients With Early and Intermediate Hepatocellular Carcinoma Underwent Curative Liver Resection.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide; however, accurate prognostic tools are still lacking. We aimed to identify immunohistochemistry (IHC)-based signature as a prognostic classifier to predict recurrence and survival in patients with HCC at Barcelona Clinic Liver Cancer (BCLC) early- and immediate-stage. In total, 567 patients who underwent curative liver resection at two independent centers were enrolled. The least absolute shrinkage and selection operator regression model was used to identify significant IHC features, and penalized Cox regression was used to further narrow down the features in the training cohort (n = 201). The candidate IHC features were validated in internal (n = 101) and external validation cohorts (n = 265). Three IHC features, hepatocyte paraffin antigen 1, CD34, and Ki-67, were identified as candidate predictors for recurrence-free survival (RFS), and were used to categorize patients into low- and high-risk recurrence groups in the training cohort (P < 0.001). The discriminative performance of the 3-IHC_based classifier was validated using internal and external cohorts (P < 0.001). Furthermore, we developed a 3-IHC_based nomogram integrating the BCLC stage, microvascular invasion, and 3-IHC_based classifier to predict 2- and 5-year RFS in the training cohort; this nomogram exhibited acceptable area under the curve values for the training, internal validation, and external validation cohorts (2-year: 0.817, 0.787, and 0.810; 5-year: 0.726, 0.662, and 0.715; respectively). The newly developed 3-IHC_based classifier can effectively predict recurrence and survival in patients with early- and intermediate-stage HCC after curative liver resection.

Hormone Receptor and Human Epidermal Growth Factor Receptor 2 Detection in Invasive Breast Carcinoma: A Retrospective Study of 12,467 Patients From 19 Chinese Representative Clinical Centers.

INTRODUCTION: We evaluated the current status of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) detection in invasive breast carcinoma (IBC) in various laboratories across China. MATERIALS AND METHODS: The Breast Pathology Study Group of the Chinese Society of Pathologists collected HR and HER2 data from 12,467 IBC cases from 19 representative clinical centers in China. The data from every center were compared with the pooled data from the other centers. RESULTS: The assessment of HR status showed that the overall positive estrogen receptor (ER) and progesterone receptor (PR) rates were 71.7% and 63.7% (range, 60.9%-87.9% and 43.9%-84.8%), respectively. The ER results in 3 centers and the PR results in 6 centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). Of the 12,467 cases, 62.4% were ER+/PR+, 9.3% were ER+/PR-, 1.3% were ER-/PR+, and 27.0% were ER-/PR-. The assessment of HER2 status showed that the overall positive rate of HER2 (with a definition of immunohistochemistry [IHC] 3+ or IHC2+/ in situ hybridization-positive) was 24.7% (range, 13.7%-35.7%) in each center. Three centers were outside the 99.5% confidence interval and were considered to be outliers (P < .0005). The proportion of HER2 IHC3+ was 21.1%. The positive rates of HER2 gene amplification in IHC 0/1+, 2+, 3+ cases were 2.0%, 17.6%, and 85.9%, respectively. CONCLUSIONS: As the largest study of HR and HER2 status in Chinese patients with IBC, the data from the present study have indicated that the overall rates of HR and HER2 were comparable to those reported in previous studies. However, the rates varied among the laboratories. Individual centers had not met the target values, and they need to improve the detection.

APOBEC3B High Expression in Gastroenteropancreatic Neuroendocrine Neoplasms and Association With Lymph Metastasis.

PURPOSE: Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3B (APOBEC3B) is known as a source of mutations in multiple cancers. Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are a group of heterogeneous tumors. However, the expression and significance of APOBEC3B in GEP-NENs remains unclear. MATERIALS AND METHODS: A total of 158 cases of GEP-NENs, including 78 cases of biopsy or endoscopic submucosal dissection resection specimens and 83 cases of surgical resection specimens were collected in this study. The cases were grouped according to tumor classification grade, including 42 cases of neuroendocrine tumors G1 (NET G1), 36 cases of NET G2, 36 cases of NET G3, 44 cases of neuroendocrine carcinoma (NEC). All of the 158 tumors were immunohistochemically studied using a polyclonal antibody against APOBEC3B. We evaluated APOBEC3B expression in GEP-NENs and investigated the relationships among the immunoreactivity of APOBEC3B, clinical and pathologic features, such as age, sex, tumor site, Ki67 cell proliferation index, and lymph metastasis. RESULTS: A total of 33 cases (78.6%) of NET G1 showed high expression of APOBEC3B. A total of 28 cases (77.8%) of NET G2 demonstrated high expression of APOBEC3B. In NET G3 and NEC cases, the positive rates were 52.8% and 2.3%, respectively. The expression of APOBEC3B in NETs was significantly higher than that in NECs, NET G1 and NET G2 were higher than NET G3, and the difference was statistically significant. APOBEC3B high expression cases have lower lymph node metastasis rate, lower Ki67 cell proliferation index. CONCLUSIONS: In this study, APOBEC3B is highly expressed in GEP-NETs and is a predictor of lymph node metastasis in NET G3 and NEC cases. These findings might provide new insights into the biological mechanisms of GEP-NENs tumorigenesis and progression.

Identifying Two Common Types of Breast Benign Diseases Based on Multiphoton Microscopy.

Multiphoton microscopy has attracted increasing attention and investigations in the field of breast cancer, based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG). However, the incidence of breast benign diseases is about 5 to 10 times higher than breast cancer; up to 30% of women suffer from breast benign diseases and require treatment at some time in their lives. Thus, in this study, MPM was applied to image fibroadenoma and fibrocystic lesion, which are two of the most common breast benign diseases. The results show that MPM has the capability to identify the microstructure of lobule and stroma in normal breast tissue, the interaction of compressed ducts with surrounding collagen fiber in fibroadenoma, and the architecture of cysts filled with cystic fluid in fibrocystic disease. These findings indicate that, with integration of MPM into currently accepted clinical imaging system, it has the potential to make a real-time diagnosis of breast benign diseases in vivo, as well as breast cancer.

Monitoring the progression from intraductal carcinoma to invasive ductal carcinoma based on multiphoton microscopy.

Intraductal carcinoma is a precancerous lesion of the breast and the immediate precursor of invasive ductal carcinoma. Multiphoton microscopy (MPM) was used to monitor the progression from intraductal carcinoma to invasive ductal carcinoma, which can improve early detection of precursor lesions and halt progression to invasive neoplastic disease. It was found that MPM has the capability to reveal the qualitative changes in features of cells, structure of basement membranes, and architecture of collagens during the development from intraductal carcinoma to invasive ductal carcinoma, as well as the quantitative alterations in nuclear area, circle length of basement membrane, and collagen density. Combined with intra-fiberoptic ductoscopy or transdermal biopsy needle, MPM has the potential to provide immediate histological diagnosis of tumor progression in the field of breast carcinoma.

Monitoring morphological alterations during invasive ductal breast carcinoma progression using multiphoton microscopy.

Morphological alteration of cells and matrices is critical for tumor initiation and progression. Monitoring these alterations during tumor progression is vitally important for making real-time histological diagnoses of tumor staging. In this study, 20 pairs of normal and cancerous human breast tissues were imaged by multiphoton microscopy (MPM), and nuclear area and collagen density were quantified by LSM 5 software (version 3.2). Comparison of MPM images from normal breast tissue with low- and high-grade invasive ductal carcinoma (IDC) lesions clearly showed changes in both cellular features and extracellular matrix architecture during IDC development. Moreover, analysis of nuclear area and collagen density established a quantitative link between these two morphological features and progression of IDC. Present results demonstrated that MPM can provide both qualitative and quantitative evaluations of tumor progression. With additional development, this technique has the potential to make real-time histological diagnoses of tumor staging and guide development of efficacious clinical therapies.

Expression of microsomal prostaglandin E2 synthase-1 and its role in human hepatocellular carcinoma.

Microsomal prostaglandin E2 synthase-1 (mPGES1), an inducible enzyme similar to cyclooxygenase-2, functions downstream of cyclooxygenase-2 in the synthesis of prostaglandin E2. It contributes to carcinogenesis in a variety of tumors. Here, mPGES1 expression was assessed using immunohistochemistry of tissue microarrays containing a total of 100 hepatocellular carcinoma (HCC) tissue samples, 100 peritumoral liver tissue samples, and 13 normal liver tissue samples. The expression of mPGES1 was significantly increased in the HCC tissue samples (P < .001), relative to normal liver tissue. Second, there was a significant positive correlation between mPGES1 expression and the Barcelona Clinic Liver Cancer stage (P < .001) in HCC tissue samples. This correlation was also observed with encapsulation (P = .004) and portal vein thrombosis (P < .001). In addition, the lentiviral vector (Lv-mPGES1-shRNA), which down-regulates mPGES1, inhibited tumor growth in an HCC animal model. Taken together, mPGES1 expression was associated with multiple malignant characteristics and enhanced tumorigenesis in HCC and may serve as an important clinical and pharmacologic biomarker.

[Role of mPGES-1 in the occurrence, progression, metastasis and invasion of hepatocellular carcinoma].

OBJECTIVE: To study the expression of mPGES-1 in hepatocellular carcinoma (HCC), observe the effect of MK886 on down-regulation of mPGES-1 gene expression on the biology of human hepatocarcinoma cell line HepG2 and to investigate its significance in the occurrence, progression, metastasis and invasion. METHODS: HCC tissues, para-carcinoma tissues, far-carcinoma tissues and normal liver tissues were collected. The expressions of mPGES-1 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The proliferation, adherence, migration and invasion abilities of HepG2 cells interfered by MK886 were assessed by MTT and transwell technique respectively. RESULTS: The expression of mPGES-1 in HCCs was higher than that in normal liver tissues (P < 0.01), which increased following histological grade. Furthermore, mPGES-1 expression level was higher in the capsule invasion and metastasis tumor than in primary locus. A significant dose-dependent down-regulation of expressions of mPGES-1 gene mRNA and protein were observed in HepG2 cells when MK886 was given for 48 h (F = 140.402, P < 0.01; a'= 0.00714, P < 0.01). Compared with the control group, the growth inhibitory rate of HepG2 cell was observed significantly time and dose-dependent when MK886 was given. The rate of adhesion cells in experimental groups were 85.3% ± 1.3%, 70.5% ± 1.5% and 45.8% ± 2.4%, respectively, less than that in control group 100.0% ± 0 (F = 626.313, P < 0.01). The migration cells was 92.47 ± 1.90, 62.63 ± 1.96 and 37.33 ± 0.83 respectively in the experimental groups after 24 h, lower than that in the control group 128.93 ± 2.60 (F = 1253.805, P < 0.01). The invasion assay revealed that the invading cells were 41.67 ± 1.30, 25.47 ± 1.30 and 13.93 ± 1.66 in the experimental groups, in contrast to 55.67 ± 2.08 in control group after 24 h. The difference between these groups was significant (F = 372.615, P < 0.01). The numbers of adhesion, migration and invasion of HepG2 cells were dose-dependent in MK886 groups. CONCLUSION: Over-expression of mPGES-1 was associated with the tumorigenesis and progression of HCC. The down-regulation of mPGES-1 gene expression might indicated the decrease of the invasion and metastasis of HCC.