Cadherin- and Rigidity-Dependent Growth of Lung Cancer Cells in a Partially Confined Microenvironment.

During tumor development, cancer cells constantly confront different types of extracellular barriers. However, fundamental questions like whether tumor cells will continue to grow against confinement or away from it and what key factors govern this process remain poorly understood. To address these issues, here we examined the growth dynamics of human lung epithelial carcinoma A549 cells partially confined in micrometer-sized cylindrical pores with precisely controlled wall stiffness. It was found that, after reaching confluency, the cell monolayer enclosed by a compliant wall was able to keep growing and pushing the boundary, eventually leading to a markedly enlarged pore. In contrast, a much reduced in-plane growth and elevated strain level among cells were observed when the confining wall becomes stiff. Furthermore, under such circumstance, cells switched their growth from within the monolayer to along the out-of-plane direction, resulting in cell stacking. We showed that these observations can be well explained by a simple model taking into account the deformability of the wall and the threshold stress for inhibiting cell growth. Interestingly, cadherins were found to play an important role in the proliferation and stress buildup within the cell monolayer by aggregating at cell-cell junctions. The stiff confinement led to an elevated expression level of cadherins. Furthermore, inhibition of N-cadherin resulted in a significantly suppressed cell growth under the same confining conditions.

Long-term use of oral nucleos(t)ide analogues for chronic hepatitis B does not increase cancer risk - a cohort study of 44 494 subjects.

BACKGROUND: Patients with chronic hepatitis B (CHB) need long-term antiviral treatment with nucleos(t)ide analogues (NA). Animal studies suggest that some NA may increase cancer risk, but human data are lacking. AIM: To investigate cancer risks in patients with or without NA treatment. METHODS: We conducted a territory-wide cohort study using the database from Hospital Authority in Hong Kong. The diagnosis of CHB and various malignancies was based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes between 2000 and 2012. Patients exposed to any of the oral NA for CHB were included. The primary outcome was incident cancers. A 3-year landmark analysis, with follow-up up to 7 years, was used to evaluate the relative risk of cancers in treated and untreated patients. RESULTS: A total of 44 494 patients (39 712 untreated and 4782 treated) were included in the analysis. During 194 890 patient-years of follow-up, hepatocellular carcinoma developed in 402 (1.0%) untreated patients and 179 (3.7%) treated patients, while other cancers developed in 528 (1.3%) and 128 (2.7%) patients respectively. After propensity score weighting, treated patients had similar risks of all malignancies [weighted hazard ratio (wHR): 1.01, 95% CI: 0.82-1.25, P = 0.899], lung/pleural cancers (wHR: 0.82, 95% CI: 0.52-1.31, P = 0.409) and urinary/renal malignancies (wHR: 1.04, 95% CI: 0.38-2.81, P = 0.944) when compared with untreated patients. CONCLUSIONS: Oral nucleos(t)ide analogue treatment does not appear to increase cancer risk in patients with chronic hepatitis B. Given the beneficial effect on liver outcomes, our data support the current practice of long-term anti-viral therapy.

Statins reduce the risk of liver decompensation and death in chronic viral hepatitis: a propensity score weighted landmark analysis.

BACKGROUND: Decompensated liver disease due to portal hypertension leads to significant morbidity and mortality. Statins can modulate intrahepatic vascular tone, but the clinical significance remains uncertain. AIM: To determine the effects of statin use on the risk of liver decompensation and death among patients with chronic viral hepatitis. METHODS: We conducted a population wide cohort study using a hospital based database from the Hong Kong Hospital Authority. Adults with chronic viral hepatitis without prior liver decompensation were identified from 2000 to 2012 by International Classification of Diseases, Ninth Revision, Clinical Modification, diagnostic codes. Statin use was defined as a cumulative defined daily dose of >28. Landmark analysis was used to overcome immortal time bias. Propensity score weighting was further performed to minimise baseline confounders. Primary outcome was a composite of portal hypertension related liver decompensation events, with adjustment for death as a competing risk. RESULTS: A total of 69 184 patients with chronic viral hepatitis (2053 statin users and 67 131 statin non-users) were identified for the 2-year landmark analysis. After propensity score weighting of 23 baseline covariates, statin use was associated with a significant reduction in composite liver decompensation events (HR: 0.55; 95% CI: 0.36-0.83; P = .005), ascites (HR: 0.57; 95% CI: 0.36-0.92; P = .02), and a dose-dependent decrease in death (HR: 0.87; 95% CI: 0.76-0.99; P = .035) relative to no statin use. CONCLUSIONS: Patients with chronic viral hepatitis who used statins have a reduced risk of liver decompensation and death compared to non-users in this propensity score weighted landmark analysis.

Laboratory parameter-based machine learning model for excluding non-alcoholic fatty liver disease (NAFLD) in the general population.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects 20%-40% of the general population in developed countries and is an increasingly important cause of hepatocellular carcinoma. Electronic medical records facilitate large-scale epidemiological studies, existing NAFLD scores often require clinical and anthropometric parameters that may not be captured in those databases. AIM: To develop and validate a laboratory parameter-based machine learning model to detect NAFLD for the general population. METHODS: We randomly divided 922 subjects from a population screening study into training and validation groups; NAFLD was diagnosed by proton-magnetic resonance spectroscopy. On the basis of machine learning from 23 routine clinical and laboratory parameters after elastic net regulation, we evaluated the logistic regression, ridge regression, AdaBoost and decision tree models. The areas under receiver-operating characteristic curve (AUROC) of models in validation group were compared. RESULTS: Six predictors including alanine aminotransferase, high-density lipoprotein cholesterol, triglyceride, haemoglobin A1c , white blood cell count and the presence of hypertension were selected. The NAFLD ridge score achieved AUROC of 0.87 (95% CI 0.83-0.90) and 0.88 (0.84-0.91) in the training and validation groups respectively. Using dual cut-offs of 0.24 and 0.44, NAFLD ridge score achieved 92% (86%-96%) sensitivity and 90% (86%-93%) specificity with corresponding negative and positive predictive values of 96% (91%-98%) and 69% (59%-78%), and 87% of overall accuracy among 70% of classifiable subjects in the validation group; 30% of subjects remained indeterminate. CONCLUSIONS: NAFLD ridge score is a simple and robust reference comparable to existing NAFLD scores to exclude NAFLD patients in epidemiological studies.

Oral nucleos(t)ide analogues reduce recurrence and death in chronic hepatitis B-related hepatocellular carcinoma.

BACKGROUND: In patients with chronic hepatitis B (CHB)-related hepatocellular carcinoma (HCC), high viral load was associated with tumour recurrence and deaths. AIMS: To investigate the effect of nucleos(t)ide analogues (NA) on the clinical outcomes after different HCC treatments. METHODS: A territory-wide cohort study was conducted using the database from Hospital Authority. We identified CHB patients with HCC by International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes in 2000-2012. HCC treatments, NA use and laboratory parameters were retrieved. The primary endpoint was HCC recurrence and death. A 3-month landmark analysis was used to evaluate the primary outcome in patients with or without NA treatment. RESULTS: A total of 2198 CHB patients (1230 NA-untreated and 968 NA-treated) with HCC, receiving at least one type of HCC treatment were included in the analysis. At a median follow-up of 2.8 (IQR 1.4-4.9) years, tumour recurrence and death occurred in 451 (36.7%) and 578 (47.0%) untreated patients; and in 216 (22.3%) and 301 (31.1%) NA-treated patients respectively. NA therapy reduced the risk of overall HCC recurrence [adjusted sub-hazard ratio (SHR) 0.63, 95% confidence interval (CI) 0.49-0.80; P < 0.001]. The effect was most obvious in patients undergoing resection (SHR = 0.58, 95% CI = 0.37-0.91, P = 0.018). The possibility of NA therapy reducing the risk of death (HR = 0.82, 95% CI = 0.64-1.03, P = 0.092), is most obvious in resection subgroup (HR = 0.64, 95% CI = 0.41-0.99, P = 0.050) but insignificant in the other treatment groups. CONCLUSION: Our findings show that nucleos(t)ide analogues treatment reduces the risk of HCC recurrence in patients with chronic hepatitis B treated by surgical resection.

Quantification of skeletal muscle necrosis in the lower extremities using 99Tcm pyrophosphate with single photon emission computed tomography.

No non-invasive quantitative method is currently available for measuring the amount of regional skeletal muscle necrosis in humans. A threshold method using 99Tcm pyrophosphate (PPi) with single photon emission computed tomography (SPECT) has been reported to quantify muscle necrosis successfully in a canine model. This method has been modified to measure muscle necrosis in the lower extremities of human patients. Modifications include adding background subtraction and using the contralateral unaffected lower extremity as background region. A region of interest was also manually drawn around the affected lower extremity muscle to exclude the tibia. In order to identify the group of patients with no significant necrosis and with diffuse hyperaemia, nine control subjects were studied. The variance of the counts within the pixels of the lower extremities was 132 (S.D. = 27). A variance of greater than 159 was considered as being significant for necrosis. Thirteen patients were studied. Six patients had significant muscle necrosis (462 ml, S.D. = 280 ml). All except one patient (who had a small amount of muscle necrosis) developed foot drop. Seven patients had no significant necrosis. All patients had normal ankle dorsiflexion. It is possible to quantify regional muscle necrosis using PPi with SPECT. Volume of necrosis determined by this method can also predict clinical outcome.

Quantitation of skeletal muscle necrosis using 99Tcm-pyrophosphate with SPECT in a canine model.

Patients with acute arterial occlusion may suffer from significant muscle necrosis. It is important to determine the amount of regional muscle necrosis for prognostic purposes and evaluation of therapy. We have used technetium-99m-pyrophosphate (PPi) with single photon emission computed tomography (SPECT) to quantitate muscle necrosis. A canine gracilis muscle ischaemia model was used. PPi was injected 1.5 h before the muscle preparation was removed from the dog. SPECT was performed on the muscle preparations. An automatic edge detection program using the threshold technique determined the number of pixels with significant PPi activity in each transaxial section. Volume was calculated by summing the number of pixels with significant PPi activity and multiplying by the voxel size. The results were compared with quantitation using computerized planimetry with nitroblue tetrazolium staining (correlation coefficient = 0.93). PPi with SPECT is an accurate method for quantiating muscle necrosis.

Scintigraphic findings in congenital lipodystrophy.

The scintigraphic findings of a patient with characteristic clinical, laboratory, and radiographic features of congenital lipodystrophy were studied. Bone scan showed uniform increased bone uptake of Tc-99m MDP with markedly enhanced peri-articular activity and very prominent renal activity. Liver-spleen scan showed marked hepatosplenomegaly. This combination of scintigraphic abnormalities is unique in congenital lipodystrophy and constitutes a very interesting constellation of findings. Observed scintigraphic findings are compatible with previously described radiographic findings.

Determination of depth distribution of internal mammary lymph nodes on lateral lymphoscintigraphy.

A method using a calibrated ruler for measuring lymph node depth on lateral lymphoscintigrams is described. The accuracy of the method was confirmed with a plexiglass phantom. At the time of internal mammary lymphoscintigraphy, lateral images were obtained in 201 patients. A total of 545 lymph nodes were visualised and categorised into 13 depth intervals. The average lymph node depth was 3.0 +/- 1.1 cm with a range of 1.0-7.1 cm. Average lymph node depth was classified according to body weight with an average depth of 2.5, 3.1 and 3.7 cm for patients weighing less than 60, 60-75 and more than 75 kg, respectively. Lateral lymphoscintigrams demonstrated normal anatomical communication between internal mammary and anterior mediastinal lymph nodes in 3.5% of patients.

Characterization of a human B-lymphocyte carcinoma cross-reacting antigen (BLCa) in B lymphocytes identified by two murine monoclonal antibodies.

The murine monoclonal antibody (MAb) MA6 is selectively reactive against a large variety of human B lymphocytes including those in early stages of B-cell differentiation such as committed progenitors of B lymphocytes, pre-B lymphocytes, Burkitt lymphoma cells, and those at later stages of differentiation such as peripheral blood B lymphocytes and myeloma cells. The major antigen identified by this antibody on such B lymphocytes (BLCa) is a 55-kDa glycoprotein or a group of similar glycoproteins, with the MA6-reactive determinant localized on the carbohydrate moiety. On isoelectric focusing, this antigen exhibits a degree of charge microheterogeneity, migrating as a diffused band with an average isoelectric point at pH 5.7. BLCa was also identified by another murine MAb, MA5. The antigenic determinant recognized by this antibody is also localized on the carbohydrate moiety of the molecule, but, unlike the MA6-reactive determinant, it is shared by other glycoproteins from different types of cells.

Characterization of a murine monoclonal-antibody-defined B-lymphocyte carcinoma cross-reacting antigen (BLCa) from nasopharyngeal carcinoma tissues.

Biopsies were obtained from 12 patients suspected of having nasopharyngeal carcinoma (NPC). A portion of the tissue was submitted for histopathology and another for Western blotting using a murine monoclonal antibody (MAb), MA6. Touch smears of the tissues were also prepared immediately prior to extraction and Western blotting for immunoenzymic staining. The results showed that a B-lymphocyte carcinoma cross-reacting antigen (BLCa), or an antigen similar to it, was the major antigen in the tumor tissue recognized by MA6. The antigen was detected in tissues from 8 patients, of whom 7 had confirmed NPC and one had eskimoma, but not in tissue from the remaining 4 patients who did not have histologically confirmed malignancy. Immunocytology showed that tumour cells were present in the touch smears from all but one of the tumour patients but not in the other patients, and that the tumour cells comprised the large majority of the MA6-reactive cell population. The other MA6-reactive cell types present included certain weakly reactive epithelial cells and occasional lymphoid cells, presumably B lymphocytes. However, these cell types were similarly distributed between the tissues obtained from patients with or without malignant diseases. It was concluded, therefore, that the tumour cells in these tissues are the principal source of BLCa and, as such, the antigen may constitute an objective and reliable marker of NPC.

Involvement of major histocompatibility complex class II antigen in Epstein-Barr virus-mediated B cell proliferation.

Five MHC class II monoclonal antibodies costimulated proliferation of cord blood leukocytes with Epstein-Barr virus. These agonistic antibodies were of different isotypes, but all of them were either specific for or cross-reacting with HLA-DR. The other MHC class II antibodies, including three that were specific for HLA-DQ and one that was specific for HLA-DP and also those that were specific for MHC class I or leukocyte common antigen, were not costimulatory. The agonistic effect of different MHC class II antibodies was additive, such that costimulation by different antibodies combined significantly exceeded that achieved by either of these antibodies alone. Spent culture media of B cell lines also costimulated B cell proliferation with the virus. Although MHC class II antibodies augmented the effects of suboptimal concentration of the conditioned media, their combined effects did not exceed the maximum costimulation achieved by either the antibodies or the spent culture media alone. These results raised the possibility that MHC class II antigen may contain distinct functional domains involved in the regulation of B cell progression.

Identification of clinically significant skeletal muscle necrosis by single photon emission computed tomography.

Ischemia-reperfusion damage to skeletal muscle may cause serious local as well as systemic complications, its impact predominantly related to the quantity of ischemic muscle in the lower extremity. To date, there has been no noninvasive method of estimating that quantity. The authors used single photon emission computed tomography (SPECT) to quantify the volume of muscle that takes up technetium-99 pyrophosphate above a baseline threshold. Compared with the standard technique of staining slices of the muscle with nitroblue tetrazolium they found a close correlation using SPECT (r = 0.88, p less than 0.01, n = 19) in the canine model. In humans, this clinically applicable noninvasive technique may allow the surgeon to document accurately the extent of muscle necrosis in the lower extremity, to anticipate the impact of an ischemia-reperfusion injury and evaluate methods of reducing the extent of post-ischemic skeletal muscle necrosis.

A shared antigenic determinant between human B lymphocytes and carcinomas (BLCa).

We have raised a murine IgM monoclonal antibody (MA6) against immunoprecipitate obtained by reacting the serum of an NPC patient with extract of the Burkitt lymphoma cell line, Raji. It reacts against an antigen (BLCa) which is broadly represented on human B lymphoid tissues and cell lines but is different from the functional B-cell markers such as surface immunoglobulins, Ia products, Fc and complement receptors. BLCa was found to occur on lymphoid cell lines representing all stages of B-cell differentiation. These included the pre-B and null-cell lines, Nalm 6 and Reh, the EBV-transformed lymphoid cell lines and the myeloma cell line. MA6 was reactive against all the Burkitt lymphoma cell lines, whether or not these harbored the EBV genomes, with the exception of P3HR-I. The antibody was found to be selectively reactive against a proportion of peripheral blood B lymphocytes and to stain the B-cell-rich primary follicles and mantle zones of secondary follicles in the lymph node. However, MA6 was not reactive against cell lines of T-lymphocyte, myeloid, monocyte fibroblast or epithelial origin. It did, nevertheless selectively stain tumor cells from a variety of carcinoma tissues originating from different anatomical sites, including the nasopharynx.

A phase I dose escalation trial of yttrium-90 microspheres in the treatment of primary hepatocellular carcinoma.

METHODS: Ten patients with primary hepatocellular carcinoma were treated with intraarterial instillation of yttrium-90 (Y-90) microspheres, including eight men and two women (median age, 52 years; range, 29-69 years). Four patients were treated at a targeted hepatic dose of 50 Gy, two at 75 Gy, and four at 100 Gy. RESULTS: In 8 of the 10 patients, there was a significant concentration of Y-90 in localized tumor masses with tumor-to-liver perfusion ratios from 1.0:1-10.0:1. No patient had a complete or partial response, but 10 patients had stable disease (median duration, 10 weeks; range, 5-64 weeks). The median survival was 18 weeks (range, 2-150 weeks), and three patients lived longer than 1 year. Significant bone marrow or hepatic toxicity was not seen. One patient had a radiation-induced duodenal ulcer that required surgical management. CONCLUSIONS: Intraarterial instillation of Y-90 microspheres appears to be safe and deserves additional evaluation to determine whether there is meaningful activity in patients with primary hepatocellular carcinoma.

Evaluation of monoclonal antibodies for the detection of exfoliative nasopharyngeal carcinoma cells.

Exfoliative cells were aspirated from 15 patients suspected of having nasopharyngeal carcinoma (NPC) and showing the presence of lesions or other abnormalities in the nasopharynx. They were tested for binding with a 125I monoclonal antibody (MAb) (MA6) which is selectively reactive against human B lymphocytes and a variety of carcinomas. A positive result was obtained from 6/9 patients with, and from 0/5 patients without, histologically confirmed disease. One patient with eskimoma also gave a negative binding result. Cytology was specific but less sensitive, tumour cells being detected in 3 of the patients with confirmed disease. Immunocytology using MA6 was limited, like cytology, by poor recovery of the tumour cells and the results were in complete concordance with cytology. The other MAbs used were raised against carcinoembryonic antigen (CEA) and a carcinoma cell line (Ca2), respectively. The latter was not reactive against the NPC tumour cells while the CEA antibody was not sufficiently selective to be useful.

CDw40 and BLCa-specific monoclonal antibodies detect two distinct molecules which transmit progression signals to human B lymphocytes.

Two monoclonal antibodies (mAb), MA6 and G28-5, have the common property of detecting markers expressed on both B lymphocytes and carcinomas: BLCa (B lymphocyte carcinoma cross-reacting antigen) and CDw40 (Bp50). A comparison of the reactivity of these mAb revealed that MA6 and G28-5 detect distinct epitopes with different cell line and tissue distributions. L cell transfectants expressing CDw40 were not bound by MA6 anti-BLCa, but were bound by G28-5 anti-CDw40. G28-5 or a CDw40-specific heterantiserum could not block the migration of BLCa, while MA6 antibody could. These results indicate that CDw40 and BLCa are distinct surface molecules. Both G28-5 anti-CDw40 and MA6 anti-BLCa mAb could provide progression signals for B cells activated by appropriate B cell activators such as phorbol esters or anti-immunoglobulin; however, only G28-5 anti-CDw40 and not MA6 was co-stimulatory with the anti-CD20 competence signal, demonstrating a clear difference in the CDw40 and BLCa-mediated progression signals. Apparently, these molecules, although structurally distinct, have related functions in B cell activation.