Intraoperative in vivo confocal endomicroscopy of the glioma margin: performance assessment of image interpretation by neurosurgeon users.

Objectives: Confocal laser endomicroscopy (CLE) is an intraoperative real-time cellular resolution imaging technology that images brain tumor histoarchitecture. Previously, we demonstrated that CLE images may be interpreted by neuropathologists to determine the presence of tumor infiltration at glioma margins. In this study, we assessed neurosurgeons' ability to interpret CLE images from glioma margins and compared their assessments to those of neuropathologists. Methods: In vivo CLE images acquired at the glioma margins that were previously reviewed by CLE-experienced neuropathologists were interpreted by four CLE-experienced neurosurgeons. A numerical scoring system from 0 to 5 and a dichotomous scoring system based on pathological features were used. Scores from assessments of hematoxylin and eosin (H&E)-stained sections and CLE images by neuropathologists from a previous study were used for comparison. Neurosurgeons' scores were compared to the H&E findings. The inter-rater agreement and diagnostic performance based on neurosurgeons' scores were calculated. The concordance between dichotomous and numerical scores was determined. Results: In all, 4275 images from 56 glioma margin regions of interest (ROIs) were included in the analysis. With the numerical scoring system, the inter-rater agreement for neurosurgeons interpreting CLE images was moderate for all ROIs (mean agreement, 61%), which was significantly better than the inter-rater agreement for the neuropathologists (mean agreement, 48%) (p < 0.01). The inter-rater agreement for neurosurgeons using the dichotomous scoring system was 83%. The concordance between the numerical and dichotomous scoring systems was 93%. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 78%, 32%, 62%, and 50%, respectively, using the numerical scoring system and 80%, 27%, 61%, and 48%, respectively, using the dichotomous scoring system. No statistically significant differences in diagnostic performance were found between the neurosurgeons and neuropathologists. Conclusion: Neurosurgeons' performance in interpreting CLE images was comparable to that of neuropathologists. These results suggest that CLE could be used as an intraoperative guidance tool with neurosurgeons interpreting the images with or without assistance of the neuropathologists. The dichotomous scoring system is robust yet simple and may streamline rapid, simultaneous interpretation of CLE images during imaging.

Intraoperative in vivo confocal laser endomicroscopy imaging at glioma margins: can we detect tumor infiltration?

OBJECTIVE: Confocal laser endomicroscopy (CLE) is a US Food and Drug Administration-cleared intraoperative real-time fluorescence-based cellular resolution imaging technology that has been shown to image brain tumor histoarchitecture rapidly in vivo during neuro-oncological surgical procedures. An important goal for successful intraoperative implementation is in vivo use at the margins of infiltrating gliomas. However, CLE use at glioma margins has not been well studied. METHODS: Matching in vivo CLE images and tissue biopsies acquired at glioma margin regions of interest (ROIs) were collected from 2 institutions. All images were reviewed by 4 neuropathologists experienced in CLE. A scoring system based on the pathological features was implemented to score CLE and H&E images from each ROI on a scale from 0 to 5. Based on the H&E scores, all ROIs were divided into a low tumor probability (LTP) group (scores 0-2) and a high tumor probability (HTP) group (scores 3-5). The concordance between CLE and H&E scores regarding tumor probability was determined. The intraclass correlation coefficient (ICC) and diagnostic performance were calculated. RESULTS: Fifty-six glioma margin ROIs were included for analysis. Interrater reliability of the scoring system was excellent when used for H&E images (ICC [95% CI] 0.91 [0.86-0.94]) and moderate when used for CLE images (ICC [95% CI] 0.69 [0.40-0.83]). The ICCs (95% CIs) of the LTP group (0.68 [0.40-0.83]) and HTP group (0.68 [0.39-0.83]) did not differ significantly. The concordance between CLE and H&E scores was 61.6%. The sensitivity and specificity values of the scoring system were 79% and 37%. The positive predictive value (PPV) and negative predictive value were 65% and 53%, respectively. Concordance, sensitivity, and PPV were greater in the HTP group than in the LTP group. Specificity was higher in the newly diagnosed group than in the recurrent group. CONCLUSIONS: CLE may detect tumor infiltration at glioma margins. However, it is not currently dependable, especially in scenarios where low probability of tumor infiltration is expected. The proposed scoring system has excellent intrinsic interrater reliability, but its interrater reliability is only moderate when used with CLE images. These results suggest that this technology requires further exploration as a method for consistent actionable intraoperative guidance with high dependability across the range of tumor margin scenarios. Specific-binding and/or tumor-specific fluorophores, a CLE image atlas, and a consensus guideline for image interpretation may help with the translational utility of CLE.

Confocal laser imaging in neurosurgery: A comprehensive review of sodium fluorescein-based CONVIVO preclinical and clinical applications.

Given the established direct correlation that exists among extent of resection and postoperative survival in brain tumors, obtaining complete resections is of primary importance. Apart from the various technological advancements that have been introduced in current clinical practice, histopathological study still remains the gold-standard for definitive diagnosis. Frozen section analysis still represents the most rapid and used intraoperative histopathological method that allows for an intraoperative differential diagnosis. Nevertheless, such technique owes some intrinsic limitations that limit its overall potential in obtaining real-time diagnosis during surgery. In this context, confocal laser technology has been suggested as a promising method to have near real-time intraoperative histological images in neurosurgery, thanks to the results of various studies performed in other non-neurosurgical fields. Still far to be routinely implemented in current neurosurgical practice, pertinent literature is growing quickly, and various reports have recently demonstrated the utility of this technology in both preclinical and clinical settings in identifying brain tumors, microvasculature, and tumor margins, when coupled to the intravenous administration of sodium fluorescein. Specifically in neurosurgery, among different available devices, the ZEISS CONVIVO system probably boasts the most recent and largest number of experimental studies assessing its usefulness, which has been confirmed for identifying brain tumors, offering a diagnosis and distinguishing between healthy and pathologic tissue, and studying brain vessels. The main objective of this systematic review is to present a state-of-the-art summary on sodium fluorescein-based preclinical and clinical applications of the ZEISS CONVIVO in neurosurgery.

Intraoperative confocal laser endomicroscopy for brain tumors - potential and challenges from a neuropathological perspective.

Confocal laser endomicroscopy (CLE) represents a new non-invasive in vivo imaging technique that holds considerable promise in neurosurgery and neuropathology. CLE is based on the principle of optical sectioning which uses pinholes placed in the light path to selectively image photons of a specific focal plane by filtering out photons above and below the focal plane. Potential indications of CLE in neurosurgery and neuropathology include intraoperative tumor diagnosis and staging as well as assessment of tumor resection margins notably in the case of diffusely infiltrating gliomas. CLE-based tumor analysis in near-real time may also have a significant impact on future tumor resection strategies. We here discuss the technical features of CLE, its potential for wide-field imaging, its role in comparison to established histological techniques for intraoperative tumor assessment and its position in digital pathology and telepathology. Based on our group's experience with a commercially available confocal laser endomicroscope (ZEISS CONVIVO), we critically address the current state of intraoperative CLE in brain tumor surgery, the applicability of classical histological criteria and the strategies required to further improve the diagnostic accuracy of CLE. We finally discuss how a widespread use of CLE in neurosurgery may modify the role of neuropathologists in intraoperative consultation, generating both new opportunities and new challenges.

Epithelial cell types and their proposed roles in maintaining the mucosal barrier in human chagasic-megacolonic mucosa.

Patients suffering from chagasic megacolon must have an intact mucosal barrier as they survive this chronic disease for decades. A key structure of the mucosal barrier are epithelial cells. Vasoactive-intestinal-peptide (VIP)-positive nerve fibres are involved in influencing, e.g., epithelial cell proliferation, mucus secretion (e.g., mucin 2 and trefoil factor 3 of goblet cells) and inflammation or autoimmunity, all putative and/or known factors altered in chagasic megacolon. We analyzed qualitatively and quantitatively goblet cells, their specific markers, such as mucin 2 (MUC2) and trefoil factor 3 (TFF3) and enterocytes, the relation of VIP-immunoreactive nerve fibres to the epithelia, the distribution of gelsolin, a protein involved in chronic inflammation processes in the epithelia, and the proliferation rate of epithelial cells by combined 4',6-diamidino-2-phenylindole (DAPI) and phosphohistone-H3 (PHH3) staining. Goblet cells were the dominating epithelial cell type. They accounted for 38.4% of all epithelial cells in controls and changed to 58.9% in the megacolonic parts. In contrast to the overall expression in goblet cells of control epithelia, TFF3 was confined to goblet cells at the base of the crypts whereas MUC2 was found only in luminal goblet cells. Gelsolin-positive goblet cells were predominantly recognized within the controls. Finally, the mean value of mitosis increased from 1.5% within the controls up to 2.6% in the anal parts of the chagasic sepcimens. Taken together, increased cell proliferation, preponderance of goblet cells, differential MUC 2, and TFF 3 expression might all be factors maintaining an intact mucosal barrier within chagasic megacolon.

Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells.

HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1-/- HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV- HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection.

NDRG2 and NDRG4 Expression Is Altered in Glioblastoma and Influences Survival in Patients with MGMT-methylated Tumors.

AIM: The N-myc down-regulated gene (NDRG) family is a group of genes that have predominantly tumor-suppressive effects. The goal of this study was to investigate the expression of NDRG2 and NDRG4 in surgical specimens of human glioblastoma and in normal brain tissue, and to search for correlations with overall (OS) and progression-free survival (PFS). MATERIALS AND METHODS: Samples from 44 patients (31 males, 13 females; mean age±SD=57.4±15.7 years) with primary (n=40) or recurrent glioblastoma (n=4) were analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry, with dimensionless semiquantitative immunoreactivity score (IRS), ranging from 0-30] for expression of NDRG2 and NDRG4. Five non-tumorous autopsy brain specimens were used as controls. RESULTS: On the protein level, expression of NDRG2 was significantly down-regulated in glioblastoma (IRS=3.5±3.0 vs. 8.8±3.3; p=0.001), while expression of NDRG4 was significantly up-regulated (IRS=5.4±3.7 vs. 0.75±0.4 vs, p<0.001). There was no statistically significant difference in PFS between a group of 15 patients with glioblastoma with MGMT methylation and enhanced expression of NDRG4 mRNA who were treated with adjuvant radiochemotherapy (temozolomide and 60 Gy) and a group of patients with low expression of NDRG4 mRNA [10 (range=5.5-14.2) months vs. 21 (range=10.7-31.3) months] (p=0.13). CONCLUSION: Expression of both NDRG2 and NDRG4 genes is significantly altered in glioblastomas. PFS among the patients with glioblastoma with MGMT methylation treated with radiochemotherapy differed significantly in high-expression groups compared to patients without MGMT methlation and without radiochemotherapy (p<0.05).

The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

Post-translational modifications of chromatin components are significantly involved in the regulation of tumor suppressor gene and oncogene expression. Connective tissue growth factor (CTGF) is an epigenetically regulated growth factor with functions in angiogenesis and cell-matrix interactions and plays a pivotal role in hepatocellular carcinoma (HCC). The pharmacologic inhibition of histone and protein deacetylases represents a new approach to interfere with pathways of apoptosis and angiogenesis. We investigated the effect of the pan-deacetylase inhibitor panobinostat (LBH589) on human HCC cell lines HepG2 (p53wt) and Hep3B (p53null) and in a subcutaneous xenograft model and explored the influence on angiogenesis. Specimens were characterized by quantitative real-time PCR. Protein was separated for western blotting against CTGF, VEGF, VEGF receptor-1 (VEGFR-1/FLT-1), VEGF receptor-2 (VEGFR-2/KDR), MAPK and phospho-MAPK. In vivo, HepG2 cells were xenografted to NMRI mice and treated with daily i.p. injections of 10 mg/kg panobinostat. After 1, 7 and 28 days, real-time PCR was performed. Immunohistochemistry and western blotting were examined after 28 days. An increased significant expression of CTGF was only seen after 24 h treatment with 0.1 µM panobinostat in HepG2 cells and Hep3B cells, whereas after 72 h treatment CTGF expression clearly decreased. In the xenografts, treatment with panobinostat showed a minimal CTGF expression after 1 day and 4 weeks, respectively. In vitro as well as in vivo, VEGF was not affected by panobinostat treatment at any time. In conclusion, panobinostat influences extracellular signaling cascades via CTGF-dependent pathways.

Ileal neuroendocrine tumors show elevated activation of mammalian target of rapamycin complex.

BACKGROUND: Neuroendocrine tumors (NETs) of the ileum are sporadic tumors derived from submucosal gastrointestinal stem cells. They often show clinical symptoms only after hepatic metastasation when curative therapy is limited or impossible. In this study, we analyzed the expression of the candidate genes mammalian target of rapamycin (mTOR), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and death domain-associated protein (DAXX) to investigate the specific oncogenetics and potential therapeutic options for ileal NETs. METHODS: In a prospective database, all patients who underwent surgical removal of a NET of the ileum between 2001 and 2011 were specified. Expression analysis was performed for mTOR, ATRX, and DAXX by immunohistochemistry of paraffin-embedded tumor samples. To evaluate the results the immunoreactive score was applied. Normal tissue and tumor tissue were analyzed for the comparison of gene expression levels using quantitative-real-time polymerase chain reaction for ATRX and mTOR genes. Results were correlated under pathologic and clinical aspects. RESULTS: A total of 69 patients were admitted to the study. Positive cytosolic expression of the potential oncogene mTOR was immunohistochemically detected in 76.2% of the human probes. A loss of nuclear ATRX expression was detected in 13.0% of the samples. A nonexpression of the DAXX-protein in cell nuclei was not found (0%). Gene transcript levels did not show a significant alteration in ileal NETs in comparison with normal tissue. CONCLUSIONS: mTOR is overexpressed in ileal NETs. Additionally, the loss of ATRX expression was registered, thus underlying a tumorigenic role in a subgroup of these tumors. To enable potential therapeutic application of mTOR inhibitors, further trials with larger study groups are needed.

The pan-deacetylase inhibitor panobinostat suppresses the expression of oncogenic miRNAs in hepatocellular carcinoma cell lines.

Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa-miR-19a, hsa-miR-19b1 and the corresponding precursors were down-regulated by panobinostat in TP53(-/-) Hep3B and TP53(+/+) HepG2 cell lines; hsa-miR30a-5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT-qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance-based real-time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti-cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets.

The role of sphingosine kinase isoforms and receptors S1P1, S1P2, S1P3, and S1P5 in primary, secondary, and recurrent glioblastomas.

Sphingosine-1-phosphate (S1P), the corresponding kinases SphK1-2, and receptors S1P1-3 and S1P5 are involved in cell survival and growth. Pathway components are overexpressed in many tumors including glioblastoma. Previous studies showed that the expression of SphK1 influenced survival of glioblastoma patients, yet the roles of SphK1-2 and receptors S1P1-3 and S1P5 have not been investigated in different forms of glioblastoma. Samples from 59 patients (37 males, 22 females, age 55.1 ± 17.1 years) suffering from primary (n = 35), recurrent (n = 18), and secondary (n = 6) glioblastomas were analyzed using quantitative real-time PCR and immunohistochemistry for expression levels of SphK1 and SphK2 and S1P1-3 and S1P5. Sixteen autopsy nontumorous brain specimens were used as controls. Expression data was correlated with clinical data and patient survival. All markers were overexpressed in the glioblastoma specimens compared to the non-neoplastic brain tissue. SphK1 and all S1P receptors were expressed in increasing order of magnitude from primary, up to recurrent and secondary glioblastomas, with values of up to 44-fold compared to normal brain tissue. In contrast, SphK2 levels were highest in primary tumors (25-fold). Expression of the sphingosine signaling pathway components was influenced by radio/radiochemotherapy in distinct ways. Immunohistochemistry for SphK1 and S1P1 confirmed the overexpression in glioblastoma. Uni- and multivariate survival analyses identified S1P5 messenger RNA levels as an independent prognostic factor of survival. The sphingosine pathway is overexpressed in glioma. Its components show distinct expression patterns in the tumor subgroups. S1P5 is identified as an independent prognostic factor in multivariate analysis, and this pathway promises to be a candidate for targeted therapies.

Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon--a quantitative immunohistochemical study.

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas' disease.

Cerebrospinal fluid ferritin--unspecific and unsuitable for disease monitoring.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage is sometimes difficult to diagnose radiologically. Cerebrospinal fluid (CSF) ferritin has been proposed to be highly specific and sensitive to detect hemorrhagic central nervous system (CNS) disease. We analyzed here the specificity of CSF ferritin in a large series of various CNS diseases and the influence of serum ferritin. MATERIALS AND METHODS: CSF ferritin, lactate, protein and total cell count were analyzed in 141 samples: neoplastic meningitis (n=62), subarachnoid hemorrhage (n=20), pyogenic infection (n=10), viral infection (n=10), multiple sclerosis (n=10), borreliosis (n=5) and normal controls (n=24). Cerebrospinal fluid ferritin was measured with a microparticle immunoassay. In addition, serum and CSF ferritin were compared in 18 samples of bacterial and neoplastic meningitis. RESULTS: In CNS hemorrhage, median ferritin was 51.55µg/L (sensitivity: 90%) after the second lumbar puncture. In neoplastic meningitis, the median CSF ferritin was 16.3µg/L (sensitivity: 45%). Interestingly, ferritin was higher in solid tumors than that in hematological neoplasms. In 90% of pyogenic inflammation, ferritin was elevated with a median of 53.35µg/L, while only 50% of patients with viral infection had elevated CSF ferritin. In ventricular CSF, median ferritin was 163µg/L, but only 20.6µg/L in lumbar CSF. Ferritin was normal in multiple sclerosis and borreliosis. CONCLUSIONS: Ferritin was elevated not only in hemorrhagic disease, but also in neoplastic and infectious meningitis. Ferritin was not a reliable marker of the course of disease. The influence of serum ferritin on CSF ferritin is negligible. We conclude that elevated CSF ferritin reliably, but unspecifically indicates severe CNS disease.

Laser resection of lung tissue: heat accumulation from adjacent laser application and how to cool it down.

BACKGROUND: Heat accumulation might induce thermal damage of the surrounding lung tissue, especially when multiple lesions are resected in one session. The present study aimed to investigate whether heat accumulates in the immediate vicinity of the resection surface and leads to thermal damage of the lung parenchyma, and what is the most effective cooling strategy in this situation. MATERIALS AND METHODS: In normothermic perfused paracardial swine lobes (n = 6), four punctiform laser lesions forming a square were created. Each lesion was lasered at a power of 100 W for 5 seconds. Two test conditions with square sides of either 1.0 or 0.5 cm were compared. Temperatures were recorded immediately after completing the laser procedure in the square center and in the corners using a thermal camera and continued during the cooling process at 10-second intervals until normothermia (37°C). We examined two cooling methods: rinsing with ice-cold (4°C) Ringer solution during the laser procedure (group B, n = 6) or submerging the lung in ice-cold water for 5 seconds immediately after laser application (group C, n = 6). In the control group A (n = 6), there was no cooling. RESULTS: In the 0.5 cm squares, mean temperature in the center immediately after laser application was 103.17 ± 8.56°C, significantly higher than in the corners (76.39 ± 2.87°C, p < 0.05). Normothermia in the quadrant corners was reached after 81 ± 14 and after 108 ± 29 seconds in the centers. Tissue in the square center revealed histological signs of thermic cell damage. In the 1.0 cm squares, mean temperature in the center was 64 ± 5°C, and in the corners was 77 ± 3.1°C (p < 0.05). Normothermia was regained after 93 ± 22 seconds in the center and 120 ± 21 seconds in the corners. Histological examination in the 1.0-quadrant centers revealed no signs of thermic cell damage. Submerging the lobe into ice-cold water lowered the temperature rapidly to under 40°C, and normothermia was regained after 75 ± 1.3 seconds. CONCLUSION: Laser application to the lung parenchyma causes considerable heat accumulation in closely related lesions. To prevent such cell damage, a distance of at least 1.0 cm between laser targets should be maintained. If no topical cooling method applied, sufficient time for spontaneous tissue cooling before additional laser application should be provided. The most effective cooling strategy against heat accumulation is submerging in ice-cold water for at least 5 seconds.

Airtightness of lung parenchyma without a closing suture after atypical resection using the Nd:YAG Laser LIMAX 120.

OBJECTIVES: Lung metastases can be non-anatomically resected with a Nd:YAG Laser. It is recommended that the resected lung surface be sealed by slowly resorbable sutures. However, the lung tissue may be restricted by the sutures once it is re-ventilated. Thus, it was analysed whether the lung parenchyma is airtight after laser resection without suturing the defect. METHODS: The pulmonary artery of unimpaired paracardial lung lobes of freshly slaughtered pigs (mean weight 46 g) was cannulated and rinsed out via a hypotonic saline-heparin solution (5000 IE) until the perfusate was clear of body fluid. The lobular bronchus was connected to an airtight ventilation tube (Fa. VYGON 520 3.5 oral tube) and ventilated pressure-controlled (PEEP + 5 cm H2O, P1 = 20 cm H2O, frequency = 10/min) via a respirator. All lobes were perfused with Ringer solution at 42°C at normothermia and normotonia. In group 1 (n = 8), an atypical peripheral parenchymal resection (average resected surface: 2 × 2 cm(2)) and in group 2 (n = 8), a deep atypical parenchymal resection (average resected surface: 4 × 4 cm(2)) were performed with the Nd:YAG Laser LIMAX 120 (output power at 100 watts). After post-resection ventilation of 15 min, the resection surface was tested for airtightness and burst pressure. RESULTS: All group 1 lobes tested airtight under pressure-controlled ventilation. The mean burst pressure was 34.4 mbar (SD ± 3.2 mbar). Six lobes of group 2 were also completely airtight. The remaining two lobes, however, revealed a serious parenchymal leak (score 3). This was caused by the cross-opening of a segmental bronchus, although the surrounding lung parenchyma was also airtight. The mean burst pressure of these lobes was 31.7 mbar (SD ± 4.08 mbar). There was no significant difference between the two groups (P = 0.12). CONCLUSIONS: Peripheral lung defects after Nd:YAG Laser resection might not be sutured, since the laser-induced vaporization of the lung parenchyma seems to be initially airtight. These experimental data warrant confirmation in a controlled clinical study.

Endoplasmic reticulum stress plays a pivotal role in cell death mediated by the pan-deacetylase inhibitor panobinostat in human hepatocellular cancer cells.

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

Enhancement of the antiproliferative activity of gemcitabine by modulation of c-Met pathway in pancreatic cancer.

Pancreatic-ductal-adenocarcinoma (PDAC) is amongst the most lethal malignancies, mainly because of its metastatic spread and multifactorial chemoresistance. Since c-Met is a marker of pancreatic-cancer-stem-cells (CSC), playing a key role in metastasis and chemoresistance, this study evaluated the therapeutic potential of the novel c-Met/ALK inhibitor crizotinib against PDAC cells, including the Capan-1-gemcitabine-resistant cells (Capan-1-R). Crizotinib inhibited PDAC cell-growth with IC50 of 1.5 µM in Capan-1-R, and synergistically enhanced the antiproliferative and proapoptotic activity of gemcitabine, as detected by sulforhodamine-B-assay, flow cytometry and combination-index method. Capan-1-R had higher expression of the CSC markers CD44+/CD133+/CD326+, but their combined expression was significantly reduced by crizotinib, as detected by quantitative-RT-PCR and FACS-analysis. Similarly, Capan-1-R cells had significantly higher protein-expression of c-Met (≈2-fold), and increased migratory activity, which was reduced by crizotinib (e.g., > 50% reduction of cell-migration in Capan-1-R after 8-hour exposure, compared to untreated-cells), in association with reduced vimentin expression. Capan-1-R had also significantly higher mRNA expression of the gemcitabine catabolism-enzyme CDA, potentially explaining the higher CDA activity and statistically significant lower levels of gemcitabine-nucleotides in Capan-1-R compared to Capan-1, as detected by Liquid-chromatography-massspectrometry. Conversely, crizotinib significantly reduced CDA expression in both Capan-1 and Capan-1-R cells. In aggregate, these data show the ability of crizotinib to specifically target CSC-like-subpopulations, interfere with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with gemcitabine, supporting further studies on this novel therapeutic approach for PDAC.

The pan-deacetylase inhibitor panobinostat modulates the expression of epithelial-mesenchymal transition markers in hepatocellular carcinoma models.

Deacetylase inhibitors (DACis) represent a novel therapeutic option for human cancers by classically affecting proliferation or apoptosis. Since transdifferentiation and dedifferentiation play a key role in carcinogenesis, we investigated the epigenetic influence on the molecular differentiation status in human hepatocellular carcinoma (HCC) models. Markers of differentiation, including cytokeratin (Ck) 7, Ck8, Ck18, Ck19, Ck20, vimentin, sonic hedgehog homolog (SHH), smoothened (Smo), patched (Ptc), glioma-associated oncogene homolog 1 (Gli1), CD133, octamer-binding transcription factor 4 (Oct4) and ß-catenin, were examined in the human HCC cell lines HepG2 and Hep3B in vitro and in vivo (xenograft model) using quantitative real-time PCR and immunohistochemistry following treatment with the pan-DACi panobinostat (LBH589). Compared to untreated controls, treated HepG2 xenografts, and to a lesser extent cell lines, demonstrated a significant increase of differentiation markers Ck7 and Ck19 (classical cholangiocellular type) and Ck8 and Ck18 (classical HCC type), and a decreased level of dedifferentiation markers vimentin (mesenchymal) and SHH/Ptc (embryonic), paralleled with a more membranous expression of ß-catenin. These findings were dose-dependently correlated with tumor size, necrosis rate, microvessel density and mitosis/Ki-67-associated proliferation rate. Our results demonstrate that the differentiation status of human HCC cells is influenced by the pan-DACi panobinostat, indicating that this treatment may influence the epithelial-mesenchymal transition (EMT) status related to metastasis and aggressiveness.

Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition.

Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 µM gemcitabine. Surviving cells were collected after 1, 3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, ß-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by wound­healing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3­fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and ß-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC(50) from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proli-feration in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.

SIVmac239-Nef down-regulates cell surface expression of CXCR4 in tumor cells and inhibits proliferation, migration and angiogenesis.

AIM: To evaluate if the lentiviral accessory protein Nef can down-regulate the C-X-C chemokine receptor type 4 (CXCR4) in tumor cells and affect tumor cell proliferation, migration and angiogenesis. MATERIALS AND METHODS: HeLa-(ACC) cells, which according to genotype analysis are virtually identical to the cervical cancer-derived HeLa cell line, were transfected with Nef from SIV(mac239) and expression levels of cell surface CXCR4 were monitored by flow cytometry. Real-time proliferation and migration of cells was measured with the xCELLigence system or with the in vitro scratch assay. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. RESULTS: Cell surface down-regulation of CXCR4 was observed in HeLa-(ACC) cells after Nef transfection, as well as in the monkey kidney-derived COS-7 cell line after co-transfection of CXCR4 and Nef. Proliferation, as well as migration, of Nef-transfected HeLa-(ACC) cells appeared to be significantly reduced. In vitro tube formation was markedly lowered after Nef transfection, and CXCR4 knockdown with siRNA. CONCLUSION: SIV-Nef could serve as an interesting tool to study the biological behavior of CXCR4-expressing tumor cells and could be helpful in the discovery of new therapeutic approaches for the treatment of CXCR4-positive tumors.