Compromised Flap Salvage With Closed Incision Negative Pressure Therapy Under Real-Time Indocyanine Green Fluorescence Assessment.

BACKGROUND: Skin flap transfer is a commonly used technique by surgeons; however, compromised blood flow may result in flap ischemia and necrosis. We describe the use of closed incision negative pressure therapy (ciNPT) to help manage skin flap reconstructions with indocyanine green fluorescence angiography (ICG-FA) to assess perfusion of the flaps before and after ciNPT. METHODS: Three female and 5 male patients underwent various skin flap reconstructions, including local flaps, pedicled flaps, and propeller flaps, for wound defects related to trauma, infection, or cancer. After flap setting and suturing, ciNPT (-125 mm Hg) was applied to the closed incision for 7 days. Perfusion was assessed using ICG-FA before applying ciNPT and again at 24 hours later. The Shapiro-Wilk test and Wilcoxon signed rank test were used in statistical analysis. RESULTS: Initial postoperative survival was observed for all skin flaps; however, 1 flap failed after 2 weeks due to uncontrolled infection. The remaining 7 flaps healed well without any surgical revision. All patients were initially determined to have impaired flap perfusion; however, skin flap perfusion was significantly higher after ciNPT than before ciNPT in each case (P = 0.012). CONCLUSIONS: This study showed good healing outcomes for skin flap reconstructions without complications, despite the fact that each flap had compromised flap perfusion to some extent during the surgery. This case series is novel in that it used laser-assisted ICG-FA to provide a real-time assessment of skin flap perfusion before and after ciNPT.

Validation of Enhancing Effects of Curcumin on Radiotherapy with F98/FGT Glioblastoma-Bearing Rat Model.

Glioblastoma, the most common and aggressive brain tumor with low survival rate, is difficult to be cured by neurosurgery or radiotherapy. Mounting evidence has reported the anti-inflammatory and anticancer effects of curcumin on several types of cancer in preclinical studies and clinical trials. To our knowledge, there is no platform or system that could be used to effectively and real-timely evaluate the therapeutic efficacy of curcumin for glioblastoma multiforme (GBM). In this study, we constructed a lentivirus vector with triple-reporter genes (Fluc/GFP/tk) and transduced into rat F98 glioblastoma cells to establish an orthotopic F98/FGT glioma-bearing rat model. In the model, the therapeutic efficacies for curcumin alone, radiation alone, and their combination were evaluated via noninvasive bioluminescent imaging and overall survival measurements. At the cell level, curcumin is capable of causing a G2/M cell cycle arrest and sensitizing the F98 cells to radiation. In animal model, curcumin synergistically enhances the effects of radiotherapy on suppressing the growth of both transplanted glioma cells and in situ brain tumors, and extending the overall survival periods longer than those of curcumin alone and radiation alone treatments. In conclusion, we have demonstrated that curcumin may serve as a novel radiosensitizer to combine with radiotherapy using the triple-reporter F98/FGT animal model for effective and simultaneous evaluation of therapeutic efficacy.

MGMT promoter methylation in non-neoplastic brain.

O(6)-methylguanine-DNA-methyltransferase (MGMT) is mainly regulated by cytosine-guanine island promoter methylation that is believed to occur only in neoplastic tissue. The present study was undertaken to investigate whether methylation occurs also in non-neoplastic brains by collecting 45 non-neoplastic brains from autopsies and 56 lobectomy specimens from epileptic surgeries. The promoter methylation status of MGMT was studied by methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), while protein expression was studied by immunohistochemical stain (IHC). The methylation rates, as determined by MSP and PSQ, were 3.0 % (3/101) and 2.9 % (2/69), respectively. Of note, no case had positive result concomitantly from both MSP and PSQ (3 were MSP+/PSQ- and 2 were MSP-/PSQ+), and all the positive samples were further confirmed by cloning and Sanger sequencing. All the methylated cases, except for those having indeterminate IHC results from autopsy specimens, revealed no loss of MGMT protein expression and similar staining pattern to that of the unmethylated cases. In conclusion, the current study demonstrated that MGMT promoter methylation could occur in a low percentage of non-neoplastic brains but did not affect the status of protein expression, which could be regarded as a normal variation in non-neoplastic brains.

Prognosis of glioblastoma with faint MGMT methylation-specific PCR product.

Methylation-specific polymerase chain reaction (MSP) for the promoter methylation status of O(6)-methylguanine-DNA-methyltranferase (MGMT) gene theoretically provides a positive or negative result. However, the faint MSP product is difficult to interpret. The aim of this study was to evaluate the significance of faint MSP product in glioblastoma (GBM). Critical concentrations of methylated control DNA, i.e., 100, 1, 0.5 and 0 % were run parallel with 116 newly diagnosed GBMs in order to standardize the interpretation and to distinguish positive (+), equivocal (±), and negative (-; unmethylated) results. Cases with the faint MSP product and its intensity between those of 1 and 0.5 % DNA controls were considered equivocal (±). MGMT methylation quantifications were also determined by quantitative real-time MSP (qMSP) and pyrosequencing (PSQ), and protein expression was detected by immunohistochemistry. There were significant correlations between MSP and all the aforementioned studies. The concordance rates between the MSP+ and qMSP+ cases, as well as the MSP- and qMSP- cases were 100 %, and the MSP± cases comprised 76.5 % of qMSP+ cases and 23.5 % of qMSP- cases. PSQ study showed that heterogeneous methylation was more frequently encountered in the MSP± cases. Multivariate analyses disclosed that although the overall survival of the MSP± cases was indistinct from that of the MSP+ cases, its progression free survival was significantly worse and was indistinct from that of the MSP- cases. In conclusion, GBMs with faint MGMT MSP products should be distinguished from MSP+ cases as their behaviors were different.

Molecular diagnostic algorithm for epidermal growth factor receptor mutation detection in Asian lung adenocarcinomas: comprehensive analyses of 445 Taiwanese patients with immunohistochemistry, PCR-direct sequencing and Scorpion/ARMS methods.

BACKGROUND AND OBJECTIVE: Therapeutic responses of lung adenocarcinoma patients to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) are closely associated with activating mutations within the EGFR tyrosine kinase domain. Screening activating EGFR mutations prior to selection for therapeutic strategy has been considered extremely valuable for clinical management of lung adenocarcinoma patients in Asian countries including Taiwan, where the EGFR mutation rate is higher than in the rest of the world. Currently there is no consensus on the method of choice to assess EGFR mutations in tumour tissue. METHODS: We enrolled 445 lung adenocarcinoma patients for analysis of tumour EGFR mutations using polymerase chain reaction (PCR)-direct sequencing, scorpion/amplified refractory mutation system (ARMS) technology and immunohistochemistry with mutation-specific antibodies. RESULTS: Two hundred forty-five patients (245/445; 55%) were found to harbour activating EGFR mutations using PCR-direct sequencing method, with a majority of patients (233/245; 95%) carrying exon 19 deletion or p.L858R point mutations. One hundred three of 200 patients were negative for EGFR mutations from PCR-direct sequencing were further analysed using Scorpion/ARMS technology. Up to 30% of the PCR-direct sequencing negative patients turned out to be positive in the Scorpion/ARMS EGFR mutation tests. For immunohistochemistry analysis of EGFR mutations, the p.E746_A750del specific antibody showed a sensitivity of 57% and a specificity of 100% for exon 19 deletions while the p.L858R point mutation specific antibody showed a sensitivity of 68% and a specificity of 95%. CONCLUSIONS: Based on this study, we proposed an algorithm for comprehensive and efficient testing of EGFR mutations on lung adenocarcinoma patients in Asia.

Factors Affecting Treatment with Life-Saving Interventions, Computed Tomography Scans and Specialist Consultations.

Background: Emergency treatments determined by emergency physicians may affect mortality and patient satisfaction. This paper attempts to examine the impact of patient characteristics, health status, the accredited level of hospitals, and triaged levels on the following emergency treatments: immediate life-saving interventions (LSIs), computed tomography (CT) scans, and specialist consultations (SCs). Methods: A multivariate logistic regression model was employed to analyze the impact of patient characteristics, including sex, age, income and the urbanization degree of the patient's residence; patient health status, including records of hospitalization and the number of instances of ambulatory care in the previous year; the Charlson Comorbidity Index (CCI) score; the accredited level of hospitals; and the triaged level of emergency treatments. Results: All the patient characteristics were found to impact receiving LSI, CT and SC, except for income. Furthermore, a better health status was associated with a decreased probability of receiving LSI, CT and SC, but the number of instances of ambulatory care was not found to have a significant impact on receiving CT or SC. This study also found no evidence to support impact of CCI on SC. Hospitals with higher accredited levels were associated with a greater chance of patients receiving emergency treatments of LSI, CT and SC. A higher assigned severity (lower triaged level) led to an increased probability of receiving CT and SC. In terms of LSI, patients assigned to level 4 were found to have a lower chance of treatment than those assigned to level 5. Conclusions: This study found that several patient characteristics, patient health status, the accredited level of medical institutions and the triaged level, were associated with a higher likelihood of receiving emergency treatments. This study suggests that the inequality of medical resources among medical institutions with different accredited levels may yield a crowding-out effect.

The Association of MMP7 Promoter Polymorphisms With Gastric Cancer.

BACKGROUND/AIM: Few studies have examined the genetic role of matrix metalloproteinases (MMPs) to early detection or prediction in gastric cancer development. In this study, the contribution of MMP7 promoter (A-181G and C-153T) polymorphic genotypes to gastric cancer risk in Taiwanese was investigated for the first time. MATERIALS AND METHODS: A total of 121 cases and 363 controls were enrolled and their MMP7 genotypes at A-181G and C-153T were examined by polymerase chain reaction-restriction fragment length polymorphism methodology using genomic DNA from serum. RESULTS: The GG genotype at MMP7 A-181G was found to represent a risk factor for gastric cancer, especially among smokers. No individual with variant genotype carrier at MMP7 C-153T was found among this Taiwanese population. CONCLUSION: The G allele of MMP7 A-181G may serve as an early predictor for gastric cancer risk in Taiwanese; other gastric cancer markers are still urgently needed.

Therapeutic Efficacy Evaluation of Pegylated Liposome Encapsulated With Vinorelbine Plus 111In Repeated Treatments in Human Colorectal Carcinoma With Multimodalities of Molecular Imaging.

BACKGROUND/AIM: In precision therapy, liposomal encapsulated chemotherapeutic drugs have been developed to treat cancers by achieving higher drug accumulation in the tumor compared to normal tissues/organs. MATERIALS AND METHODS: We developed a novel chemoradiotherapeutic approach via nanoliposomes conjugated with vinorelbine (VNB) and 111In (111In-VNB-liposome) and examined their pharmacokinetics, biodistribution, maximum tolerance dose, and toxicity in a NOD/SCID mouse model. RESULTS: Pharmacokinetic results showed that the area under the curve (AUC) of PEGylated liposomes was about 17-fold higher than that of the free radioisotope. Tumor growth inhibition by 111In-VNB-liposome was significantly higher than that of the control (p<0.05). CONCLUSION: The tumors in NOD/SCID mice bearing HT-29/tk-luc xenografts were significantly suppressed by 111In-VNB-liposomes. The study proposed repeated treatments with a novel liposome-mediated radiochemotherapy and validation of therapeutic efficacy via imaging.

Proteomic Analysis of Metastasis-Specific Biomarkers in Pancreatic Cancer: Galectin-1 Plays an Important Metastatic Role in Pancreatic Cancer.

Cancer metastasis is the major cause of death in pancreatic cancer. We have established a pair of pancreatic ductal adenocarcinoma cell line, PANC1 and invasive PANC1-I5, as a model system toinvestigate the metastatic mechanism as well as potential therapeutic targets in pancreatic cancer. We used proteomic analysis based on two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to examine the global protein expression alterations between PANC1 and PANC1-I5. Proteomic study revealed that 88 proteins are differentially expressed between PANC1-I5 and PANC1 cells, and further functional evaluations through protein expression validation, gene knockout, migration and invasion analysis revealed that galectin-1 is one of the potential players in modulating pancreatic cancer metastasis. To conclude, we have identified numerous proteins might be associated with pancreatic cancer invasiveness in the pancreatic cancer model.

Sorafenib pretreatment enhances radiotherapy through targeting MEK/ERK/NF-κB pathway in human hepatocellular carcinoma-bearing mouse model.

Patients with unresectable hepatocellular carcinoma (HCC) usually have poor prognosis because current monotherapy including surgery, chemotherapy and radiotherapy (RT) are not effective. Combination therapy may be effective to overcome this clinical problem. Here, we proposed the combination of sorafenib and RT, which have been applied in HCC treatment, could improve the treatment outcome of HCC. Our previous study showed that sorafenib could suppress the expression of NF-κB which is related to the chemo- and radio-resistance. Nevertheless, the expression of NF-κB is oscillatory and is affected by the treatments. Thus, understanding the oscillation of NF-κB expression would be beneficial for determining the optimal treatment schedule in combination therapy. Here established Huh7/NF-κB-tk-luc2/rfp cell line, in which NF-κB indicates a NF-κB promoter, was utilized to noninvasively monitor the expression of NF-κB overtime in vitro and in vivo. The results show that pretreatment of sorafenib with RT suppresses the expressions of NF-κB and its downstream proteins induced by radiation through downregulation of phosphorylated extracellular signal-regulated kinase (pERK) most significantly compared with other treatment schedules. The results were further verified with Western blotting, EMSA, and NF-κB molecular imaging. These findings suggest that pretreatment of sorafenib with RT may be the ideal treatment schedule for the treatment of HCC.

Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model.

The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2'-fluoro-2'-deoxy-5-iodo-1-ß-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases.

Characteristic Comparison Between 131I-Interferon-α and 131I-Interferon-α-Immunoglobulin-Fc Hybrid Protein in Rats Using Molecular Imaging.

BACKGROUND/AIM: Interferon-α (IFN-α) is produced to act locally and transiently with a relatively short circulation half-life in vivo. Hybridization of IFN-α with human immunoglobulin Fc, renamed as IFN-α-Fc, may overcome this limitation. In the present study, (131)I-IFN-α-Fc and (131)I-IFN-α were compared in the aspects of stability, pharmacokinetics, tissue distribution and molecular imaging quality in an animal model. MATERIALS AND METHODS: Both IFN-α-Fc and IFN-α were labelled with (131)I. Biodistributions and pharmacokinetics of both labelled proteins in Sprague-Dawley rats were assayed. Micro-single-photon emission computed tomography/computed tomography was used to non-invasively monitor the longitudinal distribution of both proteins. RESULTS: (131)I-IFN-α-Fc was shown to have higher stability than (131)I-IFN-α in whole blood, plasma, kidney, liver and stomach from the biodistribution study. The area under curve analyzed from plasma in the phomacokinetics study was 10-fold higher for (131)I-IFN-α-Fc than for (131)I-IFN-α. At 0-1 h post tail-vein injection, both labelled proteins are mainly accumulated in the kidneys and liver. Notably, (131)I-IFN-α-Fc is degraded more slowly than (131)I-IFN-α. CONCLUSION: We demonstrated that (131)I-IFN-α-Fc has longer blood circulation time and better biostability than (131)I-IFN-α, suggesting the potential application of the immunoglobulin Fc-conjugated cytokine for long-term treatment of diseases.

Using [¹8F]FBAU for imaging brain tumor progression in an F98/tk-luc glioma-bearing rat model.

1-(2-Deoxy-2-[18F]fluoro-ß-D-arabinofuranosyl)-5-bromouracil ([18F]FBAU), a substitute for thymine, has been reported as an effective reporter probe by which to trace cellular metabolism with its positron emission. In the present study, a rat xenograft model bearing F98 glioma transfected with dual reporter genes, herpes simplex virus type 1 thymidine kinase (HSV1-tk) and firefly luciferase (luc) was used for monitoring tumor progression by multimodalities of molecular imaging using [18F]FBAU and D-luciferase as probes. Rat F98 glioma cells were transfected with the pC1-tk-IRES-luc vectors. The selected stable clone was renamed as the F98/tk-luc cell line. Fischer 344 male rats bearing orthotropic F98/tk-luc gliomas in the left brain were used. On day 13 post tumor inoculation, biodistribution, positron emission tomography (PET), magnetic resonance imaging (MRI) and ex vivo autoradiography were performed. The surviving fraction of F98/tk-luc cells treated with 15 µM ganciclovir (GCV) was 15.9%, and the uptake of [131I]FIAU in these cells was significantly enhanced when compared with F98 cells. The correlation coefficient of tumor volume vs. the bioluminescence in the F98/tk-luc glioma-bearing rats was 0.90. The biodistribution showed that the accumulation ratios of [18F]FBAU for glioma-to-normal brain were 9.16, 14.24, 5.7 and 13.7 at 30, 60, 90 and 120 min post i.v. injection, respectively. Consistent tumor enhancement of [18F]FBAU/PET imaging was also noted from 30-90 min post injection. Ex vivo autoradiography also confirmed significant [18F]FBAU uptake in tumors. In conclusion, [18F]FBAU may be used as a PET probe for monitoring glioma progression in animal models and may have potential for clinical use as well.

Curcumin synergistically enhances the radiosensitivity of human oral squamous cell carcinoma via suppression of radiation-induced NF-κB activity.

The anticancer effect of curcumin has been widely reported. However, whether curcumin can enhance the radiosensitivity of human oral squamous cell carcinoma (OSCC) remains to be elucidated. The aim of the present study was to evaluate the efficacy of curcumin combined with radiation against OSCC. SAS cells were transfected with the luciferase gene (luc) and named SAS/luc. NF-κB/DNA binding activity, the surviving fraction and NF-κB-regulated effector protein expression were determined by electrophoretic mobility shift assay, clonogenic survival assay and western blotting, respectively. The therapeutic efficacy was evaluated in SAS/luc tumor-bearing mice by caliper measurement and bioluminescence imaging. Curcumin enhanced SAS/luc radiosensitivity through the inhibition of radiation-induced NF-κB activity and expression of effector proteins both in vitro and in vivo. With 4 Gy or greater radiation doses, synergistic effects of curcumin were observed. The combination group (curcumin plus radiation) had significantly better tumor control compared with that of curcumin or radiation alone. No significant body weight change of mice was found throughout the entire study. In conclusion, curcumin is a radiosensitizer against OSCC with negligible toxicity.

Exclusion of histiocytes/endothelial cells and using endothelial cells as internal reference are crucial for interpretation of MGMT immunohistochemistry in glioblastoma.

We evaluated the predictive value of O6-methylguanine-DNA methyltransferase (MGMT) protein expression and MGMT promoter methylation status in glioblastomas (GBM) treated with temozolomide (TMZ) in a Taiwan medical center. Protein expression by immunohistochemical analysis (IHC) and MGMT promoter methylation detected by methylation-specific polymerase chain reaction (MSP) were performed in a series of 107 newly diagnosed GBMs. We used endothelial cells as an internal reference for IHC staining because the staining intensities of the MGMT-expressing cells in different specimens varied considerably; a positive result was defined as the staining intensity of the majority of tumor cells similar to that of the adjacent endothelial cells. Immunostainings for microglial/endothelial markers were included as part of the MGMT IHC evaluation, and in cases that were difficult to interpret, double-labeling helped to clarify the nature of reactive cells. The MGMT protein expression was reversely associated with MGMT promoter methylation status in 83.7% of cases (MSP/IHC and MSP/IHC; Pearson r=-0.644, P<0.001). Twenty-two of 24 (91.7%) IHC tumors did not respond to TMZ treatment. Combining MSP and IHC results, all the 15 MSP/IHC GBMs were TMZ resistant. The MGMT status detected by either IHC or MSP was significantly correlated with the TMZ treatment response (both P<0.001) and survival of GBM patients (both P<0.05).