Plant-based model for the visual evaluation of electroporated area after irreversible electroporation and its comparison to in-vivo animal data.

Electroporation (EP) is widely used in medicine, such as cancer treatment, in form of electrochemotherapy or irreversible electroporation (IRE). For EP device testing, living cells or tissue inside a living organism (including animals) are needed. Plant-based models seem to be a promising alternative to substitute animal models in research. The aim of this study is to find a suitable plant-based model for visual evaluation of IRE, and to compare the geometry of electroporated areas with in-vivo animal data.For this purpose, a variety of fruit and vegetables were selected and visually evaluated after 0/1/2/4/6/8/12/16/24 h post-EP. Apple and potato were found to be suitable models as they enabled a visual evaluation of the electroporated area. For these models, the size of the electroporated area was determined after 0/1/2/4/6/8/12/16/24 h. For apples, a well-defined electroporated area was visual within two hours, while in potatoes it reached a plateau after eight hours only. The electroporated area of apple, which showed the fastest visual results was then compared to a retrospectively evaluated swine liver IRE dataset which had been obtained for similar conditions. The electroporated area of the apple and swine liver both showed a spherical geometry of comparable size. For all experiments, the standard protocol for human liver IRE was followed. To conclude, potato and apple were found to be suitable plant-based models for the visual evaluation of electroporated area after irreversible EP, with apple being the best choice for fast visual results. Given the comparable range, the size of the electroporated area of the apple may be promising as a quantitative predictor in animal tissue. Even if plant-based models cannot completely replace animal experiments, they can be used in the early stages of EP device development and testing, decreasing animal experiments to the necessary minimum.

Adjuvant durvalumab for esophageal squamous cell carcinoma after neoadjuvant chemoradiotherapy: a placebo-controlled, randomized, double-blind, phase II study.

BACKGROUND: We evaluated the efficacy of adjuvant durvalumab after neoadjuvant concurrent chemoradiotherapy (CCRT) in patients with esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: This randomized, double-blind, phase II study included patients with ESCC who underwent curative surgery after neoadjuvant CCRT. Patients were randomized to receive either durvalumab (20 mg/kg/i.v. every 4 weeks for 12 months) or placebo in a 1:1 ratio and were stratified by age and pathologic tumor stage. The primary endpoint was disease-free survival (DFS). RESULTS: Between March 2016 and June 2018, 86 patients were randomized to the durvalumab (n = 45) or placebo (n = 41) arm. The median follow-up duration was 38.7 months. There was no difference in DFS [hazard ratio (HR) 1.18, 95% confidence interval (CI) 0.62-2.27, P = 0.61] or overall survival (HR 1.08, 95% CI 0.52-2.24, P = 0.85) between the two arms. Subgroup analysis was performed for patients for whom the post-CCRT programmed death-ligand 1 (PD-L1) expression profile could be assessed (n = 54). In the PD-L1-positive group, based on tumor proportion score ≥1%, durvalumab was associated with longer overall survival compared with the placebo (36-month survival rate: 94% versus 64%; HR 0.42, 95% CI 0.10-1.76), while in the PD-L1-negative group, it was associated with shorter overall survival (42% versus 55%; HR 1.53, 95% CI 0.48-4.83), showing the tendency of interaction between post-CCRT PD-L1 status and adjuvant durvalumab therapy for overall survival (interaction P = 0.18). CONCLUSIONS: We failed to demonstrate that adjuvant durvalumab improved survival after neoadjuvant CCRT in patients with ESCC. However, post-CCRT PD-L1 expression could predict the survival of patients who receive adjuvant durvalumab after neoadjuvant CCRT, which needs to be validated.

Dynamic and subtype-specific interactions between tumour burden and prognosis in breast cancer.

We investigated the relationship between the prognostic importance of anatomic tumour burden and subtypes of breast cancer using data from the Korean Breast Cancer Registry Database. In HR+/HER2+ and HR-/HER2-tumours, an increase in T stage profoundly increased the hazard of death, while the presence of lymph node metastasis was more important in HR+/HER2+ and HR-/HER2+ tumours among 131,178 patients with stage I-III breast cancer. The patterns of increasing mortality risk and tumour growth (per centimetre) and metastatic nodes (per node) were examined in 67,038 patients with a tumour diameter ≤ 7 cm and < 8 metastatic nodes. HR+/HER2- and HR-/HER2- tumours showed a persistent increase in mortality risk with an increase in tumour diameter, while the effect was modest in HER2+ tumours. Conversely, an increased number of metastatic nodes was accompanied by a persistently increased risk in HR-/HER2+ tumours, while the effect was minimal for HR-/HER2- tumours with > 3 or 4 nodes. The interactions between the prognostic significance of anatomic tumour burden and subtypes were significant. The prognostic relevance of the anatomic tumour burden was non-linear and highly dependent on the subtypes of breast cancer.

Association of low skeletal muscle mass with the presence of advanced colorectal neoplasm: integrative analysis using three skeletal muscle mass indices.

AIM: This study aimed to evaluate an association between colorectal neoplasm (CRN) and skeletal muscle mass using three widely accepted skeletal muscle mass indices (SMIs) in a large population at average risk. METHOD: We performed a cross-sectional study using a screening colonoscopy database of 33 958 asymptomatic subjects aged 40-75 years. Appendicular skeletal muscle mass (ASM) was measured using a bioelectrical impedance analyser. ASM adjusted for height squared (ASM/ht2 ), weight (ASM/wt) and body mass index (ASM/BMI) were used as indices for muscle mass. Logistic regression models were used to evaluate the association between SMIs and CRN. RESULTS: In a multivariable-adjusted model, the risk of an advanced CRN increased linearly with decreasing quartiles for all three SMIs. The adjusted odds ratios (ORs) for advanced CRN in quartiles 1, 2 and 3 of ASM/wt compared with that in quartile 4 were 1.279, 1.196 and 1.179, respectively (Ptrend  = 0.017); for ASM/BMI, ORs were 1.307, 1.144 and 1.091, respectively (Ptrend  = 0.002); and for ASM/ht2 , ORs were 1.342, 1.169 and 1.062, respectively (Ptrend  = 0.002). The risk of distally located advanced CRN was higher in quartile 1 than in quartile 4 for all three SMIs (ASM/wt, OR = 1.356; ASM/BMI, OR = 1.383; ASM/ht2 , OR = 1.430). CONCLUSION: Our study demonstrated that low skeletal muscle mass was consistently associated with the presence of advanced CRN in a population at average risk regardless of the operational definition of the SMI, and it was particularly associated with distal advanced CRN.

IL6 receptor blockade preserves articular cartilage and increases bone volume following ischemic osteonecrosis in immature mice.

OBJECTIVE: Juvenile ischemic osteonecrosis (JIO) of the femoral head is one of the most serious hip disorders causing a permanent deformity of the femoral head in childhood. We recently reported that interleukin 6 (IL6) is predominantly increased in the hip synovial fluid of patients with JIO and that articular chondrocytes are primary source of IL6. This study investigated whether an inhibition of IL6 receptor improves cartilage preservation and bone healing in JIO. METHOD: A small animal model (i.e., 6-week-old mouse) of JIO was treated with either saline or tocilizumab, an IL6 receptor blocker, for 6 weeks. RESULTS: TUNEL-positive chondrocytes in the articular cartilage were reduced by the tocilizumab treatment, concomitant with the increase in cartilage matrix. The levels of a cartilage anabolic marker Sox9 was significantly increased in the articular cartilage of mice treated with tocilizumab. Micro-CT assessment showed tocilizumab treatment significantly increased trabecular epiphyseal bone volume (P = 0.001, n = 10), thickness (P = 0.007) and number (P = 0.014) and decreased bone separation (P = 0.002) and its deformity (P = 0.003). A bone formation marker, BMP2, and an angiogenic marker, vascular endothelial growth factor (VEGF), were both significantly increased by tocilizumab treatment under hypoxia using human chondrocytes while the bone resorption marker, RANKL/OPG ratio, was reduced. CONCLUSION: Tocilizumab treatment following ischemic osteonecrosis has cartilage anabolic effect and increases bone volume in JIO mouse model. The findings lead to a possible application of tocilizumab for preclinical study using a large animal model of JIO and a clinical trial to validate this treatment.

Determination of the proliferative fractions in differentiating hematopoietic cell lineages of normal bone marrow.

Because of the proven prognostic value of Ki-67 as a proliferation marker in several types of solid cancers, our goal is to develop and validate a multiparameter flow cytometric assay for the determination of the Ki-67 expression in hemato-oncological diseases. The aim of the present study was to establish the reference values for the fraction of Ki-67 positive cells in and during maturation of individual hematopoietic cell lineages present in normal bone marrow. Aspirates derived from femoral heads of 50 patients undergoing a hip replacement were used for the flow cytometric quantification of Ki-67 expression in the different hematopoietic cell populations of healthy bone marrow. Furthermore, the proliferative index was investigated in detail for the maturation steps during erythro-, myelo-, and monopoiesis using recently described immunophenotypic profiles in combination with a software-based maturation tool. Reference values for the proliferative index were established for different relevant hematopoietic cell populations in healthy bone marrow. During maturation, the size of the Ki-67 positive fraction was the highest in the most immature compartment of the myeloid, monocytic, and erythroid cell lineages, followed by a steady decline upon cell maturation. While proerythroblasts showed a proliferative activity of almost 100%, the myelo- and monoblast showed a lower proliferative index of on average of 50%, indicating that a relatively large proportion of these cells exist in a quiescent state. In conclusion, we can state that when using a novel combination of immunophenotypic markers, the proliferation marker (Ki-67) and a software-based maturation tool, it was possible to determine the proliferative fractions in the diverse hematopoietic cell lineages in bone marrow, in particular during maturation. Using this approach, the proliferative indices for the normal myelo-, mono-, and erythropoiesis were determined, which can be used as a reference in future studies of hematologic malignancies originating from bone marrow. © 2018 International Society for Advancement of Cytometry.

Interregional correlations of glucose metabolism between the basal ganglia and different cortical areas: an ultra-high resolution PET/MRI fusion study using 18F-FDG

Basal ganglia have complex functional connections with the cerebral cortex and are involved in motor control, executive functions of the forebrain, such as the planning of movement, and cognitive behaviors based on their connections. The aim of this study was to provide detailed functional correlation patterns between the basal ganglia and cerebral cortex by conducting an interregional correlation analysis of the 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) data based on precise structural information. Fifteen participants were scanned with 7-Tesla magnetic resonance imaging (MRI) and high resolution research tomography (HRRT)-PET fusion system using 18F-FDG. For detailed interregional correlation analysis, 24 subregions of the basal ganglia including pre-commissural dorsal caudate, post-commissural caudate, pre-commissural dorsal putamen, post-commissural putamen, internal globus pallidus, and external globus pallidus and 80 cerebral regions were selected as regions of interest on the MRI image and their glucose metabolism were calculated from the PET images. Pearson's product-moment correlation analysis was conducted for the interregional correlation analysis of the basal ganglia. Functional correlation patterns between the basal ganglia and cerebral cortex were not only consistent with the findings of previous studies, but also showed new functional correlation between the dorsal striatum (i.e., caudate nucleus and putamen) and insula. In this study, we established the detailed basal ganglia subregional functional correlation patterns using 18F-FDG PET/MRI fusion imaging. Our methods and results could potentially be an important resource for investigating basal ganglia dysfunction as well as for conducting functional studies in the context of movement and psychiatric disorders.

Solid pseudopapillary neoplasm of the pancreas with liver metastasis initially misinterpreted as benign haemorrhagic cyst.

Solid pseudopapillary neoplasm (SPN) of the pancreas is considered a low-malignant neoplasm with a good prognosis. However, 5% to 15% of patients with SPNs develop metastatic disease, most commonly in the liver. Metastatic hepatic malignancies that show pseudocystic features are rare. Here we describe the case of a middle-aged female with a cystic liver metastasis from SPN. To the best of our knowledge, SPN with a single cystic liver metastasis has not been described, although these tumours frequently undergo haemorrhagic-cystic degeneration. Thus, in these patients the marked cystic change could be misinterpreted as a benign lesion.

DNA methylation analysis for the diagnosis of thyroid nodules - a pilot study with reference to BRAF(V) (600E) mutation and cytopathology results.

OBJECTIVE: Promoter hypermethylation and the BRAF(V) (600E) mutation are both involved in thyroid tumorigenesis. We conducted a pilot study on the diagnosis of thyroid nodules by analysis of promoter hypermethylation status with reference to BRAF(V) (600E) mutation and cytopathology results using formalin-fixed, paraffin-embedded (FFPE) tissues and liquid-based preparation (LBP) thyroid fine needle aspiration (FNA) samples to predict more reliably the possibility of papillary carcinoma. METHODS: We initially performed MethyLight analysis for 30 genes that are known to be hypermethylated in malignancies using 164 papillary carcinomas and 77 benign tissue samples. Five genes selected from the tissue analysis were subsequently analysed in 75 surgically proven benign and 66 surgically proven papillary carcinoma LBP FNA samples. Samples that showed two or more positive results among the five genes were classified as methylation positive. We also analysed the BRAF(V) (600E) mutation status of the FNA samples. RESULTS: We identified five genes that were significantly hypermethylated in malignant tissues: PTGS2, HOXA1, TMEFF2, p16 and PTEN. With respect to diagnostic potential, results obtained using the BRAF(V) (600E) mutation test combined with cytological examination were not significantly different from those obtained with cytological examination only. Combining methylation analyses with cytological examination or performing all three tests for diagnoses did not improve significantly the negative predictive values and sensitivity, but a significant decrease in positive predictive value and specificity was observed. CONCLUSION: Further studies are needed on larger samples to assess the potential value of methylation analysis of thyroid FNA.

Additive effects of Artemisia capillaris extract and scopoletin on the relaxation of penile corpus cavernosum smooth muscle.

The objective was to investigate the cellular effect and action mechanism of Artemisia capillaris extract (ACE) and its component, scopoletin, on penile corpus cavernosum smooth muscle (PCCSM). In vitro study with PCCSM, the precontracted PCCSM with phenylephrine was treated with ACE or scopoletin. Cyclic nucleotides in the perfusate were measured by radioimmunoassay and expression of protein and mRNA of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase in the perfused PCCSM were measured by western blot and real-time PCR, respectively. The interaction of ACE or scopoletin with udenafil was also evaluated. ACE and scopoletin exerted a significant and concentration-dependent relaxation in PCCSM. The perfusion with ACE or scopoletin significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and the perfusion with ACE or scopoletin increased the expression of eNOS mRNA and protein. Furthermore, ACE or scopoletin enhanced udenafil-inducing relaxation in PCCSM. ACE and scopoletin relaxed the PCCSM mainly by activating nitric oxide-cGMP system and cAMP pathway and they may be additive therapeutic candidates for ED patients who do not completely respond to udenafil.

Clinical impact of BK virus surveillance on outcomes in kidney transplant recipients.

BACKGROUND: The objective of this study was to investigate the clinical impact of BK virus surveillance on graft injury in kidney transplantation. METHODS: BK viremia in kidney transplant recipients was evaluated by use of plasma quantitative polymerase chain reaction. The prevalence of BK viremia and BK virus-associated nephropathy (BKVAN) and the clinical impact of BK viremia on graft outcomes were assessed. RESULTS: This study took place between January 2008 and June 2013. A total of 213 kidney transplant recipients were included. The prevalence of BK viremia and high BK viremia (≥1 × 10(4) copies/mL) was 66.7% (142/213) and 17.4% (37/213), respectively. A diagnosis of BKVAN was confirmed by means of allograft biopsy in 9 patients (4.2%). The estimated glomerular filtration rate after transplantation was similar in both the low BK viremia (<1 × 10(4) copies/mL) and non-BK viremia groups but was significantly lower in the high BK viremia group after 18 months. In receiver operating characteristic curve analysis, the area under the curve value of plasma polymerase chain reaction was 0.980. We found that a viral load >92,850 copies/mL was able to predict BKVAN with 89% sensitivity and 94.6% specificity. The risk factors for viral loads ≥1 × 10(4) copies/mL were cytomegalovirus infection, steroid pulse therapy, and acute rejection. CONCLUSIONS: High BK viremia was associated with poor graft function after kidney transplantation. The serial monitoring of BK viremia in kidney transplant recipients was helpful in predicting BKVAN and might prevent further progression.

The association of uterine cervical microbiota with an increased risk for cervical intraepithelial neoplasia in Korea.

Recent studies have suggested potential roles of the microbiome in cervicovaginal diseases. However, there has been no report on the cervical microbiome in cervical intraepithelial neoplasia (CIN). We aimed to identify the cervical microbiota of Korean women and assess the association between the cervical microbiota and CIN, and to determine the combined effect of the microbiota and human papillomavirus (HPV) on the risk of CIN. The cervical microbiota of 70 women with CIN and 50 control women was analysed using pyrosequencing based on the 16S rRNA gene. The associations between specific microbial patterns or abundance of specific microbiota and CIN risk were assessed using multivariate logistic regression, and the relative excess risk due to interaction (RERI) and the synergy index (S) were calculated. The phyla Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, Tenericutes, Fusobacteria and TM7 were predominant in the microbiota and four distinct community types were observed in all women. A high score of the pattern characterized by predominance of Atopobium vaginae, Gardnerella vaginalis and Lactobacillus iners with a minority of Lactobacillus crispatus had a higher CIN risk (OR 5.80, 95% CI 1.73-19.4) and abundance of A. vaginae had a higher CIN risk (OR 6.63, 95% CI 1.61-27.2). The synergistic effect of a high score of this microbial pattern and oncogenic HPV was observed (OR 34.1, 95% CI 4.95-284.5; RERI/S, 15.9/1.93). A predominance of A. vaginae, G. vaginalis and L. iners with a concomitant paucity of L. crispatus in the cervical microbiota was associated with CIN risk, suggesting that bacterial dysbiosis and its combination with oncogenic HPV may be a risk factor for cervical neoplasia.

Detection of Peripheral Arterial Disease Within the Foot Using Vascular Optical Tomographic Imaging: A Clinical Pilot Study.

OBJECTIVE: Vascular optical tomographic imaging (VOTI) is a novel imaging modality that is capable of detecting hemoglobin concentrations in tissue. VOTI is non-invasive, non-ionizing and does not require contrast injection. This technology was applied to the diagnosis of peripheral arterial disease (PAD) within lower extremities of diabetic patients with calcified arteries. This could be of substantial benefit as these patients suffer from comorbidities such as arterial incompressibility, which complicates diagnosis and monitoring. METHODS: Forty individuals (10 non-diabetic patients with PAD, 10 diabetic patients with PAD, and 20 healthy volunteers) were enrolled in a diagnostic pilot study using the VOTI system. The patients were imaged during at high pressure cuff occlusion. RESULTS: The VOTI system was capable of quantifying the blood volume changes within the foot during the thigh cuff occlusion and outputting diagnostic parameters, such as change in hemoglobin concentration, enabling the assessment of foot perfusion. This study resulted in a statistically significant difference between the healthy cohort and both the non-diabetic and the diabetic PAD cohorts (p = .006, p = .006). Receiver operating characteristic (ROC) curve analysis showed that PAD diagnosis could be made with over 80% sensitivity or specificity depending on the characteristic cutoff point. In addition, VOTI was capable of providing the locations of under-perfused regions within the foot and evaluating the severity of arterial disease, even within diabetic patients with calcified arteries, who are traditionally difficult to diagnose. CONCLUSION: VOTI can effectively diagnose PAD independently of arterial compressibility, making it very useful for assessing vascular disease in diabetic patients.

Impact of CD34+ cell dose in children who receive unrelated PBSCT with in vivo T-cell depletion for hematologic malignancies.

PBSCs are increasingly being chosen as the mode of donation among unrelated donors. Pediatric patients, in particular, may receive very high CD34(+) and CD3(+) doses during unrelated PBSCT. In this work, we analyzed survival and GVHD outcomes in a cohort of 81 children who received unrelated PBSCT with uniform antithymocyte globulin (ATG)-based in vivo T-cell depletion for treatment of hematologic malignancy, with emphasis on the impact of cell dose on transplant outcomes. EFS was 61.5±5.6%, with higher CD34(+) dose (>10.0 × 10(6)/kg) and lower patient risk status predicting improved survival in multivariate study. Cumulative incidence of relapse was 30.2±5.2%; a low CD34(+) dose was the only significant factor for relapse. Neither CD34(+) nor CD3(+) dose was a significant determinant of acute or chronic GVHD. Importance of CD34(+) dose was reaffirmed in a subcohort of younger patients who received greater median cell doses than the overall cohort. In summary, for children who received unrelated PBSCT with ATG-based T-cell depletion for treatment of hematologic malignancy, the CD34(+) dose was the most important factor for relapse and EFS, and neither the CD34(+) nor the CD3(+) dose influenced incidence of acute or chronic GVHD.

Evolutionary Dynamics of a Highly Pathogenic Type 2 Porcine Reproductive and Respiratory Syndrome Virus: Analyses of Envelope Protein-Coding Genes.

Porcine reproductive and respiratory syndrome virus (PRRSV) has long been an economically devastating swine viral disease. The recent emergence of a highly pathogenic type 2 PRRSV with high mobility and mortality in China, spreading in Vietnam, Laos, and Thailand has placed neighbouring countries at risk. This study applied a codon-based extension of the Bayesian relaxed clock model and the fixed effects maximum-likelihood method to investigate and compare the evolutionary dynamics of type 2 PRRSV for all of known structural envelope protein-coding genes. By comparing the highly pathogenic type 2 PRRSV clade against the typical type 2 PRRSV clade, this study demonstrated that the highly pathogenic clade evolved at high rates in all of the known structural genes but did not display rapid evolutionary dynamics compared with typical type 2 PRRSV. In contrast, the ORF3, ORF5 and ORF6 genes of the highly pathogenic clade evolved in a qualitatively different manner from the genes of the typical clade. At the population level, several codons of the sequence elements that were involved in viral neutralization, as well as codons that were associated with in vitro attenuation/over-attenuation, were predicted to be selected differentially between the typical clade and the highly pathogenic clade. The results of this study suggest that the multigenic factors of the envelope protein-coding genes contribute to diversifying the biological properties (virulence, antigenicity, etc.) of the highly pathogenic clade compared with the typical clade of type 2 PRRSV.

Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma.

Mice in which lung epithelial cells can be induced to express an oncogenic Kras(G12D) develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of Kras(G12D) tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured Kras(G12D) tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.