Analysis of Epstein-Barr Virus (EBV) and PD-L1 Expression in Nasopharyngeal Carcinoma Patients in a Non-Endemic Region.

BACKGROUND: The purpose of this study was to evaluate the status of Epstein-Barr virus (EBV) infection and the expression of programmed cell death ligand-1 (PD-L1) in tumor samples from patients with nasopharyngeal carcinoma (NPC). METHODS: Evaluation of EBV infection was performed through the detection of EBV-encoded small ribonucleic acids (EBER) by in situ hybridization, and PD-L1 expression was performed through immunohistochemistry. RESULTS: In total, 124 samples were evaluated for EBER and 120 for PD-L1 expression. A total of 86.3% of cases were positive for EBER and 55.8% were positive for PD-L1. There was a correlation between EBER positivity and the presence of undifferentiated carcinoma histology (p = 0.007) as well as the absence of tobacco history (p = 0.019). There was a correlation between PD-L1 expression and EBER positivity (p = 0.004). There was no statistically significant difference between overall survival (OS) and EBER (p = 0.290) or PD-L1 (p = 0.801) expression. CONCLUSIONS: This study corresponds to one of the largest cohorts of NPC in a non-endemic region. Phase III studies with checkpoint inhibitors are ongoing and may provide more data about the role of PD-L1 expression in this disease.

EGF+61 A>G polymorphism does not predict response to first-generation EGFR tyrosine kinase inhibitors in lung cancer patients.

Epidermal growth factor (EGF) and its receptor (EGFR) play a paramount role in lung carcinogenesis. The polymorphism in the EGF promoter region EGF+61A>G (rs4444903) has been associated with cancer susceptibility, but its role in lung cancer patients treated with tyrosine kinase inhibitors (TKIs) remains unknown. Here, we aimed to evaluate the predictive and prognostic role of EGF+61A>G SNP in lung cancer from Brazilian EGFR-mutated TKI-treated patients. Herein, patients carrying EGFR-sensitizing mutations submitted to TKI treatment (gefitinib/erlotinib) were analyzed (n = 111) for EGF+61A>G genotype by TaqMan genotyping assay. TKI treatment was classified as partial response (PR), stable disease (SD), and disease progression (DP), according to RECIST1.1. Association analysis was assessed by chi-square and Fisher's test (univariate) and multinomial model (multivariate) and survival analysis by Kaplan-Meier method and log-rank test. The EGF+61A>G genotype frequencies observed were: AA = 31.5% (n = 35), AG = 49.6% (n = 55) and GG = 18.9% (n = 21). The allelic frequencies were 56.3% for A, and 43.7% for G and the population was in Hardy-Weinberg equilibrium (P = 0.94). EGF+61A>G codominant model (AA vs. AG vs. GG) was associated with a response to TKIs (P = 0.046), as well as a recessive model (AA vs. AG + GG; P = 0.023). The multinomial regression showed an association between the codominant model (AG) and recessive model (AG + GG) with SD compared with DP (P = 0.01;OR = 0.08; 95% CI = 0.01-0.60 and P = 0.02;OR = 0.12; 95% CI = 0.20-0.72, respectively). No association between genotypes and progression-free or overall survival was observed. In conclusion, the EGF+61 polymorphism (AG and AG + GG) was independently associated with stable disease in lung cancer patients although it was not associated with the overall response rate to first-generation TKIs or patient outcome.

Comparative evaluation of video-based on-line course versus serious game for training medical students in cardiopulmonary resuscitation: A randomised trial.

PURPOSE: To estimate the effect size of a serious game for cardiopulmonary resuscitation (CPR) training in comparison with a video-based on-line course in terms of learning outcomes among medical students before simulation-based CPR using a manikin. METHODS: Participants were 45 first-year medical students randomly assigned to CPR self-training using either a video-based Apple Keynote presentation (n = 22) or a serious game developed in a 3D learning environment (n = 23) for up to 20 min. Each participant was evaluated on a written, multiple-choice test (theoretical test) and then on a scenario of cardiac arrest (practical test) before and after exposure to the self-learning methods. The primary endpoint was change in theoretical and practical baseline scores during simulated CPR. This study was conducted in 2017. RESULTS: Both groups improved scores after exposure. The video group had superior performance in both the theoretical test (7.56±0.21 vs 6.51±0.21 for the game group; p = 0.001) and the practical test (9.67±0.21 vs 8.40±0.21 for the game group; p < 0.001). However, students showed a preference for using games, as suggested by the longer time they remained interested in the method (18.57±0.66 min for the game group vs 7.41±0.43 for the video group; p < 0.001). CONCLUSIONS: The self-training modality using a serious game, after a short period of exposure, resulted in inferior students' performance in both theoretical and practical CPR tests compared to the video-based self-training modality. However, students showed a clear preference for using games rather than videos as a form of self-training.

Nuclear morphometric analysis in tissue as an objective tool with potential use to improve melanoma staging.

Alterations in nuclear size and shape are commonly observed in cancers, and its objective evaluation may provide valuable clinical information about the outcome of the disease. Here, we applied the nuclear morphometric analysis in tissues in hematoxylin and eosin-digitized slides of nevi and melanoma, to objectively contribute to the prognostic evaluation of these tumors. To this, we analyzed the nuclear morphometry of 34 melanomas classified according to the TNM stage. Eight cases of melanocytic nevi were used as non-neoplastic tissues to set the non-neoplastic parameters of nuclear morphology. Our samples were set as G1 (control, nevi), G2 (T1T2N0M0), G3 (T3T4N0M0), G4 (T1T2N1M1), and G5 (T3T4N1M1). Image-Pro Plus 6.0 software was used to acquire measurements related to nuclear size (variable: Area) and shape (variables: Aspect, AreaBox, Roundness, and RadiusRatio, which were used to generate the Nuclear Irregularity Index). From these primary variables, a set of secondary variables were generated. All the seven primary and secondary variables related to the nuclear area were different among groups (Pillai's trace P<0.001), whereas Nuclear Irregularity Index, which is the variable related to nuclear shape, did not differ among groups. The secondary variable 'Average Area of Large Nuclei' was able to differ all pairwise comparisons, including thin nonmetastatic from thin metastatic tumors. In conclusion, the objective quantification of nuclear area in hematoxylin and eosin slides may provide objective information about the risk stratification of these tumors and has the potential to be used as an additional method in clinical decision making.

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation.

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5'-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.

GRPR antagonist protects from drug-induced liver injury by impairing neutrophil chemotaxis and motility.

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.

Quantitative histopathology identifies patients with thin melanomas who are at risk for metastases.

This small exploratory study was designed to test the hypothesis that thin melanoma lesions contain nuclei of two similar phenotypes, in different proportions. In lesions likely to progress to metastatic disease, one of these phenotypes predominates. Histopathological sections from 18 cases of thin melanomas which did not progress to metastasis, and from 10 cases which did progress were imaged and digitized at high resolution, with a total of 2084 and 1148 nuclei, respectively, recorded. Five karyometric features were used to discriminate between nuclei from indolent and from potentially metastatic lesions. For each case, the percentage of nuclei classified by the discriminant function as having come from a potentially metastatic lesion was determined and termed as case classification criterion. Standard histopathological criteria, such as ulceration and high mitotic index, indicated in this material the need for intensive therapy for only one of the 10 participants, as compared with 7/10 identified correctly by the karyometric measure. Using a case classification criterion threshold of 40%, the overall accuracy was 86% in the test set. The proportion of nuclei of an aggressive phenotype may lend itself as an effective prognostic clue for thin melanoma lesions. The algorithm developed in this training set appears to identify those patients at high risk for metastatic disease, and demonstrates a basis for a further study to assess the utility of prognostic clues for thin melanomas.

Computer-assisted teaching of skin flap surgery: validation of a mobile platform software for medical students.

The purpose of this study was to develop and validate a multimedia software application for mobile platforms to assist in the teaching and learning process of design and construction of a skin flap. Traditional training in surgery is based on learning by doing. Initially, the use of cadavers and animal models appeared to be a valid alternative for training. However, many conflicts with these training models prompted progression to synthetic and virtual reality models. Fifty volunteer fifth- and sixth-year medical students completed a pretest and were randomly allocated into two groups of 25 students each. The control group was exposed for 5 minutes to a standard text-based print article, while the test group used multimedia software describing how to fashion a rhomboid flap. Each group then performed a cutaneous flap on a training bench model while being evaluated by three blinded BSPS (Brazilian Society of Plastic Surgery) board-certified surgeons using the OSATS (Objective Structured Assessment of Technical Skill) protocol and answered a post-test. The text-based group was then tested again using the software. The computer-assisted learning (CAL) group had superior performance as confirmed by checklist scores (p<0.002), overall global assessment (p = 0.017) and post-test results (p<0.001). All participants ranked the multimedia method as the best study tool. CAL learners exhibited better subjective and objective performance when fashioning rhomboid flaps as compared to those taught with standard print material. These findings indicate that students preferred to learn using the multimedia method.

Interrelationship of clinical, histomorphometric and immunohistochemical features of oral lesions in chronic paracoccidioidomycosis.

BACKGROUND: This study aimed to analyze the oral lesions of chronic paracoccidioidomycosis concerning their histomorphometric, immunohistochemical, and clinical features in a standardized sample. METHODS: Fifty biopsy specimens of oral lesions of chronic paracoccidioidomycosis were submitted to hematoxylin and eosin (H&E), Grocott-Gomori and immunohistochemical staining. Data regarding disease duration and size and number of oral lesions, as well as erythrocytes, leukocytes, lymphocytes, hematocrit, hemoglobin, and erythrocyte sedimentation rate, were collected from medical charts. Granuloma density and number and diameter of buds and fungal cells, and IL-2, TNF-alpha and IFN-gamma expression, as well as clinical and hematological features, were quantified and correlated. RESULTS: Bud diameter was significantly greater in intermediate density granulomas compared to higher density granulomas. The other variables (number of buds, number and diameter of fungi, expression of IL-2, TNF-alpha and IFN-gamma, and clinical and hematological features) did not significantly change with the density of granulomas. There was a positive correlation between bud number and fungal cell number (r = 0.834), bud diameter and fungal cell diameter (r = 0.496), erythrocytes and number of fungi (r = 0.420), erythrocytes and bud number (r = 0.408), and leukocytes and bud number (r = 0.396). Negative correlation occurred between number and diameter of fungi (r = -0.419), bud diameter and granuloma density (r = -0.367), TNF-alpha expression and number of fungi (r = -0.372), and TNF-alpha expression and bud number (r = -0.300). CONCLUSION: The histological, immunological, and clinical features of oral lesions evaluated did not differ significantly between patients in our sample of chronic paracoccidioidomycosis. TNF-alpha levels were inversely correlated with intensity of infection.

Comparison of effects of clodronate and zoledronic acid on the repair of maxilla surgical wounds - histomorphometric, receptor activator of nuclear factor-kB ligand, osteoprotegerin, von Willebrand factor, and caspase-3 evaluation.

BACKGROUND: The aim of this study was to compare clodronate and zoledronic acid regarding their influence on the repair of surgical wounds in maxillae (soft tissue wound and tooth extraction) and their relation to osteonecrosis. MATERIAL AND METHODS: Thirty-four Wistar rats were allocated into three groups according to the treatment received: (i) 12 animals treated with zoledronic acid, (ii) 12 animals treated with clodronate and (iii) 10 animals that were given saline solution. All animals were subjected to tooth extractions and surgically induced soft tissue injury. Histological analysis of the wound sites was performed by means of hematoxylin-eosin (H&E) staining and immunohistochemical staining for receptor activator of nuclear factor-kB ligand (RANKL), osteoprotegerin (OPG), von Willebrand factor, and caspase-3. RESULTS: The zoledronic acid group showed higher incidence of non-vital bone than did the clodronate group at the tooth extraction site. At the soft tissue wound site, there were no significant differences in non-vital bone between the test groups. RANKL, OPG, von Willebrand factor, and caspase-3 did not show significant differences between the groups for both sites of surgical procedures. CONCLUSION: Both of the bisphosphonates zoledronic acid and clodronate are capable of inducing maxillary osteonecrosis. Immunohistochemical analysis suggests that the involvement of soft tissues as the initiator of osteonecrosis development is less probable than has been pointed out.

Increased tissue factor expression and poor nephroblastoma prognosis.

PURPOSE: There is potential interaction between malignant cell growth and the coagulation pathway. Recent studies suggest that tissue factor, a primary initiator of the extrinsic coagulation pathway, is expressed in various solid tumors in association with increased angiogenesis. To our knowledge we report for the first time the detection of tissue factor expression by immunohistochemistry in Wilms tumors and its correlation with clinical outcomes. MATERIAL AND METHODS: Tissue factor expression detected by immunohistochemistry was assessed in 41 formalin fixed, paraffin embedded Wilms tumor cases treated at university hospitals. We correlated findings with tumor recurrence and cancer specific survival. RESULTS: Positive immunohistochemistry detection of tissue factor was observed in 88.3% of the tumors analyzed. Tissue factor on immunohistochemistry was associated with tumor recurrence and survival (p = 0.01 and 0.02, respectively). Increased immunohistochemical detection of tissue factor was the most important risk factor for recurrence and mortality in our population on bivariate and multivariate analysis. CONCLUSIONS: Tissue factor is a promising research subject as a prognostic factor for Wilms tumor. More studies are needed to clarify the mechanisms by which tissue factor affects cancer progression and outcome, and its potential role as a therapeutic target.

Digital morphometric characterization of mucosal surface lesion patterns under magnification colonoscopy.

OBJECTIVE: To study quantified mucosal surface patterns of digitized colonoscopic images obtained with 17-40x magnification and chromoscopy of hyperplastic, adenomatous lesions and adenocarcinomas confirmed by histology. STUDY DESIGN: Morphometric measures were performed on at least 10 image files of each lesion and compared to normal mucosa at the same distance and area from each image. Thirty-seven hyperplastic lesions, 42 adenomas and 26 carcinomas from 105 patients were studied. Three morphometric characteristics were evaluated in lesions and normal mucosal surface area from each case: pit counts per area, pit diameter and pit area. RESULTS: Pit counts per area and pit area of lesions as compared to the normal mucosa of the same cases showed statistically significant differences in the 3 groups of lesions studied (p < 0.001). CONCLUSION: Real-time morphometric evaluation is feasible during videocolonoscopy with magnification roughly equivalent to a 5x microscopic field. Automated evaluation of morphometric characteristics as part of Bayesian Belief Network software may prove a useful information source for the colonoscopist's diagnostic decision.

Angiogenesis in non-small cell lung cancer: microvessel area in needle biopsy in vascular tumor density.

OBJECTIVE: To evaluate angiogenesis in non-small cell lung cancer (NSCLC) through immunohistochemistry with CD34 and computerized image analysis comparing the microvessel area in needle biopsies and surgical specimens. STUDY DESIGN: Core biopsies and surgical specimens from 28 patients with NSCLC were reviewed. Microvessels were highlighted by immunohistochemistry with anti-CD34. Tumor microvessel area was measured in digital photographs from hot spots of all samples. RESULTS: Average microvessel area among core samples was 5,093.6 microm2 (range, 233.4-17,916.8) and among surgical specimens was 3,599.3 microm2 (range, 376.9-9,514.0). There was strong correlation between overall microvascular area in biopsies and surgical specimens (r = 0.7; p = 0.0001). Mean area of core specimens was used to divide cases into groups of low and high vascular areas. A slightly stronger correlation was observed between biopsies and surgical specimens with low vascular areas (r = 0.84; p = 0.0001). CONCLUSION: There is strong correlation of microvessel area in core biopsies and respective surgical specimens in NSCLC. These data indicate that core specimens could be used to assess the extent of angiogenesis in NSCLC in the pretreatment phase.

Nuclear chromatin texture in rectal carcinoma. Prognostic value.

OBJECTIVE: To investigate the prognostic value of nuclear features in rectal carcinoma. STUDY DESIGN: High-resolution imagery of 3,635 nuclei from 51 patients operated on for rectal cancer at various Dukes' stages was digitally recorded. A set of 93 features descriptive of the spatial and statistical distribution of nuclear chromatin was computed for each nucleus to derive a digital signature. Karyometric features were analyzed for correlation with progression of disease and death. RESULTS: Multivariate analysis of main karyometric features in comparison with cancer staging demonstrated that total optical density and clumpness, as well as average nuclear signature, had significant prognostic value in predicting cancer-related death. CONCLUSION: Digital signature seems to have a role as prognostic factor in rectal cancer. The method could be a useful parameter in deciding whether to perform adjuvant therapy in particular subgroups of patients, independently of tumor staging. However, these observations need to be substantiated with additional studies, including larger numbers of patients.

[Digital morphometric and stereologic analysis of small intestinal mucosa in well-nourished and malnourished children with persistent diarrhea]. / Estudo morfométrico e estereológico digital da mucosa do intestino delgado de crianças eutróficas e desnutridas com diarréia persistente.

OBJECTIVE: To test the hypothesis that the proximal small intestines of children with persistent diarrhea present morphometric and stereologic changes proportional to their nutritional status, using microscope images stored in a computer. METHODS: Cross-sectional study with 65 pediatric patients, whose ages ranged from 4 months to 5 years, with persistent diarrhea for over 14 days. The nutritional assessment was performed according to the z-scores for weight/age (W/A), weight/height (W/H) and height/age (H/A) ratios, divided into: well-nourished = z > or =2SD and malnourished = z<2SD; well-nourished = z > or =2SD, nutritional risk = z<1SD and malnourished = z<-2SD; and continuously, in descending order, using the NCHS charts. After obtaining the computer images using the software Scion Image, villous height, crypt depth, mucosal thickness, total mucosal thickness, and villous/crypt ratio were measured in the fragments of the small intestinal mucosa, enlarged 100 times. When images were enlarged 500 times, enterocyte height, nuclear height and brush-border height were measured. Stereologic analysis was performed using cycloid arcs. RESULTS: For W/A, W/H and H/A z-scores, divided into two nutritional status categories, no statistically significant difference was observed in regard to villous height, crypt depth, mucosal thickness, total mucosal thickness and villous/crypt ratio. Enterocyte height presented the most significant difference between well-nourished and malnourished groups, for W/A and W/H ratios, with a 500x enlargement, although this difference was not statistically significant. When z-scores were subdivided into three nutritional status categories, a digital morphometric analysis showed a statistically significant difference for villous/crypt ratio between the well-nourished and slightly malnourished group and the well-nourished and mild to severe malnourished group (p=0.048). The villous/crypt ratio was higher among well-nourished children. Using the Spearman coefficient, the variables enterocyte height, height of enterocyte nucleus and brush-border height presented a clear association with the W/A ratio (r=0.25; p=0.038), W/H ratio (r=0.029; p=0.019). The height of the enterocyte and the brush-border height were associated with W/H ratio. CONCLUSION: The observed associations between nutritional status and the analyzed small intestinal mucosa variables showed a positive correlation with patients' weight. Although these associations were of a slight to moderate magnitude, we observed a tendency of enterocyte size reduction, as well as a reduction in the size of its nucleus and brush-border, as the level of malnutrition increases.

Nuclear chromatin texture in rectal cancer. Relationship to tumor stage.

OBJECTIVE: To characterize, by morphometric and chromatin texture analysis, a series of rectal carcinomas classified according to Dukes staging. STUDY DESIGN: High-resolution imagery of 6,001 nuclei from 51 specimens of rectal carcinoma and 22 specimens of normal rectal tissue was digitally recorded. A set of 93 features descriptive of the spatial and statistical distribution of nuclear chromatin was computed for each nucleus to form a characteristic signature. RESULTS: Rectal carcinomas were significantly different from normal rectum in their digital signature. Eleven karyometric features, such as nuclear area and total optical density, were clearly different between the groups, with significant differences found in analysis of 8 of those features. The most distinctive pattern in lesion signatures in comparison with normal rectal tissue was observed at Dukes' stage D. However, the highest average signature values were seen at Dukes' stage B. The lesion signatures and total optical density observed in cancer specimens deviated markedly from values in the normal group. CONCLUSION: Chromatin texture signature proved to be a useful method of identifying and characterizing nuclear differences between rectal carcinoma and normal rectal tissue.