Diagnostic Utility of Next-Generation Sequencing in Circulating Free DNA and a Comparison With Matched Tissue in Gallbladder Carcinoma.

Mutation detection for therapy monitoring in cell-free DNA (cfDNA) is used clinically for some malignancies. Gallbladder carcinoma (GBC) presents a diagnostic challenge and has limited late-stage treatment options. To our knowledge, this novel study examines, for the first time, genomic alterations in cfDNA from GBC to assess diagnostic accuracy and therapeutic options. The concordance of somatic genomic changes in cfDNA and DNA from paired tumor tissue was analyzed. Paired serum and tissue samples from 40 histologically proven GBC, 20 cholecystitis, and 4 normal (noninflamed gallbladder) controls were included. Targeted next-generation sequencing with a 22-gene panel (Colon and Lung Cancer Research Panel v2, Thermo Scientific) in cfDNA and tumor tissue with high depth and uniform coverage on ION Personal Genome Machine (ION, PGM) was performed. A spectrum of 223 mutations in cfDNA and 225 mutations in formalin-fixed paraffin-embedded tissue DNA were identified in 22 genes. Mutations ranged from 1 to 17 per case. In cfDNA frequent alterations were in TP53 (85.0%), EGFR (52.5%), MET (35%) CTNNB1, SMAD4, BRAF (32.5%), PTEN (30%), FGFR3 and PIK3CA (27.5%), NOTCH1 (25.0%), and FBXW7 and ERBB4 (22.5%). At least one clinically actionable mutation was identified in all cfDNA samples. Paired samples shared 149 of 225 genetic abnormalities (66.2%). Individual gene mutation concordance ranged from 44.44% to 82.0% and was highest for EGFR (82.0%), BRAF and NOTCH1 (80.0%), TP53 (73.08%), MET (72.22%), and ERBB4 (71.42%) with a significant level of correlation (Spearman r = 0.91, P ≤ .0001). The sensitivity and specificity of the TP53 gene at the gene level was the highest (94.44% and 100.0%, respectively). Overall survival was higher for ERBB4 and ERBB2 mutant tumors. The adenocarcinoma subtype revealed specific genetic changes in ERBB4, SMAD4, ERBB2, PTEN, KRAS, and NRAS. NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery to guide treatment decisions. Targeted therapy identified in GBC included SMAD4, ERBB2, ERBB4, EGFR, KRAS, BRAF, PIK3CA, MET, and NRAS.

Toxic potential assessment of hair dye developer 2,4,5,6-tetraaminopyrimidine sulfate exposed under ambient UVB radiation.

Synthetic cosmetics, particularly hair dyes, are becoming increasingly popular among people of all ages and genders. 2,4,5,6-tetraaminopyrimidine sulfate (TAPS) is a key component of oxidative hair dyes and is used as a developer in several hair dyes. TAPS has previously been shown to absorb UVB strongly and degrade in a time-dependent manner, causing phototoxicity in human skin cells. However, the toxic effects of UVB-degraded TAPS are not explored in comparison to parent TAPS. Therefore, this research work aims to assess the toxicity of UVB-degraded TAPS than TAPS on two different test systems, that is, HaCaT (mammalian cell) and Staphylococcus aureus (a bacterial cell). Our result on HaCaT has illustrated that UVB-degraded TAPS is less toxic than parent TAPS. Additionally, UVB-exposed TAPS and parent TAPS were given to S. aureus, and the bacterial growth and their metabolic activity were assessed via CFU and phenotype microarray. The findings demonstrated that parent TAPS reduced bacterial growth via decreased metabolic activity; however, bacteria easily utilized the degraded TAPS. Thus, this study suggests that the products generated after UVB irradiation of TAPS is considered to be safer than their parent TAPS.

A study on expression of programmed death ligand-1 in small cell lung carcinoma and correlation with clinicopathological parameters.

Small cell lung carcinoma (SCLC) is characterized by rapid growth and an aggressive clinical course. Standard therapy regimes have limited effects on disease course; therefore the prognosis of SCLC is poor. In the current study, the frequency of programmed death ligand 1 (PD-L1) expression in SCLC and its correlation with clinico-pathological features were evaluated. The study included 100 cases of SCLC wherein testing for PD-L1 was done with the SP263 clone on the Ventana benchmark XT system. Cases with > 1% PD-L1 expression in tumour cells or immune cells were categorized as positive. PD-L1 expression was identified in 14% of cases using the cut-off of ≥ 1%. The tumour proportion score was 10% and the immune proportion score was 9.78% using a cut-off of ≥ 1%. PD-L1 positive expression was more frequent in the male population with age > 40 years. All the patients with positive PD-L1 expression were smokers. In the PD-L1 positive group, presence of necrosis was identified in 71.4% of cases and when compared with the PD-L1 negative subgroup this finding was statistically significant (p = 0.010). Personalized targeted therapy for cases of SCLC is still under evaluation. The use of immunotherapeutic targets, such as PD-L1, may help to define a new treatment strategy for SCLC. Development of new treatment strategies may improve prognosis and survival.

Tattoo inks are toxicological risks to human health: A systematic review of their ingredients, fate inside skin, toxicity due to polycyclic aromatic hydrocarbons, primary aromatic amines, metals, and overview of regulatory frameworks.

Today, tattooing has become very popular among people all over the world. Tattooists, with the help of tiny needles, place tattoo ink inside the skin surface and unintentionally introduce a large number of unknown ingredients. These ingredients include polycyclic aromatic hydrocarbons (PAHs), heavy metals, and primary aromatic amines (PAAs), which are either unintentionally introduced along with the ink or produced inside the skin by different types of processes for example cleavage, metabolism and photodecomposition. These could pose toxicological risks to human health, if present beyond permissible limits. PAH such as Benzo(a)pyrene is present in carbon black ink. PAAs could be formed inside the skin as a result of reductive cleavage of organic azo dyes. They are reported to be highly carcinogenic by environmental protection agencies. Heavy metals, namely, cadmium, lead, mercury, antimony, beryllium, and arsenic are responsible for cancer, neurodegenerative diseases, cardiovascular, gastrointestinal, lungs, kidneys, liver, endocrine, and bone diseases. Mercury, cobalt sulphate, other soluble cobalt salts, and carbon black are in Group 2B, which means they may cause cancer in humans. Cadmium and compounds of cadmium, on the other hand, are in Group 1 (carcinogenic to humans). The present article addresses the various ingredients of tattoo inks, their metabolic fate inside human skin and unintentionally added impurities that could pose toxicological risk to human health. Public awareness and regulations that are warranted to be implemented globally for improving the safety of tattooing.

Kirsten rat sarcoma virus protein overexpression in adenocarcinoma lung: Association with clinicopathological and histomorphological features.

CONTEXT: Lung cancer is the leading cause of cancer-related deaths worldwide. The constitutive activation of multiple signaling pathways is the major cause of carcinogenesis. AIMS: The study evaluates the frequency of Kirsten rat sarcoma virus (KRAS) protein overexpression and correlates with clinicopathological and histomorphological features in non-small cell lung carcinoma (NSCLC)-adenocarcinoma. SETTINGS AND DESIGN: Tertiary hospital-based retrospective and prospective case series included 100 cases of NSCLC-adenocarcinoma. MATERIALS AND METHODS: The basic panel of Immunohistochemistry including Napsin-A, thyroid transcription factor-1 (TTF-1), and markers for squamous differentiation, p-40 was used in formalin-fixed paraffin-embedded tissue blocks. The KRAS monoclonal antibody (9.13, Thermo Fisher Scientific, USA) was used. STATISTICAL ANALYSIS USED: The IBM-Statistical Package for the Social Sciences (SPSS) (SPSS, International Business Machines Corporation, New York, NY, USA) analysis software, version 16 was used for all statistical calculations. RESULTS: KRAS protein expressed in 28.0% (28/100) cases. Cases were grouped as KRAS positive and negative. TTF-1 and Napsin-A were expressed in 89.25% (n = 25) and 92.86% (n = 26) cases, respectively. Stage IV clinical disease was identified in 55% of cases, and 36.84% of cases had a mean survival between 6 and 12 months. In KRAS positive group, the most common pattern of cellular arrangement was acinar/loose clusters pattern present in 64.29% (n = 21) and 75.0% (n = 18) cases followed by the solid pattern present in 42.86% of cases (n = 12), respectively. Necrosis was identified in 57.14% (n = 16) cases. Mucin pattern was present in 32.14% of cases (n = 9), which was significantly different when compared with the KRAS negative group (P = 0.036). CONCLUSIONS: This finding may imply that KRAS mutations may not be entirely triggered by alterations induced by carcinogens in smoke. KRAS gene is frequently mutated in pulmonary tumors. It should be tested in NSCLC owing to its predictive and prognostic effects.

Comparison of laboratory-developed test & validated assay of programmed death ligand-1 immunohistochemistry in non-small-cell lung carcinoma.

Background & objectives: Inhibitors of immune checkpoint regulators, programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), improve outcome in advanced non-small-cell lung carcinoma (NSCLC). Tumours expressing PD-L1 protein are more likely to benefit from this targeted therapy. Multiple concurrent clinical trials evaluating different anti-PD-1/PD-L1 therapies have validated five different immunohistochemistry (IHC) assays using varied antibody clones and staining conditions. This study was aimed at identification of a single harmonized PD-L1 assay for tumour tissue conservation and cost-effectiveness in patients with NSCLC. Methods: The performance of low-cost, manual, laboratory-developed technique (LDT) PD-L1 IHC assay using the easily available SP142 clone was compared with trial validated Ventana SP263 IHC performed on automated Ventana staining platform on tumour sections of NSCLCs. Results: Eighty cases of NSCLC were included. SP263 and SP142 stained both tumour cells and immune cells. The concordance rate of tumour cell staining was about 76 per cent, with SP263 detecting more tumour cells in 16 per cent of cases. The concordance rate of immune cell staining was only 61 per cent, with SP142 detecting more immune cells in 24 per cent of cases. The sensitivity, specificity, positive and negative predictive values of manual SP142 LDT assay against gold standard SP263 Ventana assay were 70, 94, 86 and 86 per cent, respectively, at positivity thresholds of ≥1 per cent tumour cell staining. Interpretation & conclusions: The study findings suggested that LDT using SP142 clone showed only moderate concordance with SP263 Ventana assay, and the two assays were not interchangeable. More such validation studies need to be done to generate information that can complement patient therapy in cases of NSCLC.

Congenital Pulmonary Airway Malformation - 19-Year Experience from a Tertiary Care Center in India.

Background: Congenital pulmonary airway malformation (CPAM) is a collection of non-hereditary, developmental anomalies. Our aim was to analyze the histological profiles and prevalence of CPAMs diagnosed in our center. Methods: A retrospective study of all CPAMs diagnosed from January 1999 to May 2018 from a general hospital pathology service was performed. Results: There were 79 cystic lesions encountered in fetuses, neonates, and children, 15 of which were CPAMs {5/2372 (0.21%) autopsies and 10/216026 (0.0046%) surgical resections}. The male:female ratio was 1:1.14. Gestational age of antenatal cases ranged from 22 to 32 weeks, postnatal ages ranged from 7 days to 15 years (mean 2.9 years). The cases were right-sided (8/15;53.3%), left-sided (4/15;26.7%) and bilateral (3/15,20%). Seven (46.7%), 4 (26.7%),3 (20%) and 1 (6.7%) were types 1, 2, 3 and 4, respectively. None of the surgical cases had postoperative mortality or morbidity. Conclusions: Prompt recognition and surgical resectability resulted in normal growth and symptom free survival in our postnatally diagnosed patients. Mortality in antenatally diagnosed fetuses remains high (5/11;45%).

Clinical relevance of PD-L1 expression in gallbladder cancer: a potential target for therapy.

AIMS: Programmed death-ligand 1 (PD-L1), a potential target for immune checkpoint inhibitors in various solid neoplasms, has been studied in very few cases of Gall Bladder Carcinoma (GBC). The current study aimed to evaluate PD-L1 expression at primary and metastatic sites of GBC, and its associations with standard prognostic clinicopathological parameters, as well as with overall survival. METHODS AND RESULTS: One hundred and seventy-four cases of GBC were evaluated for PD-L1 expression by the use of the SP263 clone in tissue microarrays. Clinicopathological characteristics and survival data were correlated with PD-L1 expression analysed at different cut-offs of ≥1%, ≥10% and ≥50% in tumour cells and tumour-infiltrating lymphocytes (TILs). The mean age of patients was 49.9 years, and the male/female ratio was 1:2.9. Of the cases, 73.6% presented with stage 3/4 disease. Tumour cells expressed PD-L1 in 23.0% of cases, and TILs expressed PD-L1 in 24.1% of cases. At a cut-off of 10%, 14.9% of cases expressed PD-L1, and at a cut-off of 50%, 7.5% of cases expressed PD-L1. Significant associations were seen between tumour proportion score and histological type (P = 0.004), histological grade (P = 0.004), nuclear grade (P = 0.008), nodal metastasis (P = 0.051), higher stage (P = 0.058), and TILs (P < 0.001). Tumour size, growth pattern, the presence of necrosis and lymphovascular emboli showed no significant associations with PD-L1 in tumour cells or TILs. In synchronous paired samples from primary and metastatic lymph nodes, discordantly higher PD-L1 expression was evident in lymph nodes. Overall survival was not associated with PD-L1 expression (P = 0.546). CONCLUSION: PD-L1 does not appear to be a prognostic marker or influence survival in GBC patients. However, PD-L1 expression occurs in one of four GBCs, supporting the future possibility of immune-modulation therapy to improve the dismal overall survival.

Fulminant leptomeningeal carcinomatosis from a malignant melanoma arising in a cerebellopontine epidermoid cyst: A rare case with diagnostic pointers.

A malignant component in an epidermoid cyst is rare. We report an exceptionally rare case of a malignant melanoma arising in an epidermoid cyst located in the cerebellopontine (CP) angle. A 26-year-old woman presented with headache, vomiting, ataxia and difficulty in swallowing over the previous 3 months. The radiological finding suggested an epidermoid cyst and the lesion was excised. The histopathology confirmed a CP angle epidermoid cyst. Within 1 month of discharge, she developed hydrocephalus for which a ventriculo-peritoneal shunt was performed. Postoperatively she developed weakness in lower limbs. A contrast-enhanced MRI was done which showed dilated CSF cisternal spaces with a small enhancing lesion in the pineal region and enhancement of meninges extending to the spinal cord. Re-exploration showed gelatinous material with gross adhesions in the CP angle cistern. A dural biopsy was done which showed sheets of poorly differentiated tumor cells which expressed S100 and Melan A and were immunoreactive with Human Melanoma Black (HMB)-45 antibody, consistent with the diagnosis of malignant melanoma. Histology of the excised epidermoid cyst was re-evaluated in deeper sections and showed scattered atypical melanocytes in the basal layer of the epidermis which were highlighted with HMB-45 antibody. The patient expired within 3 days of the second procedure due to respiratory failure. A very aggressive fulminant course of the disease was evident after surgery for the epidermoid cyst. Treatment options are limited. Criteria for identification of malignancy in an intracranial epidermoid cyst were identified in our case retrospectively and have been highlighted.

PD-L1 immuno-expression assay in thymomas: Study of 84 cases and review of literature.

BACKGROUND AND AIMS: Programmed death ligand 1 (PD-L1), an immune check point inhibitor, is known to be expressed in several malignancies and is being considered as a prognostic factor and a potential immunotherapeutic target. The aim of this study was to characterize PD-L1 expression in thymomas and to determine correlation with clinicopathological features and previously published studies in the literature. METHODS: Tissue microarrays were prepared from selected blocks of thymomas and immunohistochemistry (IHC) for PD-L1 was performed. Cases were considered as PD-L1 positive or negative depending on whether the percentage of stained thymic epithelial cells were <25 or >25%. Results were compared clinically and with previously published studies using Google and Pubmed search engines. RESULTS: Of 84 cases of thymoma, 69 (82.1%) revealed PD-L1 positivity in >25% cells. 94.23% of type B thymoma subtypes (B1/B2/B3) were PD-L1 positive (P < 0.001). There was no correlation of PD-L1 with age, gender, myasthenia gravis, the tumor size or stage of disease. Nine studies were available in the literature; most of which showed PD-L1 expression in higher stage and B subtype however percentage positivity varied from 53.7% to over 90%. CONCLUSIONS: PD-L1 expression is frequent in type B (B1/B2/B3) thymomas. It can be easily evaluated by IHC even on small biopsies in unresectable cases, thereby enabling improved clinical evaluation as well as prognostic stratification of patients. It will serve as a potential indicator for benefit from anti-PD-L1 antibody immunotherapy in thymomas.

Molecular mechanism of viral resistance to a potent non-nucleoside inhibitor unveiled by molecular simulations.

Recently, we reported on a potent benzimidazole derivative (227G) that inhibits the growth of the bovine viral diarrhea virus (BVDV) in cell-based and enzyme assays at nanomolar concentrations. The target of 227G is the viral RNA-dependent RNA polymerase (RdRp), and the I261M mutation located in motif I of the RdRp finger domain was found to induce drug resistance. Here we propose a molecular mechanism for the retained functionality of the enzyme in the presence of the inhibitor, on the basis of a thorough computational study of the apo and holo forms of the BVDV RdRp either in the wild type (wt) or in the form carrying the I261M mutation. Our study shows that although the mutation affects to some extent the structure of the apoenzyme, the functional dynamics of the protein appear to be largely maintained, which is consistent with the retained functionality of this natural mutant. Despite the binding site of 227G not collapsing or undergoing drastic structural changes upon introduction of the I261M substitution, these alterations reflect crucially on the binding mode of 227G, which is significantly different from that found in wt RdRp. In particular, while in the wt system the four loops lining the template entrance site embrace 227G and close the template passageway, in the I261M variant the template entrance is only marginally occluded, allowing in principle the translocation of the template to the interior of the enzyme. In addition, the mutated enzyme in the presence of 227G retains several characteristics of the wt apoprotein. Our work provides an original molecular picture of a resistance mechanism that is consistent with published experimental data.

Panel of serum long non-coding RNAs as potential non-invasive biomarkers for gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a common malignancy and is usually diagnosed in the late stages of the disease. The identification of new effective early diagnostic biomarkers could represent an effective approach in reducing mortality in GBC. Altered expression of long non-coding RNAs (lncRNAs) is believed to be associated with the emergence and development of GBC. Our study aims to identify the expression of a range of circulating lncRNAs, including HOTAIR, ANRIL, H19, CCAT1 and MEG3, in matched serum and tissues of GBC for diagnosis and its association with clinicopathological features. The case and control study included matched serum and tissues from 63 GBC, 19 cholecystitis (CC), and 46 normal controls (NC). RNA extraction and cDNA synthesis from serum and fresh tissue match were performed using commercially available kits. Relative expression was assessed using SYBR Green real-time quantitative polymerase chain reaction. Circulating lncRNA levels including HOTAIR, ANRIL and H19 were upregulated in serum samples, while MEG3 and CCAT1 were downregulated in GBC compared to controls. The trend towards upregulation and downregulation was comparable in the tissue. HOTAIR and MEG3 levels were significantly different between serum CC and early-stage GBC (p = 0.0373, 0.0020), while H19 was significantly upregulated comparing early-stage GBC to advanced-stage GBC (p = 0.018). The expression of ANRIL was significant with M stage (p = 0.0488), H19 with stage (p = 0.009), M stage (p=<0.0001) & stage (0.009) and CCAT1 with M stage (0.044). When distinguishing GBC and NC, AUC for HOTAIR was 0.75, ANRIL 0.78, H19 0.74, CCAT1 0.80 and 0.96 for MEG3. The combination sensitivity for lncRNAs ranged from 84.13% (CI: 72.74-92.12%) to 100.0% (CI: 94.31-100.0%). Significant diagnostic value in discriminating pathologic stage was observed for ANRIL and MEG3 (p = 0.022, p = 0.0005). LncRNA show a significant change in expression in GBC and in discrimination of early stage from late-stage disease. The detection of 2 lncRNAs in panels, in coordination with radiology, could represent a potential serum-based biomarker for early-stage GBC diagnosis.

Role of thoracoscopy in diagnosis of tubercular pleural effusion in low ADA setting- A prospective, observational study.

BACKGROUND: Tubercular Pleural effusion (TBPE) is one of most common extrapulmonary tuberculosis. It can be difficult to diagnose due to low sensitivity of pleural fluid smear, culture and CBNAAT. Diagnosis of TBPE is then dependent on the level of pleural fluid Adenosine Deaminase (ADA). Thoracoscopic pleural biopsy gives definite diagnosis specially in Low Pleural fluid ADA setting. AIMS AND OBJECTIVE: This study was planned to find out the prevalence of tubercular etiology in patients of exudative pleural effusion with low ADA (ADA <40 IU/L). MATERIAL AND METHODS: A Prospective, observational study was carried out in a tertiary teaching institute in north India. Total 142 patients of pleural effusion with low ADA were enrolled. All patients underwent rigid thoracoscopy for confirmation of their diagnosis. RESULTS: Out of 142 patients, male were 78 (55%) and female were 64 (45%). Mean age of patients were 57.4 years. Tuberculosis was diagnosed as a cause of effusion in 22 (15.5%) out of 142 patients. Majority of TBPE patients had pleural thickening as thoracoscopic finding. Mean ADA level in TBPE was 27.36 ± 11.6 as compared to 18.55 ± 9.02 in non tubercular pleural effusion patients and this difference was significant statistically (P- 0.002). CONCLUSION: The diagnosis of patients having exudative, low ADA pleural effusion can be very easily confirmed by thoracoscopy guided pleural biopsy which has a very high diagnostic yield.

A multicentre study to evaluate the diagnostic performance of a novel CAD software, DecXpert, for radiological diagnosis of tuberculosis in the northern Indian population.

Tuberculosis (TB) is the leading cause of mortality among infectious diseases globally. Effectively managing TB requires early identification of individuals with TB disease. Resource-constrained settings often lack skilled professionals for interpreting chest X-rays (CXRs) used in TB diagnosis. To address this challenge, we developed "DecXpert" a novel Computer-Aided Detection (CAD) software solution based on deep neural networks for early TB diagnosis from CXRs, aiming to detect subtle abnormalities that may be overlooked by human interpretation alone. This study was conducted on the largest cohort size to date, where the performance of a CAD software (DecXpert version 1.4) was validated against the gold standard molecular diagnostic technique, GeneXpert MTB/RIF, analyzing data from 4363 individuals across 12 primary health care centers and one tertiary hospital in North India. DecXpert demonstrated 88% sensitivity (95% CI 0.85-0.93) and 85% specificity (95% CI 0.82-0.91) for active TB detection. Incorporating demographics, DecXpert achieved an area under the curve of 0.91 (95% CI 0.88-0.94), indicating robust diagnostic performance. Our findings establish DecXpert's potential as an accurate, efficient AI solution for early identification of active TB cases. Deployed as a screening tool in resource-limited settings, DecXpert could enable early identification of individuals with TB disease and facilitate effective TB management where skilled radiological interpretation is limited.

Pattern of care and clinical outcome of patients with carcinoma endometrium and the impact of central histopathological review on management: A tertiary cancer centre experience.

PURPOSE/OBJECTIVE: Endometrial carcinoma (EC) is the third most common gynecological malignancy in India. Recent PORTEC-3 analysis emphasized the role of central histopathological review. We aimed to retrospectively analyze the demographic and histopathological characteristics of EC patients treated at our institute and assess the impact of the central histopathological review on management and also analyze clinical outcomes in this cohort of patients. MATERIALS AND METHODS: Data of 75 EC patients treated at our center between 2013 and 2022 were retrieved from our departmental archives. Patients were analyzed for demographic details, histopathological findings, details of surgery and histopathology (HPE), results of a review of HPE, adjuvant treatment details, and clinical outcomes. All patients with HPE outside of our institute were reviewed at our center prior to initiation of treatment. In cases of discordance, patients were discussed in the multidisciplinary tumor board for the final treatment decisions. Patients were staged as per International Federation of Gynaecology and Obstetrics 2018. RESULT: The median age was 57 years (range: 37-74 years). Twenty-seven patients with HPE reported from the outside center were reviewed at our institute and changes were observed in 26 patients (96.3%). HPE review changes were observed in terms of histological grade, histological type, myometrial invasion, and lymph node involvement in five (18.5%), three (11.1%), seven (25.9%), and three (42.8%), respectively. HPE review leads to changes in the management of 19/26 patients. Stage distribution was I: II: III in 48 (64%): 9 (13.3%): 18 (24%) patients, respectively. The median external beam radiotherapy dose was 50 Gray (range: 45-50.4 Gray at 1.8-2 Gray per fraction). The median brachytherapy dose for patients treated with brachytherapy alone was 7 Gray each for three sessions and in combination with EBRT was 6 Gray each in two sessions. At a median follow-up of 51 months (range: 6-116 months), seven (9.3%) patients developed distant metastasis, two (2.7%) patients had local plus distant metastasis, and two (2.7%) patients had local recurrence. The overall survival and disease-free survival rates at 3 years were 93.5% and 86.7%, respectively. CONCLUSION: EC patients treated at our center have excellent local control rates with a combination of external beam radiotherapy and brachytherapy. The central histopathological review may result in changes impacting patient management and should be routinely done prior to initiation of treatment in EC.

Different molecular mechanisms of inhibition of bovine viral diarrhea virus and hepatitis C virus RNA-dependent RNA polymerases by a novel benzimidazole.

The virus-encoded RNA-dependent RNA polymerase (RdRp) has emerged as a primary target in the search for selective inhibitors of Flaviviridae. Recently, we reported on the selective inhibition, in cell-based assays, of both BVDV (EC50 = 0.80 ± 0.06 µM) and HCV (EC50 = 1.11 ± 0.15 µM) by 2-{1-[2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl]ethylidene}hydrazinecarbothioamide (227G). Here we show that, in enzyme assays with recombinant enzymes, 227G inhibits, in a dose-dependent manner, the RdRp of both BVDV (IC50 = 0.0020 ± 0.0004 µM) and HCV (IC50 = 0.40 ± 0.04 µM). Furthermore, we report on the selection and molecular analysis of a BVDV-resistant mutant, characterized by the presence of the I261M mutation. By applying a multilevel computational approach, we identified different 227G binding sites on the two RdRps. They were further validated by the good agreement between the calculated affinities and those extrapolated from IC50 values. Our findings suggest different molecular mechanisms of inhibition of the HCV and BVDV RdRps by 227G and indicate the importance of understanding ligand-enzyme interactions at the molecular level for the rational design of new and more potent leads.

Next Generation Sequencing in Vitellointestinal Duct Adenoma: Existence of Adenoma-Carcinoma Sequence or too Early to Predict?

Patent vitello-intestinal duct with adenoma is rare presentation. We report a case of a 1-month-old boy presenting with intermittent passage of stool and blood from the umbilicus since birth. On local examination polypoidal mass measuring 1×1 cm was seen protruding from umbilicus with faecal discharge. Ultrasound was performed which revealed a tubular hyperechoic structure, extending from umbilicus to part of small intestine measuring 30 ×30 mm and clinical diagnosis of patent vitello-intestinal duct was given, exploratory laparotomy, excision with umbilicoplasty done, and send for histopathological examination. On histopathological examination, patent vitello-intestinal duct adenoma was rendered and next generation sequencing (NGS) was performed revealing somatic mutation of KRAS (NM_033360.4; c.38G>A; p.Gly12Asp). To our knowledge, this is the first report of the adenoma in patent vitello-intestinal duct with NGS analysis. This case emphasizes the importance of thorough microscopic examination of resected patent vitello-intestinal duct and mutational analysis of the early lesions.

Anorectal balloon cell melanoma: a rare variant.

Balloon cell melanoma is a rare presentation of malignant melanoma, usually on the skin, with less than 100 cases reported. Mucosal BCM is even rarer, with only one case of anorectal BCM reported in English literature. The diagnosis is based on the histopathologic findings of a tumor composed of large, foamy melanocytes, with or without pigmentation, and confirmed by immunohistochemical studies showing expression for melanocytic markers. The foam cell appearance of the tumor cells and the lack of melanin pigment lead to a diagnostic dilemma, mostly when presented at an unusual location. Herein, we report a case of balloon cell melanoma at the anorectal junction in a 73-year-old male patient complaining of constipation and bleeding per rectum. Surgical resection was performed with no evidence of recurrence after three years of close follow-up. We believe this case will raise awareness among the medical community to consider this tumor a differential diagnosis in rectal masses.

Concomitant Non-V600E BRAF and KRAS Mutations in Colorectal Carcinoma by Next-Generation Sequencing: A Distinct Subtype.

The RAS-RAF-MEK-ERK signaling cascade is the most frequently affected signaling pathway in colorectal cancer. BRAFV600E mutations serve as a drug-treatable hotspot and KRAS mutations as a predictor of susceptibility to anti-epidermal growth factor receptor therapy. Concomitant non-V600E BRAF and KRAS mutations may coexist and are rarely reported in the literature. We report a patient of colorectal carcinoma with inguinal lymph node metastases harboring mutations at the KRAS and BRAF non-V600E mutation codon detected by next-generation sequencing with an emphasis on clinical, pathological, and therapeutic implications of the mutation and review of the literature.

Programmed Death Ligand 1 Expression in Lung Adenocarcinoma: An Analysis of the Histomorphological Features.

Background: Immunotherapy is now a vital target therapy in the advanced cases of lung adenocarcinoma. The outstanding result of therapies with medications that inhibit the interaction of programmed death ligand 1 with programmed death protein 1 has revolutionarized prognostic treatment regimes. Aims: The study was undertaken with the objectives to analyze the detailed histomorphological features of adenocarcinoma lung with programmed death ligand-1 (PD-L1) expression in tumor cells and to compare the histomorphological features with PD-L1 negative group. Materials and Methods: The present study is a retrospective case series with 100 cases of non-small cell lung cancer-adenocarcinoma phenotype in which testing for PD-L1 had been done using immunohistochemistry. Detailed histomorphological analysis and comparison was performed for both the PD-L1 positive and negative phenotype. Results: Histomorphological features of 25 cases with positive PD-L1 positivity in the tumor cells and 75 cases that were negative for PD-L1 were analyzed. The most frequent pattern in the category that was PD-L1 positive was singly scattered cells or loose clusters present in 84% cases followed by solid nests that was identified in 60% cases. The presence of solid nests in the PD-L1 positive category was statistically significant (P = 0.018). Mucin was identified in 24% cases, and tumor necrosis was documented in 60% cases with PD-L1 positivity. In the cluster that was PD-L1 positive, 92% cases had moderate-to-severe nuclear pleomorphism. Conclusion: The identification of histomorphological patterns and characteristics may aid in triaging cases that have the likelihood to harbor PD-L1-positive phenotype, which has predictive and prognostic outcome.