Circulating free DNA integrity index and promoter methylation of tumor suppressor gene P16, DAPK and RASSF1A as a biomarker for oropharyngeal squamous cell carcinoma.

Circulating free DNA (cfDNA) is in use for the non-invasive diagnosis of tumors. Methylation of tumor suppressor genes (TSGs) is an early event in carcinogenesis and may serve as tumor biomarker. We have investigated cfDNA integrity and methylation of tumor suppressor genes P16, DAPK and RASSF1A in serum cfDNA of oropharyngeal squamous cell carcinoma (OPSCC) comparing paired serum and tumor tissue samples to evaluate their diagnostic use. Prospective case-control study, paired serum and tissue samples from 56 OPSCC, and 15 normal controls (NC). Sybr green Quantitate real time PCR was used for cfDNA quantification through amplification ALU 115 and 247 fragments. Promoter methylation of was analyzed in paired samples using methylation specific PCR. There was significantly high cfDNA integrity in OPSCC compared to normal control (p = < 0.0001). The cfDNA integrity values were significantly higher and associated with nodal status (p = 0.016). The AUC for cfDNA integrity was 0.967. The P16, DAPK and RASSF1 promoters were significantly hypermethylated in serum of OPSCC compared to NC with high concordance in tissue (up to 96.55 %). The gene promoter methylation of P16 was associated with smoking (p = 0.030), RASSF1A with stage (p = 0.011). The combination of ALU115 with cfDNA integrity and combination of gene methylation increases diagnostic sensitivity. In followup samples the cfDNA change was not different. Liquid biopsy approach including cfDNA integrity, methylation profiling in cfDNA, in combination or separately can assist in the diagnosis of OPSCC along with radio diagnostic scan. Serum changes represent changes in tissue with very high concordance.

Immunophenotypic characterization of normal and abnormal plasma cells in bone marrow of newly diagnosed multiple myeloma patients.

Background: Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. Materials and Methods: 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Results: Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Conclusion: Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8-10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.

Pheochromocytoma disguised as gestational hypertensive disease during pregnancy: A case report.

Pheochromocytoma is often diagnosed prior to pregnancy. Sometimes, the disease may be diagnosed for the first-time during pregnancy masking itself as a hypertensive disease in pregnancy. Early diagnosis and timely, appropriate management reduce possible maternal and fetal complications. We identified a case of pheochromocytoma during pregnancy misdiagnosed as preeclampsia.

Clinicopathological Challenges in Tumors of the Nasal Cavity and Paranasal Sinuses: Our Experience.

Background and objective Nasal and paranasal lesions are one of the most common otorhinolaryngological presentations encountered in clinical practice. Common presenting symptoms of these lesion range from nasal blockades, facial swellings, pain, nasal discharge, and epistaxis to orbital and ear symptoms. Diagnosis can be tricky as these symptoms are common in inflammatory conditions and tumors. The aim of our study was to observe the epidemiology and clinical pathological findings in patients with nasal and paranasal masses presenting to our institute and discuss the challenges in proper diagnosis and management due to similar presentations, and the role of histopathological examination (HPE) and immunohistochemistry (IHC) in overcoming these challenges. Methods The IPD records of 396 patients were taken up for the study. All the specimens were sent in 10% neutral buffered formalin for examination as biopsy for diagnosis or after surgical excision. After adequate fixation, the biopsy specimen was submitted for routine processing, followed by paraffin embedding, and stained with hematoxylin and eosin (H&E). Special stains like periodic acid-Schiff (PAS) and Ziehl-Neelsen (ZN) stains for acid-fast bacilli (AFB) were used as required. IHC was performed in the required samples. IHC markers were performed on representative paraffin-embedded sections according to the streptavidin-biotin immunoperoxidase technique as needed. The findings were noted, and histology was correlated with clinical presentations and investigations, tabulated, and statistically analyzed using SPSS Statistics (IBM, Armonk, NY). Results Of note, 67.92%% were non-neoplastic lesions whereas 18.18% came out to be benign neoplasms and 13.88% were malignant lesions on HPE. Nasal obstruction was the most common presenting symptom (73.23% of patients) followed by nasal mass (64.14% of cases). Inflammatory sinonasal polyps were the most prominent cases in our study, accounting for 41.16% of all lesions; 18.68% fungal RS (mucormycosis) were seen in our study and 4.54% were cases of juvenile nasopharyngeal angiofibroma (JNA). The majority of benign neoplasms encountered were Schneiderian papilloma or inverted papilloma (06.81%). Eighteen (4.54%) cases of squamous cell carcinomas (SCC) were seen in our study and 2.77% (n=11) cases were of adenoid cystic carcinoma. Of 18 cases of SCC, moderately differentiated SCC carcinoma accounted for 10 cases followed by poorly differentiated SCC (5/18) and nonkeratinizing SCC (3/18). IHC for p40 was performed in all the cases of nonkeratinizing SCC, which showed strong and diffuse nuclear positivity. Conclusion The nasal cavity is the site of the most varied presentation of tumors in the upper respiratory tract. Mass in the nose and paranasal sinus (PNS) form a heterogeneous group of lesions with varied histopathological features. The proximity of the area to the eyes and brain warrants early definitive diagnosis so that the lesion is treated before it can involve important and vital centers. Even though malignant nasal tumors have a very low incidence, they cause a lot of morbidity due to their long course and frequent local recurrences. Nasal tumors tend to become polypoidal. Epithelioid papilloma of the nasal cavity often resembles a nasal polyp. Clinical diagnosis can be challenging due to similar presentations and appearances, and hence histological examination is a vital tool for the timely diagnosis of such patients.

Dynamics of cell-free DNA in predicting response in adult diffuse glioma on chemoradiotherapy.

BACKGROUND: Adult diffuse glioma (ADG) is a heterogeneous primary brain tumor with a variable prognosis and treatment response. Tissue biomarkers and molecular genetic profiling form an integral part of diagnosis and prognostication. However, obtaining tissue in inoperable locations and diagnosis of recurrence can be an issue. Cell-free DNA (cfDNA) may help to meet these challenges in the management of ADG. OBJECTIVES: The study aimed to serially quantify cfDNA in ADG on chemoradiation and to correlate mutational profiling of the cfDNA with biopsy. MATERIAL AND METHODS: The study group comprised of histopathological confirmed ADG (n = 50), including grade II, III and IV glioma, and controls (n = 25). Serum cfDNA was extracted using ChargeSwitch gDNA 1 mL Serum Kit (Invitrogen, USA) and quantified using SYBR based quantitative polymerase chain reaction (qPCR). Next-generation sequencing (NGS) was performed in 07 pre-operative and 05 post-operative cfDNA and tumor biopsy DNA on an Ion personal genome machine (IonPGM) with an in-house designed NGS panel (including TP53, ATRX, and IDH1 and IDH2). RESULTS: In patients with ADG, the pre-radiotherapy cfDNA level was significantly higher (Median; 113.46 ng/mL) than normal controls (Median; 74.37 ng/mL), (p = 0.048). Non-responders had significantly higher cfDNA levels (Median; 184.4 ng/mL), than responders (Median; 68.12 ng/mL), (p = 0.023). TP53 gene mutation was most common in both pre-operative and post-operative cfDNA samples. CONCLUSION: Pre-radiotherapy cfDNA levels are associated with clinical outcomes independent of other prognostic factors. Targeted NGS in pre-operative cfDNA matches the results of IHC analysis with high concordance, and it may be helpful in inoperable cases or ADG recurrence.

Genomic profiling of gallbladder carcinoma: Targetable mutations and pathways involved.

Gallbladder cancer (GBC) carries a poor prognosis and frequently present in late stages of disease. Identification of targetable molecular changes for selecting appropriate treatment and identifying prognostic and therapeutic subsets is required. Next-generation sequencing (NGS) using a frequently mutated for GBC may provide a reference for clinical management. The primary aim of the current study was to analyze the frequency of individual genetic alterations in GBC and to correlate with clinicopathological characteristics. A retrospective study was conducted using 22 gene panel examined NGS based approach for the detection of actionable genomic mutations in 37 GBC patients. The genetic and clinicopathological characteristics were analyzed. The number of mutations in cases was ranged from 1 to 15. A total of 171 mutations were identified in FFPE tissue DNA. The most common alterations were TP53 (90.90%), SMAD4 (60.60%), NOTCH1(45.45)& ERBB2 (45.45%), PIK3CA (33.33%) and MET (30.30)& PTEN (30.30%). Among the 22 gene panel, the TP53 gene was associated with histopathological differentiation (p = 0.0001), ERBB4 & ALK mutation was associated with necrosis (p = 0.012, 0.027), EGFR mutation was associated with mucin status (p = 0.023) and ERBB2 gene mutation was associated with T stage (p = 0.036). The study provides an overview of the genetic alterations in GBC patients.Targetable mutations are present in 89.91% cases of GBC which include SMAD4, NOTCH1, ERBB2&4, PIK3CA, MET, PTEN, EGFR, KRAS and BRAF. Metastatic disease was associated with high frequency of CTNNB1, KRAS and NRAS gene mutations.

Comparison of circulating DNA in malignant neoplasia from diverse locations: Investigating a diagnostic role.

CONTEXT: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. DESIGN: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. RESULTS: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood-brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1-Q3) of 349.22 ng/ml (19.87-1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38-624.44), 778.50 (589.88-1864.35), 348.73 (194.67-483.61), 386.27 (47.88-959.67), and 74.12 (49.66-120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. CONCLUSION: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.

Safety of NR-INF-02, an Extract of Curcuma Longa Containing Turmerosaccharides, in Healthy Volunteers: A Randomized, Open-label Clinical Trial.

CONTEXT: Turmeric (Curcuma longa) is a common medicinal plant used in traditional medicine that also has been scientifically validated for its antioxidant, anti-arthritic, anticancer, analgesic, and anti-inflammatory properties. Researchers have still not much explored the beneficial effects of the curcuminoid-free portion of turmeric. NR-INF-02 is a proprietary, patented aqueous extract of Curcuma longa comprising turmerosaccharides with a novel phytochemical composition. OBJECTIVE: The study intended to evaluate the safety and tolerability of NR-INF-02 in healthy adult volunteers at doses of 1000 and 2000 mg, administered for 84 days. DESIGN: The study employed a randomized, open label, two-arm, parallel-group design. SETTING: The trial was carried at 2 sites, the Meenakshi Multispecialty Hospital in Chennai, Tamil Nadu, India and the Vijaya Super Specialty Hospital in Nellore, Andhra Pradesh, India. PARTICIPANTS: Participants were healthy adult, male or female volunteers, aged 18-65 years with a body mass index of ≥18.5 kg/m2 and ≤ 24.9 kg/m2 and a body weight of at least 55 kg for men and 48 kg for women. INTERVENTION: Participants were randomly divided into 2 groups with 24 participants each for a total of 48 participants. They received either 1000 or 2000 mg of NR-INF-02 for 84 days. OUTCOME MEASURES: The incidence of adverse events and the changes from baseline in clinical laboratory parameters-including hematological, biochemical, and urinalysis parameters-were assessed at baseline, at day 42, and postintervention at day 84 as primary endpoints for safety. Secondary endpoints were the changes in vital signs and the difference in the results of an electrocardiogram (ECG) between baseline and days 42 and 84. RESULTS: The NR-INF-02 at doses of 1000 and 2000 mg demonstrated a 4.17% and 20.83% incidence of adverse events (AEs), respectively. The AEs were mild to moderate and were either probably or possibly related, but not definitively, related to treatment. A detailed examination of hematological, biochemical, and urological parameters and of ECG results and vital signs didn't indicate any untoward effects for any participant. CONCLUSION: The study found NR-INF-02 to be safe and tolerable at both tested doses for the given duration of the trial for healthy adult volunteers.

Identification of Potential Targets for Thymidylate Synthase and Amp-C ß-lactamase from Non-alkaloidal Fractions of Moringa oleifera Leaves.

AIM: The aim of present investigation is to identify the potential targets for Thymidylate Synthase and Amp-C ß-lactamase from non-alkaloidal fractions of Moringa oleifera leaves. BACKGROUND: Bioactive constituents from medicinal plants, either as pure compounds or as crude forms, provide vast opportunities for new drug discoveries. Due to an increasing demand for chemical diversity in screening programs, seeking therapeutic drugs from natural products, mainly from edible plants, has grown throughout the world. Moringa oleifera has an impressive range of medicinal uses with high nutritional value. Therefore, this medicinal plant has been used widely in traditional Indian medicine for anti-inflammation, anticancer and antibacterial infections. OBJECTIVES: The primary objective is to identify the phytoconstituents present in the maximum proportion in non-alkaloidal fractions of ethanolic leaf extract of Moringa oleifera. Then, the identified phytoconstituents were used to ensure the potential target molecules for binding affinity towards the target proteins viz. Thymidylate Synthase (1HVY) and Amp-C beta-lactamase (1FSY) by docking analysis. METHODS: In present investigation, ethanolic extract of Moringa leaves was prepared and then fractionated on the basis of presence/absence of alkaloids. The antimicrobial activity of different fractions of ethanolic leaf extract was evaluated against various pathogens. Later, after this, bioactive molecules present in the non-alkaloidal fractions of ethanolic leaf extract were accomplished through GC-MS analysis, and finally, the identified phytocompounds were analyzed through docking studies to evaluate their affinity for target proteins viz. Thymidylate Synthase (1HVY) and Amp-C ß-lactamase (1FSY). RESULTS: The antimicrobial activity of non-alkaloidal fractions of ethanolic leaf extract was evaluated against various pathogens which exhibited significant antimicrobial activity. Twenty phytocompounds were identified as gas chromatogram of non-alkaloidal fractions (chloroform and ethyl acetate) of leaf extract of M. oleifera; Four most prominent compounds having highest peak area percentage were identified as Ethane, 1,1,2,2-tetrachloro, (46.45%) 2-Propanone, 1,1,3-trichloro, (13.77%) Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl (17.87%) and 2,4-Dichlorodiphenylsulfone (17.64%). Other notable compounds were 9,12-Octadecadienoic acid (Z,Z) (14.06%), Oleic acid, 3- (octadecyloxy)propyl ester (12.41%), Fluoranthene (6.98%), Phenol, 2,4-bis( 1,1-dimethylethyl) (4.16%) and Phthalic acid, butyl nonyl ester (3.47%). Only, five compounds viz. 2,6-Bis(1,1- dimethylethyl)phenol(C1), Dodecamethylcyclohexasiloxane(C2), Chlorodimethylethylsilane(C3), Fluoranthene(C4) and Hexadecanoic acid, methyl ester(C5) showed the maximum interaction with 1HVY with highest docking score of -178.51Kcal/mol, - 231.65Kcal/mol, -129.18Kcal/mol, - 173.10Kcal/mol and -220.78Kcal/mol, respectively. In addition, three compounds viz. Dodecamethylcyclohexasiloxane( C2), Fluoranthene(C4) and Hexadecanoic acid, methyl ester(C5) showed the maximum interaction with 1FSY with highest docking score of -137.23Kcal/mol, -54.34Kcal/mol and -153.84Kcal/mol, respectively. CONCLUSION: Moringa plant may provide incredible capabilities to develop pharmacological products. The present finding demonstrated that Moringa oleifera is an excellent plant candidate to be used for improving the health of communities.

Circulating free DNA as a marker of response to chemoradiation in locally advanced head and neck squamous cell carcinoma.

CONTEXT: Liquid biopsy has moved from bench to bedside as a non-invasive biomarker for early diagnosis and monitoring treatment response. OBJECTIVE: This study investigated the role of circulating free DNA (cfDNA) as a diagnostic marker in locally advanced head and neck squamous cell carcinoma (HNSCC) and in monitoring response to chemoradiation therapy. MATERIALS AND METHODS: Serum was collected from treatment naïve, histopathologically diagnosed tumors in 24 HNSCC cases and 16 normal controls. CfDNA levels were quantified using ß globin gene amplification. RESULTS: The cfDNA level was significantly elevated in HNSCC (992.67 ± 657.43 ng/mL) as compared to healthy controls (60.65 ± 30.42 ng/mL, P = <0.001). The levels of cfDNA did not significantly correlate with TNM stage, lymph node involvement and grade. In responders, percentage decrease in cfDNA levels was 9.57% and 29.66%, whereas in nonresponders percentage increase was 13.28% and 24.52% at the end of three months of follow-up. CONCLUSION: Our study adds to the evidence that cfDNA levels are significantly higher in HNSCC cases and provides some evidence that levels increase with tumor progression. CfDNA may be a promising prospective non-invasive marker to predict response in patients undergoing chemo-radiotherapy.

Salmonella Typhi: a rare cause of parotid abscess.

The incidence of extraintestinal infection caused by Salmonella spp has been increased during the past decade. Here we report a case of a parotid abscess caused by Salmonella enterica subspecies enterica serotype Typhi (S Typhi) in an individual without any significant abnormality of the parotid gland. A 68-year-old man presented to the surgical department with high-grade intermittent fever associated with painful swelling over the right side of the face, extending into the neck. An ultrasound of the neck revealed an abscess of the right parotid gland. S Typhi was isolated from the pus drained from the parotid gland. The patient was treated with intravenous followed by oral cephalosporin for a period of 7 days. This case gives an insight into one of the rarer aetiological agents causing parotid abscess.

Long-term results of a single injection of intravitreal dexamethasone as initial therapy in diabetic macular edema.

Purpose: To evaluate the long-term safety and efficacy of the intravitreal dexamethasone implant in the treatment of diabetic macular edema (DME) as initial therapy. Methods: A hospital-based prospective, non-comparative case study of recently detected DME patients was conducted between July 2016 and December 2017, in which30 eyes of 30 patients were studied. Presenting vision, age, gender, duration of diabetes, general and ocular examination, intraocular pressure, indirect ophthalmoscopy, fundus fluorescein angiography, optical coherence tomography (OCT), and blood sugar levels were noted. Patients with increased central macular thickness (CMT) received an intravitreal dexamethasone implant as initial therapy. All were followed up at 1 week, 1 month, 3 months, 6 months, and 1 year, and the findings were recorded and analyzed using SPSS software. Results: 30 eyes of 30 patients were studied which included 22 males and 8 females. The mean age of presentation was 58.7 ± 4.45 years. The mean decrease in CMT following intravitreal dexamethasone was 269.27 ± 112.002, 253.5 ± 108.294, and 286.73 ± 143.395 µm at the end of 3, 6, and 12 months, respectively, and the mean improvement in visual acuity (VA) was 2.27 ± 1.70 lines at 3 months, 2.27 ± 1.83 lines at 6 months, and 1.17 ± 2.00 lines at 12 months. Out of 30 cases, 4 had persistent DME and 6 had recurrence of DME at completion of 1 year of follow-up. Conclusion: Intravitreal dexamethasone as initial therapy in the treatment of DME is both safe and efficacious in the reduction of CMT and improvement of vision and can be considered as primary therapy for DME.

Interaction with zinc oxide nanoparticle kinetically traps α-synuclein fibrillation into off-pathway non-toxic intermediates.

α-Synuclein is an intrinsically disordered amyloidogenic protein associated with Parkinson's disease (PD). The monomeric α-synuclein transition into amyloid fibril involves multiple steps, which are affected by several intrinsic and extrinsic factors. This increases complexities in development of targeted therapeutics against the pathological intermediate(s). Several studies have been dedicated to find an effective molecule to inhibit the detrimental amyloidogenesis. In recent years, metal oxide nanoparticle interfaces have shown direct effects on protein conformation, hence may be adopted as an alternative potential therapeutic approach against amyloidosis. In this context, our study explores the zinc oxide nanoparticle (ZnONP) with negative surface potential interface interaction with α-synuclein, and subsequent impact of the interaction on the protein fibrillation and the fibril-mediated cytotoxicity. N-terminus amphipathic "KA/TKE/QGV" repeating motifs in α-synuclein primarily interact with the ZnONP interface that enthalpically drives initial adsorption of the protein onto the interface. Whereas, subsequent bulk-protein adsorption onto the hard-corona is entropically driven, leading into flocculation of the complex. The flocs appear as amorphous mesh-like morphology in TEM micrographs, as opposed to the typical fibrils formed by the wild-type protein. Interestingly, α-synuclein in complex with ZnONP shows significantly lowered cytotoxicity against the IMR32 and THP-1 cells in-vitro, as compared to fresh α-synuclein.

Diagnostic Value of Circulating Free DNA Integrity and Global Methylation Status in Gall Bladder Carcinoma.

The current study investigates the role of circulating free DNA (cfDNA) as a liquid biopsy in diagnosis gall bladder carcinoma (GBC) utilizing levels of long DNA fragments (ALU247) derived from tumor necrosis, short apoptotic fragments (ALU115) denoting total cfDNA and cfDNA integrity denoting ratio of ALU247 and ALU115. The global methylation status of cfDNA was also estimated with the hypothesis that these parameters provide a diagnostic distinction between cancer and non-cancer subjects, with higher or altered values favoring presence of malignancy. Study group included 60 cases of GBC and 36 controls including diseased controls (cholecystitis) and healthy subjects. Median levels of ALU115, ALU247 and cfDNA integrity were significantly different in GBC at 1790.88, 673.75, 0.4718 vs. controls at 840.73, 165.03, 0.1989 ng/ml respectively. Global DNA methylation was not significantly different between GBC at 0.679% and controls at 0.695%. The sensitivity and specificity of ALU 247 in discriminating GBC from controls was highest with a sensitivity, specificity and diagnostic accuracy of 80.0%, 86.1% and 82.2% respectively. Global DNA methylation showed lowest sensitivity of 55.0% and specificity of 50.0%. Clinico-pathological parameters showing significant association with cfDNA integrity, on ROC curve analysis, showed significant diagnostic discrimination of the tumor stage, lymphovascular invasion, disease stage and grade histology. This is a first time analysis of ALU115, ALU247 and cfDNA integrity in the diagnosis of GBC and confirms that the combination of ALU247 and cfDNA integrity provides good sensitivity, specificity and diagnostic accuracy in discriminating GBC from controls as well correlates with aggressive disease parameters.

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.

Investigation of targetable predictive and prognostic markers in gallbladder carcinoma.

BACKGROUND: Gallbladder carcinoma, uncommon in most of the world, is frequent in South East Asia. It presents in advanced stages with limited therapeutic options. We investigated targetable predictive and prognostic Markers in 268 cases including 233 primary site lesions and 35 metastatic gallbladder carcinoma. METHODS: EGFR, VEGF, HER2/Neu and p53 were assessed using immunohistochemistry. HER2/Neu was validated in a subset by fluorescent in situ hybridization (FISH) using tissue microarray (TMA). Broader spectrum of gene variation was screened in NGS in representative FFPE tissue using the Ion AmpliSeq cancer hotspot panel V2. RESULTS: Mean age was 49.5 years with female predominance (77.6%). Histological types included 221 cases of adenocarcinoma not otherwise specified (NOS), 23 invasive papillary carcinoma, 11 mucinous adenocarcinoma, 8 adenosquamous, 1 signet ring, 3 neuroendocrine and 1 undifferentiated carcinoma. Majority (76.1%) presented with stage 3/4 disease. Overall positive expression of p53 was 44.8%, VEGF 79.4%, HER2/Neu 27.3% and EGFR 34.6%. Intratumoral heterogeneity was evident in HER2/Neu. Marker expression was not significantly associated with stage, grade, type and metastasis except VEGF which correlated with histological type (P=0.018) and tumor grade (P=0.027). NGS using Ion AmpliSeq cancer hotspot panel V2 revealed multiple non synonymous mutations, most frequent being TP53 and CDKN2A mutations and mutations in MET, KDR, PIK3CA, VHL, MPL, HER2 and SMARCB1 genes. CONCLUSIONS: Predictive targetable markers like HER2/Neu, VEGF and EGFR are expressed in high proportion of gallbladder carcinoma. Significant expression of RAS pathway molecules suggests that interactions take place among the different members of the ErbB family during tumor development.

EpCAM-based Flow Cytometric Detection of Circulating Tumor Cells in Gallbladder Carcinoma Cases

Purpose: Liquid biopsy has entered the arena of cancer diagnostics in the past decade and detection of circulating tumor cells (CTC) is one diagnostic component. CTCs in gallbladder cancer (GBC) have hitherto not been comprehensively analysed. Methods and Results: The current study focused on the diagnostic role of CTCs in 27 cases of treatment-naive GBC and 6 normal controls as well as 6 cases of cholecystitis. An EasySep kit featuring negative immunomagnetic bead separation and flow cytometric detection of EpCAM positive and CD45 negative cells revealed CTCs in 25 of the 27 cases. At a cut-off point of ≥1, the CTC count discriminated GBC from controls with a sensitivity, specificity and diagnostic accuracy of 92.6%, 91.7% and 92.3%, respectively. CTC levels in turn correlated significantly with clinico-pathological parameters of cases in terms of known prognostic indicators, with significant diagnostic potential at a cut-off point of >4, to discriminate disease stage I and II vs. III and IV GBC. With a cut-off of >3, the CTC count discriminated tumor stages I and II vs. III and IV and at >6 CTCs could discriminate metastatic vs. non metastatic GBCs with a sensitivity, specificity and diagnostic accuracy of 55. 6%, 100.0% and 85.2, respectively. A review of CTC in pancreatico-biliary malignancies is included. Conclusion: Detection and quantification of CTCs may serve as a non-invasive biomarker for GBC diagnosis in correlation with radiological studies.

Quantification of Circulating Free DNA as a Diagnostic Marker in Gall Bladder Cancer.

Gall bladder Carcinoma (GBC) is the fifth most common cancer of the digestive tract and frequently diagnosed in late stage of disease. Estimation of circulating free DNA (cfDNA) in serum has been applied as a "liquid biopsy" in several deep seated malignancies. Its value in diagnosis of gall bladder carcinoma has not been studied. The present study was designed to assess the role of cfDNA in the diagnosis of GBC and correlate levels with the TNM stage. Serum was collected from 34 patients with GBC and 39 age and sex matched controls including 22 cholecystitis and 17 healthy individuals. Serum cfDNA levels were measured through quantitative polymerase chain reaction (qPCR) by amplification of ß-globin gene. Performance of the assay was calculated through the receiver operating characteristic (ROC) curve. The cfDNA level was significantly lower in healthy controls and cholecystitis (89.32 ± 59.76 ng/ml, 174.21 ± 99.93 ng/ml) compared to GBC (1245.91 ± 892.46 ng/ml, p = <0.001). The cfDNA level was significantly associated with TNM stage, lymph node involvement and jaundice (0.002, 0.027, and 0.041, respectively). Area under curve of ROC analysis for cancer group versus healthy and cholecystitis group was 1.00 and 0.983 with sensitivity of 100 %, 88.24 % and specificity of 100 % respectively. Quantitative analysis of cfDNA may distinguish cholecystitis and gall bladder carcinoma and may serve as new diagnostic, noninvasive marker adjunct to imaging for the diagnosis of GBC.