Clinicopathological and prognostic significance of VEGF, PDGF-B, and HER2/neu expression in gallbladder cancer.

AIM: Gallbladder cancer (GBC) is usually diagnosed in advanced stages with poor survival. The molecular mechanisms of GBC still remain unexplored. Several angiogenesis factors play a pivotal role in tumor progression. We aimed to study the expression of VEGF, PDGF-B, and human epidermal growth factor receptor 2 (HER2/neu) and its association with clinicopathological features and survival in GBC. MATERIALS AND METHODS: VEGF, PDGF-B, and HER2/neu expression was studied by immunohistochemistry (IHC) after histological evaluation in 91 GBC cases. The relationship between these markers and clinicopathological features and survival was explained through the Cox regression model and Kaplan-Meier method. RESULTS: VEGF, PDGF-B, and HER2/neu overexpressed in 45, 79, and 68% GBC cases, respectively. VEGF was significantly overexpressed in GBC without gall stones (GS) (p = 0.007) and with moderately and poorly differentiated tumors (p = 0.012). HER2/neu was significantly overexpressed in GBC with GS (p = 0.022). Median overall survival (OS) was 39 months (95% CI: 23-55). In univariate analysis, histological type (adenocarcinoma and papillary) vs. others (signet ring/mucinous/adenosquamous) (p = 0.004), depth of tumor infiltration (p = 0.017), distant metastasis (p = 0.012), and adjuvant therapies (chemotherapy/radiotherapy) (p = 0.083) were associated with poor prognosis. Multivariate survival analysis showed histological type (p = 0.004) and distant metastasis (p = 0.032) to be independent prognostic factors for OS. Histological type (p = 0.002), distant metastasis (p = 0.003), and depth of tumor infiltration (T3-T4) (p = 0.012) showed poor median survival. Poor survival was seen in VEGF and HER2/neu positive cases. CONCLUSION: Overexpression of VEGF, PDGF-B, and HER2/neu might be possible prognostic biomarkers in GBC. Poor survival of VEGF and HER2/neu positive cases indicates the possibilities of using their blockers as therapeutic agents.

CRISPR screening identifies BET and mTOR inhibitor synergy in cholangiocarcinoma through serine glycine one carbon.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Microparticle-Delivered Cxcl9 Prolongs Braf Inhibitor Efficacy in Melanoma.

Patients with BRAF-mutant melanoma show substantial responses to combined BRAF and MEK inhibition, but most relapse within 2 years. A major reservoir for drug resistance is minimal residual disease (MRD), comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically "cold" after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines as strongly upregulated initially and steeply decreasing as the immune response faded. Therefore, we hypothesized that sustaining chemokine signaling could impair MRD maintenance through increased recruitment of effector T cells. We found that intratumoral administration of recombinant Cxcl9 (rCxcl9), either naked or loaded in microparticles, significantly impaired MRD relapse in BRAF-inhibited tumors, including several complete pathologic responses after microparticle-delivered rCxcl9 combined with BRAF and MEK inhibition. Our experiments constitute proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of first-line treatment methods.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Risk factors for gallbladder cancer development in northern India: A gallstones-matched, case-control study.

Background & objectives: A high incidence of gallbladder cancer (GBC) is observed in northern India. This study was aimed to identify the factors involved in developing GBC in this region. Methods: A gallstones-matched, case-control study was conducted in northern India. Ninety nine patients with GBC and gallstones (33 men and 66 women, mean age of 51.4 yr) comprised the case group, while 99 patients with cholelithiasis (40 men and 59 women, mean age of 45.7 yr) comprised the control group. All participants were interviewed to complete 183 questionnaire items that included 105 food items. Potential risk factors were identified using a multivariate analysis adjusted for age and sex. Significant risk factors were identified using a stepwise logistic-regression analysis. Results: Age (≥50 yr), education (illiterate), socioeconomic status (≤below poverty line), bowel habits (≤once a day), hypertension history, hypotensive drug use, non-vegetarian diet, use of firewood for cooking, tap water drinking, hand pump water drinking and high consumption of coffee and sweets were identified as the potential risk factors. In women, factors included menarche (<13 yr), number of pregnancies (≥3 pregnancies) and parity (≥3 babies). Of these factors, age, education, bowel habits, tap water drinking and multiple pregnancy and/or multiparity were identified as significant risk factors, whereas a high consumption of coffee and sweets or hypotensive drug use and/or hypertension history were protective factors. Interpretation & conclusions: Poor bowel habits and drinking unsafe water appear to be the main risk factors for developing GBC. These are, however, modifiable factors which are capable of decreasing the risk of GBC in the north Indian population.

Carcinogen Metabolism Pathway and Tumor Suppressor Gene Polymorphisms and Gallbladder Cancer Risk in North Indians: A Hospital-Based Case-Control Study.

BACKGROUND: Carcinogen metabolism pathway and tumor suppressor gene polymorphisms have been reported to be associated with increased gallbladder cancer risk. However, the association of genetic variants and gallbladder cancer risk in Indians are not well studied. We examined whether genetic polymorphisms of metabolic enzymes cytochrome P450 1A1 and glutathione S-transferase and tumor suppressor gene p53 (TP53) are associated with an increased risk of gallbladder cancer in North Indians. METHODS: This hospital-based case-control study was conducted in 96 gallbladder cancer patients with gallstones (cases) and 93 cholelithiasis patients (controls) at the Sanjay Gandhi Postgraduate Institute of Medical Sciences in Lucknow, India from July 2014 through May 2017. Genomic DNA was extracted from white blood cells of each patient using a simple salting-out procedure. The genotypic frequencies of CYP1A1 rs4646903, CYP1A1 rs1048943, and TP53 rs1042522 polymorphisms were investigated using TaqMan SNP Genotyping Assay and GSTM1 and GSTT1 polymorphisms were analyzed using the multiplex PCR assay. RESULTS: The frequency of CC genotype of TP53 rs1042522 polymorphism was 27.1% (26/96) in cases and 12.9% (12/93) in controls. The CC genotype was associated with an increased risk of gallbladder cancer in North Indians (age- and sex-adjusted odds ratio, 2.81; 95% confidence interval, 1.19-6.61; P = 0.02). No significant differences in genotypic and allelic frequencies of the metabolic pathway gene polymorphisms were found between cases and controls. CONCLUSIONS: Our data provide preliminary evidence that the CC genotype of the TP53 rs1042522 polymorphism may be associated with an increased risk of gallbladder cancer in North Indians.

Gastrointestinal Stromal Tumor: Genotype Frequency and Prognostic Relevance.

BACKGROUND: Genotyping has an important role in the prognosis and prediction of response to tyrosine kinase inhibitor therapy. KIT exon 11 deletions serve as an adverse prognostic marker. Gastrointestinal stromal tumor (GIST) genotype has been described in developed countries; however, data from India are lacking. The aim of this study was to determine the genotype frequency and its prognostic relevance. MATERIALS AND METHODS: Eighty consecutive cases of resected GIST were evaluated for histologic and immunohistochemical findings. Mutation analysis for exons 9, 11, 13, and 17 of KIT and 12 and 18 of PDGFRA was carried out by PCR-Sanger sequencing. Genotypes were correlated with risk groups, recurrence, and imatinib therapy. RESULTS: Forty-seven of 80 cases (58.7%) showed mutations, including 30 cases (37.5%) in KIT exon 11, 9 cases (11.2%) in KIT exon 9, and 8 cases (10%) in PDGFRA exon 18. Codon 557-558 deletion was present in 15 cases. D842E was the most common in PDGFRA, with similar histologic features as D842V. KIT exon 11 deletion had higher mitotic rate, larger tumor size, high-risk stratification, and lower recurrence-free survival. Recurrences were seen in 12 (16.4%) patients. Nine patients (75%) with recurrence were on imatinib therapy. CONCLUSIONS: GIST genotype frequency is lower in Indians. KIT exon 11 deletion is associated with poor prognosis compared with wild-type and other missense mutations. D842E is a common PDGFRA mutation in Indian patients. Patients with a wild genotype are not suitable candidates for imatinib therapy. Genotyping can serve as an important prognostic marker.

Molecular Characteristics of Large Parathyroid Adenomas.

INTRODUCTION: The clinical entity of large parathyroid adenomas (LPTAs) has not been well defined. It is speculated that LPTAs would have biochemical, histological, and molecular characteristics different from small adenomas. Our study aimed to find out occurrence of atypia and carcinomas in large parathyroid lesions and the presence of distinct molecular abnormalities in LPTAs. MATERIALS AND METHODS: We divided the parathyroid lesions into large (>7 g, i.e., LPTAs) and small (<7 g) adenomas. We performed parafibromin, APC (adenomatous polyposis coli), galectin 3, and PGP9.5 (protein gene product 9.5) analysis by immunohistochemistry in adenomas without atypia, atypical adenomas, and carcinomas. RESULTS: Mean serum calcium, alkaline phosphatase, and intact PTH were significantly higher in large parathyroid tumor group. The presence of both atypical adenoma and carcinoma was higher in large parathyroid tumor group. There was higher percentage of atypia in patients with LPTAs >10 g (33%), and 68% of tumors showed at least one marker suggestive of malignancy in this group. Detailed analysis of immunohistochemical features of LPTA >10 g revealed that six patients showed complete loss of parafibromin immunoreactivity (out of these four showed atypia), while seven showed partial loss. In histopathologically proven malignancy (n = 9), six patients showed complete loss of parafibromin staining, 5 (55%) APC negativity, and 45% showed both galectin 3 and PGP9.5 positivity. Three out of these showed all IHC markers s/o malignancy, and all of them had evidence of metastases or recurrence. 32% of atypical adenoma and 13% of atypical adenoma showed complete loss of parafibromin staining, however none developed metastases or recurrence in follow-up (median follow-up 40 months). Loss of parafibromin staining (complete or partial) was higher in LPTA group (56%) than that in small adenoma (39%); however, it was not statistically significant. APC, galectin 3, and PGP9.5 markers suggestive were higher in LPTA group but were not significant. CONCLUSION: LPTAs may show some morphological and immunohistochemical features suggestive of malignancy and can be considered a separate entity. However, the immunohistochemical markers are unable to clearly segregate those LPTAs that may show premalignant potential. Further, we would like to recommend that LPTAs showing complete parafibromin loss together with atypia should be kept under close follow-up.

Alternative methods in toxicology: CFU assays application, limitation and future prospective.

Blood is a fluid connective tissue which plays a vital role for normal body function. It consist different type of blood cells which is continuously reproduce inside the bone marrow from hematopoietic system. Xenobiotics could be specifically toxic to the hematopoietic system and they can cause hematological disorders by disturbing the normal functions. In vitro hematopoietic colony-forming cell assays play a crucial role to evaluate potential toxic effects of new xenobiotics and also helpful in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. Use of these assays in conjunction with, high-throughput screening reduces the cost and time associated with these assays. This article provides a critical view over in vitro hematopoietic colony-forming cell assays in assessment of hematotoxicity.

Matrix metalloproteinase 7 expression in ampullary carcinoma.

BACKGROUND: Matrix metalloproteinase 7 (MMP7) has largely been studied in pancreatic cancer which is the most common component of periampullary cancer in the western population. In India, the ampullary carcinoma is seen as the most common periampullary cancer in resected pancreaticoduodenectomies. We aimed to study the expression of MMP7 and its correlation with clinicopathological features in ampullary cancer. MATERIALS AND METHODS: Consecutive cases of all ampullary cancer in a 3-year period were reviewed for histological differentiation (intestinal and pancreatobiliary) by morphology and immunohistochemistry (CDX2, MUC2, cytokeratin 20 [CK20], MUC1, cytokeratin 7 [CK7], and cytokeratin 17 [CK17]). All cases were stained for MMP7 and expression was correlated with histological variables, differentiation, and overall survival. RESULTS: There were a total of 91 ampullary carcinomas (36 intestinal, 44 pancreatobiliary and 6 other types). Ampullary carcinoma showed MMP7 expression in 63.7% cases. Two-third of intestinal type and half of the pancreatobiliary type cancers showed MMP7 expression. MMP7 expression was significantly higher in low pathological T-stage of total ampullary carcinomas; however, it was seen more commonly in higher overall stage of the pancreatobiliary type compared to intestinal type of ampullary carcinoma. Overall survival in patients with MMP7 expression was lower compared to MMP7 negative patients. CONCLUSIONS: This is the first study on MMP7 expression in ampullary cancer. MMP7 expression was seen in nearly 64 % of ampullary cancer and showed a significant correlation with low pathological (T-) stage and high overall stage with a shorter survival. MMP7 can be explored as a target for MMP inhibitor therapy in the future.

Potential pharmacological interventions against hematotoxicity: an overview.

Various treatment regimens, including chemotherapy, are known to induce heavy oxidative stress on the system, which in turn leads to adverse effects on healthy tissues. Blood being prone to oxidative stress is affected the most. At this juncture, it might not be prudent to anticipate having chemotherapeutic agents with no hematotoxicity; the best way forward is to look for potential anti-hematotoxic compounds, which could be supplemented to exposed patients, thus reducing the toxic burden on blood cells. We mined existing literature for reviewing possible interventions against hematotoxicity and figured that there is a great lacuna in this field in terms of not having such useful information at one place. This review presents the possible entities based on their antioxidant potentials, including their mechanistic pathways.

Toxicogenomics of Phenylhydrazine Induced Hematotoxicity and its Attenuation by Plumbagin from Plumbago zeylanica.

BACKGROUND: High regenerative and proliferative capacity of blood and its components renders it to be at higher risk of adverse drug reactions (ADRs) which are manifested in several treatment regimens against various ailments such as cancers, viral diseases, and several metabolic disorders. OBJECTIVE: It is prudent to come up with some therapeutic entity that can prevent this damage and protects the blood from these ADRs. MATERIALS AND METHODS: We examined protective effects of Plumbago zeylanica (PZ) and its active constituent plumbagin (PL) on Sprague Dawley (SD) rats using a phenylhydrazine (Phz) induced hematotoxicity model. Hemoglobin (Hgb), red blood cells (RBCs), mean corpuscular volume, mean corpuscular Hgb (MCH), MCH concentration (MCHC), leukocytes and platelets were studied. Anti-oxidant enzymes superoxide dismutases 2 and 3 (SODs) and nuclear erythroid 2 p45-related factor 1 and 2 (Nfer-1 and 2) were also studied using quantitative real-time polymerase chain reaction (PCR). RESULTS: In Phz treated rats, the positive hematotoxic response was obtained in terms of deviated endpoints of blood indices. In PLtreated groups protective response was obtained in terms of normal endpoints of blood indices. In PCR studies, we observed the similar trend. Thus, it can be postulated that PL exerts its protective effects via modulation of anti-oxidant enzymes. CONCLUSION: The study proves that PL can be employed against combatting the ADRs associated with several therapeutic treatment regimens. Similar studies employing such pharmacological entities and their combinations may further prove to be effective against ADRs, especially in the context of blood cells. SUMMARY: Hematotoxicity is generally encountered in various therapeutic regimens as ADRs (Adverse Drug Reactions). Plumbagin, an active constituent of plant Plumbago zeylanica is tested for its anti-hematotoxic potential in Phenylhydrazine induced hematotoxicity model in Sprague dawley rats. In vivo, in-vitro and molecular studies confirmed the peremptory actions of PL. It was revealed in our studies that the anti-hematotoxic actions of Plumbagin are due to its capacity to modulate anti-oxidant enzyme system.