Lymph Node Metastasis in Gastrointestinal Carcinomas: A View from a Proteomics Perspective.

Lymph node metastasis (LNM) is one of the major prognostic factors in human gastrointestinal carcinomas (GICs). The lymph node-positive patients have poorer survival than node-negative patients. LNM is directly associated with the recurrence and poor survival of patients with GICs. The early detection of LNM in patients and designing effective therapies to suppress LNM may significantly impact the survival of these patients. The rapid progress made in proteomic technologies could be successfully applied to identify molecular targets for cancers at high-throughput levels. LC-MS/MS analysis enables the identification of proteins involved in LN metastasis, which can be utilized for diagnostic and therapeutic applications. This review summarizes the studies on LN metastasis in GICs using proteomic approaches to date.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

Prognostic significance of tumour budding, tumour-stroma ratio and desmoplastic stromal reaction in gall bladder carcinoma.

AIMS AND METHODS: The prognostic role of tumour budding (TBd) and its interaction with the stromal microenvironment has gained a lot of attention recently, but remains unexplored in gall bladder cancer (GBC). We aimed to study the interrelationship of TBd by International Tumour Budding Consensus Conference scoring system, tumour-stroma ratio (TSR) and desmoplastic stromal reaction (DSR) with the conventional clinicopathological prognostic factors, mortality and overall survival (OS) in 96 patients of operated GBC. RESULTS: Higher age, high TNM stage, lymphovascular and perineural invasion, positive resection margins, higher TBd score, low TSR and immature DSR were significantly associated with worse OS. However, on multivariate analysis, only metastases, positive resection margins and TSR <50% proved to be independent prognostic factors. The TBd score of stroma-rich tumour group (6.40±4.69) was significantly higher than that of stroma-poor group (2.77±3.79, p≤0.001). The TBd score of immature and intermediate DSR groups was significantly higher than that of mature group (p≤0.001 and p=0.002, respectively). There was a strong interobserver agreement for TBd score, TSR and type of DSR (Cohen's Kappa=0.726 to 0.864, p≤0.001). Stroma-rich tumours were significantly associated with immature DSR and fibrotic DSR with high TSR (p≤0.001). CONCLUSION: A high TBd, low TSR and immature DSR were significantly associated with several high-risk clinicopathological parameters and poor OS in GBC. These novel, simple, reproducible and cost-effective parameters may be included in the routine reporting checklist for GBC as additional prognostic parameters that can substratify the high-risk patients.

Proteomic profiling of cell line-derived extracellular vesicles to identify candidate circulatory markers for detection of gallbladder cancer.

Gallbladder cancer (GBC) is the sixth most common gastrointestinal tract cancer with a very low overall survival and poor prognosis. Profiling of cancer-derived extracellular vesicles (EVs) is an emerging strategy for identification of candidate biomarkers for the detection and prognosis of the disease. The aim of the study was to analyse the protein content from GBC cell line- derived EVs with emphasis on proteins which could be used as candidate biomarkers for the detection of GBC. NOZ and OCUG-1 cell lines were cultured and EVs were isolated from conditioned media. LC-MS/MS analysis of total EV proteins led to the identification of a total of 268 proteins in both the cell lines. Of these, 110 proteins were identified with ≥2 unique peptides with ≥2 PSMs in at least two experimental and technical replicate runs. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database was used to perform bioinformatics analysis of 110 proteins which showed 'cell adhesion molecule binding', 'integrin binding', 'cadherin binding' among the top molecular functions and 'focal adhesion' to be among the top pathways associated with the EV proteins. A total of 42 proteins including haptoglobin (HP), pyruvate kinase (PKM), annexin A2 (ANXA2), thrombospondin 1 (THBS1), were reported to be differentially abundant in GBC tissue. Of these, 16 proteins were reported to be differentially abundant in plasma and plasma-derived EVs. We infer these proteins to be highly important to be considered as potential circulatory biomarkers for the detection of GBC. To check the validity of this hypothesis, one of the proteins, haptoglobin (HP) as a representative case, was analysed in plasma by quantitative Enzyme- linked immunosorbent assay (ELISA) and we observed its increased levels in GBC in comparison to controls (p value= 0.0063). Receiver operating characteristic (ROC) curve analysis for GBC vs controls showed an Area under the ROC Curve (AUC) of 0.8264 for HP with 22% sensitivity against 100% specificity. We propose that HP along with other candidate proteins may be further explored for their clinical application.

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of the gastrointestinal tract with a poor prognosis. It is important to understand the molecular processes associated with the pathogenesis of early stage GBC and identify proteins useful for diagnostic and therapeutic strategies. Here, we have carried out an iTRAQ-based quantitative proteomic analysis of tumor tissues from early stage GBC cases (stage I, n=7 and stage II, n=5) and non-tumor controls (n=6) from gallstone disease (GSD). We identified 357 differentially expressed proteins (DEPs) based on ≥ 2 unique peptides and ≥ 2 fold change with p value < 0.05. Pathway analysis using the STRING database showed, 'neutrophil degranulation' to be the major upregulated pathway that includes proteins such as MPO, PRTN3, S100A8, MMP9, DEFA1, AZU, and 'ECM organization' to be the major downregulated pathway that includes proteins such as COL14A1, COL1A2, COL6A1, COL6A2, COL6A3, BGN, DCN. Western blot and/or IHC analysis confirmed the elevated expression of MPO, PRTN3 and S100A8 in early stage of the disease. Based on the above results, we hypothesize that there is an increased neutrophil infiltration in tumor tissue and neutrophil degranulation leading to degradation of extracellular matrix (ECM) proteins promoting cancer cell invasion in the early stage GBC. Some of the proteins (MPO, MMP9, DEFA1) associated with 'neutrophil degranulation' showed the presence of 'signal sequence' suggesting their potential as circulatory markers for early detection of GBC. Overall, the study presents a protein dataset associated with early stage GBC.

Plasma-derived candidate biomarkers for detection of gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types-5'-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma.

BACKGROUND: Early diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of 'tumor-associated antigens (TAAs) that elicit humoral response' in early stage GBC patients. METHODS: Total protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins. RESULTS: Image analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed 'high' expression levels in 72% of GBC cases whereas all the controls showed 'low' expression levels. CONCLUSIONS: The study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

Does IgG4 level evaluation in pancreatic mass play role in avoiding major surgery in uncertain presentation: A case report.

A 66-year-old male presented with chief complaints of anorexia associated with mild dull, intermittent epigastric pain for 6 months. The patient was a known diabetic on oral hypoglycemic and on routine checkup was found to have deranged liver function profile. On radiology, an ill-defined hypoechoic enhancing lesion involving head, neck, and uncinate process of pancreas was noted. Whipple's pancreaticodudenectomy was done and reported as IgG4-related autoimmune pancreatitis. Later, IgG (slightly) and IgG4 were found to be markedly raised. We report this case to highlight the importance of IgG4 evaluation prior to major surgery in uncertain pancreatic mass.

Mucoepidermoid Carcinoma of the Gallbladder: A Case-Based Study of an Extremely Rare Tumor Highlighting the Role of Immunohistochemical Profiling.

Mucoepidermoid carcinoma (MEC) is rarely reported in the hepatobiliary system. In this article, we detail the first case of MEC arising from the gallbladder, presenting in a 50-year-old male. Imaging and gross examination showed a large mass in the gallbladder fossa infiltrating the liver. This microscopically displayed the classical morphology of MEC, which was confirmed on dual immunostain for p63 and CK7 and histochemical staining for mucin. MUC and p53 immunoexpression patterns suggested a poor prognosis. The patient succumbed to the disease with liver metastasis within 6 months. The cytomorphology of the metastatic lesion is also presented. Differences in the stem cell niches in the gallbladder and the rest of the biliary tract may possibly indicate the reason for the rarity of this tumor in the gallbladder. A possibly aggressive biological behavior of the gallbladder MEC necessitates its distinction from entities like the adenosquamous carcinoma.

Segmental dilatation of ileum in a young patient presenting with anemia.

Segmental dilatation of ileum (SDI) is a less known uncommon entity with a confusing clinical scenario and no definite etiopathogenesis. The preoperative diagnosis is of exclusion. However, it has an excellent prognosis after surgery. We describe a case of a young patient who presented with anemia without any overt gastrointestinal (GI) bleed. Thorough radiological examinations were needed to reach the diagnosis of SDI which was confirmed postoperatively.

A Study of the Patients Suffering from Tuberculosis and Tuberculosis-diabetes Comorbidity in Revised National Tuberculosis Control Program Centers of Northern Madhya Pradesh, India.

BACKGROUND: Diabetes mellitus (DM) is recognized as an important risk factor to tuberculosis (TB). India has high TB burden, along with rising DM prevalence. AIM: This study was conducted to document the coexistence of DM and TB in persons with established TB under the Revised National Tuberculosis Control Program. TYPE OF STUDY: This was a cross-sectional, descriptive observational study conducted at selected Directly Observed Therapy center in Gwalior North Central India. MATERIALS AND METHODS: A total of 550 patients with confirmed diagnosis of TB and on treatment were recruited. The study participants were screened for DM and diagnoses were made on the basis of the World Health Organization criteria. Clinical parameters were compared between persons with DM and those without DM. RESULTS: DM/TB co-morbidity was noted in 85 individuals and these made up 15.4% of the study population. The mean age was higher in DM patients with TB (43.4 ± 15.4 vs. 33.1 ± 16.2 years, P = 0.000); the mean duration of symptoms of TB with DM was more (124 ± 16.4 vs. 107.49 ± 10.3 days). Multinomial logistic regression analysis showed that increasing age, positive family history of diabetes, sedentary occupation, and presence of pulmonary TB were significantly associated with diabetes among TB patients. CONCLUSIONS: Diabetes is an important co-morbid feature to be sought in patients with TB. This study re-echo the need to raise awareness on screening for DM in persons with TB.

Laparoscopic removal of a needle from the pancreas.

Foreign bodies inside the pancreas are rare and usually occur after the ingestion of sharp objects like fish bone, sewing needle and toothpick. Most of the ingested foreign bodies pass spontaneously through the anus without being noticed but about 1% of them can perforate through the wall of stomach or duodenum to reach solid organs like pancreas or liver. Once inside the pancreas they can produce complications like abscess, pseudoaneurysm or pancreatits. Foreign bodies of pancreas should be removed by endoscopic or surgical methods. We hereby report our experience of successful removal one a sewing needle from pancreas.

Mass-forming xanthogranulomatous cholecystitis masquerading as gallbladder cancer.

BACKGROUND: Mass-forming xanthogranulomatous cholecystitis (XGC), an uncommon inflammatory pathology of gallbladder (GB), masquerades as gallbladder cancer (GBC) and diagnosis is often a histological surprise. METHODS: A retrospective analysis of prospectively collected database of patients with GB mass operated between August 2009 and September 2012 was conducted to determine clinical, radiological, and intraoperative findings that might aid in the preoperative diagnosis of mass-forming XGC and ascertain their optimal management strategy. RESULTS: Of the 566 patients with GB mass and suspected GBC, 239 were found to be inoperable on preoperative workup and 129 patients had unresectable disease on staging laparoscopy/laparotomy. Of the 198 with resectable disease, 31 were reported as XGC on final histopathology (Group A), while 167 were GBC (Group B). Of these 31 patients, six with an intraoperative suspicion of benign pathology underwent cholecystectomy with segments IVb and V resection, and frozen section histopathology. Twenty-five underwent radical cholecystectomy, with (n = 10) or without (n = 15) adjacent organ resection. In comparison, anorexia and weight loss were significantly more in Group B (p = 0.001 and <0.001). Intraoperatively, empyema and associated gallstones were more common in Group A (p = 0.011 and <0.001). On computed tomography (CT) of the abdomen, continuous mucosal line enhancement and intramural hypodense bands were significantly more in Group A (p < 0.001 and 0.025). While CT abdomen revealed one or more features suggestive of XGC in 64.5 % (20/31) of patients in Group A, 11(35.5 %) did not have any findings suggestive of XGC on imaging. CONCLUSION: Mass-forming XGC mimics GBC, making preoperative and intraoperative distinction difficult. While imaging findings can help in suspecting XGC, definitive diagnosis require histopathological examination. Presence of typical radiological findings, however, can help in avoiding extended radical resection in selected cases.

Analysis of patients attending a private psychiatric clinic.

BACKGROUND: Private psychiatric clinics provide help for the bulk of our population, but they have not been evaluated systematically. It is time that we analyze the functioning of these set-ups to improve functioning. This paper is a modest attempt to analyze one such clinic. AIMS: To analyze the diagnostic distribution of the clients attending the clinic and to study the pattern of follow-up. MATERIALS AND METHODS: All records from April 1(st) 1997 to 31(st) March 1999 were analyzed. This is a descriptive study. RESULTS AND CONCLUSIONS: A total of 3077 new patients visited the clinic in this period. The male:female ratio was 54:46. Diagnostic distribution revealed the following: schizophrenia 20%, affective disorders 40%, OCD 8%, dissociative disorders 5.5% and anxiety disorders around 4.5%. The pattern of visits revealed that 50% of the patients dropped out after the first visit. Another 25% did so in next the two visits.

Liver hydatid with HIV infection: an association?

Various opportunistic infections have been reported in patients with human immunodeficiency virus (HIV) infection. Although there are a few reports of echinococcal infection of the lung, spine, and brain, a hydatid cyst of the liver has never been described. In our experience of treating over 150 cases of hydatid cysts of the liver, we identified three patients with large, multifocal hydatid cysts who also had an HIV infection. The current article describes one such patient and discusses the possible host immune-parasite interaction to ascertain if HIV positivity results in increased susceptibility and severity of echinococcal infection.