The Smad family and its role in pancreatic cancer.

One of the major signaling pathways that determine the tumor aggression and patient outcome in pancreatic cancer is the transforming growth factor-beta (TGF-ß) pathway. It is inactivated at various levels in pancreatic cancer and plays a dual role in tumor initiation and progression. The Smad family of proteins transduce signals from the TGF-ß superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. This review discusses the structure, function and regulation of various participating Smad family members, and their individual roles in determining the progression and outcome of pancreatic cancer patients, with a special emphasis on Smad4.

A comprehensive examination of Smad4, Smad6 and Smad7 mRNA expression in pancreatic ductal adenocarcinoma.

BACKGROUND: Smad4, Smad6 and Smad7 are important molecules in TGF-beta pathway, which plays an important role in pancreatic ductal adenocarcinoma (PDAC) biology. Aims : This study examined the expression profiles of Smad4, Smad6 and Smad7 mRNA in patient samples of PDAC and their relationship to Smad protein expression, SMAD4 gene mutations, clinicopathological parameters and patient survival. SETTINGS AND DESIGN: Surgically resected, paired normal and tumor tissues of 25 patients of PDAC were studied. MATERIALS AND METHODS: Protein and mRNA levels were assessed by immunohistochemistry and RT-PCR, respectively. STATISTICAL METHODS: Statistical analysis was done using Student's t-test, Pearson's chi-square test, Spearman's Rank Correlation, Pearson's Correlation test and Kaplan-Meier Logrank test. RESULTS: While there was a highly significant difference in the protein levels of all three Smads in tumor as compared to normal samples, mRNA levels were significantly different only for Smad4. Protein levels did not correlate significantly with mRNA levels for any of the three Smads. The mRNA levels of Smad4 and Smad6, Smad4 and Smad7, and Smad6 and Smad7 in tumor samples showed a significant positive correlation. The relationship of Smad4 mRNA expression to SMAD4 gene status and Smad4 protein expression was discordant and there was no significant correlation between mRNA expression and clinicopathological parameters and patient survival. CONCLUSION: The absence of concordance between SMAD4 gene status, mRNA expression and Smad4 protein expression suggests the presence of other regulatory mechanisms in Smad4 transcription and translation in PDAC.

Traumatic diaphragmatic hernia: tertiary centre experience.

BACKGROUND: Traumatic diaphragmatic hernia (TDH) resulting from traumatic diaphragmatic rupture (TDR) may not be easily detected and can lead to significant morbidity and mortality. PATIENTS AND METHODS: A retrospective case note analysis was performed of all patients treated for TDR at a major teaching hospital between March 2003 and March 2008. The aetiological factors, associated injuries, management and outcome were analysed. RESULTS: Twenty-seven patients were studied (24 males, 3 females) and their ages ranged from 16 to 72 years (median 35 years). TDR was left-sided in 85% and right-sided in 15%. Aetiology was blunt trauma in 81% and 19% had penetrating injury. Associated injuries were present in 81%. The most common approach for repair was transabdominal (89%); additional thoracotomy was needed in 11%. Herniation of abdominal contents was present in 85% and herniation of more than one organ was present in 57%. The diaphragmatic rent was repaired primarily in 89% using nonabsorbable sutures. Post-operative pulmonary complications occurred in 52% of patients. Three patients (11%) died. CONCLUSION: Left-sided blunt traumatic diaphragmatic rupture was more common than right-sided rupture. The most commonly herniated organs were the stomach and colon. Most ruptures could be repaired by an abdominal approach, which also allowed a complete exploration of the abdominal organs. Careful attention should be given to associated intra-abdominal injuries. Most of the defects were repaired directly using nonabsorbable sutures.

High frequency of loss of allelic integrity at Wilms' tumor suppressor gene-1 locus in advanced breast tumors associated with aggressiveness of the tumor.

BACKGROUND: The product of Wilms' tumor suppressor gene (WT1), a nuclear transcription factor, regulates the expression of the insulin-like growth factor (IGF) and transforming growth factor (TGF) systems, both of which are implicated in breast tumorigenesis and are known to facilitate angiogenesis. In the present study, WT1 allelic integrity was examined by Loss of Heterozygosity (LOH) studies in infiltrating breast carcinoma (n=60), ductal carcinoma in situ (DCIS) (n=10) and benign breast disease (n=5) patients, to determine its possible association with tumor progression. METHODS: LOH at the WT1 locus (11p13) as determined by PCR-RFLP for Hinf1 restriction site and was subsequently examined for its association with intratumoral expression of various growth factors i.e. TGF-beta1, IGF-II, IGF-1R and angiogenesis (VEGF and Intratumoral micro-vessel density) in breast carcinoma. RESULTS: Six of 22 (27.2%) genetically heterozygous of infiltrating breast carcinoma and 1 of 4 DCIS cases showed loss of one allele at WT1 locus. Histologically, the tumors with LOH at WT1 were Intraductal carcinoma (IDC) and were of grade II and III. There was no correlation in the appearance of LOH at WT1 locus with age, tumor stage, menopausal status, chemotherapy status and lymph node metastasis. The expression of factor IGF-II and its receptor, IGF-1R was significantly higher in carcinoma having LOH at WT1 locus. A positive correlation was observed between the TGF-beta1, VEGF expression and IMD scores in infiltrating carcinoma. CONCLUSIONS: The current study indicates that the high frequency of loss of allelic integrity at Wilms' tumor suppressor gene-1 locus in high-graded breast tumors is associated with aggressiveness of the tumor.

Abberant crypt foci -- importance in colorectal carcinogenesis and expression of p53 and mdm2: a changing concept.

BACKGROUND: The presence of aberrant crypt foci (ACF) represents an important preneoplastic condition of the large intestine. The ACF are used extensively as the biological end point to study the effects of diet, biological agents and chemotherapeutic agents as they undergo distinct morphological and genetic changes in response to these factors. Till date, in human ACF, K-ras, beta-catenin, carcinoembryonic antigen (CEA) and adenomatous polyposis coli (APC) protein expression have been investigated, but the expression of late markers of colon carcinogenesis have not been studied in great detail. AIMS: To study the significance of ACF in carcinogenesis, the incidence of ACF in colorectal carcinoma (CRC) patients and the expression profiles of the tumor repressor protein p53 and the murine double minute (mdm2) protein in ACF. METHODS AND MATERIALS: The pattern of expression, histology and immunohistochemistry of the p53 and mdm2 proteins were studied in 32 colectomy specimens. Careful scoring was carried out using standard criteria. The prevalence of ACF, types of ACF, surrounding mucosal changes and the relation of ACF with tumor morphology and aggressiveness were determined, as was any difference in the staining pattern between hyperplastic and dysplastic ACF. RESULT: Aberrant crypt foci were present in a large number of cases (84.4%), of which 65% were hyperplastic. All hyperplastic and dysplastic ACF tested positive for p53 in the nucleus. Both the nucleus and cytoplasm tested positive for mdm2 irrespective of the degree of dysplasia. No correlation was noted between ACF and tumor histology. CONCLUSION: The p53 and mdm2 proteins are expressed during the very early stages of mucosal changes in ACF, irrespective of histological changes, but the pattern of expression can differ depending on geographical barriers, genetic factors, diet and environmental conditions. This results suggests that the conventional adenoma-carcinoma sequence is not completely correct. The detection of ACF and p53 and mdm2 expression can help to curtail the disease in its early stage by targeted therapy.

Expression of interferon-gamma- inducible protein-10 and its receptor CXCR3 in chronic pancreatitis.

AIM: The role of CXC chemokine, interferon-gamma-inducible protein-10 and its receptor CXCR3 in pathophysiology of chronic pancreatitis (CP) is not very clear. Hence, this study was carried out to analyze the expression of CXCL10 and CXCR3 in CP tissues. METHODS: Pancreatic tissues from 25 histopathologically graded CP cases (11 alcohol associated CP, 5 confirmed idiopathic and 9 of undefined nature) and 10 normal cases were studied. Tissues were subjected to real-time PCR, immunohistochemistry, and Western blot analysis for CXCL10 and CXCR3 expression. RESULTS: Real-time (RT)-PCR revealed increased expression of CXCL10 (13-fold) and CXCR3 (7-fold) in CP tissue. The immunohistochemistry and Western blot analysis of the same showed significant increased protein expression and correlated well with the histopathological grades. The CXCL10 was localized mainly in the cytoplasm of pancreatic acinar cells and expression increased from grade I to grade II and declined in grade III while no expression was recorded in normal. The CXCR3 was expressed strongly at the acinar cell membrane in CP as compared to normal. Further, comparative analysis by semiquantitative RT-PCR analysis was performed for other CXC/CC chemokines (CXCL9, CXCL11, CCL3, CCL4, CCL5) and receptor (CCR5) which revealed their upregulation in the diseased state. CONCLUSION: The existence of CXCL10 and CXCR3 with other CXC/CC chemokine signature in CP is suggestive of their vital role in the progression of chronic inflammation.

Intratumoral FOXP3 expression in infiltrating breast carcinoma: Its association with clinicopathologic parameters and angiogenesis.

The activity of T regulatory cells (Tregs) is known to be closely associated with the expression of forkhead/winged helix transcription factor, FOXP3. To determine, whether accumulation and activation of intratumoral Tregs help in the progression of breast carcinoma, we have analyzed the intratumoral expression of FOXP3 in invasive breast carcinoma and compared it with its level in ductal carcinoma in situ (DCIS) and adjacent normal tissue with the main aim of using this factor as marker of tumor progression. Intratumoral FOXP3 levels were correlated with the levels of transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF, an invasogenic and angiogenic growth factor) and intratumoral microvessel density (IMD, a prognostic marker for angiogenesis). We also analyzed whether FOXP3 gene expression correlated with other clinicopathological variables like age, tumor stage, histological grade and lymph node metastasis. Infiltrating cancers had higher FOXP3 transcription (7.43+/-3.44) than did ductal carcinoma in situ (4.27+/-1.97, p<0.05) and normal tissues (3.51+/-1.22, p<0.001). Intratumoral FOXP3 expression was significantly higher in patients with stage III disease (TNM classification) compared to patients who had stage II disease (p=0.037). In infiltrating carcinoma, a significant positive correlation between FOXP3 expression and TGF-beta1 expression was noted (p<0.001). Furthermore, a positive correlation between FOXP3 expression with VEGF expression and IMD values was also detected, however, statistically that was non-significant. A linear association of intratumoral FOXP3 expression with invasion, size and vascularity suggests a utility of FOXP3, an indicator of Treg activity as a marker of tumor progression and metastasis in breast carcinoma.

Primary duodenal neoplasms: a retrospective clinico-pathological analysis.

AIM: To analyze the clinico-pathological spectrum of primary duodenal neoplasms. METHODS: A total of 55 primary duodenal neoplasms reported in the last 10 years after excluding ampullary and periampullary tumors were included in the study. Clinical details were noted and routine hematoxylin and eosin stained paraffin sections were studied for histological subtyping of the tumors. RESULTS: On histopathological examination primary duodenal neoplasms were categorized as: epithelial tumor in 27 cases (49.0%) including 10 cases of adenoma, 15 cases of adenocarcinoma, and 2 cases of Brunner gland adenoma; mesenchymal tumor in 9 cases (16.3%) consisting of 4 cases of gastrointestinal stromal tumor, 4 cases of smooth muscle tumor and I case of neurofibroma; lymphoproliferative tumor in 12 cases (21.8%), and neuroendocrine tumor in 7 cases (12.7%). CONCLUSION: Although non-ampullary/periampullary duodenal adenocarcinomas are rare, they constitute the largest group. Histopathological examination of primary duodenal tumors is important for correct histological subtyping.

Comparison of multidetector computed tomographic colonography and conventional colonoscopy for detection of colorectal polyps and cancer.

BACKGROUND: Computed tomographic colonography (CTC) is a new technique for detecting colonic neoplasms. Data on the utility of this method in the Indian population are limited. METHODS: Forty-two patients with symptoms of colonic disease underwent CTC and conventional colonoscopy (CC) within one week of each other and the findings at these two investigations were compared. RESULTS: The entire colon could be evaluated in 38 patients on CTC and in 23 patients on CC. Of the 19 patients who had incomplete CC, 14 had occlusive colonic lesions. Of the 86 lesions detected on CC, 76 (88.4%) were correctly identified on CTC with regard to location and size. CTC was false negative for 10 lesions and false positive for 5 lesions in 3 patients. The sensitivity and specificity of CTC were 65% and 77%, respectively, for lesions 1-5 mm; 97% and 83% for 6-9 mm-sized lesions; and 100% and 100% for lesions 10 mm or larger. Extracolonic findings were seen in 24 of 42 patients (57%). CONCLUSIONS: CTC is reliable for detecting lesions 6 mm or larger in size. It permits evaluation of the region proximal to an occlusive growth, which is often not possible with CC.

Evaluation of gall bladder carcinoma with dynamic magnetic resonance imaging and magnetic resonance cholangiopancreatography.

The objective of this study is to evaluate the efficacy of dynamic MRI with magnetic resonance cholangiopancreatography (MRCP) in the preoperative assessment of gall bladder carcinoma. Magnetic resonance imaging and MRCP were carried out in 15 patients with gall bladder carcinoma before surgery and the imaging findings correlated with surgical and pathological findings. Gall bladder carcinoma manifested as focal or diffuse wall thickening in 73% (11/15) and as a mass replacing the gall bladder in 27% (4/15). All tumours showed enhancement in the early phase, which persisted into the delayed phase. The sensitivity and specificity of MRI with MRCP in detecting hepatic invasion, lymph node metastasis and bile duct invasion was 87.5 and 86%, 60 and 90%, and 80 and 100%, respectively. Magnetic resonance imaging correctly diagnosed duodenal invasion in only 50% and in none of the two patients with peritoneal metastasis. In conclusion, dynamic MRI with MRCP is an accurate and a reliable method of showing gall bladder carcinoma and in assessing its local and regional extent as part of preoperative assessment.

MDCT in the staging of gallbladder carcinoma.

OBJECTIVE: The purpose of our study was to determine the utility of dual-phase MDCT with 3D reconstruction in the staging and resectability of gallbladder carcinoma. SUBJECTS AND METHODS: Twenty-seven consecutive patients with suspected gallbladder carcinoma on clinical examination and routine sonography were prospectively analyzed with dual-phase MDCT. Of these patients, only 20 who underwent a laparotomy for extended cholecystectomy or a palliative surgery were included in the study. Three-dimensional volume-rendered reconstruction was used for evaluation of the vascular invasion and anatomy. The staging and resectability as determined on CT were compared with preoperative findings. RESULTS: On the basis of the CT findings, eight tumors were resectable and 12 were unresectable. On surgery, 11 tumors were found to be resectable and the remaining were unresectable. Overstaging by CT occurred in three patients due to overassessment of duodenal infiltration. CT had a sensitivity of 72.7%, a specificity of 100%, and an accuracy of 85% for determining resectability of gallbladder carcinoma. For the diagnosis of hepatic and vascular invasion by the tumor, there was 100% correlation between CT and surgery. Vascular variations were found in six of the 11 patients who underwent radical cholecystectomy. CONCLUSION: Dual-phase MDCT with 3D reconstruction is a comprehensive imaging technique for staging gallbladder carcinoma and determining the vascular road map before surgery.

Xanthogranulomatous cholecystitis.

Twelve cases of xanthogranulomatous cholecystitis are reported. The clinical presentation was similar to chronic cholecystitis. All patients had associated gallstones. The diagnosis was achieved at histopathological examination of the resected gall bladders and none had any focus of malignancy. Cholecystectomy was curative.

Clinical relevance of intracellular cytokines IL-6 and IL-12 in acute pancreatitis, and correlation with APACHE III score.

Pro-inflammatory cytokines are involved in the pathogenesis of acute pancreatitis (AP). Here, we measure and correlate clinically the percentages of peripheral blood mononuclear cells (PBMC) that contain interleukin (IL)-6 and IL-12 and compare these with acute physiology and chronic health evaluation (APACHE III) scores in 30 patients with AP. Severity of AP is determined according to the Atlanta criteria. Patients with severe AP (n = 15) had significantly higher IL-6 values compared to those with mild AP (n = 15). IL-12 levels correlated well with aetiological factors (alcohol and biliary pathology) in patients with AP. Correlation was seen between IL-6 value and APACHE score in severe AP. A score of 30 points was used as the cut off between mild (<30) and severe (>30) cases, with a sensitivity of 80% and specificity of 100%. Cut off percentages for IL-6- and IL-12-positive PBMCs were >25% (positive predictive value [PPV]: 100%) and >9% (PPV: 70%), respectively. Based on these results, it would seem logical to use both APACHE III score and IL-6 percentage to assess severity in patients with AP.

Pneumobilia--clue to an unusual cause of diarrhea.

A 65-year lady presented with diarrhea and weight loss of six months duration. Initial evaluation suggested that malabsorption was the possible underlying mechanism for the diarrhea. Work up for the common etiologies of malabsorption was non-contributory. Presence of pneumobilia raised the suspicion of a bilio-enteric fistula, which was subsequently confirmed on barium enema and endoscopic cholangio-pancreaticography to be a cholecystocolic fistula. At surgery, a fistulous tract from the fundus of the gallbladder was found to be communicating with the hepatic flexure. Fistulectomy with cholecystectomy resulted in prompt relief of symptoms. Cholecystocolic fistula (CCF) is a rare biliary fistula with diverse presentation.

Pericholecystic adhesions in single v multiple gallstones and their consequences for laparoscopic cholecystectomy.

BACKGROUND: Adhesions are amongst the common reasons for open conversion of laparoscopic cholecystectomy. It is not clear whether this problem is more common with single or multiple gallstones. PATIENTS AND METHODS: The clinical records of 110 patients with chronic cholelithiasis harboring multiple stones in the gallbladder (multiple stone group; MSG) and 45 patients with single stones in the gallbladder (SSG) undergoing laparoscopic cholecystectomy were analyzed for differences in the clinical presentation and outcome with special reference to the incidence of pericholecystic adhesions, size of the stones, and their implications for conversion and complications. RESULTS: Patients in the SSG had a significantly higher incidence of dense pericholecystic adhesions in the region of the porta hepatis (P = 0.003). Eleven patients in the SSG (24.4%) were converted to open cholecystectomy. Dense pericholecystic adhesions around the porta hepatis alone contributed to nine of these conversions (81.8%). The size of the stones was significantly greater (P < 0.001) in those patients of the SSG who required conversion to open cholecystectomy. Thirteen patients of the MSG (11.8%) required conversion to open cholecystectomy. Dense pericholecystic adhesions alone contributed to conversion in four cases (30.7%), and the size of the gallstones was not significantly different (P = 0.981) in patients with or without conversion to open cholecystectomy. There was no difference in the clinical presentation or complications in the two groups of patients. CONCLUSION: Dense adhesions in the porta hepatis are significantly more common in patients with single stones and are the most common reason for open conversion.

Two-phase dynamic contrast-enhanced computed tomography with water-filling method for staging of gastric carcinoma.

The objective of the study was to evaluate the usefulness of two-phase incremental contrast-enhanced computed tomography (CT) with water-filling method in the preoperative assessment of stomach carcinoma. Dynamic CT was done in 20 patients with biopsy-proven gastric cancers and the findings were compared with surgery. A 150-ml ionic contrast medium was administered intravenously at a flow rate of 3.5 ml/s. CT scanning was done at 45 s (early phase) and 3 min (equilibrium phase) after the start of intravenous injection. Primary tumors were detected in all the cases. The overall accuracies of dynamic CT in determining depth of tumor invasion, pancreatic invasion, adjacent liver infiltration, and lymph node metastasis were 85% (17/20), 90% (18/20), 85% (17/20), and 69% (13/19), respectively. The early phase of dynamic CT is highly accurate in determining the depth of tumor invasion of advanced gastric cancers but has low sensitivity in the detection of nodal metastasis. The delayed phase scans do not add any additional information and hence, should not be routinely used for staging.

Expression of activation, adhesion molecules and intracellular cytokines in acute pancreatitis.

Adhesion and activation molecules as well as cytokines play an important role in an immune scenario. In acute pancreatitis, we have studied some of these in order to evaluate dysregulation. For this we took peripheral blood mononuclear cells and pancreatitis tissue cells. We analysed activation markers like CD69, CD25 and HLA-DR and found a marked elevation of CD69 as well as CD25 in both peripheral blood cells and tissue mononuclear cells when compared to controls. In PBMC-CD69: P<0.01 and CD25: P<0.01; in tissue-CD69: P<0.001 and CD25: P<0.001. The HLA-DR levels, however, were reduced in the disease state (in acute pancreatitis patient blood (P<0.01) and tissue cells (P<0.001)). The adhesion molecules showed unanimous rise in the blood and the tissue samples. In blood samples, CD11a: P<0.05 and CD11b: P<0.05 and tissue samples CD11a: P<0.01 and CD11b: P<0.01and CD54 in peripheral blood (P<0.05) and tissue (P<0.01) of AP was high as compared to controls. By simultaneous flowcytometric analysis, we determined the co-expression of a surface marker (CD4/CD8/CD14) and intracellular cytokine (TNF-alpha and IFN-gamma) in individual cells. The IFN-gamma producing CD8+T cells were elevated in pancreatic tissue (P<0.01). TNF-alpha producing cell numbers were significantly higher in tissue cells than in blood and also in CD8+ T cells (P<0.001). We conclude that monocyte function is affected in AP as shown by reduced HLA-DR numbers and lowered TNF-alpha producing cells. Moreover, the CD8+T cells appear to play an important role in cytokine synthesis at the effector site.

Gastrointestinal stromal tumors the assessment of malignant potential.

GISTS are the largest category of non-epithelial neoplasms of stomach and small bowel. Numerous immunohistochemical, ultrastructural and flow cytometry studies have been carried out for evaluation of prognostic factors which could predict malignant behaviour of these neoplasms. Tumor size of 5 cm and mitosis of 2/10 hpf were suggested as two important parameters which could predict the chances of recurrence and clinically aggressive course. The aim of this study is to examine predictive value of these two important parameters in assigning the tumors as high, intermediate and low risk groups. Using these two parameters we categorized 30 cases of GIST over a period of 6 years (1990-95) into low, intermediate and high risk groups and examined other features of these cases. Based on these two parameters alone we found that 4 cases each in low and intermediate group could be assigned to a higher risk group clinically as there were presence of adjacent organ infiltration, lymphatic emboli, serosal nodules, lymph node metastasis and transmural infiltration. Hence, other features like hemorrhage, necrosis and anaplasia should also be included in risk assessment. Metaplastic tissues like bone, cartilage and adipose tissues were seen only in high-risk categories.