Focused panel sequencing points to genetic predisposition in non-cirrhotic intrahepatic portal hypertension patients in India.

OBJECTIVE: Non-cirrhotic intrahepatic portal hypertension (NCIPH), a portal microangiopathy affecting small portal vein radicles, is a disease of Indian sub-continent. NCIPH appears to be a complex disease with interactions between inherited and acquired factors, though the exact pathophysiological mechanism is unknown. We aimed at investigating the genetic variants that might contribute to susceptibility to NCIPH. METHODS: In this case-control study, we analyzed genes associated with microangiopathy-VWF-ADAMTS13 (von Willebrand factor and its cleavase enzyme - a disintegrin and matrix metalloprotease with thrombospondin type-1 motifs member 13) and alternative complement system vitamin B12 metabolism and with familial NCIPH. RESULT: Eighty-four Indian patients with liver biopsy-proven NCIPH (cases) and 103 healthy controls (matched for residential region of India) were included in the study. Targeted next-generation sequencing (NGS) panel, comprising 11 genes of interest, was done on 54 cases. Genotyping of selected variants was performed in 84 cases and 103 healthy controls. We identified variants in MBL2, CD46 and VWF genes either associated or predisposing to NCIPH. We also identified a single case with a novel compound heterozygous mutation in MBL2 gene, possibly contributing to development of NCIPH. CONCLUSION: In this first of a kind comprehensive gene panel study, multiple variants of significance have been noted, especially in ADAMTS13-VWF and complement pathways in NCIPH patients in India. Functional significance of these variants needs to be further studied.

Development of insulin resistance preceded major changes in iron homeostasis in mice fed a high-fat diet.

Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) have been associated with dysregulation of iron metabolism. The basis for this association is not completely understood. To attempt to investigate this, we studied temporal associations between onset of insulin resistance (IR) and dysregulated iron homeostasis, in a mouse model of T2DM. Male C57Bl/6 mice (aged 8 weeks) were fed a high-fat diet (HFD; 60% energy from fat) or a control diet (CD; 10% energy from fat) for 4, 8, 12, 16, 20 and 24 weeks. Development of IR was documented, and various metabolic, inflammatory and iron-related parameters were studied in these mice. HFD-feeding induced weight gain, hepato-steatosis and IR in the mice. Onset of IR occurred from 12 weeks onwards. Hepatic iron stores progressively declined from 16 weeks onwards. Accompanying changes included a decrease in hepatic hepcidin (Hamp1) mRNA expression and serum hepcidin levels and an increase in iron content in the epididymal white adipose tissue (eWAT). Iron content in the liver negatively correlated with that in the eWAT. Factors known to regulate hepatic Hamp1 expression (such as serum iron levels, systemic inflammation, and bone marrow-derived erythroid regulators) were not affected by HFD-feeding. In conclusion, the results show that the onset of IR in HFD-fed mice preceded dysregulation of iron homeostasis, evidence of which were found both in the liver and visceral adipose tissue.

Pancreatic Adenocarcinoma With Synchronous Colonic Metastases.

Metastases from pancreatic malignancy are commonly known to occur in the regional lymph nodes, liver, lung, and peritoneum. Synchronous or metachronous metastasis from the pancreas to the colon is rare, with only 6 cases reported in the literature. We report a man who was found to have adenocarcinoma on biopsies from synchronous lesions in the colon and the pancreas. The immunohistochemistry report revealed the diagnosis of a primary pancreatic malignancy with synchronous colonic metastases.

Gastric IgG4-related disease presenting as a mass lesion and masquerading as a gastrointestinal stromal tumor.

IgG4-related disease of the stomach is a rare disorder, and only a few cases have been reported. We present two cases that were identified over a 2-month period in our center. Two male patients aged 52 and 48 years presented with mass lesion in the stomach, which were clinically thought to be gastrointestinal stromal tumor, and they underwent excision of the lesion. Microscopic examination revealed marked fibrosis, which was storiform in one case, associated with diffuse lymphoplasmacytic infiltration and an increase in IgG4-positive plasma cells on immunohistochemistry. Serum IgG4 level was markedly elevated. Although rare, IgG4-related disease should be considered in the differential diagnosis of gastric submucosal mass lesions.

What makes non-cirrhotic portal hypertension a common disease in India? Analysis for environmental factors.

In India, an unexplained enteropathy is present in a majority of non-cirrhotic intrahepatic portal hypertension (NCIPH) patients. Small intestinal bacterial contamination and tropical enteropathy could trigger inflammatory stimuli and activate the endothelium in the portal venous system. Groundwater contaminated with arsenic is an environmental factor of epidemic proportions in large areas of India which has similar consequences. Von Willebrand factor (a sticky protein) expressed by activated endothelium may promote formation of platelet microthrombi and occlusion of intrahepatic portal vein branches leading to NCIPH. Environmental factors linked to suboptimal hygiene and sanitation, which enter through the gastrointestinal (GI) tract, predispose to platelet plugging onto activated endothelium in portal microcirculation. Thus, NCIPH, an example of poverty linked thrombophilia, is a disease mainly affecting the lower socio-economic strata of Indian population. Public health measures to improve sanitation, provide clean drinking water and eliminate arsenic contamination of drinking water are urgently needed. Till such time as these environmental factors are addressed, NCIPH is likely to remain 'an Indian disease'.

Histological and immunohistochemical study of hepatoblastoma: correlation with tumour behaviour and survival.

BACKGROUND: Hepatoblastoma (HB) has different histological subtypes, with varying prognosis. Though the survival has drastically improved, subsets of patients are not responsive to therapy. Therefore, it becomes important to determine the factors which affect the behaviour of the tumour. This study was aimed to look at the histopathological subtypes and compare with immunohistochemical (IHC) expression of CK19, beta-catenin and EpCAM and survival. METHODS: This study included 55 cases of HB. IHC expression of CK19, beta-catenin and EpCAM were correlated with histological subtypes, tumour behaviour, response to chemotherapy and survival. RESULTS: Most common epithelial subtype was fetal (43.2%) and mixed epithelial (54.8%) in pre- and post-chemotherapy groups respectively. Microvascular invasion (MVI) was present in 14/33 resected tumours. CK19 expression was seen in 54.2% and 72.2% of embryonal subtype, nuclear beta-catenin expression in 48.7% and 57.1% and EpCAM in 100% and 82.1% of tumours in pre- and post-chemotherapy groups, respectively. Fetal subtype had a lesser chance of MVI, recurrence, metastasis and death. Beta-catenin expression was associated with lower event free survival (EFS) and EpCAM with ≥50% viable tumour following chemotherapy (P=0.04). Age at diagnosis ≤2 years, male sex, alpha-fetoprotein <10,000 IU/mL following chemotherapy, solitary tumour (P=0.001), size ≤5 cm, pretreatment extent of disease (PRETEXT) I&II, mitosis ≤2/10 high power fields (hpf), viable tumour <50% (P=0.04) and absent nuclear expression of beta-catenin, predicted a higher EFS rate. CONCLUSIONS: Beta-catenin expression is associated with lower EFS and EpCAM expression with tumour viability. Multifocality and viable tumour ≥50% were significant factors predicting lower EFS. These factors should be included in the prognostication of HBs.

ADAMTS13 missense variants associated with defective activity and secretion of ADAMTS13 in a patient with non-cirrhotic portal hypertension.

BACKGROUND: Non-cirrhotic intrahepatic portal hypertension (NCIPH) is characterized by thrombotic microangiopathy of the portal venous system, low ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13), and high vWF (von Willebrand factor) levels. This study aimed to screen for ADAMTS13 mutations, focusing on the CUB domain, in these patients. METHODS: Prospectively recruited NCIPH patients and healthy volunteers underwent tests for plasma vWF-ADAMTS13 balance. Sanger sequencing of the CUB domain of ADAMTS13 was done in a subset of the NCIPH patients, and the detected mutation was screened for in all the study participants. Next-generation sequencing of clinically relevant exome and liver immunostaining for ADAMTS13 was done in patients with detected ADAMTS13 mutation. RESULTS: Plasma vWF-ADAMTS13 balance was significantly altered in 24 NCIPH patients (Child's class A:23, B:1) as compared to 22 controls. On initial sequencing of the CUB domain (17 cases and 3 controls), one NCIPH patient showed a rare missense variant (SNV) at position c.3829C >T resulting in p.R1277W (rs14045669). Subsequent RFLP analysis targeted to the R1277W variant did not detect this in any other NCIPH patient, nor in any of the 22 controls. The NCIPH patient with the R1277W variant had severe ADAMTS13 deficiency, consistently high vWF, other missense SNVs in ADAMTS13, vWF, and complement genes. Immunostaining of his liver biopsy revealed globules of ADAMTS13 within stellate cells. CONCLUSIONS: We report missense variants in ADAMTS13, vWF, and complement genes in a patient with NCIPH who had decreased secretion and activity of ADAMTS13 protein. Further studies are needed in NCIPH patients in this regard.

Risk factors for non-alcoholic fatty liver disease are common in patients with non-B non-C hepatocellular carcinoma in India.

AIM OF THE STUDY: The aim of the study was to analyze the prevalence of risk factors for non-alcoholic fatty liver disease (NAFLD) in patients with non-B non-C hepatocellular carcinoma (HCC). METHODS: Between June 2012 and November 2014, patients with HCC, negative for hepatitis B surface antigen and hepatitis C virus antibody, were included in this study. All patients were assessed for risk factors for NAFLD such as diabetes mellitus (DM), hypertension, dyslipidemia, metabolic syndrome, and obesity. RESULTS: Forty-seven patients with non-B non-C HCC (males, 37; age, 60±10 years; mean±SD) were studied. Model for end-stage liver disease score was 11±4. Twenty-five patients were in Child's class A. History of significant alcohol intake was noted in 11 (23%) patients. Prevalence of risk factors for NAFLD were obesity 24 (51%), DM 22 (47%), metabolic syndrome 21 (45%), hypertension 16 (34%), and dyslipidemia 13 (28%). Forty (85%) patients had at least one risk factor for NAFLD. The mean duration of at least one NAFLD risk factor was 7.5 years, prior to diagnosis of HCC. Thirteen (28%) patients were positive for anti-HBc; however, none of the study patients had detectable HBV DNA in blood. CONCLUSIONS: Eighty-five percent of the patients with non-B non-C HCC had at least one risk factor for NAFLD. None of the study patients had occult hepatitis B infection. NAFLD is emerging as the major etiological contributing factor for non-B non-C HCC in India.

Malignant melanocytic neoplasm of pancreas with liver metastasis: Is it malignant melanoma or clear cell sarcoma?

Malignant melanocytic neoplasm, usually seen in soft tissues, is rare in a visceral location and presents as a diagnostic dilemma. We present a case of pancreatic malignant melanocytic neoplasm with liver metastasis. A 58-year-old man presented with left upper abdominal swelling and loss of appetite. Imaging revealed a large mass arising from the pancreatic tail, and this was diagnosed as malignant neoplasm with melanocytic differentiation on biopsy with the possible differentials of malignant melanoma, clear cell sarcoma (CCS), and perivascular epithelioid cell neoplasm. The patient underwent distal pancreatectomy and splenectomy for the same. Follow-up imaging 6 months later showed a metastatic liver lesion, for which he also underwent a liver resection. BRAF mutational analysis was found to be negative. Both CCS and malignant melanoma have similar morphological features and melanocytic differentiation, but each harbors a distinct genetic background. Differentiation of both has diagnostic and therapeutic implications.

Neuroendocrine neoplasms of liver - A 5-year retrospective clinico-pathological study applying World Health Organization 2010 classification.

AIM: To study the clinicopathological characteristics of neuroendocrine neoplasms (NEN) on liver samples and apply World Health Organization (WHO) 2010 grading of gastroenteropancreatic (GEP) NEN. METHODS: Clinicopathological features of 79 cases of NEN of the liver diagnosed between January 2011 to December 2015 were analyzed. WHO 2010 classification of GEP NEN was applied and the tumors were graded as G1, G2 or G3. Two more categories, D1/2 (discordant 1/2) and D2/3 (discordant 2/3) were also applied. The D1/2 grade tumors had a mitotic count of G1 and Ki-67 index of G2. The D2/3 tumors had a mitotic count of G2 and Ki-67 index of G3. The follow up details which were available till the end of the study period (December 2015) were collected. RESULTS: Of the 79 tumors, 16 each were G1 and G2, and 18 were G3 tumors. Of the remaining 29 tumors, 13 were assigned to D1/2 and 16 were D2/3 grade. Male preponderance was noted in all tumors except for G2 neoplasms, which showed a slight female predilection. The median age at presentation was 47 years (range 10-82 years). The most common presentation was abdominal pain (81%). Pancreas (49%) was the most common site of primary followed by gastrointestinal tract (24.4%) and lungs (18%). Radiologically, 87% of the patients had multiple liver lesions. Histopathologically, necrosis was seen in only D2/3 and G3 tumors. Microvascular invasion was seen in all grades. Metastasis occurred in all grades of primary NEN and the grades of the metastatic tumors and their corresponding primary tumors were similar in 67% of the cases. Of the 79 patients, 36 had at least one follow up visit with a median duration of follow up of 8.5 mo (range: 1-50 mo). This study did not show any impact of the grade of tumor on the short term clinical outcome of these patients. CONCLUSION: Liver biopsy is an important tool for clinicopathological characterization and grading of NEN, especially when the primary is not identified. Eighty-seven percent of the patients had multifocal liver lesions irrespective of the WHO grade, indicating a higher stage of disease at presentation. Follow up duration was inadequate to derive any meaningful conclusion on long term outcome in our study patients.

Arsenicosis, possibly from contaminated groundwater, associated with noncirrhotic intrahepatic portal hypertension.

BACKGROUND AND AIMS: Idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH), a chronic microangiopathy of the liver caused by arsenicosis from use of contaminated groundwater, was reported from Asia. This study aimed to see, if in the twenty-first century, arsenicosis was present in NCIPH patients at our hospital and, if present, to look for groundwater contamination by arsenic in their residential locality. METHODS: Twenty-seven liver biopsy proven NCIPH patients, 25 portal hypertensive controls with hepatitis B or C related cirrhosis and 25 healthy controls, matched for residential locality, were studied. Eighty-four percent to 96 % of study subjects belonged to middle or lower socioeconomic category. Arsenicosis was looked for by estimation of arsenic levels in finger/toe nails and by skin examination. Arsenic levels in nails and in ground water (in NCIPH patients with arsenicosis) was estimated by mass spectrometry. RESULTS: Nail arsenic levels were raised in five (10 %) portal hypertensive study subjects [two NCIPH patients (both had skin arsenicosis) and three portal hypertensive controls]. All of these five patients were residents of West Bengal or Bangladesh. Skin arsenicosis was noted in three NCIPH patients (11 %) compared to none of disease/healthy controls. Ground water from residential locality of one NCIPH patient with arsenicosis (from Bangladesh) showed extremely high level of arsenic (79.5 µg/L). CONCLUSIONS: Arsenicosis and microangiopathy of liver, possibly caused by environmental contamination continues in parts of Asia. Further studies are needed to understand the mechanisms of such 'poverty-linked thrombophilia'.

Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.

Hepatic Fe overload has often been reported in patients with advanced alcoholic liver disease. However, it is not known clearly whether it is the effect of alcohol that is responsible for such overload. To address this lacuna, a time-course study was carried out in mice in order to determine the effect of alcohol on Fe homoeostasis. Male Swiss albino mice were pair-fed Lieber-DeCarli alcohol diet (20 % of total energy provided as alcohol) for 2, 4, 8 or 12 weeks. Expression levels of duodenal and hepatic Fe-related proteins were determined by quantitative PCR and Western blotting, as were Fe levels and parameters of oxidative stress in the liver. Alcohol induced cytochrome P4502E1 and oxidative stress in the liver. Hepatic Fe levels and ferritin protein expression dropped to significantly lower levels after 12 weeks of alcohol feeding, with no significant effects at earlier time points. This was associated, at 12 weeks, with significantly decreased liver hepcidin expression and serum hepcidin levels. Protein expressions of duodenal ferroportin (at 8 and 12 weeks) and divalent metal transporter 1 (at 8 weeks) were increased. Serum Fe levels rose progressively to significantly higher levels at 12 weeks. Histopathological examination of the liver showed mild steatosis, but no stainable Fe in mice fed alcohol for up to 12 weeks. In summary, alcohol ingestion by mice in this study affected several Fe-related parameters, but produced no hepatic Fe accumulation. On the contrary, alcohol-induced decreases in hepatic Fe levels were seen and may contribute to alcohol-induced suppression of hepcidin.

Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy.

Tissue Xpert™ MTB/Rif assay is of limited use in diagnosing peritoneal tuberculosis in patients with exudative ascites.

BACKGROUND: Xpert™ MTB/Rif is a multiplex hemi-nested real-time PCR-based assay to detect presence of M. tuberculosis within 2 hours of sample collection. The present study aimed at assessing efficacy of Xpert™ MTB/Rif assay for diagnosing peritoneal tuberculosis. METHODS: Patients with exudative ascites, fluid negative for acid-fast bacilli on auramine O fluorescence staining and unyielding fluid cytology for malignant cells, were included. Ultrasound-guided omental biopsy samples were obtained in all. Xpert™ MTB/Rif assay on tissue samples was assessed against a composite "reference" standard for diagnosis of peritoneal tuberculosis, defined as presence of any of the three-culture showing M tuberculosis, granulomatous inflammation on histology or resolution of ascites with 2 months of antitubercular therapy. RESULTS: During January 2012-July 2013, 28 patients (age:43 ± 15 years; mean ± SD; male:20) were recruited. Serum ascitic albumin gradient was <1.1 in all except in four patients with underlying cirrhosis. Twenty-one of the 28 patients had peritoneal TB as diagnosed by composite reference standard (histology:18; culture:4; treatment response:3). Seven patients (25%) had an alternative diagnosis (metastatic carcinoma 2, adenocarcinoma 2, mesothelioma 2, and systemic lupus erythematous 1). Xpert™ MTB/Rif assay was positive in 4/21 patients with peritoneal tuberculosis and in none of the 7 patients with alternative diagnosis. Thus, sensitivity, specificity, positive, and negative predictive values for tissue Xpert™ MTB/Rif assay in diagnosing peritoneal tuberculosis were 19% (95% C.I: 6% to 42%), 100% (95% C.I: 59% to 100%), 100% (40% to 100%), and 29% (95% C.I: 13% to 51%), respectively. INTERPRETATION AND CONCLUSION: Tissue Xpert™ MTB/Rif assay was of limited use in diagnosing peritoneal tuberculosis.

Cancer stem cells in hepatocellular carcinoma--an immunohistochemical study with histopathological association.

BACKGROUND & OBJECTIVES: Cancer stem cells (CSCs) may be responsible for tumour recurrence and resistance to chemotherapy in hepatocellular carcinoma (HCC). This study was carried out to evaluate the association between histological parameters and liver CSCs (LCSC) in HCC, and to compare distribution of liver CSCs in HCC associated with and without hepatitis B virus (HBV) infection. METHODS: Seventy nine tumours (49 surgical resections from 46 patients, and 30 from autopsy) were reviewed. Immunohistochemical staining for the LCSC marker EpCAM (epithelial cell adhesion molecule), liver progenitor cell (LPC) markers CK19 (cytokeratin 19) and neural cell adhesion molecule (NCAM) were performed and were associated with histological features of tumour behaviour. RESULTS: Thirty three tumours (41.8%) showed positive staining for EpCAM. CK19 and NCAM expression were seen in 26 (32.9%) and four (5.1%) tumours, respectively. The expression of EpCAM and CK19 was significantly associated with each other ( P<0.001). EpCAM expression was significantly associated with clinical and histological features indicating aggressive tumour behaviour, including younger age of onset, higher serum alpha foetoprotein (AFP) levels, tumour cell dedifferentiation, increased mitotic activity, and vascular invasiveness. There was no significant difference in expression of EpCAM, CK19 and NCAM between HBV positive and negative HCC. INTERPRETATION & CONCLUSIONS: The LCSC marker EpCAM was expressed in less than half of HCC, was independent of HBV aetiology, and was strongly associated with clinical and histological features of aggressive tumour behaviour. Positive staining for CK19 suggests a possible LPC origin of the EpCAM positive HCCs.

Clinicopathological profile and management of severe autoimmune hepatitis.

BACKGROUND: Severe autoimmune hepatitis is an entity which has been rarely reported in the Indian literature. We describe here the clinicopathological profile and treatment of severe autoimmune hepatitis (SAH) which is to the best of our knowledge the first report from India addressing this illness. METHODS AND RESULTS: Between September 2010 and March 2013, 13 patients seeking treatment at our centre were diagnosed as SAH and treated with steroids. Jaundice along with coagulopathy was the presenting symptom in all these patients. Ascites was present in ten and encephalopathy in 6 patients. The median serum IgG was 2135 mg/dl (range: 1122-5490).Significant titers of autoantibodies were present in all patients except one. Transjugular liver biopsy in 9 patients showed characteristic features of SAH such as extensive bridging necrosis and moderate to dense portal inflammation. With corticosteroid therapy, 10 patients survived while three died. In those who survived, biochemical improvement was seen as early as seven days with excellent long-term remission. CONCLUSIONS: Clinical suspicion supported by liver biopsy and autoimmune serology led to the diagnosis of SAH in a cohort of patients with unexplained liver failure. Corticosteroids were beneficial in majority of patients affording excellent results and this could be predicted by early reduction in serum bilirubin within 7-15 days.

Investigation into celiac disease in Indian patients with portal hypertension.

BACKGROUND: There is limited data on celiac disease in patients with cryptogenic cirrhosis or idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH). Our objective was to evaluate for celiac disease in patients with portal hypertension in India. METHODS: Consecutive patients with portal hypertension having cryptogenic chronic liver disease (cases) and hepatitis B- or C-related cirrhosis (controls) were prospectively enrolled. We studied tissue transglutaminase (tTG) antibody and duodenal histology in study patients. RESULT: Sixty-one cases (including 14 NCIPH patients) and 59 controls were enrolled. Celiac disease was noted in six cases (including two NCIPH patients) as compared to none in controls. In a significant proportion of the remaining study subjects, duodenal biopsy showed villous atrophy, crypt hyperplasia, and lamina propria inflammation, not accompanied by raised intraepithelial lymphocytes (IELs); this was seen more commonly in cases as compared to controls. An unexpectedly high rate of tTG antibody positivity was seen in study subjects (66 %) of cases as compared to 29 % in controls (p-value < 0.001), which could indicate false-positive test result. CONCLUSION: In this study, 10 % of patients with unexplained portal hypertension (cryptogenic chronic liver disease) had associated celiac disease. In addition, an unexplained enteropathy was seen in a significant proportion of study patients, more so in patients with cryptogenic chronic liver disease. This finding warrants further investigation.