C.A.F. Escanhoela

Viral hepatitis in patients infected with human immunodeficiency virus

From 1992 to 1995 we studied 232 (69% male, 87% Caucasian) anti-human immunodeficiency virus (anti-HIV) positive Brazilian patients, through a questionnaire; HIV had been acquired sexually by 50%, from blood by 32%, sexually and/or from blood by 16.4% and by an unknown route by 1.7%. Intravenous drug use was reported by 29%; it was the most important risk factor for HIV transmission. The alanine aminotransferase quotient (qALT) was >1 for 40% of the patients, 93.6% had anti-hepatitis A virus antibody, 5.3% presented hepatitis B surface antigen, 44% were anti-hepatitis B core antigen positive and 53.8% were anti-hepatitis C virus (anti-HCV) positive. The anti-HCV test showed a significant association with qALT>1. Patients for whom the probable HIV transmission route was blood had a 10.8 times greater risk of being anti-HCV positive than patients infected by other routes. Among 30 patients submitted to liver biopsy, 18 presented chronic hepatitis.

Fine Needle Aspiration Biopsy for Improving the Diagnostic Accuracy of Cut Needle Biopsy of Focal Liver Lesions

OBJECTIVE To determine the value of fine needle aspiration biopsy (FNAB) in comparison to cut needle biopsy (CNB) for the diagnosis of malignancy of focal liver lesions. STUDY DESIGN A retrospective analysis was conducted on 68 FNAB and 49 CNB procedures performed on 62 patients with focal liver lesions. RESULTS Cytology permitted a diagnosis of the lesion in 78% of cases. When punctures with insufficient material were excluded (11), the diagnostic accuracy of FNAB was 93%. For the 49 patients who underwent both procedures, FNAB and CNB had the same diagnostic accuracy, 78%, when considered separately and of 88% when considered in combination. Sensitivity, specificity and positive predictive value were similar for the 2 techniques. The negative predictive value was 64% for FNAB and CNB used separately and reached 78% when the 2 techniques were combined. There were no complications during the execution of FNAB and CNB. CONCLUSION FNAB is an effective and safe method for the diagnosis of focal hepatic lesions, with diagnostic accuracy similar to that of CNB. When the 2 techniques are combined, the accuracy of the diagnosis of malignancy of focal liver lesions increases.

Chronic liver disease in kidney recipients with hepatitis C virus infection

Abstract: Background:  The prevalence of anti‐hepatitis C virus (HCV) positive test is higher among patients in dialysis and in kidney recipients than in general population. Hepatitis C virus infection is the main cause of chronic liver disease in renal transplant patients. Liver biopsy and virological analysis were performed to clarify the grade of liver damage in kidney recipients.

Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

Hepatic inflammatory pseudotumor: A case series

INTRODUCTION Inflammatory pseudotumor (IPT) is a rare lesion consisted of inflammatory and myofibroblastic cells. These lesions may be found in different organs. There are less than 300 described cases. PRESENTATION OF CASE Case 1. 64-year-old cirrhotic male with a palpable epigastric mass. CT showed a lesion in liver segments 2 and 3 and left hepatic artery aneurism. Percutaneous embolization and wide spectrum antibiotics were tried, however the lesion grew. Left lateral hepatectomy was performed, and HIPT diagnosed. The patient died due to multiple organ dysfunction. Case 2. 30-year-old male with abdominal pain and fever. CT showed a hepatic hilar lesion. Surgical resection was performed after an ineffectual antibiotic trial, and HIPT was confirmed. The patient is doing well. Case 3. 73-year-old female with abdominal pain and fever. CT showed a 7cm lesion in the left liver lobe. Unrewarding cancerous screening was performed, and unsuccessful antibiotic course was tried. Resection was performed, and HIPT diagnosed. The patient is doing well. Case 4. 50-year-old cirrhotic male with abdominal pain. CT showed a segment 6 lesion and portal vein thrombosis. Considering cancer as the first hypothesis and the MELD score of 9, segmentectomy was performed. HIPT was the final diagnosis. The patient died due to abdominal sepsis. DISCUSSION HIPT is a lesion with a vast list of differential diagnosis. Antibiotics are the first line of therapy, although surgery is often necessary. Overall prognosis is good, although comorbidities may worsen it. CONCLUSION HIPT is a rare and misleading entity.

Predicting Survival After Liver Transplantation Using Up-to-Seven Criteria in Patients With Hepatocellular Carcinoma

BACKGROUND Liver transplantation is a controversial issue in the initial management of hepatocellular carcinoma (HCC). The aim of this study was to demonstrate that up-to-seven could be useful as a model in evaluating potential candidates for liver transplantation for HCC. METHODS Between January 1997 and December 2010, 84 patients undergoing liver transplantation were analyzed for HCC. Pathologic variables included the number of lesions, maximal tumor diameter, vascular invasion, and tumor grade. The following variables were recorded: Age, gender, recurrence of HCC, survival, donor age, incidental, Milan criteria, Model for End-Stage Liver Disease score, and alpha-fetoprotein (AFP) levels. To evaluate variables we used the Kaplan-Meier method, Cox regression and Kolmogorov-Smirnov test. RESULTS There were 68 (80.9%) patients within up-to-seven criteria and 16 (19.1%) beyond this criteria. AFP levels were 96.4 versus 464.3 ng/dL in patients within versus beyond up-to-seven criteria. The median diameter of the largest nodule was 22.2 versus 32.0 mm in patients within versus beyond up-to-seven criteria, respectively. The median sum of nodules was 1.8 and 5.6 nodules in patients within versus beyond up-to-seven criteria. Vascular invasion was present in 11 (13.0%) patients within versus 7 (8.3%) beyond. Recurrence was observed in 9 (10.7%) patients and only 3 were beyond up-to-seven criteria, 2 had vascular invasion, and another showed poor differentiation. Post liver transplantation survival rates were 87.7%, 74.5%, and 65.3% at 1, 3, and 5 years in patients within up-to-seven versus 80.7%, 51.7%, and 32.1% beyond (P = .03). Similar results were observed when we applied Milan criteria or expanded Milan criteria. The largest nodule was the only predictive factor after proportional hazard Cox regression application (Beta = 0.037; exponent = 1.0377; Wald = 4.542; P = .03). In the present study, up-to-seven criteria could be useful to evaluate potential liver transplant candidates due to HCC.

Useful properties of undifferentiated mesenchymal stromal cells and adipose tissue as the source in liver-regenerative therapy studied in an animal model of severe acute fulminant hepatitis

BACKGROUND AIMS End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. METHODS AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbeccos modified Eagles medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbeccos modified Eagles medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. RESULTS All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. CONCLUSIONS AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.

Improved Detection of Helicobacter pylori DNA in Formalin-fixed Paraffin-embedded (FFPE) Tissue of Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection (LCM)

Dear Editor, Helicobacter DNA has been detected in hepatic tissues from patients with various hepatobiliary diseases, mainly cirrhosis and hepatocellular carcinoma (HCC). Although the role of Helicobacter spp. in pathogenesis of these diseases remains unclear, the available data suggest that Helicobacter infection may play a role in hepatic carcinogenesis [1]. Considering that HCC is one of the most common malignancies with more than 500.000 new tumors diagnosed annually [2], further studies related to H. pylori and development of HCC have fundamental importance on the understanding of its pathogenesis. Formalin-fixed paraffin-embedded (FFPE) tissue represents an extraordinary source for molecular studies as genomic DNA can be extracted from this sample. However, DNA extraction from FFPE tissues is challenging because nucleic acids are commonly fragmented and cross-linked with proteins. Furthermore, methods of DNA extraction from FFPE tissue are generally laborious and time-consuming. Laser capture microdissection (LCM) is a recently developed technique for isolation of pure populations of cells from tissue sections by microscopic visualization. Because of its high precision and accuracy, LCM has been employed in cancer-related studies. In this work, we used LCM technique to improve the detection of H. pylori in FFPE liver from patients with HCC. With this aim, six H. pylori-positive samples detected by polymerase chain reaction (PCR) with H. pylori-specific 16S rRNA primers were selected. The sequence of the sense primer (JW21) was 5′-GCGACCTGCTGGAACATTAC-3′(position 691-710) and the antisense primer (JW22) was 5′-CGTTAGCTCCATTACTGGAGA-3′ (position 829-809) [3]. Tissue samples were cut on 0.17 mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then routine staining with carbol fuchsin was performed [4]. Thereafter, stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then ejected into the Eppendorf tube cap by a single laser shot (Fig. 1C,D). After microdissection, a digestion buffer was added into Eppendorf for DNA extraction. The crude lysate was directly employed as template for PCR [4]. The samples were further amplified using H. pylori 16S rRNA primers as previously described [3], and amplicons were identified by sequence analysis. Microorganisms resembling H. pylori were observed in hepatic sinus from HCC samples (Fig. 1A,B). The number of cocci was greater than of bacilli as previously described [5]. PCR results showed that all six microdissected samples were positive for 16S rRNA gene and showed 98% similarity to 16S rRNA gene of H. pylori by sequence analysis (GeneBank accession number CP003419.1). Nevertheless, we cannot exclude the possibility of cross-reaction of these primers with other Helicobacter spp. These results demonstrated that LCM can be extensively applied for identification of H. pylori in FFPE liver tissue of HCC patients. Considering that bacteria were mainly found in peritumoral tissue, this technique was highly effective for obtaining a targeted bacterial population within a selected area in the HCC tissue. Beyond that, LCM simplified the H. pylori detection because extracted DNA was used directly as a template for PCR amplification. Further studies will be performed to isolate H. pylori from other tissues using LCM technique.

Prognostic Factors for Hepatocellular Carcinoma Recurrence: Experience With 83 Liver Transplantation Patients

INTRODUCTION Orthotopic liver transplantation (OLT) is a rational therapeutic option for early-stage hepatocellular carcinoma (HCC) providing a potential cure and improving survival. METHODS This retrospective study of a longitudinal cohort used an electronic database collected prospectively from September 1997 to May 2010. The variables were gender, age (years), and alpha-fetoprotein (AFP) level (ng/mL). In explanted livers we observed: microvascular or macrovascular invasion, number of nodules and their largest size, Edmondson-Steiner histological differentiation, incidental tumor transarterial chemoembolization (TACE), Milan criteria, and previous down-staging. RESULTS Five of 83 (6.0%) subjects including 68 (82%) males with a mean time to diagnosis of 9 months experienced tumor relapses. Mean patient age at HCC recurrence was 55.3 years for male and 44.6 years for female subjects. Vascular invasion was detected in 17/83 (20.5%) subjects, namely 2% of macrovascular invasion, and 52.5% with expanded Milan criteria due to an increased number and size of nodules in the explanted livers. An incidental tumor was observed in 29.5% of cases. Preoperative TACE treatment was performed in 13 (15.6%) patients. None of the patients who had a HCC recurrence had undergone TACE. AFP level at the time of recurrence was around 1,900 ng/mL. The predictive factor for mortality was nodule size (P=.04; hazard ratio=0.0269; confidence interval [CI], 95% 0.0094-0.299). CONCLUSION Patients with relapses showed the worst survival and tumor size was a predictive factor for recurrence.

Extragastrointestinal Stromal Tumor and Liver Transplantation : Case Report and Review

The occurrence of de novo malignant neoplasias has been shown in postransplant patients under imunosuppression. It is the second leading cause of late death in liver transplant recipients. The greatest incidence is seen in cancers associated with chronic infection by human papilloma virus, skin cancers, oropharyngeal, and gastrointestinal (GI) malignancies. GI stromal tumors (GISTs) are the most common mesenchymal tumors of the GI tract. Rare cases are identified outside the GI tract are collectively known as extragastrointestinal stromal tumors (EGISTs). We present an EGIST case in a liver transplantation patient. A 64-year-old man underwent liver transplantation because of cirrhosis (hepatitis B virus and alcoholism) and hepatocellular carcinoma. Histopathologic findings revealed 2 trabecular hepatocellular carcinomas: a 3.5-cm-diameter lesion located at segment VIII and another 2-cm one at segment V. Seven months later, he noticed a hardened, mobile, painless, 3-cm subcutaneous nodule in the perineum localized in the right lateral quadrant 2 cm distant from the anus. A surgical resection with 1 cm margin yielded a histopathology report of a 5.0 x 3.0 cm spindle cell stromal tumor. The immunohistochemical profile was compatible with a GIST, with 5 mitosis per 50 high-powered fields. This tumor is extremely rare after liver transplantation but has shown a good outcome up to now.