Venancio Avancini Ferreira Alves

Pathologic diagnosis of early hepatocellular carcinoma : a report of the international consensus group for hepatocellular neoplasia

Advances in imaging techniques and establishment of surveillance protocols for high-risk populations have led to the detection of small hepatic nodules in patients with chronic liver diseases, particularly those with cirrhosis or chronic hepatitis caused by hepatitis B or C viruses. These nodules, comprising a broad range of diagnostic entities—some benign and some with malignant potential—are currently defined histologically, and their clinical management often depends on the ability to make a reliable histologic diagnosis. Evidence accumulated in the last two decades strongly favors the existence of a sequence of events in hepatic nodules that precedes the emergence of hepatocellular carcinoma (HCC),1-10 and these lesions are recognized as precursors of HCC. However, from the beginning of their recognition, there has been considerable confusion concerning nomenclature and diagnostic approaches to these hepatic nodules. To clarify these issues, an International Working Party (IWP) of the World Congresses of Gastroenterology proposed a consensus nomenclature and diagnostic criteria for hepatocellular nodular lesions in 1995.11 The IWP classified nodular lesions found in chronic liver disease into large regenerative nodule, lowgrade dysplastic nodule (L-DN), high-grade dysplastic nodule (H-DN), and HCC; this nomenclature has been widely adopted. In addition, the IWP introduced the concept of dysplastic focus as a cluster of hepatocytes with features of early neoplasia (in particular small cell change or iron-free foci in a siderotic background) measuring less than 0.1 cm, and defined small HCC as a tumor measuring less than 2 cm. More recent studies support the division of small HCC into two clinico-pathological groups that have been termed early HCC and progressed HCC. Early HCC has a vaguely nodular appearance and is well differentiated. Progressed HCC has a distinctly nodular pattern and is mostly moderately differentiated, often with evidence of microvascular invasion.12 Early HCC has a longer time to recurrence and a higher 5-year survival rate compared with progressed HCC.13 Small lesions with malignant potential have only subtle differences from the surrounding parenchyma, making them difficult to assess reproducibly. Differences in the application of diagnostic criteria between Western and Eastern pathologists has been a persistent difficulty in research and clinical management of these lesions.14 In order to obtain a refined and up-to-date international consensus on the histopathologic diagnosis of nodular lesions, such as dysplastic nodules and early HCC, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in April 2002 in Kurume, Japan. The group has met several times up to July 2007 under the auspices of the Laennec Liver Pathology Society. The ICGHN is currently comprised of 34 pathologists and two clinicians from 13 countries. It includes most members of the original IWP who are still active and all the participants from the first ICGHN meeting. This consensus document summarizes the results of our meetings.

Lung Pathology in Fatal Novel Human Influenza A (H1N1) Infection

RATIONALE There are no reports of the systemic human pathology of the novel swine H1N1 influenza (S-OIV) infection. OBJECTIVES The autopsy findings of 21 Brazilian patients with confirmed S-OIV infection are presented. These patients died in the winter of the southern hemisphere 2009 pandemic, with acute respiratory failure. METHODS Lung tissue was submitted to virologic and bacteriologic analysis with real-time reverse transcriptase polymerase chain reaction and electron microscopy. Expression of toll-like receptor (TLR)-3, IFN-gamma, tumor necrosis factor-alpha, CD8(+) T cells and granzyme B(+) cells in the lungs was investigated by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS Patients were aged from 1 to 68 years (72% between 30 and 59 yr) and 12 were male. Sixteen patients had preexisting medical conditions. Diffuse alveolar damage was present in 20 individuals. In six patients, diffuse alveolar damage was associated with necrotizing bronchiolitis and in five with extensive hemorrhage. There was also a cytopathic effect in the bronchial and alveolar epithelial cells, as well as necrosis, epithelial hyperplasia, and squamous metaplasia of the large airways. There was marked expression of TLR-3 and IFN-gamma and a large number of CD8(+) T cells and granzyme B(+) cells within the lung tissue. Changes in other organs were mainly secondary to multiple organ failure. CONCLUSIONS Autopsies have shown that the main pathological changes associated with S-OIV infection are localized to the lungs, where three distinct histological patterns can be identified. We also show evidence of ongoing pulmonary aberrant immune response. Our results reinforce the usefulness of autopsy in increasing the understanding of the novel human influenza A (H1N1) infection.

Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas

Tumour cells are known to be highly glycolytic, thus producing high amounts of lactic acid. Monocarboxylate transporters (MCTs), by promoting the efflux of the accumulating acids, constitute one of the most important mechanisms in the maintenance of tumour intracellular pH. Since data concerning MCT expression in colorectal carcinomas (CRC) are scarce and controversial, the present study aimed to assess the expressions of MCT1, 2, and 4 in a well characterized series of CRC and assess their role in CRC carcinogenesis. CRC samples (126 cases) were analyzed for MCT1, MCT2, and MCT4 immunoexpression and findings correlated with clinico-pathological parameters. Expression of all MCT isoforms in tumour cells was significantly increased when compared to adjacent normal epithelium. Remarkably, there was a significant gain of membrane expression for MCT1 and MCT4 and loss of plasma membrane expression for MCT2 in tumour cells. Plasma membrane expression of MCT1 was directly related to the presence of vascular invasion. This is the larger study on MCT expression in CRC and evaluates for the first time its clinico-pathological significance. The increased expression of these transporters suggests an important role in CRC, which might justify their use, especially MCT1 and MCT4, as targets in CRC drug therapy.

Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms.

The distinction of hepatocellular carcinoma (HCC) from metastatic tumor in the liver often presents a diagnostic challenge that carries significant impact on prognostication and therapy. The number of diagnostically useful immunohistochemical markers of hepatocytes is limited to hepatocyte paraffin antigen (HepPar-1), polyclonal carcinoembryonic antigen, and CD10, with α-fetoprotein and glypican-3 labeling HCCs. Arginase-1 (Arg-1) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea. We used immunohistochemistry to compare the sensitivity of Arg-1 to that of HepPar-1 in 151 HCCs. We found that the overall sensitivities of Arg-1 and HepPar-1 are 96.0% and 84.1%, respectively. The sensitivities of Arg-1 in well, moderately, and poorly differentiated HCCs are 100%, 96.2%, and 85.7%, respectively, whereas, in comparison, HepPar-1 demonstrated sensitivities of 100%, 83.0%, and 46.4% for well, moderately, and poorly differentiated tumors, respectively. There were no HCCs in our study that were reactive for HepPar-1 but nonreactive for Arg-1. We also examined Arg-1 expression in nonhepatocellular tumors, including many that are potential mimics of HCC (renal cell carcinomas, neuroendocrine tumors, melanomas, gastric adenocarcinomas, and adrenocortical carcinomas) and found that only 2 non-HCC tumors were reactive for Arg-1. Arg-1 represents a sensitive and specific marker of benign and malignant hepatocytes that may ultimately prove to be a useful diagnostic tool in routine surgical pathology practice.

A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma

BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is a well recognized complication of advanced NASH (non-alcoholic steatohepatitis). We sought to produce a rat model of NASH, cirrhosis and HCC. METHODS Adult Sprague-Dawley rats, weighing 250-300g, were fed a choline-deficient, high trans-fat diet and exposed to DEN in drinking water. After 16 weeks, the animals underwent liver ultrasound (US), sacrifice and assessment by microscopy, immunohistochemistry and transmission electron microscopy (TEM). RESULTS US revealed steatosis and focal lesions in 6 of 7. All had steatohepatitis defined as inflammation, advanced fibrosis and ballooning with Mallory-Denk bodies (MDB) with frank cirrhosis in 6. Areas of more severe injury were associated with anti-CK19 positive ductular reaction. HCC, present in all, were macro-trabecullar or solid with polyhedral cells with foci of steatosis and ballooned cells. CK19 was positive in single or solid nests of oval cells and in neoplastic hepatocytes. TEM showed ballooning with small droplet fat, dilated endoplasmic reticulum and MDB in non-neoplastic hepatocytes and small droplet steatosis in some cancer cells. CONCLUSIONS This model replicated many features of NASH including steatohepatitis with ballooning, fibrosis, cirrhosis and hepatocellular carcinoma. Oval cell proliferation was evident and the presence anti-CK 19 positivity in the cancer suggests oval cell origin of the malignancy.

Steroidogenic Factor 1 Overexpression and Gene Amplification Are More Frequent in Adrenocortical Tumors from Children than from Adults

BACKGROUND Steroidogenic factor 1 (SF-1) is a key determinant of endocrine development and function of adrenal cortex. SF-1 overexpression and gene amplification were previously demonstrated in a small group of pediatric adrenocortical tumors. OBJECTIVE Our objective was to determine the frequency of SF-1 protein expression and gene amplification in a large cohort of pediatric and adult adrenocortical tumors. PATIENTS SF-1 protein expression was assessed in a cohort of 103 adrenocortical tumors from 36 children and 67 adults, whereas gene amplification was studied in 38 adrenocortical tumors (17 from children). METHODS Tissue microarray, multiplex ligation-dependent probe amplification, and quantitative real-time PCR were used. RESULTS A strong nuclear SF-1 expression was detected by tissue microarray in 56% (20 of 36) and 19% (13 of 67) of the pediatric and adult adrenocortical tumors, respectively (P = 0.0004). Increased SF-1 copy number was identified in 47% (eight of 17) and 10% (two of 21) of the pediatric and adult adrenocortical tumors, respectively (P = 0.02). All adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five (29%) pediatric adrenocortical tumors without SF-1 amplification. The frequency of SF-1 overexpression and gene amplification was similar in adrenocortical adenomas and carcinomas. CONCLUSION We demonstrated a higher frequency of SF-1 overexpression and gene amplification in pediatric than in adult adrenocortical tumors, suggesting an important role of SF-1 in pediatric adrenocortical tumorigenesis.

Differential reactivity for galectin-3 in Hürthle cell adenomas and carcinomas

Hürthle cell carcinomas behave as the most aggressive variant of differentiated thyroid carcinoma of follicular origin, with frequent recurrences and higher morbidity. Its differential diagnosis with Hürthle cell adenoma remains a problem for the clinician and for the pathologist. The vertebrate lectins, galectin-1 and galectin-3 have been implicated in the regulation of cellular growth, differentiation, and malignant transformation in thyroid neoplasms. Galectin-3, a β-galactoside binding protein, has been recently found to be highly expressed in papillary and follicular carcinomas. The current study was undertaken to investigate immunohistochemical reactivity for galectin-3 of thyroid specimen tissues with Hürthle cell adenomas (n=14) and carcinomas (n=17), follicular (n=14) and papillary (n=11) carcinomas, colloid goiter (n=30), Hashimoto’s thyroiditis (n=11), follicular adenoma (n=9), and normal thyroid tissues (n=18). Follicular (78.5%) and papillary (82.0%) carcinomas were frequently reactive for galectin-3, more often when some Hürthle cells were present. There was no galectin-3 immunostaining in any of the specimens from Hashimoto’s thyroiditis, colloid goiters or normal thyroid samples, whereas only one case of follicular adenoma was found positive (11.1%). By contrast, galectin-3 immunostaining in Hürthle cell carcinomas was significantly higher (59%) than in Hürthle cell adenomas (7.1%, p<0.05). These results suggest that galectin-3 may potentially serve as a marker in difficult differential diagnosis cases involving Hürthle cell adenomas and Hürthle cell carcinomas.

Comparison of manual and automated methods of liquid-based cytology. A morphologic study.

OBJECTIVE To evaluate the morphologic characteristics of gynecologic samples prepared by 3 different methods of liquid-based cytology. STUDY DESIGN Cytologic samples from representative cases of each diagnostic category of squamous epithelial lesion, prepared by automated and manual liquid-based systems, were analyzed by 3 laboratories in the United States, Portugal and Brazil. The systems included: ThinPrep (automated, U.S. Food and Drug Administration approved; Cytyc Corp., Boxborough, Massachusetts, U.S.A.), Autocyte PREP (South American system, manual; TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) and DNACITOLIQ (manual; Digene Brazil, São Paulo, Brazil). A panel of 16 morphologic parameters was evaluated: cellularity, clean background, uniform distribution, artifacts, cellular overlapping, architectural and cytoplasmic distortion, cytoplasmic vacuolization, cellular elongation, imprecise cytoplasmic borders, folded cytoplasmic borders, nuclear hyperchromasia, coarse chromatin, prominent nucleolus, irregular nuclear borders, atypical mitosis and inflammatory infiltrate. Negative, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) cases were included. Cases without biopsies were confirmed by consensus. RESULTS Cellularity was adequate in all samples. Clean background was observed in the vast majority of samples with all liquid-based systems. Uniform distribution was frequently found in ThinPrep and Autocyte PREP samples but not in DNACITOLIQ. Artifacts were not present in DNACITOLIQ samples, rare in ThinPrep and observed in 8 (34.7%) Autocyte PREP. Cellular overlapping was observed in all 3 system samples: 11 (31.42%) cases in ThinPrep, 16 (69.56%) in Autocyte PREP and 17 (68%) in DNACITOLIQ System. Architectural and cytoplasmic distortion were present in 3 cases of HSIL (13%) and cytoplasmic vacuolization in 2 cases of LSIL and 1 HSIL of Autocyte PREP. Cellular elongation was found in 13 (56.5%) Autocyte PREP and in 5 (20%) DNACITOLIQ samples. Inflammatory infiltrate was found in all 3 system samples but with more frequency in the Autocyte PREP (69.56%) and DNACITOLIQ System (72%). CONCLUSION This study clearly indicated that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation. The choice of method will depend on price, availability and procedures involved.

High-fat diet: A trigger of non-alcoholic steatohepatitis? Preliminary findings in obese subjects

OBJECTIVE We correlated dietary profile and markers of visceral and somatic obesities in non-alcoholic fatty liver disease. METHODS Patients with histologically proven fatty infiltration of the liver (n = 25, 52 +/- 11 y of age, 64% women) underwent abdominal computed tomography, bioelectrical impedance, and anthropometric measurements. Insulin resistance was evaluated (homeostasis model assessment) and dietary intake of macronutrients was estimated by 24-h recall. Main outcome measurements were correlation of carbohydrate and fat ingestion with liver histology. RESULTS Metabolic syndrome was present in 72% of the population, and increased waist circumference and low high-density lipoprotein cholesterol occurred in 66%. Total body fat (bioimpedance) and dietary intake of lipids were higher in patients with non-alcoholic steatohepatitis (P < 0.05), but not in diabetic subjects who exhibited more steatosis than non-alcoholic steatohepatitis. Waist circumference exhibited a good correlation with homeostasis model assessment, total energy intake, and ingestion of specific fatty acids. Body mass index correlated well with somatic and visceral adiposities. CONCLUSION Energy intake and visceral adiposity were predisposing factors for fatty liver disease. Lipid input correlated with non-alcoholic steatohepatitis in the entire group and after stratification for diabetes. These findings suggest that lipid intake may play a greater role in non-alcoholic steatohepatitis than hitherto suspected.

Aggressive metastatic follicular thyroid carcinoma with anaplastic transformation arising from a long-standing goiter in a patient with Pendred’s syndrome

In this article we describe detailed pathological and molecular genetics studies in a consanguineous kindred with Pendreds syndrome. The index patient was a 53-year-old female patient with congenital deafness and goiter. Her parents were first-degree cousins. She had a large goiter (150 g) that had been present since childhood. One of her sisters and a niece are also deaf and have goiter as well. The presence of Pendreds syndrome was confirmed by a positive perchlorate test and the demonstration of a Mondini malformation. Thyroid function tests (under levothyroxine [LT4] therapy) were in the euthyroid range with a thyrotropin [TSH] level of 2.8 microU/mL (0.2-3.2), a serum total thyroxine (T4) of 90 nmol/L (54-142), and a serum total triiodothyronine (T3) of 2.7 nmol/L (0.8-2.4). Total thyroidectomy was performed, and the mass in the right lobe was found to have invaded adjacent tissues. The histopathological findings were consistent with a follicular carcinoma with areas of anaplastic transformation and lung metastasis. The patient was treated twice with 100 mCi 131iodine (3,700 MBq) and received suppressive doses of LT4. Postoperatively, the serum thyroglobulin (Tg) levels remained markedly elevated (2,352 to 41,336 ng/mL). The patient died of a sudden severe episode of hemoptysis. Sequence analysis of the PDS gene performed with DNA from the two relatives with Pendreds syndrome revealed the presence of a deletion of thymidine 279 in exon 3, a point mutation that results in a frameshift and a premature stop codon at codon 96 in the pendrin molecule. We concluded that prolonged TSH stimulation because of iodine deficiency or dyshormonogenesis in combination with mutations of oncogenes and/or tumor suppressor genes, may result in the development of follicular thyroid carcinomas that undergo transformation into anaplastic cancers. It is likely that these pathogenetic mechanisms have been involved in the development of aggressive metastatic thyroid cancer in this unusual patient with Pendreds syndrome.