Attila Zalatnai

Membrane receptors for peptides in experimental and human pancreatic cancers

Membrane receptors for [D-Trp6]-luteinizing hormone-releasing hormone ([D-Trp6-LH-RH), somatostatin (SS-14), and epidermal growth factor (EGF) were investigated in experimental N-nitrosobis-(2-oxopropyl)-amine (BOP)-induced pancreatic cancers of hamsters and in specimens of normal human pancreas and human pancreatic cancer obtained from autopsies. Membrane receptors for [D-Trp6]-LH-RH were absent in the pancreas of normal hamsters, but appeared after the carcinoma was induced with BOP. Binding capacity of SS-14 receptors was lower in membranes of BOP-induced pancreatic cancers than in the normal pancreas. In the BOP-induced pancreatic cancers, the receptors were also characterized following in vivo treatment of hamsters with microcapsules of the agonist [D-Trp6]-LH-RH, somatostatin analog RC-160, and the combination of both peptides, which resulted in significant tumor inhibition. Therapy with [D-Trp6-LH-RH and RC-160, alone or in combination, decreased the binding capacity of receptors for [D-Trp6-LH-RH, but increased Bmax for SS-14. There were no significant changes in characteristics of the EGF receptor following these therapies. Membranes from human pancreatic cancers showed binding sites for [D-Trp6]-LH-RH, but no binding was detected in normal human pancreas. The presence of receptors for LH-RH in pancreatic tumors of hamster and humans raises the intriguing possibility that LH-RH could be involved in complex interactions that contribute to the appearance of pancreatic cancer. The binding capacity of receptors for SS-14 in human pancreatic cancer membranes was lower, while Bmax for EGF2 was higher, as compared to normal pancreas. Observed changes in receptors and tumor suppression suggest that the agonist [D-Trp6-LH-RH and somatostatin analogs might exert some direct inhibitory effects on experimental pancreatic cancer of hamsters. It is possible that LH-RH agonists and somatostatin analogs could also be used for treatment of human pancreatic cancer. The presence of membrane receptors for [D-Trp6]-LH-RH, SS-14, and EGF in specimens of human pancreatic cancer also implies that this malignancy might be responsive to hormonal manipulations.

Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as αv-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n=33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffin-embedded samples (70–92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch- and G-protein-coupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffin-embedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

Molecular Aspects of Stromal-Parenchymal Interactions in Malignant Neoplasms

Carcinomas are composed of parenchymal and stromal elements, and the malignant behavior is principally dictated by the cancer cells. However, the malignant tumors not merely grow into a preexisting interstitial tissue, but they actively form a new stroma and modify their composition. Thus, the tumor stroma is significantly different from that of the neighboring tissues. Cancer cells may alter their stroma by cell-to-cell contact, soluble factors or by modification of the extracellular matrix (ECM), they induce myofibroblast differentiation and govern the desmoplastic stroma reaction. On the other hand, the stromal cells (especially the myofibroblasts) are able to modify the phenotype, invasiveness, metastatic capacity of carcinomas, typically promoting the progression. Regarding pancreatic cancer, the pancreatic stellate cells (PSCs) seem to be the key elements in the cross-talk between the parenchymal cells and the desmoplastic stroma. The tumor stroma is also rich in tumor-associated macrophages (TAM), but their role in the malignant process is contradictory and may be different in various tumor types, but most studies suggest a negative impact on the tumor growth. The relationship between the parenchymal and stromal elements is highly complex, they mutually alter their characteristics. Because the neostroma of the carcinomas largely seems to promote the invasiveness of the malignant tumors, novel therapeutic strategies are being evaluated targeting the stromal elements, with some encouraging, but still fragmentary results.

Glucagon Cell Adenomatosis: A Newly Recognized Disease of the Endocrine Pancreas

BACKGROUND Glucagon-producing tumors are either solitary neoplasms of the pancreas, occasionally associated with a glucagonoma syndrome, or multiple neoplasms associated with multiple endocrine neoplasia type 1 (MEN1). We observed a previously undescribed multicentric glucagon-producing tumor disease that is not related to MEN1. METHODS Pancreatic tissue from four patients showing multiple neuroendocrine microadenomas and in two cases also macrotumors were screened for hormones using immunohistochemical and morphometric methods. MEN1, von Hippel-Lindau, and p27 germ line and somatic mutation analysis was performed. Deletion of MEN1 (11q13), von Hippel-Lindau (3p25), and the centromere 11 and 3 gene locus was determined by fluorescence in situ hybridization. DNA copy number changes were studied using array comparative genomic hybridization. RESULTS The pancreatic tissue from the four patients contained more than 870 microadenomas and 10 macrotumors, all of which expressed exclusively glucagon and none of which showed evidence of malignancy. In addition, many islets were unusually large and showed glucagon cell hyperplasia. There was no clinical or molecular evidence of any hereditary tumor disease, and changes in the MEN1 gene were only seen in individual tumors. Array comparative genomic hybridization of one macrotumor and 20 pooled microadenomas revealed a homogeneous diploid chromosome set. CONCLUSIONS The findings are sufficiently distinctive to suggest a new neoplastic disease of the endocrine pancreas that we recommend calling glucagon cell adenomatosis. Clinically, this disease may be an incidental finding, or it may lead to a glucagonoma syndrome.

Validation of diagnostic accuracy using digital slides in routine histopathology.

BackgroundRobust hardware and software tools have been developed in digital microscopy during the past years for pathologists. Reports have been advocated the reliability of digital slides in routine diagnostics. We have designed a retrospective, comparative study to evaluate the scanning properties and digital slide based diagnostic accuracy.Methods8 pathologists reevaluated 306 randomly selected cases from our archives. The slides were scanned with a 20× Plan-Apochromat objective, using a 3-chip Hitachi camera, resulting 0.465 μm/pixel resolution. Slide management was supported with dedicated Data Base and Viewer software tools. Pathologists used their office PCs for evaluation and reached the digital slides via intranet connection. The diagnostic coherency and uncertainty related to digital slides and scanning quality were analyzed.ResultsGood to excellent image quality of slides was recorded in 96%. In half of the critical 61 digital slides, poor image quality was related to section folds or floatings. In 88.2% of the studied cases the digital diagnoses were in full agreement with the consensus. Out of the overall 36 incoherent cases, 7 (2.3%) were graded relevant without any recorded uncertainty by the pathologist. Excluding the non-field specific cases from each pathologists record this ratio was 1.76% of all cases.ConclusionsOur results revealed that: 1) digital slide based histopathological diagnoses can be highly coherent with those using optical microscopy; 2) the competency of pathologists is a factor more important than the quality of digital slide; 3) poor digital slide quality do not endanger patient safety as these errors are recognizable by the pathologist and further actions for correction could be taken.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1913324336747310.

Neuroendocrine carcinoma associated with X-linked hyper-immunoglobulin M syndrome: Report of four cases and review of the literature

X-linked hyper-immunoglobulin M syndrome (XHIGM) is a primary immunodeficiency disorder characterized by severe defects of both cellular and humoral immunity due to impaired expression of CD40 ligand on activated T lymphocytes. Patients with XHIGM usually present with a wide variety of infections caused by common and opportunistic pathogens including Pneumocystis jirovecii. In addition, subjects with XHIGM have an increased risk for hepatocellular and bile duct carcinomas, which are rarely observed in other primary immunodeficiencies. We present here clinical, immunological, and molecular findings of four patients with CD40 ligand deficiency associated with neuroendocrine carcinoma (NEC). NEC developed as a rapidly disseminated solid cancer leading to death in three patients. Data presented here and published previously suggest that CD40 ligand deficiency may predispose patients for the development of NEC. Histochemical findings suggested that CD56, in addition to cytokeratin and chromogranin A, may be a useful marker for early detection of NEC. We conclude that patients with XHIGM should be carefully followed to diagnose and treat NEC, a formidable neuroendocrine cancer.

Correlation of ultrasound attenuation and histopathological parameters of the liver in chronic diffuse liver diseases.

Objective In patients with chronic diffuse liver diseases two characteristic ultrasound patterns of bright liver, the low (DI) and the high (DII) attenuation types, are seen. Correlation was studied between liver attenuation and histopathological analysis of biopsy specimens of the same patients. Methods Ultrasound attenuation of the liver was measured quantitatively using a homogeneous tissue-equivalent reference phantom. Semiquantitative scores were used for histopathological analysis of biopsy specimens. Results One hundred and twenty-two patients were investigated; 40 of them showed a normal liver echopattern. The average attenuation was 0.68±0.03 dB/cm per MHz. Histopathological parameters were normal in most of the patients, except for four cases where a minimal amount of collagen and 14 cases where a subtle amount of lipid content could be detected, in three cases accompanied by some cells indicating inflammation. From 82 patients with bright liver, 47 showed the DI type. The average attenuation was 0.80±0.03 dB/cm per MHz. In all of these patients, significant increases of collagen content and inflammatory reaction were found. In 23 cases a negligible amount of lipid deposition could also be revealed. Thirty-five patients exhibited a DII-type bright liver. The average attenuation was 1.21±0.06 dB/cm per MHz. A significant amount of lipid deposition was detected in all cases. In 13 patients a minimal amount of collagen and in 14 patients some inflammatory cells were detected. Conclusions In livers with a normal echopattern, none or minimal pathological changes were seen. In DI-type bright liver, connective tissue dominance exists, in DII-type bright liver lipid deposition dominance. On this basis it is proved that the diagnosis of a fatty liver can be established without biopsy, if no other indication for biopsy exists.

Secondary cutaneous infiltration in B cell chronic lymphocytic leukemia.

We describe a patient presenting with B cell chronic lymphocytic leukemia (B-CLL) who subsequently developed cutaneous infiltrates. Specimens of the blood, bone marrow and cutaneous infiltrations all showed the same heavy-chain gene rearrangement. Following failure of conventional chemotherapy, and in view of the similarity of the disease to cutaneous T cell lymphoma, interferon-α therapy was employed with satisfactory results. Introduction of this cytokine to the therapeutic modalities for secondary cutaneous B-CLL would hopefully change the poor outcome of this entity, or at least could produce a better quality of life. Loss of histidine decarboxylase activity in the infiltrating cells – in contrast to circulating lymphocytes – may be associated with the transformation of B-CLL to a more aggressive infiltrative form, offering a possible explanation for tissue invasiveness. The changing character of the disease raises the possibility of a second mutational event in the course of B-CLL.

Pancreatic Carcinomas in a 60-year, Institute-based Autopsy Material With Special Emphasis of Metastatic Pattern

To the Editor: T he incidence of the pancreatic cancer is steadily rising in many countries of world; however, these figures show significant differences. In the United States, a 3-fold increase was noted between 1920 and 1978, and nowadays, it ranks fourth among cancer mortality data. This tumor type is mostly diagnosed at advanced stage because of early and rapid spread and aggressive local infiltration; therefore, only 15% to 20% of patients are resectable. The overall 5-year survival rate is less than 5%; even after pancreaticoduodenectomy, the median survival still remains poor: 12 to 18 months. In Hungary, however, the mortality rate has increased by 16-fold during the last 60 years (1948: 1.1/100,000, 2008: 18/100,000), accounting for the highest rise in Europe. The clear explanation of this finding is still lacking. One of the possible explanations of this is that the biological behavior of the tumor has been changed; therefore, we have investigated the main clinicopathologic features and the metastatic pattern of this tumor during a 60-year period in our autopsy material.