Monosomy and ring chromosome 13 in a thyroid nodular goiter-do we underestimate its relevance in benign thyroid lesions?

Classical cytogenetic examination of a thyroid nodular goiter revealed the existence of two different cytogenetically aberrant cell clones. They were characterized by monosomy 13 as the sole abnormality in one clone, and loss of one chromosome 13 and a ring chromosome that was found to consist of chromosome 13 material by fluorescence in situ hybridization in the other clone. We have concluded that during the course of karyotypic evolution, the instability of the ring chromosome has led to its loss and subsequent monosomy 13. In the literature, two cases of partial monosomy 13 have been reported in adenomatous goiters, suggesting that this abnormality characterizes a rare but distinct subgroup of benign thyroid lesions histologically presenting as adenomatous goiters. Possible target genes of these deletions are the retinoblastoma (RB1) gene locus and the MIR16-1/15A cluster. Based on similar changes in other tumors, it seems reasonable to also analyze a large number of adenomatous goiters for submicroscopic deletions of the long arm of chromosome 13.

Interphase fluorescence in situ hybridization analysis detects a much higher rate of thyroid tumors with clonal cytogenetic deviations of the main cytogenetic subgroups than conventional cytogenetics.

In benign thyroid lesions, three main cytogenetic subgroups, characterized by trisomy 7 or structural aberrations involving either chromosomal region 19q13.4 or 2p21, can be distinguished by conventional cytogenetics (CC). As a rule, these aberrations seem to be mutually exclusive. Interphase fluorescence in situ hybridization (I-FISH) analysis on benign as well as malignant thyroid neoplasias has been performed in the past, but rarely in combination with CC. In the present paper, we have analyzed 161 benign thyroid lesions both with CC and I-FISH on touch preparations by using a multi-target, triple-color FISH assay as well as dual-color break-apart probes for detection of the main cytogenetic subgroups. Within the samples, I-FISH detected tumors belonging to either of the subgroups more frequently than CC (23 vs. 11.4%), either due to small subpopulations of aberrant cells or to cryptic chromosomal rearrangements (three cases). Thus, I-FISH seems to be more sensitive than CC, particularly in the detection of subpopulations of cells harboring cytogenetic aberrations that may be overlooked by CC. In summary, I-FISH on touch preparations of benign thyroid lesions seems to be a favorable method for cytogenetic subtyping of thyroid lesions.

On the prevalence of the PAX8-PPARG fusion resulting from the chromosomal translocation t(2;3)(q13;p25) in adenomas of the thyroid.

The chromosomal translocation t(2;3)(q13;p25) characterizes a subgroup of tumors originating from the thyroid follicular epithelium and was initially discovered in a few cases of adenomas. Later, a fusion of the genes PAX8 and PPARG resulting from this translocation was frequently observed in follicular carcinomas and considered as a marker of follicular thyroid cancer. According to subsequent studies, however, this rearrangement is not confined to carcinomas but also occurs in adenomas, with considerably varying frequencies. Only five cases of thyroid adenomas with this translocation detected by conventional cytogenetics have been documented. In contrast, studies using reverse-transcription polymerase chain reaction (RT-PCR) detected fusion transcripts resulting from that translocation in an average of 8.2% of adenomas. The aim of this study was to determine the frequency of the PAX8-PPARG fusion in follicular adenomas and to use the HMGA2 mRNA level of such tumors as an indicator of malignancy. In cytogenetic studies of 192 follicular adenomas, the t(2;3)(q13;p25) has been identified in only two cases described herein. Histopathology revealed no evidence of malignancy in either case, and, concordantly, HMGA2 mRNA levels were not elevated. In summary, the fusion is a rare event in follicular adenomas and its prevalence may be overestimated in many RT-PCR-based studies.

Radio-guided parathyroidectomy in patients with primary hyperparathyroidism and concomitant multinodular goiter.

PURPOSE: We investigated possible instances where the standard bilateral neck exploration for parathyroid adenoma may be omitted in primary hyperparathyroidism (pHPT) if preoperative diagnostics for the location have been performed. METHODS: Ten patients underwent surgical treatment for pHPT and multinodular goiter between October 2006 and October 2008. Identification of the parathyroid adenomas' location with cervical ultrasound and (99m)technetium-sestamibi nuclear scanning ((99m)Tc-MIBI) was not possible in any of these patients. An extirpation of the parathyroid adenomas was performed with intraoperative use of the (99m)Tc-MIBI-guided probe technique. The median follow-up time was 17.5 months (range 2-30). RESULTS: Ten patients underwent an elective operation for solitary (n = 9) or dual (n = 1) parathyroid adenomas and concomitant thyroid disease. Definitive proof of the parathyroid adenomas was achieved in all of the patients without further neck exploration. The adenomas were 1.3 cm (range 1-2) in diameter. Calcium and parathyroid hormonal levels were reduced on the first postoperative day (P = 0.003). There were no postoperative complications. All patients were free from recurrence. CONCLUSION: The intraoperative probe technique is feasible in patients with pHPT and limited diagnostics for the location of parathyroid adenomas with concomitant goiter. This diagnostic technique identified the parathyroid adenoma in all cases, and thus rendered a bilateral neck exploration obsolete.

The two stem cell microRNA gene clusters C19MC and miR-371-3 are activated by specific chromosomal rearrangements in a subgroup of thyroid adenomas.

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias.

Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions.

BACKGROUND: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. RESULTS: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. CONCLUSION: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.

Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions.

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 microM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression.

Laparoscopic unroofing of nonparasitic liver cysts within segments VII and VIII: technical considerations.

BACKGROUND: The laparoscopic accessibility of congenital liver cysts located in the anterosuperior (VIII) and posterosuperior (VII) segments has been questioned for some time. In support of the laparoscopic approach, we here describe our minimally invasive technique in two patients with solitary congenital cysts located in the apex of liver segments VIII and VII, respectively. METHOD: Both patients were placed in the inverted Y position. Four trocars were used, their position depending on the location of the cyst. RESULTS: The segment VIII cyst was easily reached via this anterior approach, while the segment VII cyst required significant mobilization of the right liver lobe. In both cases a complete excision of the cystic roof was achieved using the harmonic scalpel. Without performing an omentoplasty no recurrences were observed after 20 and 28 months, respectively. CONCLUSION: Solitary cysts located in segments VII and VIII of the liver can be safely treated by laparoscopic unroofing. Cyst recurrences may best be prevented by a complete excision of the cystic roof with an adjacent rim of hepatic parenchyma.

Expression of glutathione S-transferases (GSTs) in human colon cells and inducibility of GSTM2 by butyrate.

The glutathione S-transferases (GSTs) are a multigene family of enzymes largely involved in the detoxification of chemicals. In animals, enhanced expression is mediated by products of gut fermentation. Of these, butyrate induces GSTP1 protein expression and GST activity in the human colon tumor cell line HT29. The aim of the following investigations was to further elucidate butyrate-modulated induction of additional colonic GSTs in HT29 and to determine baseline expression in non-transformed cells, isolated from human colorectal tissue. We measured five GST protein subunits (GSTA1/2-composed of GST A1-1, A1-2 and A2-2-GSTM1, GSTM2, GSTP1, GSTT1) by western blot, GST activity using 1-chloro-2,4-dinitrobenzene as substrate and GSTM2 mRNA expression with RT-PCR. GSTP1, followed by GSTT1, were major subunits in all colon cells. Cells isolated from colon tissue were identified to be colonocytes and colon fibroblasts, both of which also expressed substantial levels of GSTM1 and GSTM2. The inter-individual variation of GST subunits in coloncytes of 15 individuals was marked, with total GST protein per 106 cells differing by more than a factor of four. In HT29, butyrate significantly enhanced GSTA1/2 (3.5-fold), GSTM2 (not detectable in controls), GSTP1 (1.5-fold) and GST activity (1.4-fold), but not GSTM1 or GSTT1. GSTM2 mRNA expression was significantly induced after 24 ( approximately 14-fold) and 72 h treatment ( approximately 8-fold). In colon fibroblasts, butyrate (4 mM, 72 h) also induced GSTM2 protein (1.7-fold) and GST activity (1.4-fold). Colonocytes were too short lived to be used for inducibility studies. In conclusion, GSTs are expressed with high inter-individual variability in human colonocytes. This points to large differences in cellular susceptibility to xenobiotics. However, butyrate, an important luminal component produced from fermentation of dietary fibers, is an efficient inducer of GSTs and especially of GSTM2. This indicates that butyrate may act chemoprotectively by increasing detoxification capabilities in the colon mucosa.

Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal.

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.

Coagulation profiles and intraoperative substitution requirements during elective piggyback liver transplantation with prophylactic antifibrinolytic therapy.

During recent years, piggyback liver transplantation (pOLT) with preservation of the retrohepatic vena cava has been introduced in adults. The objective of this study was to evaluate hemostatic changes associated with this transplantation technique. Fifty-seven patients undergoing elective pOLT for endstage liver disease were studied. Most significant changes were observed after graft reperfusion, when PT showed a 49% decrease and activated partial thromboplastin time (aPTT) as well as TT a 2- to 3-fold prolongation. At the same time, factors of the extrinsic coagulation pathway (II, V, VII) revealed an overall 50% decline. Similar changes were observed for antithrombin III (ATIII) and fibrinogen plasma levels. However, only 42% of all patients required intraoperative substitution with coagulation components. There was an association between preoperative fibrinogen (<1.7 g/dl) and ATIII (<50%) plasma levels and the substitution requirement. Multiple linear regression showed a significant correlation between preoperative ATIII activity and intraoperative blood loss. Despite a marked impairment of hemostasis, pOLT can frequently be performed with minimized substitution therapy.

Influence of antiseptic agents on interleukin-8 release and transmigration of polymorphonuclear neutrophils in a human in vitro model of peritonitis.

BACKGROUND: The aim of this study was to evaluate the influence of taurolidine (TAU) and polyhexanid (POLY) on basic inflammatory reactions during peritonitis by using an in vitro model of human peritoneum. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) and human peritoneal mesothelial cells (HPMC; concentration: 2x10(5)/cm2) were brought on a collagen-coated filter insert with 3-microm pore size (HUVEC on the bottom, HPMC on the top), thus resulting in a two-chamber peritoneal model. After 5 days, confluence of the cells was reached, and HPMC were stimulated with 0.5 mL of TNF-alpha (10 microg/mL) for 4 h. Afterwards, 0.5 mL of TAU (1% and 2%) or 0.5 mL of POLY (0.1% and 0.2%) solution were added to the upper (HPMC) compartment. Polymorphonuclear neutrophils (PMN, 10(6)/mL) were placed in the lower compartment 1 h later. After 2 and 6 h, aliquots were taken from the upper compartment and transmigrated PMN were counted. Interleukin-8 (IL-8) concentrations were measured in both compartments by chemiluminescent enzyme immunometric assay. Expression of the adhesion molecules P-selectin and intercellular adhesion molecule-1 (ICAM-1) was assessed by immunohistochemistry. Controls were either TNF-alpha-stimulated HPMC without any antiseptic agents, or stimulated HPMC where TNF-alpha had been substituted by culture medium. Each experiment was performed in triplicate. RESULTS: Stimulation with TNF-alpha led to a time-dependent increase of IL-8 secretion to the apical compartment resulting in a gradient between both chambers, as well as to a time-dependent increase of PMN transmigration and expression of adhesion molecules. IL-8 gradients and PMN migration were significantly higher as compared to the other groups (p<0.05). After substitution of the stimulus by culture medium, significantly less IL-8 was measured in both compartments. PMN transmigration was almost absent (p<0.05). Addition of POLY and TAU led to comparable low IL-8 gradients with concomitant low PMN transmigration. The initially detected expression of adhesion molecules significantly decreased during the observation time. The IL-8 gradient in all groups correlated significantly with PMN transmigration (r=0.74226; p<0.0001). CONCLUSION: The diminished IL-8 response together with low PMN transmigration rates after addition of TAU and POLY may reflect either antiinflammatory effects or cellular damage.