The pathologic response of resected synovial sarcomas to hyperthermic isolated limb perfusion with melphalan and TNF-α: a comparison with the whole group of resected soft tissue sarcomas.

BACKGROUND: Hyperthermic isolated limb perfusion with tumor necrosis factor-α and melphalan (TM-HILP) has been successfully used to treat limb soft tissue sarcomas (STSs) with high response rates. The data on the effectiveness of HILP-TM for the treatment of STSs are mainly based on various STS types. The aim of this study was to investigate the responses of synovial sarcomas (SS) to TM-HILP. METHODS: A total of 125 TM-HILP-treated tumors (STS all), including 14 SSs, were included in the study. The tumors were subdivided into proximal and distal limb localizations. Tumor typing (using the WHO classification), resection status (using the UICC classification), and response to therapy were assessed using light microscopy. The SSs were tested for the SYT-SSX translocation using RT-PCR. The following tests were applied: a chi-squared test, a t test, and the Mann-Whitney U test. RESULTS: The SSs were localized distally more often than were the STS cohort (STS(-SS)) (85.7% vs. 32.4%) and were smaller (5.8 cm vs. 10.7 cm). There were no differences in the responder/nonresponder ratios or the mean percentages of pathological regression between the SS and STS(-SS) cohorts (74.0% vs. 76.0%). A general localization-dependent difference in the tumor responses to TM-HILP could not be detected in the STS all cohort (distal, 72.0% vs. proximal, 78.0%); however, a UICC R0 status was more often observed in proximal tumors (distal, 50.0% vs. proximal, 71.4%). There was no association between the SYT-SSX type and SS responses to TM-HILP. CONCLUSIONS: Because of the high response rates, TM-HILP is recommended for the treatment of SSs. The distal limb localization of TM-HILP-treated STSs was generally (STS all cohort) associated with fewer R0 resections.

Size-based clinical response evaluation is insufficient to assess clinical response of sarcomas treated with isolated limb perfusion with TNF-α and melphalan.

BACKGROUND: The clinical assessment of the response of sarcomas to preoperative treatment is usually defined using size-based evaluation standards. For nonresectable sarcomas, hyperthermic isolated limb perfusion with TNF-α and melphalan (TM-ILP) yields high response rates. Based on our experience, we assume that anatomic radiological response criteria are insufficient to assess the degree of regression after TM-ILP. METHODS: The clinical response of 35 sarcomas to TM-ILP was assessed by unidimensional, bidimensional, and tridimensional size-based anatomical criteria, and responders were identified according to the established thresholds. The same tumors were investigated for pathological response according to the Salzer-Kuntschik regression scale (>90% devitalization) and reviewed for cystic degeneration, hemorrhage, and predominant necrotic or fibrosclerotic regression phenotype. RESULTS: None of the clinical response criteria were able to reliably identify the pathologic responders. The extent of size changes showed no association with the pathological degree of regression. The number of clinical responders was low compared with the number of pathological responders (RECIST N = 1, WHO N = 3, volumetry N = 3, pathology N = 19). The occurrence of hemorrhage and/or cystic degeneration was more frequently observed in predominant necrotic sarcomas and was associated with an increase in tumor size after TM-ILP. Furthermore, we identified the fibrosclerotic phenotype of regression to be more significantly strongly associated with posttherapeutic shrinkage than necrosis. CONCLUSIONS: Size-based clinical response evaluation is insufficient to assess clinical response in TM-ILP-treated sarcomas. The size changes of tumors after therapy reflect the type of regression rather than the extent of destruction.

The BRAF V600E mutation in papillary thyroid carcinoma is associated with glucose transporter 1 overexpression.

BACKGROUND: The glucose transporter 1 (GLUT1) is a key protein that facilitates the extensive glucose uptake of cancer cells, and its overexpression is associated with more aggressive tumor phenotypes. In cases of BRAF mutations, GLUT1 seems to be a target of the constitutive activation of the RAF/MEK/ERK pathway. In this study, we hypothesized that the common BRAF V600E mutation was associated with GLUT1 overexpression and proliferation in papillary thyroid carcinomas (PTCs). METHODS: A total of 57 cases of paraffin-embedded PTC (31 BRAF V600E, 26 wild-type BRAF) were investigated using immunohistochemistry with antibodies against GLUT1 and Ki-67 (MK167) protein. The BRAF V600E mutations were detected using direct sequencing of genomic DNA that was isolated from formalin-fixed paraffin-embedded tumor tissues. GLUT1 expression was assessed using the Remmele immunoreactive score and subdivided into three groups (I=negative, II=weakly positive, and III=positive). The Ki-67 labeling index (Ki-67 LI) was determined by counting Ki-67-positive nuclei. RESULTS: GLUT1 expression was found in 39/57 (68.4%) samples of PTC. The occurrence of the BRAF V600E genetic variant was significantly correlated with GLUT1 overexpression (p=0.007) and showed a trend toward higher proliferation, which was indicated by Ki-67 LI (p=0.06). Moreover, GLUT1 overexpression was positively associated with Ki-67 labeling (p=0.023). CONCLUSIONS: The V600E BRAF mutation in PTC may contribute to the initiation of the glycolytic phenotype and confers growth advantages in cancer cells. Better understanding of the molecular mechanisms of cancer cell energy metabolism may lead to the implementation of targeted treatment modalities, which regulate cancer glucose uptake.

Glucose transporter 1 expression, tumor proliferation, and iodine/glucose uptake in thyroid cancer with emphasis on poorly differentiated thyroid carcinoma.

PURPOSE: Glucose transporters 1 (GLUT1) facilitates glucose uptake in cancer. An inverse relationship between I-131 and F-18 fluorodeoxyglucose (FDG) uptake in PET/CT ("flip-flop phenomenon") was described for thyroid cancers (TCs) during dedifferentiation. We investigated the relationship among GLUT1 expression, proliferation, iodine concentration, and glucose uptake in different TC types, with emphasis on "poorly differentiated thyroid carcinoma" (PDTC). METHODS: For immunohistochemistry, 95 thyroid tumors (follicular adenoma, papillary TC, follicular TC, PDTC, and anaplastic TC [ATC]) were investigated for GLUT1 expression and proliferation (Ki-67 index). For PET/CT study, 47 F-18 FDG PET/CT of patients with TC (22 PDTC), 39 corresponding I-124 PET/CT, and glucose and iodine uptake were evaluated. PTC and FTC were summarized under differentiated TC (DTC). RESULTS: Immunohistochemistry: 65% of TC expressed GLUT1. The number of GLUT1-positive TC and GLUT1 expression increased with escalating dedifferentiation/aggressiveness of TC types (P < 0.001). A correlation between proliferation and GLUT1 expression was noted (P < 0.001). PET/CT study: F-18 FDG uptake was measured in 81% of cases. Occurrence of F-18 FDG-avid cases as well as median F-18 FDG maximum standardized uptake values were lowest in DTC, intermediate in PDTC, and highest in ATC. Accordingly, numbers of iodine-avid cases and median I-124 maximum standardized uptake value featured an inverse pattern. CONCLUSIONS: Dedifferentiation in TC is accompanied by GLUT1 upregulation and increased proliferation. PDTC was found to be intermediate between DTC and ATC in terms of GLUT1 expression and F-18 FDG or I-124 uptake, suggesting that the flip-flop phenomenon occurs at a dedifferentiation stage in between. Furthermore, the results suggest that F-18 FDG PET/CT is an important imaging modality for ATC and PDTC.

Lossless compression of JPEG2000 whole slide images is not required for diagnostic virtual microscopy.

The use of lossy compression in medical imaging is controversial, although it is inevitable to reduce large data amounts. In contrast with lossy compression, lossless compression does not impair image quality. In addition to our previous studies, we evaluated virtual 3-dimensional microscopy using JPEG2000 whole slide images of gastric biopsy specimens with or without Helicobacter pylori gastritis using lossless compression (1:1) or lossy compression with different compression levels: 5:1, 10:1, and 20:1. The virtual slides were diagnosed in a blinded manner by 3 pathologists using the updated Sydney classification. The results showed no significant differences in the diagnosis of H pylori between the different levels of compression in virtual microscopy. We assume that lossless compression is not required for diagnostic virtual microscopy. The limits of lossy compression in virtual microscopy without a loss of diagnostic quality still need to be determined. Analogous to the processes in radiology, recommendations for the use of lossy compression in diagnostic virtual microscopy have to be worked out by pathology societies.

Dissecting molecular events in thyroid neoplasia provides evidence for distinct evolution of follicular thyroid adenoma and carcinoma.

Benign hypofunctional cold thyroid nodules (CTNs) are a frequent scintiscan finding and need to be distinguished from thyroid carcinomas. The origin of CTNs with follicular morphologic features is unresolved. The DNA damage response might act as a physiologic barrier, inhibiting the progression of preneoplastic lesions to neoplasia. We investigated the following in hypofunctional follicular adenoma (FA) and follicular thyroid cancer (FTC): i) the mutation rate of frequently activated oncogenes, ii) the activation of DNA damage response checkpoints, and iii) the differential proteomic pattern between FA and FTC. Both FTC and FA, which did not harbor RAS, phosphoinositide-3-kinase, or PAX/peroxisome proliferator activated receptor-γ mutations, express various proteins in common and others that are more distinctly expressed in FTC rather than in FA or normal thyroid tissue. This finding is in line with the finding of constitutive DNA damage checkpoint activation (p-Chk2, γ-H2AX) and evidence for replicative stress causing genomic instability (increased cyclin E, retinoblastoma, or E2F1 mRNA expression) in FTC but not FA. We discuss the findings of the increased expression of translationally controlled tumor protein, phosphatase 2A inhibitor, and DJ-1 in FTC compared with FA identified by proteomics and their potential implication in follicular thyroid carcinogenesis. Our present findings argue for the definition of FA as a truly benign entity and against progressive development of FA to FTC.

Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy.

CONTEXT: Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. OBJECTIVE: To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. DESIGN: Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. RESULTS: We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. CONCLUSIONS: Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

The prevalence of the c-kit exon 10 variant, M541L, in aggressive fibromatosis does not differ from the general population.

BACKGROUND: The frequency of the c-kit exon 10 variant, M541L, (c.1621 A>C) in aggressive fibromatosis (AF) was recently determined to be 2.5-fold higher than in healthy controls. It was thus hypothesised that M541L could be associated with the development of AF. METHODS: 42 cases of sporadic AF were investigated for c-kit exon 10 alterations, by traditional sequencing. Subsequently, the AF cases and 126 healthy volunteers were screened for the M541L by pyrosequencing. RESULTS: Genotype frequency of M541L in AF was 16.7% (allele frequencies: A, 0.92 and C, 0.08), which did not differ from the control group. Moreover the M541L variant was found to be not tumour specific. CONCLUSIONS: The result classifies the M541L variant of c-kit exon 10 as a single nucleotide polymorphism. Because its prevalence does not differ between the AF and general populations, an association with AF tumourigenesis is unlikely.

Increased shedding of soluble TNF-receptor 1 during hyperthermic TNF-α-based isolated limb perfusion.

PURPOSE: Hyperthermic isolated limb perfusion (HILP) with TNF-α and melphalan has high response rates in patients with soft tissue sarcomas (STS) or melanomas of the limbs. Its effectiveness is based on the destructive effect of TNF-α on the blood supply of the tumours. Shedding of soluble TNF-receptor (sTNF-R) negatively modulates the effects of TNF-α, whereas hyperthermia (HT) induces shedding. Here, we investigated whether sTNF-R shedding in response to HT occurs during HILP. MATERIAL AND METHODS: The serum levels of sTNFR-1 were measured in 23 patients with HILP by obtaining serum from the extracorporeal and central circuits. The samples were taken from the patients under normothermic (37°C) and hyperthermic (39°C) conditions. Additionally, cell cultures of HUVEC, human fibrosarcoma cells and peripheral blood cells were used to confirm the effects of HT on sTNF-R1 shedding by ELISA and western blot. RESULTS: Under HT, levels of sTNF-R1 increased 23.5% in the extracorporeal circuit, but this increase was not observed in the systemic circuit. However, we could not confirm this effect using the cell culture model, where cellular TNF-R1 and sTNF-R1 of culture supernatants, respectively, were not significantly different between NT and HT conditions. CONCLUSIONS: HT is associated with an increase of sTNF-R1 in the extracorporeal circuit of perfused limbs. Interestingly, HT does not exhibit the same effect on cells cultured in vitro. Additional studies will be aimed at determining whether our findings have an impact in the clinic by analysing the relationship between TNF-R1 shedding and tumour response to HILP.

The XbaI G>T polymorphism of the glucose transporter 1 gene modulates 18F-FDG uptake and tumor aggressiveness in breast cancer.

UNLABELLED: We investigated the relevance of single-nucleotide polymorphisms (SNPs) in the glucose transporter 1 (GLUT1) gene to the uptake of (18)F-FDG and tumor aggressiveness in breast cancer. METHODS: In 52 individuals with breast cancer, a diagnostic PET/CT scan was obtained, and the standardized uptake value was determined as a measure of (18)F-FDG uptake using a region-of-interest technique. Three GLUT1 SNPs (XbaI G>T, HpyCH4V A>T, and HaeIII T>C) were investigated in genomic DNA that was isolated from the paraffin-embedded specimens of all patients. Tumors were typed and graded according to the World Health Organization classifications. RESULTS: The GG genotype of the XbaI G>T SNP was associated with increased tumor uptake of (18)F-FDG, with a mean standardized uptake value of 11.7 (TT/GT genotypes, 5.9; P = 0.03). Furthermore, the GG genotype was positively related to enhanced tumor proliferation (mitotic count, P = 0.01). In line with this finding, the GG genotype was absent in grade 1 carcinomas and increasingly prevalent in tumors with higher malignancy (grade 2, 28.0%; grade 3, 50%; P = 0.04). CONCLUSION: This study found that the XbaI G>T SNP of the GLUT1 gene is associated with an increased (18)F-FDG uptake and a more advanced tumor grade or growth in breast cancer. Thus, this genetic variant might favor aggressive phenotypes by modulating the efficiency of cancer cells to recruit glucose and escalate growth rate, suggesting the XbaI G>T SNP as a proliferation-related prognostic factor.

MET overexpressing chordomas frequently exhibit polysomy of chromosome 7 but no MET activation through sarcoma-specific gene fusions.

Overexpression of MET and polysomy 7 was formerly demonstrated in chordomas. We investigated mesenchymal-epithelial transition factor (MET) protein expression and copy numbers of chromosome 7 in human chordomas. Furthermore, tumors were screened for gene fusions (PAX3-FKHR, ASPL-TFE3, and SYT-SSX) previously shown to be associated with MET activation in sarcomas. Tissue microarrays (TMAs) were constructed from 66 chordoma samples. MET protein expression was assessed by immunohistochemistry using an immunoreactive score (IRS, scores 0-12). fluorescence in situ hybridization (FISH) with a dual-color DNA probe (7q31) for MET amplification was performed on TMA sections and RT-PCR for PAX3-FKHR, ASPL-TFE3 (type 1 + 2), and SYT-SSX (type 1 + 2) gene fusions on punch biopsies. All tumors (n = 66) expressed MET protein. FISH analysis of 33 tumors lacked MET gene amplification but showed polysomy of chromosome 7 in 15 (45.5%) tumors (13 low and two high polysomies). Although, polysomy 7 showed an increasing incidence with escalating MET IRS, this finding was not statistically significant. PAX3-FKHR, ASPL-TFE3, or SYT-SSX gene fusions were not demonstrable (n = 52). We found MET protein expression in all chordomas. A clear influence of polysomy 7 on MET protein expression could not be statistically demonstrated for this cohort. Moreover, gene fusions with the ability to cause MET overexpression do not occur in chordomas.

Hyalinizing trabecular tumour of the thyroid-differential expression of distinct miRNAs compared with papillary thyroid carcinoma.

AIMS: To compare the expression pattern of five microRNAs (miRNAs) (146b, -181b, -21, -221, -222) of papillary thyroid carcinoma (PTC) and hyalinizing trabecular tumour of the thyroid (HTT). METHODS AND RESULTS: The expression pattern of five miRNAs known to be up-regulated in PTC was retrospectively analysed in 18 HTTs, adjacent normal thyroid tissue, 10 PTCs, 10 follicular adenomas and 10 non-toxic multinodular goitres (MNG) by reverse transcriptase-polymerase chain reaction using the TaqMan miRNA assay. Furthermore, the two common genetic alterations characteristic for PTC, the V600E mutation of the BRAF gene and RET/PTC 1 and 3 rearrangements, were determined in all HTTs. All miRNAs were significantly up-regulated in PTCs, whereas all miRNAs in HTT, normal thyroid tissue, adenomas, and MNGs were down-regulated. Calculating relative changes in gene expression, a 510-fold change of miRNA 146b between PTC and HTT could be observed followed by fold changes between 6.4 and 29 in the remaining miRNAs (P < 0.001). All HTTs lacked BRAF mutations and RET/PTC rearrangements. CONCLUSIONS: Our findings do not support the concept that a high proportion of HTT represents a variant of PTC. It is suggested that HTTs lacking both a miRNA expression pattern characteristic for PTC and RET/PTC rearrangements are re-designated as 'hyalinizing trabecular adenomas'.

Overexpression of the drug resistance-associated protein metallothionein does not correlate with response of sarcomas to isolated limb perfusion treatment.

BACKGROUND AND OBJECTIVES: Hyperthermic isolated limb perfusion with TNF-alpha and melphalan (HILP-TM) achieves high response rates in sarcomas. Melphalan resistance was previously reported to be associated with overexpression of metallothioneins (MTs). Objective of this study was to investigate the influence of MT expression on tumor responses in HILP-TM-treated soft tissue (STSs) and bone sarcomas (BS). METHODS: In primary biopsies of 41 HILP-TM-treated sarcomas (37 STSs and 4 BS), MT expression was assessed by an immunoreactive score. The pathologic response to HILP-TM was quantified in the corresponding tumor resection specimens. We studied the association of MT-IRS between histological regression (responder >90%, or non-responder

Lossy compression in diagnostic virtual 3-dimensional microscopy--where is the limit?

Data compression is inevitable to reduce the huge data amounts of virtual slides, especially in virtual 3-dimensional (3D) microscopy. Lossy compression influences the image quality and leads to recognizable compression artifacts above a compression ratio of 20:1 in JPEG2000 format. To test out whether higher compression ratios are acceptable in diagnostic pathology, we prepared virtual 3D slides of gastric biopsy specimens with or without Helicobacter pylori gastritis using 5 different compression ratios as follows: 20:1, 40:1, 50:1, 75:1, and 200:1. The virtual 3D slides were diagnosed in a blinded manner by 3 pathologists according to the updated Sydney classification. The results showed no significant differences using virtual 3D slides with any compression of up to 200:1. We conclude that compression ratios higher than those commonly used can be applied in virtual microscopy, even in diagnostic applications.

Immunocytochemical investigation of Langerin (CD207) is a valuable adjunct in the cytological diagnosis of Langerhans cell histiocytosis of the thyroid.

Langerhans cell histiocytosis (LCH) is an uncommon disease encompassing three clinically different entities: eosinophilic granuloma, Hand-Schüller-Christian disease, and Abt-Letterer-Siewe disease. Despite usually being a multisystemic disease affecting numerous different organs, involvement of the thyroid gland is extremely rare, and only a few cases in adults have been described in the literature. Herein, we present the case of a 28-year-old male patient presenting with LCH involving the skin, the skeletal system, and the thyroid gland. Fine needle aspiration (FNA) of the thyroid was performed and showed the typical Langerhans cells (LC) with foamy cytoplasm and slender nuclei with longitudinal grooves against a background of inflammatory cells with only a few eosinophilic granulocytes. Immunocytochemically, the LC showed positive staining with antibodies against CD1a and Langerin, a recently detected glycoprotein exclusively expressed in LC. Langerin is the major protein that makes up the so-called Birbeck granules, the electronmicroscopical hallmark of LC. Since LCH involvement of the thyroid is occasionally mistaken for papillary thyroid carcinoma cells, we propose that application of Langerin in combination with CD1a is a helpful diagnostic adjunct for the correct assessment of LCH affecting the thyroid gland.

Prognostic impact of intrahepatic lymphatic and microvascular involvement in cases of colorectal liver metastases.

OBJECTIVE: The purpose of this study was to evaluate the effect of intrahepatic microvascular and lymphatic infiltration on survival in cases of colorectal liver metastases. MATERIALS AND METHODS: Prospectively collected data of 331 patients were analyzed for microvascular invasion (V), lymphatic infiltration (L), and resection margins (R) with respect to overall and disease-free survival. RESULTS: One-, 3-, and 5-year overall survival rates for R0 resected patients were 89%, 64%, and 39%, respectively. The corresponding survival rates for R1 resected patients were 83%, 42%, and 24% (p < 0.001). The sole presence of microvascular invasion (V1) or lymphatic infiltration (L1) was not associated with a diminished overall survival (p > 0.05). However, patients with a combination of L1V1 had a significantly worse overall survival of 68%, 20%, and 0% when compared to L0V0 patients. This difference was not influenced by the status of the resection margin. No other parameter investigated was found to be of predictive value. CONCLUSIONS: The presence of combined lymphatic and vascular invasion (L1V1) constitutes a predictor of poor overall and disease-free survival. This subgroup of patients might benefit from adjuvant strategies such as chemotherapeutic treatment.

Evaluation of 47 soft tissue sarcoma resection specimens after isolated limb perfusion with TNF-alpha and melphalan: histologically characterized improved margins correlate with absence of recurrences.

BACKGROUND: Isolated limb perfusion (TM-ILP) is an effective limb-sparing treatment for primarily nonresectable soft tissue sarcomas (STS). Surgical margins of STS after ILP were yet not systematically studied. METHODS: In 47 patients with nonresectable STS, TM-ILP with subsequent tumor resection was performed. Surgical margins were systematically analyzed by light microscopy using the TNM and the Enneking classification. Furthermore, margins were analyzed for tumor regression in terms of improved resectability. Results were correlated with clinical and pathological parameters. RESULTS: Of 47 STS, 44 were classified as high-grade (93.6%) with a median tumor size of 10.0 cm. Primary limb-salvage rate was 85.1%. According to TNM resection margins were complete in 70.2% (R0) and incomplete in 29.8% (R1=21.3%, R2=8.5%). According to Enneking, 27.7% intralesional, 42.6% marginal, 21.3% wide, 2.1% radical, and 6.4% unclassifiable margins were found. Prior surgery and/or radiotherapy significantly decreased margin quality. Ten patients with incomplete resection (three intralesional, seven marginal) had no viable tumor at the plane of dissection, which was designated as "improved margins." Whereas those patients remained relapse free, five patients with viable tumor (not improved margins) at the resection margin had local recurrences. Poor margins were associated with local and distant recurrences and limited disease-specific survival. CONCLUSION: TM-ILP is effective for achieving limb salvage. Histopathology of surgical margins demonstrates cases with so-called "improved margins" after TM-ILP, which are related to a better outcome even in intralesionally resected tumors. Improvement of margins should be further evaluated as a potential relevant prognostic parameter.

FOXO3a: a novel player in thyroid carcinogenesis?

The forkhead box transcription factor FOXO3a has recently been identified as central mediator of the cellular response to oxidative stress inducing cell cycle arrest or apoptosis. The aim of our study was to investigate the regulation of FOXO3a in the thyroid and to determine whether alterations in FOXO3a activity occur in thyroid carcinogenesis. In vitro, we demonstrate that FOXO3a activity is negatively regulated by the PI3K/Akt cascade promoting increased phosphorylation and cytoplasmatic accumulation of FOXO3a with decreased transcription of the target genes p27kip (CDKN1B) and Bim (BCL2L11), but increased expression of GADD45A. By contrast, we show that H(2)O(2) exposure activates FOXO3a in thyrocytes with JNK (MAPK8)-mediated nuclear accumulation of FOXO3a and increased expression of the cell cycle arrest genes p27kip and GADD45A. In vivo, we observed a marked cytoplasmatic accumulation of FOXO3a in differentiated thyroid cancers versus an exclusive nuclear accumulation in follicular adenoma and normal thyroid tissue. Moreover, this cytosolic accumulation of FOXO3a correlated with an increased phospho-Akt expression in thyroid malignancies and was accompanied by decreased expression of the FOXO targets p27kip and Bim and an increase in GADD45A mRNA expression in the thyroid cancers. Our data suggest FOXO3a as a novel player of cellular stress response in the thyroid, mediating the thyrocyte's fate either to survive or to undergo apoptosis. Furthermore, PI3K-dependent FOXO3a inactivation may be a novel pathomechanism for the escape from apoptosis in thyroid cancer cells, in particular in follicular thyroid carcinoma.

Evidence for a role of the amyloid precursor protein in thyroid carcinogenesis.

We have recently found an increased expression of amyloid precursor protein (APP) in cold thyroid nodules that are difficult to classify as a truly benign thyroid neoplasm or a lesion with the potential for further dedifferentiation. Since differences in APP activity have been found in other human cancers, we asked whether thyroid carcinogenesis might be associated with an altered APP expression and function. APP regulation was studied in vitro in differentiated (FRTL-5) and dedifferentiated follicular thyroid carcinomas (FTC-133) thyroid cells after specific inhibition or activation of the cAMP-PKA, the PI3K/AKT or the protein kinase c (PKC) cascades. In vivo analysis of APP expression and downstream signalling was performed in benign and malignant thyroid tissues. We found that upregulation of APP expression and sAPP secretion is induced by TSH in differentiated thyroid cells and by insulin in thyroid cancer cells. PKC is a strong activator of APP cleavage and in FTC-133 confers prolonged release of the APP ectodomain. FTC-133 but not FRTL-5 cells show a prominent cell surface expression of the APP ectodomain, which has been suggested to function as an autocrine growth factor. Thyroid cancers are characterized by APP upregulation, increased membrane targeting of the APP ectodomain and significantly increased mRNA levels of the APP scaffold proteins JIP1, ShcA and Fe65.