Quality assurance trials for Ki67 assessment in pathology.

Ki67 is a broadly used proliferation marker in surgical pathology with an obvious need for standardization to improve reproducibility of assessment. Here, we present results of the so far only existing round robin tests on Ki67, organized annually in Germany, Austria, and Switzerland from 2010 to 2015 with up to 160 participating laboratories (QuIP). In each quality assessment trial, eight probes from each breast cancer, neuroendocrine tumor, and malignant lymphoma were compiled on a tissue microarray (TMA). TMAs were stained in the participants' laboratories with antibodies and procedures also applied in their daily routine. Participating pathologists were expected to assign Ki67 values to one of four different categories for each tumor type. All local stainings and evaluations were reassessed by the organizing panel and compared to a preset standard. On average, 95% of participants reached the benchmark of over 80% concordance rates with the Ki67 category pre-established by the panel. Automatization and type of antibody did not affect the success rate. Concordance rates differed between tumor entities being highest in each tumor type with either very high or very low labeling indices. Lower rates were seen for intermediate Ki67 levels. Staining quality improved during the observation period as did inter-observer concordance with 85% of participants achieving excellent agreement (kappa > 0.8) in the first year and over 95% in 2015. In conclusion, regular external quality assurance trials have been established as a tool to improve the reproducibility and reliability of the prognostic and predictive proliferation marker Ki67.

Hypothermic circulatory arrest with "low flow" lower body perfusion: an experimental feasibility study of microcirculatory parameters.

BACKGROUND: To avoid extended cardiopulmonary bypass (CPB), moderate temperatures are commonly accepted for hypothermic circulatory arrest (HCA), thereby jeopardizing organ protection. Distal aortic perfusion may be an option, but supportive experimental data is missing. METHODS: Eight juvenile pigs (36 ± 2 kg) were cooled to 30 °C followed by 60 min of HCA with 50 min of low flow (LF) lower body perfusion. Multimodal monitoring was used to measure overall metabolism, hemodynamics and microcirculation of the terminal ileum. The animals were observed for four hours following reperfusion. Organs were harvested for histopathological evaluation. RESULTS: During LF perfusion, initially elevated l-lactate levels decreased subsequently ( P < 0.05). Capillary blood flow decreased during cooling to 50 % baseline levels ( P = 0.03), but remained stable under LF conditions. Parameters indicative of reduced liver and kidney function were slightly elevated at the end of the experiment, but still within normal ranges. CONCLUSION: Under moderate hypothermia, low flow perfusion seems to provide adequate protection for the lower body organs. Microcirculatory parameters during perfusion as well as lactate levels within normal ranges throughout the experiments further confirm the concept.

Expression of hypoxia-inducible factor 1 alpha in thyroid carcinomas.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1 alpha and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1 alpha was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1 alpha staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1 alpha pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O(2), anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1 alpha in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1 alpha and HIF-1 alpha targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1 alpha with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

[Predictive parameters in children with biliary atresia]. / Prognostische Marker bei Kindern mit Gallengangatresie.

Most children with biliary atresia require liver transplantation, and only about 20% survive in the long term with their native livers. Prognostic factors that determine disease progression are still lacking. This retrospective survey of 85 BA patients from 1993 to 2003 was aimed to evaluate prognostic factors using the log rank test. After 5 years 40% of the patients are alive with their native livers (35/85), 26 of them with normal bilirubin (31%). Age at Kasai operation (P=0.46), degree of liver fibrosis (P=0.95), and all laboratory test results before Kasai failed to correlate with outcome. Normal levels of bilirubin 3, 6, and 12 months after Kasai and of aspartate aminotransferase with gammaGT after 6 months are associated with survival with native liver. In conclusion our data demonstrate that a lack of predictive factors must prevent primary liver transplantation in BA patients.

Primary cervical cancer truly negative for high-risk human papillomavirus is a rare but distinct entity that can affect virgins and young adolescents.

Cancer of the uterine cervix is almost exclusively associated with human papillomavirus (HPV). Carcinogenesis is slow, the minimal time from initial HPV infection to invasive carcinoma seems to be less than ten years. In order to identify rapid onset cervical cancer, we carried out a retrospective re-analysis of an extended cohort of patients with invasive cervical cancer, and reviewed cases identified within the cancer registry of Lower Saxony or using Medline or ISI data. No instances of a rapid-onset cancer or true HPV-DNA negative cancer were found among our hospital cohort of 178 women with primary cancer of the uterine cervix. Registry data identified four out of 5,878 patients who were diagnosed with primary cervical cancer at 14 to 20 years of age. They were classified as clear-cell and endometriod adenocarcinoma and tested persistently negative for high-risk HPV-DNA. Fourteen more cases of cervical cancer in virgins and very young women were identified by a Medline search, mostly with unknown histologic type or rare subtypes of adenocarcinoma. In conclusion, rare adenocarcinoma of the uterine cervix may represent an entity unrelated to HPV, thus explaining instances of rapid onset cervical cancer.

[Interlaboratory trials for quality assurance of breast cancer biomarkers in Germany]. / Ringversuche zum Nachweis von therapeutischen Zielmolekülen beim Mammakarzinom in Deutschland.

In the age of personalized medicine, and in addition to typing and grading, breast cancer pathologists are now also involved in determining biomarkers such as steroid hormone receptors and Her-2, which are of the utmost importance in adjuvant therapy. In order to assure quality of these biomarker assays, external proficiency testing has been implemented in Germany. Since 2002 trials have been conducted annually, with up to 180 participating laboratories. More than 85% of all participants achieved good results in clearly negative and positive cases seen in daily practice. If at all, discordant results were observed in the rarer low steroid-hormone receptor expressing tumors and Her-2 borderline cases (2+). Regular participation in interlaboratory testing leads to significantly improved immunohistochemical results, particularly in these problematic cases. Tissue microarrays (TMA) with 20-24 different breast cancer samples including cell lines meant that a huge number of pathologists were challenged with identical samples, providing the prerequisite for comparability. Participation is recommended for pathology departments involved in the service for breast units. The organizational frame work of the trials is described here. The confidence of cooperating disciplines in breast cancer biomarkers assessed by pathologists will be fostered by external proficiency testing as presented here.

Immunocytological detection of lymph node metastases in dogs with malignant epithelial tumours.

The aim of this study was to compare immunolabelling of cytological specimens with conventional staining in the detection of metastases in lymph nodes from dogs with carcinoma. Cytological touch imprints of 161 lymph nodes from 72 dogs, as well as 50 fine needle aspirates from 23 dogs, with malignant epithelial tumours were included in the study. Immunolabelling was performed with commercially available human antibodies. Touch imprints of all lymph nodes were labelled with broad spectrum anticytokeratins AE1/AE3 and KL1. In addition, lymph node touch imprints from dogs with primary tumours that reacted positively with the specific anticytokeratins CK7 (n=104) and CK20 (n=20) were also labelled with CK7 and CK20. Fine needle aspirates of 50 lymph nodes were examined by immunolabelling with AE1/AE3. "Reference investigations" with a combination of histological and immunohistochemical methods were performed on all lymph nodes. The immunocytological detection of lymph node metastases with the broad spectrum anti-cytokeratin AE1/AE3 in imprint smears resulted in a significant increase in sensitivity (0.99 vs 0.88 [conventional stain]) and in negative predictive value (0.99 vs 0.85) (P<0.01; t-test). Micrometastases in particular were detected more readily. Specificity (0.93 vs 0.88) and positive predictive value (0.95 vs 0.90) did not differ significantly between the two techniques. Immunolabelling with KL1 was associated with lower sensitivity and negative predictive value, indicating lack of cross-reactivity of this antibody with canine epithelial cells. In fine needle aspirates the detection of lymph node metastases, especially micrometastases, was more efficient by mean of immunolabelling with AE1/AE3 than by conventional staining. The study indicated the value of immunocytological labelling for the detection of metastases in cytological specimens of canine lymph node preparations.

Immmunohistochemical study of the blood and lymphatic vasculature and the innervation of mouse gut and gut-associated lymphoid tissue.

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.

[Improvement of breast cancer grading in punch biopsies: grading with the Ki-67 marker]. / Mammakarzinomgraduierung an Gewebestanzen: Objektivierung mit dem Proliferationsmarker Ki-67.

The grading of invasive breast cancers according to Bloom and Richardson (Nottingham modification) provides one of the most important prognostic factors in addition to size and the status of the lymph nodes. Diagnostic reproducibility has been problematic in daily practice as the required criteria for selection and extent of the grading area are frequently not present in the punch biopsies.A total of 346 cases were retrospectively used to compare routine grading from surgical preparations with an equivalently small sample from punch biopsies. In addition, a modified grading of these small samples was developed with Ki-67 immunochemistry and the measurement of core size. In the case of modified grading, 1-3 points were given for Ki-67 and average maximum core diameter. Tubule development was evaluated with 1 or 2 points. A comparison for recurrence free survival and total survival showed significant prognostic differences between 3-5 points (low risk) and 6-8 points (high risk) in uni- and multivariate analyses. The evaluation criteria for Nottingham-Bloom-Richardson grading in a small tissue sample, such as that from a punch biopsy, can hardly be fulfilled. In our series, prognostic value was only found for nodal negative cases. After modification using objective parameters such as nuclear size measurement and Ki-67 proliferation index, a small tissue sample can prove to be of significant prognostic value for nodal negative as well as nodal positive cases.

[The importance of immunohistochemistry for the diagnosis of cholangiocarcinomas]. / Bedeutung der Immunhistochemie für die Diagnose des Cholangiokarzinoms.

The correct diagnosis of intrahepatic cholangiocarcinoma (CC) is often confounded by the small size of the diagnostic specimen and the wide morphological range of carcinomas metastasising to the liver. Expression analysis of cytokeratins, glycoproteins, mucoproteins, adhesion molecules, receptors and transcription factors has been shown to be a valuable adjunct in the typing of carcinomas. For this study, the expression pattern of 30 well documented antibodies to CC and the most common metastatic adenocarcinomas of the liver were studied. CC show a rather distinct immunophenotype with co-expression of CK7, CK17, CD7 and a lack of CDX2. Although this pattern allows the separation of CC from most metastatic carcinomas, pancreatic carcinomas show a broad overlap with this expression pattern.

Pitfalls in immunohistochemical assessment of EGFR expression in soft tissue sarcomas.

BACKGROUND: New targeted cancer treatments acting against growth factor receptors such as the epidermal growth factor receptor (EGFR) necessitate selecting patients for treatment with these drugs. Besides carcinomas, soft tissue sarcomas (STS) express EGFR and might thereby be a promising target for this new therapeutic strategy. OBJECTIVE: To test and compare different EGFR antibodies to determine the frequency of EGFR expression in STS. METHODS: 302 consecutive specimens of STS were examined using the tissue microarray technique. EGFR expression levels were assessed by immunohistochemistry using five different commercially available antibodies. Gene amplification status was measured by fluorescence in situ hybridisation (FISH). Immunoreactivity and amplification status were correlated with clinicopathological features and follow up data available in 163 cases. RESULTS: EGFR expression frequency ranged between 0.3% and 52.9%, depending on the antibody and scoring method used. In all, 3.5% of the tumours showed egfr gene amplification by FISH, which correlated with EGFR expression for three antibodies. Only one antibody had independent prognostic value in multivariate analysis and correlated with an unfavourable outcome; egfr gene amplification status showed no correlation with clinical features. CONCLUSIONS: Frequency of EGFR immunopositivity in STS strongly depends on the antibody used, and only one of five antibodies tested predicted an unfavourable clinical outcome. This indicates that choice of primary antibody and scoring system have a substantial impact on the determination of EGFR immunoreactivity.

Oncofoetal fibronectin--a tumour-specific marker in detecting minimal residual disease in differentiated thyroid carcinoma.

Supposedly, thyrocyte-specific transcripts such as thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) were proposed to be useful for the diagnosis of circulating tumour cells in patients suffering from differentiated thyroid carcinoma (DTC). However, several research groups reported blood-borne Tg transcripts in healthy individuals. This study determines in particular the origin of Tg mRNA in nucleated blood cells and analyses whether other tumour-associated sequences are absent in leukocytes, but widely expressed in DTC. Therefore, expression analyses for Tg, TSH-R, cytokeratin 19 (CK 19), human telomerase reverse transcriptase (hTERT) and oncofoetal fibronectin (onfFN) were carried out using cDNAs derived from (1) leukocyte fractions, (2) 18 follicular thyroid carcinomas (FTCs) and 48 papillary thyroid carcinomas (PTCs), and (3) leukocytes of two thyrocyte-depleted individuals treated for C-cell carcinoma of the thyroid. Expression of onfFN was additionally analysed by semiquantitative RT-PCR and by quantitative fluorescence-based real-time PCR. Tg and TSH-R expression was demonstrated not only in both athyroid individuals, but in all leukocyte subgroups tested, while hTERT was absent in resting CD4+ cells and only weakly expressed in the CD8+ group. CK 19 was notable in each leukocyte population except for resting CD14(+), as well as for activated and resting CD19+ cells. All blood cell fractions proved negative for onfFN mRNA, whereas its presence in thyroid carcinoma was 78/98% (FTC/PTC). Threshold cycle values were calculated at: porphobilinogen deaminase (PBGD) = 25.95+/-0.73 (FTC)/24.55+/-5.43 (PTC) (P = 0.2878); onfFN = 25.48+/-3.15 (FTC)/21.44+/-3.44 (PTC) (*P = 0.0001). Finally, onfFN transcripts were detected in blood samples of six out of nine patients with known DTC metastases, demonstrating a reliable assay functionality. We propose that real-time RT-PCR of onfFN mRNA is superior to other markers in monitoring minimal residual disease in DTC with regard to both assay sensitivity and specificity.

Wnt-5a has tumor suppressor activity in thyroid carcinoma.

Stabilization of beta-catenin by inhibition of its phosphorylation is characteristic of an activation of the canonical Wnt/beta-catenin signaling pathway and is associated with various human carcinomas. It contrasts to an as yet incompletely characterized action of an alternative noncanonical Wnt signaling pathway on neoplastic transformation. The aim of the present study was to test the effects of a member of the noncanonical Wnt signaling pathway, Wnt-5a, in primary thyroid carcinomas and in thyroid carcinoma cell lines. Compared to normal tissue Wnt-5a mRNA expression was clearly increased in thyroid carcinomas. Immunohistochemically, a bell-shaped response was observed with low to undetectable levels in normal tissue and in anaplastic tumors whereas differentiated thyroid carcinomas showed strong positive immunostaining for Wnt-5a. Transfection of Wnt-5a in a thyroid tumor cell line FTC-133 was able to reduce proliferation, migration, invasiveness and clonogenicity in these cells. These effects of Wnt-5a are associated with membranous beta-catenin translocation and c-myc oncogene suppression and are mediated through an increase in intracellular Ca(2+) release, which via CaMKII pathways promotes beta-catenin phosphorylation. Specific inhibition of beta-catenin phosphorylation by W-7, a calmodulin inhibitor, or by KN-93, a CaMKII inhibitor, supports these findings whereas PKC inhibitors were without effect. This interaction occurs downstream of GSK-3 beta as no Wnt-5a effect was seen on the Ser(9) phosphorylation of GSK-3 beta. Our data are compatible with the hypothesis that Wnt-5a serves as an antagonist to the canonical Wnt-signaling pathway with tumor suppressor activity in differentiated thyroid carcinomas.

[Tissue-based markers of prognosis in breast cancer]. / Gewebebasierte Prognosefaktoren beim Mammacarcinom.

Precise prognostication represents one of the essential but still unsolved challenges in breast cancer pathology. There is a striking discrepancy between the plethora of suggested markers that have proved useful in mono-centre retrospective studies, including molecular expression arrays and the only small number of parameters applied in clinical decision finding. When adjuvant therapy is considered clinicians still rely predominantly on traditional parameters like staging and hormonal receptor status. Another traditional marker which has proven its strength in mono-centre studies but is compromised by subjectivity and limited reproducibility is provided by grading. We have conducted a study on how traditional grading markers can be objectified and adapted to small amounts of tissue which have become custom with the wide-spread use ob needle biopsies. A modified grading scheme replacing mitosis counting by Ki-67 immunohistochemistry and nuclear pleomorphism by digital determination of nuclear size was applied to 346 cases of breast cancer with a median follow-up of 6 years in a tissue micro-array. A highly significant correlation with overall and disease-free survival could be established in this retrospective study although not more than 1.4 square millimeters of tissue were evaluated. When combined with nodal status and progesterone receptor evaluation a subgroup free of any relapse could be identified. It is concluded that standardized and objectified application of grading as a traditional tissue-based marker of prognosis can improve its impact considerably even in limited amounts of tissue.

Prevention of peritoneal carcinomatosis from human gastric cancer cells by adjuvant-type intraperitoneal immunotherapy in a SCID mouse model.

PURPOSE: We analyzed the effect of intraperitoneal immunotherapy in an animal model mimicking locoregional dissemination of tumor cells during resection of advanced tumors. METHODS: We first established a tumor model with human gastric cancer cells (MKN-45) in the peritoneal cavity of CB-17-SCID mice. Three hours following the injection of tumor cells into the peritoneal cavity, mAb 17-1A alone and in combination with human LAK cells were given intraperitoneally at different dosages. The results were quantified by determining the weight of the peritoneal tumor masses. RESULTS: After intraperitoneal administration of 17-1A mAb, a tumor reduction could be shown (median tumor mass after 10 microg mAb: 171 microg; after 100 microg: 130 microg) when compared with the control group (632 microg). Following a combined therapy with mAb and LAK cells, a statistically significant tumor reduction could be observed (after 10 microg mAb + 20-50 x 10(6) LAK cells: 80 microg; after 100 microg mAb + 20-50 x 10(6) LAK cells: 12 microg, p = 0.0005). With specific dosages of antibody and LAK cells it was even possible to achieve complete tumor clearance. CONCLUSIONS: Intraperitoneal immunotherapy reduces the peritoneal tumor masses and can even prevent the peritoneal carcinomatosis formation.

Human papillomavirus, coinfection with Schistosoma hematobium, and cervical neoplasia in rural Tanzania.

Cervical cancer is the most common malignant tumor among women in Tanzania and other countries in tropical Africa. Genital schistosomiasis has been proposed as a possible cofactor in the genesis of this malignant disease that might contribute to its high incidence in regions where bilharzias is endemic. One hundred nine Tanzanian patients from an area with endemic bilharzias who were transferred to a gynecologic out-patient clinic were age-matched with 109 German controls. In patients and controls, separate samples were taken for cytologic assessment and human papillomavirus (HPV) DNA detection using the Hybrid Capture 2 assay (HC2) and PCR (GP5+/6 +). Samples that tested positive for HPV DNA with general primers were re-tested with HPV type-specific primers. After application of 3% acetic acid, punch biopsies were taken from any cervical lesion. Patients were interviewed for recent symptoms or clinical history suggestive of bilharzias. Urine samples from all patients were examined for the presence of schistosoma hematobium ova. Additionally six Tanzanian patients with invasive cervical cancer were included for separate analysis. Patients and controls had an identical prevalence of HPV-DNA (21.5%) using HC2. Based on PCR results with general primers, the corresponding prevalence was 34.5% for Tanzanian cases and 26.9% for German controls. A history suggestive of bilharzias and/or active schistosomiasis were associated with a significantly increased risk for infection with high-risk HPV types. We conclude that infection with Schistosoma hematobium seems to favor persistent genital HPV infection either by traumatizing the genital epithelium and/or by local immunosuppression.

[Interlaboratory trial 2000 " Immunohistochemistry" of the German Society for Pathology and the Professional Association of German Pathologists]. / Ringversuch 2000 "Immunhistochemie" der Deutschen Gesellschaft für Pathologie und des Berufsverbandes der Deutschen Pathologen.

Early in 2000 an interlaboratory trial on immunohistochemistry was held in Germany in which 172 pathologists took part. Each pathologist received one H&E stained and five unstained slides of five different tumors to reach a diagnosis based on immunohistochemical stains. Additionally, the diagnosis-independent staining quality was assessed by using a multi-tissue block. Altogether, 828 diagnoses were made, among which 57% (468) were correct. The individual steps of immunohistochemistry (tentative morphological diagnosis, choice of primary antibodies, technical staining quality, conclusions from the diagnosis and rendering a final diagnosis) were assessed independently. Although each of these steps was correlated to the correct final diagnosis, in the multivariate analysis only the tentative diagnosis, choice of primary antibodies and the conclusions drawn from individual stains were independent factors to reach the correct final diagnosis. In the diagnostic part of the interlaboratory trial, the technical quality of the immunostaining was not an independent variable to reach a correct diagnosis. In contrast, the results of the multi-tissue block proved that the immunohistochemical staining quality has to be standardized to reach reproducible results in defining the estrogen receptor expression as a basis for therapeutic decisions.

Quality assurance in immunohistochemistry: results of an interlaboratory trial involving 172 pathologists.

The practicability of quality assurance in immunohistochemistry and its integration into the diagnostic process were both tested in this Germany-wide interlaboratory trial. One hundred seventy-two pathologists received one hematoxylin and eosin and five unstained slides from five cases; all cases were selected by a panel because immunohistochemistry was required for their final diagnosis. Participants rendered a morphologic diagnosis and then substantiated it immunohistochemically. Stained slides and evaluation sheets were reviewed by the panel, and the diagnostic process was analyzed in individual steps: morphologic diagnosis, selection of antibodies, staining quality, interpretation of stained slides, conclusions, and final diagnosis. Diagnosis-independent immunohistochemical performance was tested using a multisample tissue block (30 samples) that was stained and evaluated for six common antigens. For individual cases, corresponding to their difficulty, 21-89% of the final diagnoses (altogether 57% from 828 diagnoses) were correct. In a statistical analysis, the tentative diagnosis, the interpretation of stains and conclusions drawn from immunohistochemistry, were independent factors in reaching the diagnosis. Sensitivity to detect estrogen receptors on the multisample tissue block was only 48%. However, 24% of the stains were interpreted as falsely negative. The low staining sensitivity was not correlated to the number of correct diagnoses. The major problem of applying immunohistochemistry in surgical pathology appears to be its integration into the diagnostic process and not the staining quality. Both future quality control projects and training will have to regard these integrative requirements. Multisample tissue blocks provide a promising tool to standardize quantitative immunohistochemical parameters, such as receptor or proliferation scores.

Identification of a Wnt/beta-catenin signaling pathway in human thyroid cells.

beta-Catenin is a structural component of the adherens junctions. Outside the adherens junctions a complex consisting of glycogen synthase kinase 3beta, the tumor suppressor adenomatous polyposis coli, and axin constantly targets beta-Catenin for degradation to keep levels of free beta-Catenin low. Free beta-Catenin is able to bind to transcription factors of the T cell factor/lymphoid-enhancing factor family and to stimulate transcription of target genes. This signaling function of beta-Catenin is activated by extracellular Wnt factors that bind to Frizzled receptors and induce inhibition of beta-Catenin degradation. By RT-PCR and subcloning, we observed the expression of five Wnt factors, three members of the Frizzled receptor family, and all known Disheveled isoforms in thyroid cells. Immunoprecipitation studies demonstrated the formation of the complex targeting beta-Catenin for degradation. Introduction of a degradation resistant beta-Catenin into the thyroid carcinoma cell line WRO induced appearance of monomeric beta-Catenin as shown by size fractionation and nuclear beta-Catenin immunostaining. Reporter gene assays demonstrated a stimulation of T cell factor/lymphoid-enhancing factor-mediated transcription in these cells. In ARO cells, a thyroid carcinoma cell line carrying a mutated adenomatous polyposis coli gene, monomeric beta-Catenin and nuclear immunostaining were observed. In summary, our data indicate that elements of the Wnt signaling pathway are expressed in thyroid cells and that this pathway is functionally active.