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IntroductionThe aim of this study was to compare the reproducibility of epidermal growth factor receptor (EGFR) immunohistochemistry (IHC),EGFRgene amplification analysis, andEGFRandKRASmutation analysis among different laboratories performing routine diagnostic analyses in pathology in The Netherlands, and to generate normative data.MethodsIn 2008, IHC, in-situ hybridisation (ISH) forEGFR, and mutation analysis forEGFRandKRASwere tested. Tissue microarray sections were distributed for IHC and ISH, and tissue sections and isolated DNA with known mutations were distributed for mutation analysis. In 2009, ISH and mutation analysis were evaluated. False-negative and false-positive results were defined as different from the consensus, and sensitivity and specificity were estimated.ResultsIn 2008, eight laboratories participated in the IHC ring study. In only 4/17 cases (23%) a consensus score of ≥75% was reached, indicating that this analysis was not sufficiently reliable to be applied in clinical practice. ForEGFRISH, andEGFRandKRASmutation analysis, an interpretable result (success rate) was obtained in ≥97% of the cases, with mean sensitivity ≥96% and specificity ≥95%. For small sample proficiency testing, a norm was established defining outlier laboratories with unsatisfactory performance.ConclusionsThe result of EGFR IHC is not a suitable criterion for reliably selecting patients for anti-EGFR treatment. In contrast, molecular diagnostic methods forEGFRandKRASmutation detection andEGFRISH may be reliably performed with high accuracy, allowing treatment decisions for lung cancer.

Die vorliegende Arbeit vergleicht, mittels In-situ-Hybridisierung, die Expression cGMP-abhängiger Proteinkinasen (cGKs) auf neuronaler Ebene mit der von IRAG im Hinblick auf eine eventuelle Kongruenz des Verteilungsmusters im ZNS. Die Untersuchungen zeigten eine isolierte Expression der cGKIα im cerebellären Cortex; cGKIβ hingegen war v.a. in Hippocampus und Gyrus dentatus hoch exprimiert. cGKII-Transkripte konnten u.a. deutlich im Thalamus detektiert werden. IRAG-Expression war isoliert auf einzelne thalamische Kerngebiete beschränkt, so daß sich eine Kolokalisation von cGKIβ und IRAG auf neuronaler Ebene nicht etablieren ließ. This study compares the expression of cGMP-dependent protein kinases (cGKs) and IRAG in neuronal tissue through in-situ-hybridization with regards to a potential co-localization in the CNS. The results revealed an isolated expression pattern of cGKIalpha in the cerebellar cortex; cGKIbeta however was predominantly expressed in the hippocampal region and the dentate gyrus. cGKII transcripts could clearly be detected in the thalamic region of the brain. The expression of IRAG was restricted to several thalamic nuclei so that a co-localization of cGKIbeta and IRAG in the mouse CNS could not be established.

Background Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory. Methods We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization. Results In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours. Conclusions Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

This study aimed to see HTLV-1 infection in 277 cases of malignant lymphoma (ML) in a HTLV-1indemic area by means of ISH employing a highly biotinylated concatamer probe synthesized by a couple of primers SK43 and 44 PCR for HTLV-1 provial DNA pX Tax region. The MLs comprised 115 T-cell MLs, 73 B-cell MLs, 14 Hodgkin's disease (HD) and MLs from the age group less than 20 years old (y.o.). The non-Hodgkin's MLs were categorized according to updated Kiel classification. ISH detected a signal of HTLV-1 proviral DNA including pX Tax region, showing positive stain dominantly in cytoplasm of lymphoma cells and non-neoplastic cells including intermingling small 1ymphocytes, histiocytes and epithelioid cells. ML in which lymphoma cells showed the signal was defined as ""HTLV-1-related ML"". And ML in which any cells revealed the signal was defined as ""HTLV-1-carrier ML"". HTLV-1-related MLs were found in all the subtypes of T-cell MLs (69/115, 60%), indicating its high rate (44/45, 97.8%) in T-cell pleomorphic lymphoma with histological features of adult T-cell leukemia/ lymphoma (ATLL). Even in MLs other than the T-cell MLs, HTLV-1ielated MLs were noted in 21 (29%) of B-cell MLs, 8 (57%) of HD and 4 (16%) of MLs from the age group less than 20 y.o.. HTLV-1-carrier MLs were found in 93 (81%) of T-cell MLs, 35 (49%) of B-cell MLs, 8 (57%) of HD, 13 (52%) of MLs from the age group less than 20 y.o.. HTLV-1-related T-cell MLs in which many lymphoma cells showed the signal may be ATLL, but it was unknown whether there were ATLLs in the ISH-negative cases and in HTLV-1-carrier MLs other than HTLV-1ielated MLs. Considering several technical problems in this ISH, the followings were suggested in this study. HTLV-1 may have oncogenetic effect(s) specific for ATLL and non-specific for MLs other than ATLL but ATLL development would need factor(s) other than HTLV-1 infection. There may be HTLV-1-related B-cell MLs and HD. Some of patients with B-cell MLs and in the under 20-year-old ML patients would fail to produce antibodies against HTLV-1, because higher HTLV-1 carrier rates in these patients were recognized in this study than in the serovirological studies.