Comparison of automated silver enhanced in situ hybridisation (SISH) and fluorescence ISH (FISH) for the validation of HER2 gene status in breast carcinoma according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists.

HER2 is an important tumour marker in breast cancer. However, there is controversy regarding which method reliably measures HER2 status. This study evaluates the concordance between HER2 gene amplification in invasive breast cancer determined by fluorescence in situ hybridisation (FISH) and a new silver enhanced in situ hybridisation (SISH) technique. Ninety-nine cases were analysed by direct-labelled manual FISH (PathVysion(R), Abbott/Vysis) and bright field automated SISH (INFORM(R), Ventana). For comparison, all specimens were stained by immunohistochemistry (Dako-HercepTesttrade mark and Ventana-PATHWAY(R)4B5). Evaluation was performed by five pathologists following the algorithms of the manufacturers and the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Concordance was calculated and the value of kappa statistics estimated. Overall concordance between FISH and SISH was 96.0% (kappa = 0.754, 95%CI). Discrepancies were mostly seen in tumours with intra-tumoural heterogeneity of HER2 amplification. In conclusion, HER2 gene copy status can be reliably determined by SISH. The 96% concordance with FISH fulfils the ASCO/CAP requirement of greater than 95% concordance for amplified vs non-amplified cases. There was a low inter-observer variability in the interpretation of SISH, suggesting that SISH is equally reliable in determining HER2 amplification as FISH. Because SISH combines bright field microscopy with molecular analysis and full automation, it appears to be particularly suited for routine application in surgical pathology.

Enhanced tumorigenicity of fibroblasts transformed with human herpesvirus 8 chemokine receptor vGPCR by successive passage in nude and immunocompetent mice.

The human herpes virus 8 (HHV-8)-encoded G protein-coupled chemokine receptor (vGPCR) has been implicated in the pathogenesis of Kaposi's sarcoma (KS), particularly because of its high constitutive signaling activity. Here, we used retroviral transduction to generate vGPCR-expressing 3T3 fibroblasts that are tumorigenic in nude mice, but as expected fail to induce tumors in their immunocompetent counterparts. However, tumor fragments obtained from nude mice grow progressively in immunocompetent BALB/c mice. Unexpectedly, vGPCR-expressing cells established from grafted tumor fragments gave rise to tumors in immunocompetent mice. These tumors exhibit a striking histological resemblance to KS including plump spindle cell morphology, a high degree of vascularization and brisk mitotic activity. High expression of vGPCR was confirmed in the cell lines and tumors using a newly developed vGPCR-specific monoclonal antibody. Finally, short interfering RNA directed at vGPCR abrogated or significantly delayed tumorigenesis in mice, demonstrating that the tumor development is specifically driven by vGPCR. This novel model for vGPCR-mediated oncogenesis will contribute to our understanding of the role of vGPCR in the pathogenesis of HHV-8 and may even be important in identifying critical molecular and epigenetic changes during tumor progression in vivo.

Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

[beta-Catenine as a genomic target of high-grade microsatellite instability in colorectal cancer]. / beta-Catenin als genomischer Zielort der hochgradigen Mikrosatelliteninstabilität in kolorektalen Karzinomen.

AIMS: Various inherited and acquired alterations affecting the genes and gene products of the WNT pathway appear to be involved in the different molecular routes leading to colorectal cancer (CRC). This study was initiated to investigate the prevalence of somatic mutations in the beta-catenin gene (CTNNB1) and the associated pathology in CRC with defective DNA mismatch repair. METHODS: Paraffin and/or frozen sections of 33 primary CRC (including any liver and lymph node metastases present) with high-grade microsatellite instability (MSI-H; i.e. with > or = 5 unstable microsatellite markers of 10 tested) were polytopically fractionated by microdissection. Genomic and c-DNA samples were sequenced across exons 2-4 of CTNNB1 and the expression patterns of beta-catenin (beta-C) analyzed by immunohistology and Western blotting. RESULTS: Seven somatic mutations affecting phosphorylation sites of exon 3 (2 deletions also encompassing parts of either intron 2 or exon 4 [delta X2/3 bzw. delta X3/4] and 5 missense mutations [2 x T41A, 2 x S45F, S45P]) were identified. Two mutations (delta X3/4 and S45F) were concordantly present in CRC primaries and their respective metastases whereas the S45P mutation was restricted to a hepatic metastasis. In the delta X2/3 CRC primary only a shortened 66 kD CTNNB1 gene product was present while its associated liver metastasis showed a total loss of beta-C expression. CONCLUSIONS: Both exon 3 and the entire locus coding for beta-C are somatically altered in approximately 20% of CRC with MSI-H at different stages of tumor progression. Thus CTNNB1 appears to be a genomic target for complex oncogenic mutations and deletional processes in a substantial fraction of this molecular subset of CRC.

Microsatellite alterations in serum DNA of patients with colorectal cancer.

Cell-free DNA in the blood of cancer patients has been shown to harbor microsatellite alterations frequently matching those of the primary tumors. The aim of this study was to assess the prevalence of allelic loss and instability of serum DNA microsatellites in colorectal cancers. DNA extracted from preoperative sera and microdissected tumors of 27 patients with colorectal adenocarcinoma were allelotyped for nine markers on chromosome arms 1p, 5q, 8p, 12p, 15q, 17p, 17q, and 18q. In all tumors, expression of MLH1 and MSH2 was explored immunohistochemically. Microsatellite alterations comprising loss of heterozygosity (LOH) or microsatellite instability (MSI) were present in 26 of 27 (96%) tumors and in 16 of 27 (59%) serum samples. Using stringent criteria, serum MSI was significantly (p < 0.02) more detectable than serum LOH. Of the three patients with high-grade MSI (more than two unstable loci) present in tumor and serum DNA, two had MSH2-negative tumors on immunohistochemical testing. No significant association of tumor stage or clinical outcome with serum microsatellite alterations of LOH or MSI type could be demonstrated. Although the DNA-shedding phenotype of tumors remains to be elucidated, its detection by serum DNA microsatellite analysis seems to be useful for the diagnosis and monitoring of neoplasms, including colorectal cancers with and without MSI.

Strong indication for a breast cancer susceptibility gene on chromosome 8p12-p22: linkage analysis in German breast cancer families.

Chromosomal losses involving the short arm of chromosome 8 are frequent in a variety of tumor types, including breast cancer, suggesting the presence of one or more tumor suppressor genes in this region. Previous linkage analysis and studies of loss of heterozygosity (LOH) have suggested the presence of a putative third breast cancer susceptibility gene around D8S505 at 8p12-p22. We have performed linkage analysis in two German breast cancer families, showing negative lod scores with 17q and 13q markers, using seven adjacent microsatellite markers from 8p12-p22. Incorporating LOH data from tumors of the affected family members a maximum cumulative three-point lod score of 3.30 at theta = 0.00 was obtained with D8S137 and D8S131. Our findings considerably strengthen the evidence for a third breast cancer susceptibility locus (BRCA3) mapping to the short arm of human chromosome 8.

Deletion mapping and linkage analysis provide strong indication for the involvement of the human chromosome region 8p12-p22 in breast carcinogenesis.

We have identified a high frequency of loss of heterozygosity (LOH) on the human chromosome region 8p12-p22 in a panel of microdissected familial (86% LOH) and sporadic (74% LOH) breast tumours. The two most frequently deleted regions were defined around marker D8S133 and in a broader centromeric region bounded by markers D8S137 and D8S339. We cannot unequivocally characterize the 8p12-p22 loss as an early or a late event in breast carcinogenesis. In parallel, we have performed linkage analysis in four German breast cancer families. A location score greater than 13.67 corresponding to a LOD score of 2.97 at the marker D8S137 has been obtained. Our results considerably strengthen the evidence for a breast cancer susceptibility gene(s) located on the short arm of the chromosome region at 8p12-p22.

The genomics of soluble proteins with collagenous domains: C1q, MBL, SP-A, SP-D, conglutinin, and CL-43.

The gene cluster encoding the A, B and C chains of human C1q has been localised to 1p34.1-1p36.3, on the short arm of chromosome 1. The C1q molecule, although it is not a lectin, shows certain structural and functional similarities to a group of mammalian C-type lectins which contain collagen-like regions. These lectins include the serum proteins conglutinin, mannose-binding lectin (MBL) and Collectin-43 (CL-43) and the lung surfactant proteins A and D (SP-A and SP-D). The genes for MBL, SP-A and SP-D have been mapped to human chromosome 10, with at least two expressed SP-A genes (SP-AI and SP-AII) forming a cluster with an SP-A pseudogene. Somatic cell hybrid mapping places the human SP-A and SP-D genes at 10q22-q23 while MBL is localised at 10q21. Conglutinin and CL-43 have so far only been characterised in the bovine system but if there are human analogues of these proteins it seems likely that they will also map to the long arm of chromosome 10.

Regional mapping of short tandem repeats on human chromosome 10: cytochrome P450 gene CYP2E, D10S196, D10S220, and D10S225.

Human CYP2E encodes an ethanol-inducible cytochrome P450 monooxygenase that metabolizes various carcinogens and may therefore play a role in cancer susceptibility. An intronic (GGAT)n.(CCTA)n repeat element was found to display limited polymorphism in Caucasoids and was used as a sequence-tagged site for genomic amplification from somatic cell hybrids to localize CYP2E to 10q24.3-qter; using the same panel, three microsatellite markers, D10S196, D10S220, and D10S225, were mapped to 10q21. The close synteny of CYP2E, CYP2C, and CYP17 belonging to two different cytochrome P450 families suggests a central role for the long arm of chromosome 10 in the evolution of this large gene superfamily.

Assignment of the human pulmonary surfactant protein D gene (SFTP4) to 10q22-q23 close to the surfactant protein A gene cluster.

Pulmonary surfactant consists of a complex mixture of phospholipids and several proteins essential to normal respiratory function. Two of the surfactant proteins, SP-A and SP-D, appear to have lectin-like activity relevant to the local phagocytic defense. Using polymerase chain reaction (PCR)-based somatic cell hybrid mapping, the human SP-D gene (SFTP4) was assigned to chromosome 10. A regional mapping panel was assembled and characterized using sequence tagged sites for five loci previously mapped to 10q. SFTP4, the SP-A gene (SFTP1), and the microsatellite D10S109 were placed in the interval 10q22-q23. Low-stringency PCR using the SFTP1 primer pair suggested the presence of at least two additional SP-A-related genes in the same region. With the locus for mannose-binding lectin (MBL) at 10q21, this may be indicative of this region's central role in the evolutionary history of carbohydrate-binding proteins containing collagen-like regions.

Carrier detection in families with properdin deficiency by microsatellite haplotyping.

Human properdin deficiency is an X-linked disorder strongly predisposing to meningococcal disease which has been recorded in over 50 cases of various ethnic origins. Immunochemically, total deficiency (type I), partial deficiency (type II), and deficiency due to a dysfunctional molecule (type III) can be differentiated. It is therefore most likely that the causative molecular defects will show considerable genetic heterogeneity. Analysis of the properdin locus at Xp11.3-Xp11.23 has led to the characterization of two polymorphic (dC-dA)n.(dG-dT)n repeats located approximately 15 kb downstream from the structural gene. Three families (two Scottish Caucasoid, one Tunisian Sephardic) with seven deficient individuals were investigated immunochemically and using a nonradioisotopic polymerase chain reaction-based method for microsatellite detection. Probable and definite carriers frequently showed properdin levels which were in the normal range. No recombinants between the microsatellite loci and properdin deficiency were detected, thus allowing identification of the defective allele through the generations in all three pedigrees. Haplotyping for these highly polymorphic microsatellites in close physical linkage to the properdin gene can provide rapid and nonradioactive detection of carrier status and prenatal diagnosis without extensive sequencing analysis.

Genetic deficiencies of the complement system and association with disease--early components.

Genetic deficiency of one of the early components of the classical pathway of complement (C1q, C1r, C1s, C4 and C2) is often associated with clinical symptoms and immunochemical abnormalities common in idiopathic autoimmune diseases, such as lupus erythematosus, but also with an increased incidence of various, local and generalized infections. These observations are consistent with the current view of the complement system's role in handling immune complexes and combating microbial invasion. However, the absence of absolute correlations in these experiments of nature suggests that genetic defects of the classical pathway act only epistatically to other host factors and the primary etiologies of the associated diseases. In contrast, the strong association of properdin and factor D deficiency with serious infections caused by encapsulated Gram-negative bacteria suggests a more immediate involvement of the alternative pathway in a specific segment of immunity and its pathology. This concept is also supported by the primordial role of the alternative pathway in the evolution of the complement system and the apparent lethality of factor B deficiency. The gene structures of most of these early components have now been elucidated providing the basis for detailed analyses of the defective alleles, the determination of carrier status, and prenatal diagnosis.

Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme.

Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am-, acu- double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am+ (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3- mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.

An angle-variable three-dimensional pulsed field gel electrophoresis system.

A three-dimensional pulsed field electrophoretic method based on the simultaneous application of fixed and cyclically alternating polarity fields at a right angle is described. Requiring only minimal electronic hardware it provides highly homogeneous field conditions over a large gel area and the versatility to vary the pulse vector angle. The electrophoretic parameters critical to achieve fast high resolution separation over a wide range of molecular sizes have been optimized and applied to megabase-size chromosomal DNA molecules. The empirical relationships between pulse time, field strength conditions, and resolution limits derived allow selection of coordinated experimental conditions for the separation of specific DNA size ranges.

IGM-containing immune complexes and antiphospholipid antibodies in patients with Sneddon's syndrome.

We report three patients with a Sneddon syndrome in whom predominantly small (500-900 kD) IgM-containing serum immune complexes were detectable. Furthermore, antiphospholipid antibodies and increased von Willebrand factor antigen were found in the sera of two cases. Especially the data demonstrating small circulating immune complex as suggest that Sneddon's syndrome, a rare vasculitis disorder, might immunologically be characterized by circulating IgM-containing immune complexes which, in addition, could play a role in the pathogenesis of this disease entity. The elevated antiphospholipid antibodies as well as the increased von Willebrand factor antigen in the sera of the investigated patients have to be considered as nonspecific vasculitis-associated phenomena.

Improved in vitro diagnosis of allergy to Alternaria tenius and Cladosporium herbarum.

To improve the in vitro diagnosis of mould allergy 22 children suffering from allergic asthma caused by Alternaria tenius and/or Cladosporium herbarum as proven by bronchial provocation test were investigated. Partially purified, standardized mould preparations were used in radioallergosorbent test (RAST) with conventional and new update mould discs, mould-induced histamine release and immunoblotting. Updated RAST discs were found to be superior to the old-type discs for the detection of Alternaria but not Cladosporium sensitivity. In all patients except one, specific IgE-antibodies to the respective mould were demonstrated by immunoblotting. Mould-induced histamine release failed to prove sensitization in only two patients. No differences were found comparing histamine release from whole blood with release from isolated cells. The results demonstrate a high sensitivity of in vitro tests when purified and standardized extracts are used.