MicroRNA miR-371a-3p in serum of patients with germ cell tumours: evaluations for establishing a serum biomarker.

As only 60% of the patients with germ cell tumour (GCT) express the classical markers, new markers as for example microRNAs (miRNAs) are required. One promising candidate is miR-371a-3p, but data are sparse to date. We measured serum levels of miR-371a-3p in GCT patients, in controls, and in cases with other malignancies. We also assessed the expression in other body fluids and we looked to the decline of serum miR-371a-3p levels after treatment. miR-371a-3p levels were measured by quantitative polymerase chain reaction in serum samples of 25 GCT patients, 6 testicular intraepithelial neoplasia (TIN) patients, 20 healthy males and 24 non-testicular malignancies (NTMs). Testicular vein blood (TVB) was examined in five GCT patients and five controls. Five GCT patients had serial daily measurements after orchiectomy. Five seminal plasma samples, three urine specimens and one pleural effusion fluid were processed likewise. GCT patients had significantly higher miR-371a-3p serum levels than controls and NTMs. Serum levels of controls, TINs and NTMs were not significantly different. TVB samples of GCT patients had 65.4-fold higher serum levels than peripheral blood. Malignant pleural effusion fluid had extremely high levels of miR-371a-3p, seminal plasma had strongly elevated levels by comparison with serum levels of controls. In urine of GCT patients, no miR-371a-3p expression was detected. Daily measurements after orchiectomy in stage 1 patients revealed a decline by 95% within 24 h. Serum levels of miR-371a-3p appear to be a promising specific biomarker of GCTs as is suggested by high serum levels in GCT patients, the rapid return of elevated levels to normal range after treatment, the association of serum levels with tumour bulk, the non-expression in NTMs and the much higher levels of miR-371a-3p in TVB. This potential marker deserves further exploration in a large-scale clinical study.

MicroRNAs miR-371-3 in serum as diagnostic tools in the management of testicular germ cell tumours.

BACKGROUND: miRNAs are small noncoding RNA molecules that can be released into body fluids. Germ cell tumours (GCTs) overexpress miRNAs of the miR-371-3 cluster. Thus, serum levels of these miRNAs may correlate with tumour load. METHODS: miRNAs of the miR-371-3 cluster were quantified in cubital vein blood samples of 20 GCT patients with clinical stage 1, and of 4 patients with advanced stages before and after treatment. In six patients testicular vein blood (TVB) was examined additionally. Seventeen healthy males served as controls. Likewise, expression of miRNAs in 15 matching tumour specimens was measured. RESULTS: In all patients, serum levels of miRNAs 371-3 were much higher than in controls. In stage 1, levels decreased postoperatively 336.7-fold, 7.4-fold, and 7.7-fold for miRNAs 371a-3p, 372, and 373-3p, respectively (P<0.01). Also, in those cases with advanced disease levels dropped to the normal range after completion of treatment. miR-371-3 levels in TVB exceeded those in peripheral blood in all cases. Expression of miR-371a-3p was also documented in tumour tissue. However, no correlation was found regarding the extent of miRNA expression in tissue and the values measured in matching serum. CONCLUSION: Thus, miR-371a-3p serum level appears to be a useful biomarker in GCTs.

Chromosome analyses in dogs.

Cytogenetics is the study of normal and abnormal chromosomes. Every species is characterized by a given number of chromosomes that can be recognized by their specific shape. The chromosomes are arranged according to standard classification schemes for the respective species. While pre- and postnatal chromosome analyses investigate the constitutional karyotype, tumor cytogenetics is focused on the detection of clonal acquired, tumor-associated chromosome aberrations. Cytogenetic investigations in dogs are of great value especially for breeders dealing with fertility problems within their pedigrees, for veterinarians and last but not least for the dog owners. Dogs and humans share a variety of genetic diseases, including cancer. Thus, the dog has become an increasingly important model for genetic diseases. However, cytogenetic analyses of canine cells are complicated by the complex karyotype of the dog. Only just 15 years ago, a standard classification scheme for the complete canine karyotype was established. For chromosome analyses of canine cells the same steps of chromosome preparation are used as in human cytogenetics. There are few reports about cytogenetic changes in non-neoplastic cells, involving predominantly the sex chromosomes. Cytogenetic analyses of different entities of canine tumors revealed that, comparable to human tumors, tumors of the dog are often characterized by clonal chromosome aberrations, which might be used as diagnostic and prognostic markers. The integration of modern techniques (molecular genetic approaches, adaptive computer programs) will facilitate and complete conventional cytogenetic studies. However, conventional cytogenetics is still non-replaceable.

Genetic characterization of dogs via chromosomal analysis and array-based comparative genomic hybridization (aCGH).

The results of cytogenetic and molecular cytogenetic investigations revealed similarities in genetic background and biological behaviour between tumours and genetic diseases of humans and dogs. These findings classify the dog a good and accepted model for human cancers such as osteosarcomas, mammary carcinomas, oral melanomas and others. With the appearance of new studies and advances in canine genome sequencing, the number of known homologies in diseases between these species raised and still is expected to increase. In this context, array-based comparative genomic hybridization (aCGH) provides a novel tool to rapidly characterize numerical aberrations in canine tumours or to detect copy number aberrations between different breeds. As it is possible to spot probes covering the whole genome on each chip to discover copy number aberrations of all chromosomes simultaneously, this method is time-saving and cost-effective - considering the relation of costs and the amount of data obtained. Complemented with traditional methods like karyotyping and fluorescence in situ hybridization (FISH) analyses, the aCGH is able to provide new insights into the underlying causes of canine carcinogenesis.

Cytogenetic analysis of CpG-oligonucleotide DSP30 plus Interleukin-2-Stimulated canine B-Cell lymphoma cells reveals the loss of one X Chromosome as the sole abnormality.

Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.

High-mobility group protein HMGA2-derived fragments stimulate the proliferation of chondrocytes and adipose tissue-derived stem cells.

In previous research, it was shown that recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro, opening up promising applications of this embryonic architectural transcription factor for tissue engineering, such as in cartilage repair. In this paper, we describe the development and analyses of two synthetic fragments comprising the functional AT-hook motifs of the HMGA2 protein, as well as the nuclear transport domain. They can be synthesised up to large scales, while eliminating some of the problems of recombinant protein production, including unwanted modification or contamination by the expression hosts, or of gene therapy approaches such as uncontrolled viral integration and transgene expression even after therapy. Application of one of these peptides onto porcine hyaline cartilage chondrocytes, grown in in vitro monolayer cell culture, showed a growth-promoting effect similar to that of the wild type HMGA2 protein. Furthermore, it also promoted cell growth of adult adipose tissue derived stem cells. Due to its proliferation inducing function and vast availability, this peptide is thus suitable for further application and investigation in various fields such as tissue engineering and stem cell research.

Two new cases of polysomy 13 in canine prostate cancer.

Besides man, the dog is the only known mammalian species that spontaneously develops carcinomas of the prostate with considerable frequency. For this reason, the dog is considered to be the only useful animal model for spontaneously occurring prostate malignancies in man. Cytogenetic investigations of human prostate cancers have revealed the frequent occurrence of trisomies 7, 8, and 17. Chromosome analyses of canine prostate carcinomas are rare. In this report we present 2 cases of canine prostate cancer showing a clonal polysomy 13 along with complex karyotype changes. Along with a previous report demonstrating polysomy 13 as the only karyotype deviation in a canine prostate cancer the present report supports the hypothesis that in canine prostate cancer, polysomy 13 is a recurrent cytogenetic aberration linked to the development of the disease. As human chromosomes (HSA) 8q and 4q and the canine chromosome (CFA) 13 share high homology, these results suggest that a conserved area on these chromosomes is involved in tumorigenesis in both species.

Expression of the high mobility group A1 (HMGA1) and A2 (HMGA2) genes in canine lymphoma: analysis of 23 cases and comparison to control cases.

Overexpression of high mobility group A (HMGA) genes was described as a prognostic marker in different human malignancies, but its role in canine haematopoietic malignancies was unknown so far. The objective of this study was to analyse HMGA1 and HMGA2 gene expression in lymph nodes of canine lymphoma patients. The expression of HMGA1 and HMGA2 was analysed in lymph node samples of 23 dogs with lymphoma and three control dogs using relative quantitative real-time RT-PCR. Relative quantity of HMGA1 was significantly higher in dogs with lymphoma compared with reference samples. HMGA2 expression did not differ between lymphoma and control dogs. With the exception of immunophenotype, comparison of disease parameters did not display any differences in HMGA1 and HMGA2 expression. The present findings indicate a role of HMGA genes in canine lymphoma. This study represents the basis for future veterinary and comparative studies dealing with their diagnostic, prognostic and therapeutic values.

High-mobility group B1 proteins in canine lymphoma: prognostic value of initial and sequential serum levels in treatment outcome following combination chemotherapy.

Elevated high-mobility group box 1 (HMGB1) levels have been demonstrated in different human neoplasias. Information on serum HMGB1 before and during chemotherapy is lacking, as is data pertaining to its prognostic significance. The aim of this study was to characterize serum HMGB1 level in dogs with lymphoma and to assess its influence on the outcome following chemotherapy. Serum HMGB1 concentrations were measured in 16 dogs with lymphoma before treatment (W1) and on weeks 2 (W2), 6 (W6) and 12 (W12) of treatment with chemotherapy. Initial serum HMGB1 levels were significantly higher than HMGB1concentrations in control dogs and the levels in W2, W6 and W12. HMGB1-W1 concentrations were lower in dogs achieving complete remission than that in the single dog with partial remission. The ratio W12/W6 exhibited significant influence on remission duration. In these dogs with lymphoma, serum HMGB1 was elevated in comparison with that in controls. Initial serum HMGB1 level and its modulation during treatment may possess prognostic value.

Inhibition of caspase-3 differentially affects vascular smooth muscle cell apoptosis in the concave versus convex aortic sites in ascending aneurysms with a bicuspid aortic valve.

Apoptosis of vascular smooth muscle cells (VSMCs) is involved in bicuspid aortic valve (BAV) ascending aorta aneurysms characteristically affecting the convex site. Caspase-3 is a pivotal effector of the apoptosis machinery. The aim of this study was to investigate the impact of an inhibited caspase-3 pathway on apoptosis in convex and concave sites VSMCs of ascending aortic tissue in vitro. Specimens from the convex and concave sites of ascending aortic aneurysm were collected from nine patients with BAV (mean age 58.7+/-14.8). Cultured VSMCs were characterized morphologically and immunohistochemically. Apoptosis activity was measured in VSMCs using Annexin V-APC with propidium iodide nuclear staining in flow cytometry. To investigate apoptotic modulation, caspase-3 was inhibited by N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Apoptosis was initiated by calcium chloride. Inhibition of caspase-3 with Ac-DEVD-CHO protected VSMCs against calcium chloride apoptosis significantly more in the concave site than in the convex site (25.8+/-9.8 versus 38.5+/-8.0% apoptotic cells, p=0.01). Morphological scanning using light microscopy revealed typical VSMCs. We provide evidence that VSMCs show a different behavior with respect to apoptosis in the concave versus the convex sites in BAV ascending aortic aneurysm. Inhibition of caspase-3 resulted in a significantly increased protection of VSMCs against apoptosis in the concave site compared with the convex site in ascending aortic aneurysm in BAV. These findings may have some implications on understanding aneurysmal formation and its potential modulation.

[Molecular markers in salivary gland tumors: their use in diagnostic and prognostic workup]. / Molekulare Marker in Speicheldrüsentumoren: Einsatz zur Diagnose und prognostischen Bewertung.

The molecular genetic background of salivary gland neoplasms has not been characterized in detail to date. However, interesting target genes which could be used as prognostic and diagnostic molecular biomarkers have already been identified, e.g. CRTC1-MAML2 in mucoepidermoid carcinoma, or PLAG1 and HMGA2 in pleomorphic adenoma. In particular, CRTC1-MAML2 has shown strong diagnostic and prognostic potential in recent years. One of the major advantages of molecular tumor markers is that valid results are obtained on minute cell and/or tissue samples. Due to high-throughput techniques like comparative genome hybridization (CGH), micro- or gene profiling array detection of new marker genes can be expected in the future. This is also true for the most frequent malignant salivary gland tumors after the mucoepidermoid carcinoma, i.e. adenoid cystic carcinomas and acinic cell carcinomas.

Cartilage replacement in dogs -- A preliminary investigation of colonization of ceramic matrices.

The objective of this study was to examine the behaviour of canine chondrocytes following colonisation of a beta-tricalcium phosphate (beta-TCP, Cerasorbâ, Curasan) matrix. In total, five of these cylinders were inoculated with 1.5 ml of cell suspension and subsequently incubated for about one week. In the second part of the experiment, another five Cerasorbâ cylinders were each studded with two cartilage chips of variable size and then incubated for about one week. The series of experiments were analyzed using cell staining and imaging techniques that included scanning electron microscopy. Cell migration onto the matrix was proven for both colonisation methods. It was observed that colonising the cylinders by pipetting cell suspension on them produced far better results, with respect to both growth rate and spreading of the cells, than did colonisation by studding with cartilage chips. A homogenous, surface-covering colonisation with predominantly living cells was demonstrated by scanning electron microscopy in the chondrocyte morphology. In comparison to cell-culture controls, there was a clearly better colonisation, with cells attached to both the material's primary grains and its micropores. The ceramic studied is well accepted by canine chondrocytes, and appears to be fundamentally well-suited as a matrix for bio-artificial bone-cartilage replacement. Additional qualitative analyses and a series of experiments aiming to accelerate cell proliferation are planned for subsequent studies.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent.

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

Cell-free DNA in amniotic fluid remains to be attached to HMGA2-implications for noninvasive prenatal diagnosis.

OBJECTIVE: The expression of the high mobility group protein gene HMGA2 is primarily confined to embryonic and fetal cells. The aim of this study was to determine the relative expression level of HMGA2 in cells of amniotic fluid samples. Furthermore, it should be investigated by chromatin immunoprecipitation whether or not HMGA2 is attached to cell-free DNA in amniotic fluid. METHOD: Expression levels of HMGA2 in 58 amniotic fluid samples from the second trimester were measured using quantitative real-time polymerase chain reaction (PCR). Furthermore, the presence of HMGA2, attached to cell-free DNA was tested by chromatin immunoprecipitation. RESULTS: Expression of HMGA2 was detected in all samples, but in cells of the amniotic fluid it was 161-fold higher than in cells of the urine from healthy donors. The real-time PCR with GAPDH showed a signal in all samples treated with the improved protocol of immunoprecipitation. CONCLUSION: Our data clearly show that cells of the amniotic fluid strongly overexpress HMGA2 according to their fetal origin. The fact that apparently HMGA2 remains to be attached to cell-free DNA suggests interesting new approaches in noninvasive prenatal diagnosis.

High Mobility Group Box 1-Protein expression in canine haematopoietic cells and influence on canine peripheral blood mononuclear cell proliferative activity.

High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in mammalian tissues. HMGB1 demonstrates cytokine function and induces inflammation when actively released by haematopoietic cells or passively released during cell necrosis. This study aimed at the determination of HMGB1 expression in different cell types and at the evaluation of the role of HMGB1 in PBMC proliferation. Therefore we investigated the HMGB1 mRNA expression level in different canine haematopoietic cell types and the influence of exogenous rhHMGB1 on canine PBMC proliferation. Differentiated haematopoietic blood cells showed lower relative HMGB1 expression levels compared to CD34+ haematopoietic stem cells. Relative HMGB1 expression seemed also to decrease during differentiation of CD34+ stem cells into dendritic cells. Furthermore, peripheral blood CD14+ monocytes and granulocytes showed a lower relative HMGB1 expression in comparison to CD3+ T-lymphocytes. When exogenous rhHMGB1 at low concentrations was added to single PBMC cultures an increase of proliferation was obvious. However, in higher concentrations HMGB1 lost its stimulative effect. In conclusion, HMGB1 is broadly expressed in canine haematopoietic cells with highest levels in haematopoietic stem cells. HMGB1 induced directly PBMC proliferation.

Quantitative expression analysis in peripheral blood of patients with chronic myeloid leukaemia: correlation between HMGA2 expression and white blood cell count.

The architectural transcription factor HMGA2 is highly expressed during embryogenesis but scarcely detectable in non-dividing adult cells. Previously, HMGA2 re-expression was detected in blood from CML patients by conventional RT-PCR, while blood samples from healthy volunteers were HMGA2 negative. Using the sensitive method of real-time quantitative RT-PCR, herein HMGA2 expression was detectable not only in peripheral blood from leukaemia patients but also in blood from healthy donors. Statistical analysis revealed a highly significant correlation between white blood cell count and HMGA2 transcript levels. The results indicate that up-regulation of HMGA2 expression is correlated to the undifferentiated phenotype of leukaemic cells accumulating during progression of chronic phase to blast crisis.

Anaplasma phagocytophilum in dogs in Germany.

A total number of 111 dogs were included in the present prospective study investigating the prevalence of Anaplasma phagocytophilum in dogs in Germany. Dogs were divided into two groups. Dogs of group 1 (n = 49) showed clinical and/or haematological signs seen in infections with A. phagocytophilum, whereas those of group 2 (n = 62) did not have any evidence of anaplasmosis. For each dog, an A. phagocytophilum 16S rRNA-nested polymerase chain reaction (PCR) of ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood analysis, a microscopic evaluation of a buffy coat and a serum indirect fluorescent antibody test (IFAT) were performed. Forty-eight seroreactive dogs were identified altogether, which amounts to an overall point prevalence of 43.2%. There was no significant difference between the seroreactivity to A. phagocytophilum antigens among group 1 (44.9%) and 2 (41.9%) (P > 0.5). Seven dogs (6.3%) had positive PCR results. All of them were seroreactive. Six belonged to group 1. Morulae in neutrophilic granulocytes were found in two dogs of group 1 but in none of group 2. Both dogs were seroreactive. Very high antibody titres (> or =1:1024) were detected significantly more frequently in dogs with clinical signs attributable to infection with A. phagocytophilum (group 1) than in those without (group 2) (P < 0.001). There was no significant correlation of overall positives or antibody titres to age, breed, sex, or whether the dogs were family or working dogs. Dogs with high tick infestation were significantly more often seroreactive to A. phagocytophilum than those with no or low tick infestation (P = 0.007). In conclusion, there seems to be a high risk of infection with A. phagocytophilum in Germany. Results of this study suggest that severe illness solely caused by A. phagocytophilum may be possible although definitive evidence does not exist. Very high antibody titres (>1:1024) may be associated with clinical anaplasmosis.

Evidence for a 3p25 breakpoint hot spot region in thyroid tumors of follicular origin.

Epithelial tumors of the thyroid are cytogenetically well-investigated tumors. So far, the main cytogenetic subgroups, characterized by trisomy 7 and by rearrangements of either 19q13 or 2p21, respectively, have been described. Recently, we have been able to describe the involvement of a novel gene called THADA in benign thyroid lesions with 2p21 rearrangements. Other fusion genes found in thyroid lesions are RET/PTC and PAX8/PPAR(gamma). The latter occurs in follicular thyroid carcinomas with a t(2;3)(q13;p25). Here we present molecular-cytogenetic and cytogenetic investigations on a follicular thyroid adenoma with a t(2;20;3)(p21;q11.2; p25). In this case, an intronic sequence of PPAR(gamma) is fused to exon 28 of THADA. We used BAC clones containing the genomic sequence of PPARgamma for fluorescence in situ hybridization to confirm the localization of the breakpoint within intron 2 of PPAR(gamma) . Our findings suggest that the close surrounding of PPAR(gamma) is a breakpoint hot spot region, leading to recurrent alterations of this gene in thyroid tumors of follicular origin including carcinomas as well as adenomas with or without involvement of PAX8.

[Research into the human genome driven by improved methods]. / Moderne Methoden in der Genomforschung und Humangenetik: Beispiele und Anwendungsbereiche.

The enormous progress made by research of the human genome is mainly driven by newly established or improved methods for the analysis of nucleic acids and proteins. Among the methods that have gained a wide-spread use within a comparably short time are fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) including methods for quantitative PCR, and the use of short interfering RNA (siRNA) molecules aimed at gene silencing. The increasing significance of the analysis of secondary modifications of nucleic acids and proteins (genomic imprinting by DNA methylation, posttranslational protein modification) is reflected by an increasing use of mass spectrometry for the analysis and characterization of these biomolecules. Overall, in the future the research into the human genome and the interpretation of data will further benefit from these and other refined tools.

RAS gene hot-spot mutations in canine neoplasias.

Point mutations in the cellular homologues HRAS, KRAS2, and NRAS of the viral Harvey and Kirsten rat sarcoma virus oncogenes are commonly involved in the onset of malignancies in humans and other species such as dog, mouse, and rat. Most often, three particular hot-spot codons are affected, with one amino acid exchange being sufficient for the induction of tumor growth. While RAS genes have been shown to play an important role in canine tumors such as non-small lung cell carcinomas, data about RAS mutations in canine fibrosarcomas as well as KRAS2 mutations in canine melanomas is sparse. To increase the number of tumors examined, we recently screened 13 canine fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot spots. The results were compared to the already existing data from other studies about these tumors in dogs.