Sven Hauke

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule.

The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

Fibroblast growth factor receptor (FGFR) gene amplifications are rare events in bladder cancer

Activating point mutations and protein overexpression of fibroblast growth factor receptors (FGFRs), especially FGFR3, are frequent events in bladder cancer. Little is known about gene amplifications, therefore we characterized amplification of FGFR1‐3 by fluorescence in‐situ hybridization (FISH).

Losses of 3p14 and 9p21 as shown by fluorescence in situ hybridization are early events in tumorigenesis of oral squamous cell carcinoma and already occur in simple keratosis.

Tumorigenesis of oral squamous cell carcinoma (OSCC) has been postulated to represent a multistep process driven by the accumulation of carcinogen‐induced genetic changes. Alterations of the 3p14 fragile site containing the fragile histidine triade gene and of the 9p21 tumor suppressor locus containing methylthioadenosine phosphorylase, p16 and p15 characteristically occur in oral leukoplakia, a known precursor of OSCC, and are at present considered to indicate the transition from simple keratosis (hyperplasia) to dysplasia. The aim of the study was to evaluate the occurrence of losses of 3p14 and 9p21 and to evaluate polysomies 3 and 9 in leukoplakias using highly sensitive fluorescence in situ hybridization (FISH) probes. Examining 67 leukoplakias (24 hyperplasias, 33 dysplasias, 10 in situ carcinomas), control tissues of oral mucosa from infants and adults as well as invasive carcinomas and normal epithelia of tumor patients with locus specific FISH probes targeting 3p14 and 9p21, and centromeric probes for chromosomes 3 and 9 we could demonstrate that losses of these sites appeared very early in the tumorigenesis of OSCC and were already present in the great majority of simple keratoses. Polysomy 3 occurring more frequently than polysomy 9 was characteristic of dysplasia and in situ carcinomas and thus seems to follow losses of 3p14 and 9p21 during oral squamous cell carcinogenesis.

Chromosomal rearrangements leading to abnormal splicing within intron 4 of HMGIC

Fusion of the high‐mobility group protein gene HMGIC to other genes due to chromosomal rearrangements occurs in a variety of human benign tumors. In contrast to genes clearly derived from other chromosomes, some of the ectopic sequences fused to HMGIC have been assigned to chromosome 12 by CASH (chromosome assignment using somatic cell hybrids) analyses and thus can be assumed either to result from alternative splicing or to represent true ectopic sequences derived from other genes on chromosome 12. In an attempt to identify the ectopic sequences fused to this exon, we have sequenced the entire intron 4. Four of seven ectopic sequences previously described to be fused to exon 4 of HMGIC in different tumors were found to be located within intron 4 of the gene and thus are due to abnormal splicing. As for a mechanism explaining this observation, it can be suggested that breakpoints of chromosomal aberrations not directly disrupting HMGIC may induce small genomic alterations in their vicinity and thus facilitate abnormal splicing. The latter mechanism may underlie the development of part of the neoplasms characterized by 12q14–15 rearrangements.

Molecular and clinicopathological analysis of dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous tumor of intermediate malignancy. The most remarkable cytogenetic feature of DFSP is the chromosomal translocation t(17;22)(q22;q13), causing a fusion of the platelet-derived growth factor beta chain (PDGFB) gene at 22q13, and the collagen type 1 alpha 1 (COL1A1) at 17q22. The aim of the study was to analyze the molecular characteristic of DFSP in conjunction with histopathological and clinical features. We performed fluorescence in situ hybridization (FISH) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to detect chromosomal translocations and fusion gene transcripts in 16 formalin-fixed, paraffin-embedded DFSP samples. In addition, the amplification of PDGFB was also evaluated in the 16 DFSP samples by real-time PCR. FISH analysis revealed that all the 16 samples exhibited COL1A1-PDGFB gene fusion. Eleven out of 11 informative cases (100%) showed fusion transcripts by multiplex RT-PCR analysis. Various exons of the COL1A1 gene were fused with the PDGFB gene. Among them, exon 25 was found to be more frequently involved. Real-time PCR showed that the PDGFB copy number increase in the DFSP samples was higher than in normal skin tissues (p=0.007). Values of FISH fusion signals and PDGFB DNA analysis were variable between samples, but suggested that increased values might be associated with parameters of tumor progression. Our results confirm that analysis of the COL1A1-PDGFB status by FISH and RT-PCR is a useful tool in the confirmation of a DFSP diagnosis. In addition, the analysis of PDGFB copy number status may become a useful diagnostic marker since the gene is a potential target for treatment of DFSP patients.

Sequencing of intron 3 of HMGA2 uncovers the existence of a novel exon

Aberrations affecting the gene encoding the high mobility group protein HMGA2 (formerly HMGIC) have been found in a variety of human tumors, e.g., uterine leiomyomas, lipomas, and pulmonary chondroid hamartomas. These aberrations lead to fusion genes, transcriptional up‐regulation, or aberrant transcripts of HMGA2. In the latter case, truncated transcripts consisting of exons 1 to 3 of HMGA2, encoding the three DNA‐binding domains, and ectopic sequences derived from chromosome 12 are frequent. There are several lines of evidence indicating that the biological and tumorigenic features of truncated HMGA2 derivatives, i.e., those composed of the DNA‐binding domains and a shortened acidic tail, clearly differ from those of the normal protein consisting of three DNA‐binding domains and one large acidic tail. By sequencing the complete 112 kb third intron of HMGA2, we were able to detect several of the ectopic sequences, known as fused to HMGA2. Expression studies revealed co‐expression of one of these transcripts with the normal transcript in tumors with 12q14‐15 aberrations as well as in other tumors, and in normal tissues. Thus, this transcript (HMGA2b) is flanked by an alternative terminal exon of HMGA2. Due to the loss of the part encoding the acidic tail, the expression of the latter transcript may have more striking effects than the “wild type” HMGA2 (HMGA2a) in terms of tumorigenesis. This finding clearly indicates that functional studies also should address the role of the HMGA2b transcript.

Establishment of a Multicolour Fluorescence In Situ Hybridisation-based Assay for Subtyping of Renal Cell Tumours

MATERIAL & METHODS: We composed 2 different multicolour FISH assays. Probe set 1 contained VHL gene region labelled in green, red-labelled chromosomal region 1p12, goldlabelled centromeric region of chromosome 7 and blue-labelled centromere of chromosome 17. Probe set 2 was composed of blue labelled chromosomal 2q11, gold-labelled centromere of chromosome 6 and green/orange labelled translocation probe for region 11q13. Overall 61 tumors were analyzed by FISH: 26 clear cell renal cell carcinomas (ccRCC), 10 papillary (pRCC), 13 chromophobe (chRCC) tumors and 12 renal oncocytomas (ROs). As reference to FISH analysis we carried out CGH for all tumors.

HER2 FISH results in breast cancers with increased CEN17 signals using alternative chromosome 17 probes - reclassifying cases in the equivocal category.

HER2 testing of invasive breast cancer by in‐situ hybridization guides therapy decisions. Probing HER2 and centromere of chromosome 17 (cen17) simultaneously is supposed to reveal both a potential HER2 gene amplification and polysomy 17. However, a considerable number of breast cancer patients with quasi polysomy 17 are considered ‘equivocal’, which is diagnostically meaningless. Moreover, patients with equivocal/false polysomic tumours are prevented from a potentially beneficial anti‐HER2 treatment. Here we evaluated the RAI1, D17S122 and TP53 hybridization markers to indicate true polysomy reliably and to reclassify equivocal samples accurately as HER2‐positive.

The FlexISH assay brings flexibility to cytogenetic HER2 testing

Fluorescence in‐situ hybridization (FISH) is the method of choice for quantitative human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) gene testing in invasive breast cancer. HER2 testing has great clinical impact, and is often claimed to expeditiously complete the entire diagnostic procedure for an individual patient. Against this background, the aim of this study was to evaluate the usefulness and performance of a novel dual‐colour HER2/cen17 FISH assay designed to facilitate flexible (overnight) and rapid (<2 h of hybridization) FISH.

MP30-05 DETECTION OF TRANSLOCATION TUMOURS ON RENAL CELL CARCINOMA TISSUE MICRO ARRAYS BY FISH

(i.e., younger age and high BMI) in Group 2 were pathologically diagnosed with benign tumors. CONCLUSIONS: These findings suggest that preoperative findings on enhanced CT could be the most useful information for predicting final pathological diagnosis in patients with SRMs suspicious for RCC. However, the proportion of patients with benign tumors was shown to be comparatively high in those with CT findings atypical for CCRCC; therefore, it should be recommended to consider other clinical parameters, such as age and BMI, when determining therapeutic option for those with such SRMs.