Britta Meyer

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.

HMGA2 overexpression in non‐small cell lung cancer

Lung cancer is still the leading cause of death from cancer worldwide primarily because of the fact that most lung cancers are diagnosed at advanced stages. Overexpression of the high mobility group protein HMGA2 has been observed in a variety of malignant tumors and often correlates with poor prognosis. Herein, HMGA2 expression levels were analyzed in matching cancerous and non‐cancerous lung samples of 17 patients with adenocarcinoma (AC) and 17 patients with squamous cell carcinoma (SCC) with real‐time quantitative RT‐PCR (qRT‐PCR). Transcript levels were compared to results obtained by immunohistochemistry (IHC). HMGA2 expression was detectable by qRT‐PCR in all samples tested and varied from 5422 to 16 991 545 copies per 250 ng total RNA in the carcinoma samples and from 289 to 525 947 copies in the non‐cancerous tissue samples. In 33/34 non‐small cell lung cancer (NSCLC) samples tested, an overexpression of HMGA2 was revealed with statistically highly significant differences between non‐neoplastic and tumor samples for both AC (P < 0.0001) as well as for SCC (P < 0.0001). Expression varies strongly and is increased up to 911‐fold for AC and up to 2504‐fold for SCC, respectively, with statistically significant higher increase in SCC (P < 0.05). The results presented herein indicate that HMGA2 overexpression is a common event in NSCLC and could serve as molecular marker for lung cancer.

Molecular characterization of the canine HMGB1

Due to the close similarities of numerous canine diseases to their human counterparts, the dog could join the mouse as the species of choice to unravel the genetic background of complex diseases as e.g. cancer and metabolic diseases. Accordingly, the role of the dog as a model for therapeutic approaches is strongly increasing. However, prerequisite for such studies is the characterization of the corresponding canine genes. Recently, the human high mobility group protein B1 (HMGB1) has attracted considerable interest of oncologists because of what is called its “double life”. Besides its function as an architectural transcription factor HMGB1 can also be secreted by certain cells and then acts as a ligand for the receptor for advanced glycation end products (RAGE). The binding of HMGB1 to RAGE can activate key cell signaling pathways, such as p38MAPK, JNK, and p42/p44MAPK emphasizing the important role of HMGB1 in inflammation and tumor metastasis. These results make HMGB1 a very interesting target for therapeutic studies done in model organisms like the dog. In this study we characterized the molecular structure of the canine HMGB1 gene on genomic and cDNA levels, its predicted protein, the gene locus and a basic expression pattern.

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.

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.

Novel Cryptic Rearrangements in Adult B-Cell Precursor Acute Lymphoblastic Leukemia Involving the MLL Gene

MLL (mixed-lineage-leukemia) gene rearrangements are typical for acute leukemia and are associated with an aggressive course of disease, with a worse outcome than comparable case, and thus require intensified treatment. Here we describe a 69-year-old female with adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) with hyperleukocytosis and immunophenotype CD10- and CD19+ with cryptic MLL rearrangements. G-banding at the time of diagnosis showed a normal karyotype: 46,XX. Molecular cytogenetics using multitude multicolor banding (mMCB) revealed a complex rearrangement of the two copies of chromosome 11. However, a locus-specific probe additionally identified that the MLL gene at 11q23.3 was disrupted, and that the 5′ region was inserted into the chromosomal sub-band 4q21; thus the aberration involved three chromosomes and five break events. Unfortunately, the patient died six months after the initial diagnosis from serious infections and severe complications. Overall, the present findings confirm that, by far not all MLL aberrations are seen by routine chromosome banding techniques and that fluorescence in situ hybridization (FISH) should be regarded as standard tool to access MLL rearrangements in patients with BCP-ALL.

A cryptic three-way translocation t(10;19;11)(p12.31;q13.31;q23.3) with a derivative Y-chromosome in an infant with acute myeloblastic leukemia (M5b)

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant transformation of hematopoietic precursors to a pathogenic cell clone. Chromosomal band 11q23 harboring MLL (=mixed lineage leukemia) gene is known to be involved in rearrangements with variety of genes as activating partners of MLL in different AML subtypes. Overall, an unfavorable prognosis is associated with MLL abnormalities. Here we investigated an 11-month-old male presenting with hyperleukocytosis being diagnosed with AML subtype FAB-M5b. In banding cytogenetics a der(19)t(19;?)(q13.3;?) and del(Y)(q11.23) were found as sole aberrations. Molecular cytogenetics revealed that the MLL gene was disrupted and even partially lost due to a t(10;19;11)(p12.31;q13.31;q23.3), an MLL/MLLT10 fusion appeared, and the der(Y) was an asymmetric inverted duplication with breakpoints in Yp11.2 and Yq11.23. The patient got hematopoietic stem cell transplantation from his haploidentical mother. Still three months afterwards 15% of blasts were detected in bone marrow and later the patient was lost during follow-up. The present case highlights the necessity to exclude MLL rearrangements, even when there seems to be no actual hint from banding cytogenetics.

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.

Reply to Jérôme Verine, Christophe Leboeuf and Philippe Ratajczak's Letter to the Editor re: Jimsgene Sanjmyatav, Sven Hauke, Mieczyslaw Gajda, et al. Establishment of a Multicolour Fluorescence In Situ Hybridisation-based Assay for Subtyping of Renal Cell Tumours. Eur Urol 2013;64:689–91

We appreciate the comments by Verine and colleagues regarding our paper [1]. The authors correctly state that a recent online survey by the International Society of Urological Pathology (ISUP) demonstrated that fluorescence in situ hybridization (FISH) is not commonly used in the diagnostic algorithms of practicing pathologists [2]. However, wewould conclude that this fact underscores the need to prove the impact of genetic analysis based on FISH tests, as we performed in our study. Does FISH help to overcome the limitations of pathologic classification? Because the paper was accepted as a short communication, it was impossible to present detailed data of our FISH results in each tumor sample. In our retrospective validation study, we used cases with unequivocal histopathologic diagnosis. As we discussed in the paper, it is especially useful to identify specific genetic alterations to distinguish poorly differentiated clear cell renal cell carcinoma (RCC) from variants of papillary type 2 RCC. Although there was a genetic overlap between these entities, a clear diagnosis could be made in the majority of cases on the basis of our FISH analysis. An overlap was also found between chromophobe RCC and oncocytomas. Histologically defined chromophobe RCCs were correctly identified based on FISH. In some oncocytomas, however, multiple chromosomal losses were detected in small numbers of cell nuclei in contrast to chromophobe RCCs, with >50% of altered nuclei per tumor. The oncocytomas with several chromosomal losses were independently evaluated by three pathologists, showing some interobserver variability in the classification of such tumors with equivocal FISH results. Further studies are necessary to characterize this specific subgroup of oncocytomas, which seems to be in a close relationshipwith chromophobe RCC. At the Vancouver ISUP conference, consensus was that such hybrid oncocytic chromophobe tumors should be placed, for the time being, in the chromophobe RCC category [3].