Axel Wellmann

Diagnostic and Prognostic Information in Prostate Cancer with the Help of a Small Set of Hypermethylated Gene Loci

Purpose: Our study was designed to evaluate promoter CpG island hypermethylation in the diagnosis and prognosis of prostate cancer. Experimental Design: Primary prostate cancers from 53 patients, pelvic lymph nodes, noncancerous prostate tissues, and prostate cell lines were analyzed. Real-time methylation-specific PCR was used to identify CpG island hypermethylation at five promising gene loci (i.e., GSTP1, APC, PTGS2, MDR1, and RASSF1a). Results: At three gene loci (GSTP1, APC, and PTGS1) and CpG island, hypermethylation was highly prevalent in prostate cancers (71-91%), and analysis of receiver operator curves showed that hypermethylation at these three gene loci can distinguish between prostate cancer and noncancerous prostatic tissue (i.e., benign hyperplasia) with a sensitivity of 71.1% to 96.2% and a specificity of 92.9% to 100%. Using sensitive SYBR green methylation-specific PCR technology, we observed a respective 28% and 71% hypermethylation rate at the RASSF1a and MDR1 loci in benign prostate hyperplasia, which may represent early nonaggressive carcinogenesis. Methylation characteristics in prostate cancer metastases (i.e., pelvic lymph nodes) were comparable to the respective primary cancer. Statistical analysis showed no correlation between the methylation status of a single gene locus and clinicopathologic variables (e.g., preoperative prostate specific antigen levels, Gleason score, capsular penetration, involvement of seminal vesicle, and age). In contrast, the methylation of two (GSTP1/APC; GSTP1/PTGS2) or three (GSTP1/APC/PTGS2) gene loci correlated with prognostic indicators (i.e., pathologic stage, extraprostatic extension, and Gleason score, but not with prostate specific antigen levels). Conclusions: Our data suggest that the evaluation of DNA hypermethylation at three gene loci (i.e., GSTP1, APC, and PTGS2) is of diagnostic and prognostic value in prostate cancer.

The Ets-1 transcription factor is up-regulated together with MMP 1 and MMP 9 in the stroma of pre-invasive breast cancer.

The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix‐degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets‐1 transcription factor transactivates several genes encoding matrix‐degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets‐1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up‐regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets‐1 for both angiogenesis and tumour invasion, via matrix‐degrading proteases which are already expressed during the early stages of breast carcinogenesis. Copyright

CSE1L/CAS: Its role in proliferation and apoptosis

CAS/CSE1L is the human homologue of the yeast gene CSE1. It was first cloned while searching for genes that rendered breast cancer cells resistant towards toxin induced apoptosis. Since depletion of CSE1 leads to cell-cycle arrest, CAS is thought to be involved in proliferation. CAS functions in the mitotic spindle checkpoint. CAS is located on chromosome 20q13, a locus often amplified in cancers of various origin, e.g. colonic or breast cancer. Since genetic instability is a hallmark of cancer, amplification or over expression of the CAS gene might interfere with or override its role in the mitotic spindle checkpoint. CAS is also implicated in the nuclear to cytoplasmic reshuffling of importin α, which itself is necessary for the nuclear transport of several proliferation activating proteins, transcription factors, oncogene and tumor suppressor gene products such as p53 and BRCA1. Inhibition of MEK1 mediated phosphorylation has been shown to enhance paclitaxel (Taxol) induced apoptosis in breast, ovarian, and lung tumor cell lines in-vitro. Since CAS is also phosphorylated (activated) by MEK1, and since the anti-cancer drug Taxol alters the microtubule assembly and activates pro-apoptotic signaling pathways, altering the activity/phosphorylation status of CAS via MEK1 inhibition may present a potential strategy in experimental cancer therapy.

Stromal expression of invasion-promoting, matrix-degrading proteases MMP-1 and -9 and the Ets 1 transcription factor in HNPCC carcinomas and sporadic colorectal cancers.

Hereditary nonpolyposis colorectal cancers (HNPCCs) are an important subgroup of colorectal carcinomas. Compared to sporadic variants, they present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis. This contrasts to the generally poor differentiation of the epithelial tumor component. Since matrix‐degrading proteases secreted by stromal fibroblasts contribute significantly to tumor invasion, we analyzed the stromal expression of 2 matrix metalloproteinases (MMP‐1 and ‐9) and of one of their regulators, the Ets 1 transcription factor, by both immunohistochemistry and in situ hybridization in sporadic colorectal carcinomas and HNPCC tumors. We found that MMP‐1 and ‐9 as well as Ets 1 are upregulated in the fibroblastic stroma during the development from sporadic adenomas to invasive carcinomas. HNPCC tumors exhibited a significantly lower expression of Ets 1, MMP‐1 and ‐9. These findings on the basis of lower matrix‐degrading properties of the fibroblastic tumor stroma in HNPCC tumors might help to explain why, in spite of their less differentiated phenotype, HNPCC tumors have a less invasive and metastatic potential compared to sporadic cancers.

The Human Trithorax Protein hASH2 Functions as an Oncoprotein

Regulation of chromatin is an important aspect of controlling promoter activity and gene expression. Posttranslational modifications of core histones allow proteins associated with gene transcription to access chromatin. Closely associated with promoters of actively transcribed genes, trimethylation of histone H3 at lysine 4 (H3K4me3) is a core histone mark set by several protein complexes. Some of these protein complexes contain the trithorax protein ASH2 combined with the MLL oncoproteins. We identified human ASH2 in a complex with the oncoprotein MYC. This finding, together with the observation that hASH2 interacts with MLL, led us to test whether hASH2 itself is involved in transformation. We observed that hASH2 cooperates with Ha-RAS to transform primary rat embryo fibroblasts (REF). Furthermore, transformation of REFs by MYC and Ha-RAS required the presence of rAsh2. In an animal model, the hASH2/Ha-RAS-transformed REFs formed rapidly growing tumors characteristic of fibrosarcomas that, compared with tumors derived from MYC/Ha-RAS transformed cells, were poorly differentiated. This finding suggests that ASH2 functions as an oncoprotein. Although hASH2 expression at the mRNA level was generally not deregulated, hASH2 protein expression was increased in most human tumors and tumor cell lines. In addition, knockdown of hASH2 inhibited tumor cell proliferation. Taken together, these observations define hASH2 as a novel oncoprotein.

Application of MALDI imaging for the diagnosis of classical Hodgkin lymphoma

Hodgkin lymphoma (HL) is a distinctive lymphoma subtype characterized by rareness of tumor cells [Hodgkin’s and Reed-Sternberg (HRS) cells in classical HL and lymphocytic and histiocytic cells in lymphocyte predominant HL] as well as the vast majority of the surrounding inflammatory-like cellular infiltrate. Still the onset of this highly variable disease is not completely understood. Proteome analysis can lead to the identification of potential proteins capable of elucidating malignant growth and survival in HL. Especially MALDI imaging could result in pinpointing differentially expressed proteins, which might represent potential marker molecules. In this study, we were able to distinguish between classical Hodgkin lymphoma and lymphadenitis with a sensitivity and specificity of 83.92 and 89.37%, respectively.

Downregulation of clusterin expression in testicular germ cell tumours.

Objectives: Clusterin is implicated in many biological processes including cell adhesion, apoptosis and transformation. Clusterin expression was demonstrated during sperm maturation and is overexpressed in different malignancies including breast and prostate carcinomas and anaplastic large-cell lymphomas. Methods: The aim of this study was to determine the expression of clusterin in a series of different germ cell tumours by immunohistochemistry. Twenty-two seminomas, 27 embryonal carcinomas, 22 mature and immature teratomas, 8 yolk sac tumours and 1 chorion carcinoma as well as 30 normal testes were analysed using a monoclonal antibody. Results: In normal testes strong signals were seen in the maturing germ cells and in Leydig cells. In most tumours examined no clusterin expression was detected (84%). Only a focal weak expression was seen in some teratomas (32%), embryonal carcinomas (15%) and yolk sac tumours (25%). Conclusions: Our results suggest that clusterin is associated with normal germ cell development and that the loss of clusterin expression might play a role in the malignant transformation of germ cells.

Multicenter randomized controlled trial comparing the performance of 22 gauge versus 25 gauge EUS–FNA needles in solid masses

Abstract Background and aims. Few randomized studies have assessed the clinical performance of 25-gauge (25G) needles compared with 22-gauge (22G) needles during endoscopic ultrasound-guided fine needle aspiration (EUS–FNA) biopsy of intra-abdominal lesions. We aimed to compare the diagnostic yield, as well as performance characteristics of 22G versus 25G EUS biopsy needles by determining their diagnostic capabilities, the number of needle passes as well as cellularity of aspirated tissue specimen. Methods. The study is a prospective, randomized, multicenter study. Patients were referred between January 2009 and January 2010 for diagnostic EUS including EUS-guided FNA of different lesions adjacent to the upper GI tract. All patients were randomized to EUS–FNA performed with either a 22G or 25G aspiration needle. Results. EUS–FNA was performed in 135 patients (62 patients with a 22G needle). Sensitivity and specificity of the 22G needle was 94.1% and 95.8%, respectively, and for the 25G needle 94.1% and 100%, respectively. Investigators reported better visualization and performance for the 22G needle compared to the 25G (p < 0.0001). The number of tissue slides obtained was higher for the 22G needle during the second and third needle passes (p < 0.05). We did not observe significant differences between the number and preservation status of obtained cells (p > 0.05). Conclusions. A significant difference was found between the two types of needles in terms of reduced visualization of the 25G needle and suboptimal performance rating. However, this did not impact on overall results since both needles were equally successful in terms of a high diagnostic yield and overall accuracy.

Priming with a Combination of Proangiogenic Growth Factors Enhances Wound Healing in Streptozotocin-Induced Diabetes in Mice

Background: Numerous proangiogenic growth factors have been shown to improve impaired wound healing. This study evaluated the effects of subcutaneous pretreatment with a combination of proangiogenic growth factors on wound closure, mechanical properties, vessel density, and morphology. Methods: Thirty-six Balb/c mice with streptozotocin-induced diabetes were divided into 3 groups. A mixture of VEGF (35.0 µg), bFGF (2.5 µg), and PDGF (3.5 µg) was administered subcutaneously 3, 5, and 7 days prior to wounding in the first group, whereas the second group received three doses of 3.5 µg PDGF. Wound sizes were assessed daily and the repaired tissues were harvested 7 days after wound closure. Results: Complete closure (≧95% healing of initial wound area) was reached in all proangiogenic pretreated animals by day 17, whereas the PDGF monotherapy group needed up to 20 days for complete closure. By the time of tissue harvesting on day 24, complete closure was not reached in all control animals. Punch biopsy material revealed 1.6-fold higher vessel densities in the proangiogenic combination-pretreated group than in the controls. Conclusions: Proangiogenic priming revealed several significant effects on diabetic wound healing: faster time to closure, a higher vessel density, and improved functional outcome.

The EndoRotor(®): endoscopic mucosal resection system for non-thermal and rapid removal of esophageal, gastric, and colonic lesions: initial experience in live animals.

Background and study aims: The EndoRotor® is a novel, non-thermal, automated mechanical endoscopic resection system designed to remove benign mucosal neoplastic tissue throughout the gastrointestinal tract. It uses suction pressure to pull in mucosa and rapidly and precisely cut it while automatically transporting the samples to a collection trap for later histologic evaluation. Patients and methods: To study the technical properties and therapeutic potential of this new tool, we performed multiple upper and lower gastrointestinal endoscopic mucosal resections in three healthy live pigs. Animals were anesthetized and kept artificially ventilated while two physicians performed multiple qualitative mucosal resections on various sites of the pigs’ esophagus, stomach, duodenum, and colon. Results: Rapid resection of flat and slightly elevated mucosa up to several centimeters in size/diameter was performed. No major bleeding occurred during and after resections. When used properly, no gastrointestinal wall perforations occurred during superficial resections. Perforations in the colon were only observed when the device was deliberately pushed against deeper sub-mucosal layers or when exceptional force was applied to penetrate the gastrointestinal wall. Histologic specimens showed complete mucosal removal at resection sites. The flexible catheter could be moved and directed towards most of the areas of interest in the gastrointestinal tract. Conclusion: The EndoRotor rapidly and easily resects flat and slightly elevated gastrointestinal mucosa with a short learning curve. Future studies in humans should be performed to prove its ability for large-area mucosal resections in benign conditions such as laterally spreading adenomas in the colon, or Barrett’s mucosa in the distal esophagus.