Markus C. Fleisch

Targeted and Nontargeted Effects of Ionizing Radiation That Impact Genomic Instability

Radiation-induced genomic instability, in which the progeny of irradiated cells display a high frequency of nonclonal genomic damage, occurs at a frequency inconsistent with mutation. We investigated the mechanism of this nontargeted effect in human mammary epithelial cells (HMEC) exposed to low doses of radiation. We identified a centrosome-associated expression signature in irradiated HMEC and show here that centrosome deregulation occurs in the first cell cycle after irradiation, is dose dependent, and that viable daughters of these cells are genomically unstable as evidenced by spontaneous DNA damage, tetraploidy, and aneuploidy. Clonal analysis of genomic instability showed a threshold of >10 cGy. Treatment with transforming growth factor beta1 (TGFbeta), which is implicated in regulation of genomic stability and is activated by radiation, reduced both the centrosome expression signature and centrosome aberrations in irradiated HMEC. Furthermore, TGFbeta inhibition significantly increased centrosome aberration frequency, tetraploidy, and aneuploidy in nonirradiated HMEC. Rather than preventing radiation-induced or spontaneous centrosome aberrations, TGFbeta selectively deleted unstable cells via p53-dependent apoptosis. Together, these studies show that radiation deregulates centrosome stability, which underlies genomic instability in normal human epithelial cells, and that this can be opposed by radiation-induced TGFbeta signaling.

Expression of the hyaluronan receptor RHAMM in endometrial carcinomas suggests a role in tumour progression and metastasis.

Purpose. Interactions of hyaluronic acid (HA) with its binding protein RHAMM (receptor for HA-mediated motility) have been proposed as being important in promoting tumour progression and dissemination. This comparative study was designed to investigate the RHAMM expression patterns in endometrial carcinoma.Methods. We examined a series of 89 endometrial carcinomas and 15 normal endometrial tissues by immunohistochemistry, using a RHAMM-specific polyclonal antibody. Expression of RHAMM was assessed according to the pattern and intensity within (overall cytoplasm, center/periphery of tumours) and between the tumours. The staining results were compared to the corresponding clinical data (age, menopause status, histological staining, histological grading, lymph node status).Results. RHAMM-expression was detectable in 58% of the 89 tumours [Histological stage: pT1a (8/12); pT1b (16/37); pT1c (18/26); pT2 (6/9); pT3a (4/5)] and 13% (2/15) of the normal endometrial tissues. The positivity rates for RHAMM were 100% in patients with positive lymph nodes but only 50.7% in patients with negative lymph nodes (P<0-01). Additionally, the expression pattern showed a highly significant correlation (P<0.01) with the histological grade of the tumours [G1 (6/42), G2 (33/34), G3 (13/13)] and occurrence of lymph node metastases.Conclusions. Our results suggest that RHAMM expression may enhance and improve the invasion and metastasis of endometrial carcinomas.

Hysteroscopy for asymptomatic postmenopausal women with sonographically thickened endometrium

UNLABELLED Endometrial carcinoma is the most common genital cancer in women. While patients usually present with vaginal bleeding, in 10-20% this characteristic symptom is absent. Endometrial thickness (double layer) is measured by transvaginal sonography and thickening indicates an increased risk of malignancy or other pathology (hyperplasia or polyps). OBJECTIVE We sought to correlate hysteroscopic and pathological findings in asymptomatic postmenopausal women with sonographically thickened endometrium (>6mm). STUDY DESIGN A prospective observational study in a university hospital of 304 postmenopausal women referred between 1996 and 2006 because of a sonographically thickened endometrium in the absence of abnormal bleeding, who underwent continuous flow hysteroscopy (4.5mm Storz hysteroscope) and fractionated curettage of the uterine cervix and corpus (D & C) in addition to vaginal sonography (5MHz probe). RESULTS The mean age of the women was 64.8 (range 57.7-71.9) years. Average endometrial thickness measured by ultrasound was 12mm+/-6.7mm. Hysteroscopy suggested the presence of endometrial polyps in 226 women (74.3%), simple endometrial hyperplasia in 34 (11.2%), atrophic endometrium in 18 (5.9%), complex endometrial hyperplasia in 2 (0.7%), atypical hyperplasia in 3 (1%) and leiomyoma in 9 (3.0%). In 12 women (3.9%), the hysteroscopic appearance suggested malignancy and histology revealed endometrial adenocarcinoma. All hysteroscopic results were confirmed by histological examination. CONCLUSION Hysteroscopy represents an easy, safe and effective method for the investigation of asymptomatic women with a thickened endometrium found with transvaginal ultrasound. The commonest pathology was endometrial polyps.

Women in early‐phase clinical drug trials: Have things changed over the past 20 years?

rom the Institute of Clinical Pharmacology, HELIOS Klinikum Wuppertal, University of Witten/Herdecke, Wuppertal, and Department of Obstetrics and Gynecology, Heinrich-HeineUniversity, Düsseldorf. his manuscript contains parts of the doctorate thesis of Jeanette Fleisch, MD, at the University of Witten/Herdecke, Faculty of Medicine. eceived for publication May 19, 2005; accepted Aug 16, 2005. eprint requests: Petra A. Thürmann, MD, Chair of Clinical Pharmacology, University of Witten/Herdecke, Philipp Klee-Institute of Clinical Pharmacology, HELIOS Klinikum Wuppertal, Heusnerstrasse 40, D-42283 Wuppertal, Germany. -mail: pthuermann@wuppertal.helios-kliniken.de lin Pharmacol Ther 2005;78:445-52. 009-9236/

Compartment syndrome after gynecologic operations: evidence from case reports and reviews

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Mapping mammary gland architecture using multi-scale in situ analysis

Compartment syndrome (CS) of the lower leg is a rare but severe complication of operations in the lithotomy (LT) position after urologic, gynecologic and general surgery. A delay in diagnosis and treatment can lead to loss of function and even life-threatening complications. The pathophysiology is still not fully understood but it is believed that ischemia as a result of increased compartment pressure and decreased perfusion pressure may lead to CS. The type of leg support and the intraoperative hypotension have been discussed as risk factors but evidence is mainly based on case reports and expert opinion. Studies suggest that time spent in the LT position and the addition of head-down tilt are associated with CS. As these positions are routinely applied during various gynecologic procedures, forensically CS has to be considered as a specific complication of gynecologic surgery in the LT position. Despite the low incidence there is a need for prospective studies and guidelines for its prevention. Sixteen case reports describing 19 cases of CS following gynecologic surgery in lithotomy position were found during a literature search. This review is based on 14 of these case reports (17 cases), which describe a postoperative compartment syndrome in a previously healthy leg. We summarize the reported cases and literature on CS after gynecologic procedures in order to increase awareness among medical staff and to give careful recommendations regarding perioperative management based on available information.

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

We have built a novel computational microscopy platform that integrates image acquisition, storage, processing and analysis to study cell populations in situ. This platform allows high-content studies where multiple features are measured and linked at multiple scales. We used this approach to study the cellular composition and architecture of the mouse mammary gland by quantitatively tracking the distribution and type, position, proliferative state, and hormone receptor status of epithelial cells that incorporated bromodeoxyuridine while undergoing DNA synthesis during puberty and retained this label in the adult gland as a function of tissue structure. Immunofluorescence was used to identify label-retaining cells, as well as epithelial cells expressing the proteins progesterone receptor and P63. Only 3.6% of luminal cells were label-retaining cells, the majority of which did not express the progesterone receptor. Multi-scale in situ analysis revealed that luminal label-retaining cells have a distinct nuclear morphology, are enriched 3.4-fold in large ducts, and are distributed asymmetrically across the tissue. We postulated that LRC enriched in the ventral mammary gland represent progenitor cells. Epithelial cells isolated from the ventral versus the dorsal portion of the gland were enriched for the putative stem cell markers CD24 and CD49f as measured by fluorescence activated cell sorting. Thus, quantitative analysis of the cellular composition of the mammary epithelium across spatial scales identified a previously unrecognized architecture in which the ventral-most, large ducts contain a reservoir of undifferentiated, putative stem cells.

Modifications of Laparoscopic Supracervical Hysterectomy Technique Significantly Reduce Postoperative Spotting

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.

Combination of a MDR1-targeted replicative adenovirus and chemotherapy for the therapy of pretreated ovarian cancer

STUDY OBJECTIVE The aim of this study was to estimate the benefit of excision of the endocervix during laparoscopic supracervical hysterectomy (LSH) with regard to postoperative cyclical bleeding. DESIGN Cohort study from 2 centers (Canadian Task Force classification II-2). SETTING Two surgical teams at the University of Duesseldorf Medical Center and PAN Clinic, Cologne, Germany. PATIENTS Women with menstrual bleeding disorders resistant to medical treatment, symptomatic leiomyomata, dysmenorrhea. INTERVENTION Laparoscopic supracervical hysterectomy. The uterus was transsected from the cervix with 2 techniques with and without excision of cervical canal. MEASUREMENTS AND MAIN RESULTS We evaluated 300 patients who underwent consecutive LSH procedures. In 150 patients the uterus was transsected from the cervix using a monopolar loop. In a second series of 150 patients a unipolar needle electrode was used for the uterine amputation and the excision of cervical canal. The mean duration of the transsection was 65 seconds (monopolar loop) versus 168 seconds (monopolar needle). The excision of the endocervix was performed without any complications in 148 procedures. Histologic examination of the removed tissue revealed endocervical tissue in 83.3% (n = 125), endometrium in 9.4% (n = 14), cervicoisthmic mucosa in 3.3% (n = 5), and myometrium only in 4% (n = 4). All 300 patients were contacted 12 months after surgery to inquire about bleeding status, and 282 (94%) responded. In patients who underwent excision of the endocervix, postoperative cyclical bleeding was significantly reduced compared with the control group (1.4% vs 10.7%). CONCLUSION The results of this study indicate that the routine excision of the endocervix is a quick safe procedure which allows a significant reduction of postoperative cyclical bleeding in patients who undergo LSH.

BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells

PurposeTargeted oncolytic adenoviruses capable of replication selectively in cancer cells are an appealing approach for the treatment of various cancer types refractory to conventional therapies. The aim of this study was to evaluate the effect of Ad5/3MDR1E1, a multidrug resistance gene 1 (MDR1)-targeted fiber-modified replication-competent adenovirus for the therapy of platinum-pretreated ovarian cancer in combination with cytostatic agents.MethodsMDR1-specific tumor cell killing of Ad5/3MDR1E1 was systematically evaluated in chemotherapy naïve and pretreated ovarian cancer cells in vitro. Combinations of Ad5/3MDR1E1 and cytostatic agents were studied in vivo and in vitro. An in vivo hepatotoxicity model was used to evaluate liver toxicity.ResultsWe demonstrate efficient oncolysis of Ad5/3MDR1E1 in chemotherapy-resistant ovarian cancer cells as well as therapeutic efficacy in an orthotopic mouse model. Further, combining Ad5/3MDR1E1 with paclitaxel resulted in greater therapeutic benefit than either agent alone.ConclusionThese preclinical data suggest that a fiber-modified adenovirus vector under the control of the MDR1 promoter represents a promising treatment strategy for platinum-pretreated ovarian cancer as a single agent or in combination with conventional anticancer drugs.