Dirk Watermann

Splicing Factor Tra2-β1 Is Specifically Induced in Breast Cancer and Regulates Alternative Splicing of the CD44 Gene

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.

Uterosacral ligament in postmenopausal women with or without pelvic organ prolapse

The uterosacral ligaments are thought to contribute to pelvic support. The objective of this study was to compare the structural components of these ligaments in women with and without pelvic organ prolapse (POP). We characterized uterosacral ligaments of 25 postmenopausal women with POP and 16 controls histomorphologically and immunohistochemically by quantifying their content of collagen I, III, and smooth muscle using a computerized image analysis. In 84% the uterosacral ligaments were composed of more than 20% of smooth muscle cells. There was no difference in collagen I expression and smooth muscle cell amount between women with POP and those without. In contrast, the collagen III expression was significantly related to the presence of POP (p<0.001) rather than age or parity. Our findings suggest that the higher collagen III expression might be a typical characteristic of POP patients’ connective tissue. The considerable amount of smooth muscle cells in uterosacral ligaments may provide pelvic support.

Increased expression of matrix metalloproteinase 2 in uterosacral ligaments is associated with pelvic organ prolapse

The uterosacral ligaments are an important part of the pelvic support system and connective tissue alterations are thought to contribute to the development of pelvic organ prolapse (POP). The objective of this study was to compare the expression of matrix metalloproteinases (MMPs) 1 and 2 in these ligaments in women with and without POP. We analyzed the tissue samples obtained from left and/or right uterosacral ligaments of 17 women with POP and 18 controls by immunohistochemistry. There was no difference in MMP-1 expression between women with POP and those without. In contrast, the MMP-2 expression was significantly related to the presence of POP (p=0.004) rather than to age or parity. There was no difference in MMP-1 and MMP-2 expression between left and right uterosacral ligaments in women with POP compared to controls. Our findings strongly indicate that increased MMP-2 expression in uterosacral ligaments is associated with POP.

The MSKCC nomogram for prediction the likelihood of non-sentinel node involvement in a German breast cancer population.

Objective To assess whether the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram for prediction of NSLN metastasis is useful in a German breast cancer population and whether the characteristics of the breast tumor and the sentinel lymph node (SLN) are able to predict the likelihood of non-sentinel lymph node (NSLN) metastasis. Methods A total of 545 patients with primary breast cancer and SLN examination were evaluated. The MSKCC nomogram was applied to 98 patients with a positive SLN who subsequently had completion axillary lymph node dissection (ALND). Predictive accuracy was assessed by calculating the area under the receiver-operator characteristic (ROC) curve. The collective was evaluated by correlating the prevalence of NSLN and SLN metastasis to pathological features. Results The MSKCC nomogram achieved a ROC of 0.58 indicating a bad accuracy of the nomogram. Tumor size, histology, lymphovascular infiltration, multifocality, Her-2-neu positivity, and nuclear grade correlated with the probability of SLN metastasis. Histology and primary tumor localization correlated significantly with the probability of NSLN metastasis. Conclusions The MSKCC nomogram did not provide a reliable predictive model in our study population. However, the likelihood of SLN metastasis correlated with the presumed risk factors and no obvious differences between the MSKCC population and our population could be seen. In order to achieve interinstitutional reproducibility, standardization of surgical procedure and of the pathological assessment of the SLN is desirable.

Specific induction of pp125 focal adhesion kinase in human breast cancer.

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT–PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51±0.84 (mean±standard deviation), which was strongly induced to 2.91 (±1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0±0.63 vs 2.27±0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.

Good Prediction of the Likelihood for Sentinel Lymph Node Metastasis by Using the MSKCC Nomogram in a German Breast Cancer Population

BackgroundThe sentinel lymph node (SLN) procedure could be omitted in cases of accurate prediction of very high or very low probability of SLN metastasis in early breast cancer patients. We evaluated a breast cancer nomogram, an online tool provided by the Memorial Sloan–Kettering Cancer Center (MSKCC), that predicts the likelihood of a positive lymph node.MethodsData from 545 patients with successful SLN biopsy were collected, including 118 patients with a positive sentinel lymph node. Histopathological assessment of the SLN included hematoxylin and eosin staining and/or immunohistochemistry. Predictive accuracy was assessed by calculating the area under the receiver–operator characteristic (ROC) curve.ResultsIn our collective tumor size, histology, lymphovascular infiltration, multifocality, Her-2-neu positivity, and nuclear grade correlated with the probability of SLN metastasis. The ROC of the validated nomogram in our breast cancer population revealed a value of 0.78 compared with 0.75 in the original publication.ConclusionThe MSKCC nomogram is a useful tool in our population of breast cancer patients. However, variations in the pathological assessment of the SLN between breast cancer centers worldwide might be an impediment to widespread application of the nomogram.

Surgical repair of posterior compartment prolapse : preliminary results of a novel transvaginal procedure using a four-armed polypropylene mesh with infracoccygeal and pararectal suspension

Background. Anatomical defects of the posterior vaginal compartment are a common reason for pelvic floor reconstructive surgery. The implantation of a four‐armed monofilamentous polypropylene mesh with infracoccygeal and pararectal suspension is a recently introduced innovative technique, which is believed to reduce the risk of mesh retraction and prolapse recurrences, and additionally, allows a tension‐free adjustment of the mesh. Methods. In this preliminary case series, we aimed to evaluate feasibility, intraoperative complications and short‐term follow‐up results of this novel surgical procedure in a multicentre approach. Seventy‐three patients undergoing surgery for posterior vaginal compartment prolapse were enrolled. The mean follow‐up time was 3.8 months (range: 2–6 months), and follow‐up information was available in 60/73 (82.2%) women. Results. Intraoperative complications were observed in 4.2% of cases, 2 patients with blood loss >500 ml, and one bladder injury occurring during concomitant anterior compartment surgery. Importantly, there were no intraoperative complications directly related to the implantation technique (e.g. rectum perforations), and no prolapse recurrences at follow‐up examinations 3–6 months postoperatively. Our short‐term mesh erosion rate was 3.1%. Conclusions. We conclude that this innovative procedure is a feasible and safe technique for the treatment of posterior vaginal compartment prolapse. Further prospective and multicentre trials are warranted.

Expression of nuclear splicing factors in ovarian cancer: correlations

16550 Background: Alternative splicing represents an important nuclear mechanism in the post-transcriptional regulation of gene expression, which is frequently altered during tumorigenesis. Previou...