Gerhard Breitenecker

Lymphatic microvessel density as a novel prognostic factor in early-stage invasive cervical cancer.

Few data on the influence of lymphatic microvessel density (MVD) on survival in cancer are available since until recently there was no reliable immunohistological marker for lymphatic endothelium. Using an antibody staining podoplanin, a novel marker for lymphatic endothelium, lymphatic MVD in tissue samples of 85 patients with cervical cancer classification pT1b treated by radical hysterectomy was investigated. Survival was determined using univariate and multivariate analyses. Lymphatic MVD was also compared to MVD assessed by immunostaining against factor VIII–related antigen, which is considered a marker for blood vessels. Patients with >5 lymphatic microvessels/0.25 mm2 field had significantly better overall survival (mean 91.8 months) than those with ≤5 lymphatic microvessels/field in univariate analysis (mean 113 months) (p = 0.0105, log‐rank test). In multivariate analysis, lymphatic node involvement (p =0.0183), vessel infiltration (p =0.0158) and lymphatic MVD (p =0.0269) remained independent prognostic factors. No correlation between lymphatic MVD and various clinical and histopathological parameters was observed. Correlation between lymphatic MVD and MVD assessed by immunostaining against factor VIII was only weak (p = 0.004, r = 0.312, Spearmans coefficient of correlation). Our results suggest that increased lymphatic MVD is associated with favorable prognosis in early‐stage cervical cancer.

Impact of multiple HPV infection on response to treatment and survival in patients receiving radical radiotherapy for cervical cancer

To obtain information on the incidence and the clinical significance of infection with various types of the human papillomavirus (HPV) in cancer of the uterine cervix, we retrospectively examined the HPV status of 106 patients who had received radical radiotherapy for cervical cancer stages IB to IIIB. DNA was extracted from formalin‐fixed, paraffin‐embedded biopsies and PCR was carried out to identify HPV types 16, 18, 31, 35, 33 and 45. To detect additional HPV types, consensus PCR products were cloned and sequenced. A catalyzed signal‐amplified colorimetric in situ hybridization was carried out in 84 of 106 specimens as a positive control. Response to therapy, progression‐free survival (PFS) and cervical cancer‐specific survival (CCSS) were the statistical endpoints. Survival analysis was carried out using univariate and multivariate analysis (Cox regression). Ninety‐six patients (90.6%) were HPV‐positive and 42/96 (43.7%) were positive for multiple HPV types. Eight patients had persistent disease after radiotherapy. From these 8 patients, 7 were infected with multiple HPV types and only 1 patient had an infection with a single HPV type. After a median follow up period of 50 months, patients with multiple HPV infection had a significantly shorter PFS and CCSS compared to those with single HPV infection (24.8% and 34.9% vs. 64% and 60.8%, Log rank, p < 0.01 and 0.04). In multivariate analysis, the presence of multiple HPV types (RR 1.9), node status (RR 2.3), tumor size (RR 3.2) and histologic type (RR 4.8) were independent prognostic factors of CCSS. Our results demonstrate that the presence of multiple HPV types is associated with poor response and with reduced survival in cervical cancer patients who receive radiotherapy as the primary treatment.

Prognostic value of immunohistochemically detected CD44 isoforms CD44v5, CD44v6 and CD44v7–8 in human breast cancer

We investigated the expression of CD44 isoforms containing variant exons v5, v6 and v7-8 in 115 human breast cancer specimens by means of immunohistochemistry. CD44 isoforms CD44v5, CD44v6 and CD44v7-8 were detected in 56% (n = 64), 24% (n = 28) and 15% (n = 17), respectively. In 36 specimens of axillary lymph node metastasis, expression of CD44v5, CD44v6 and CD44v7-8 was found in 94% (n = 34), 92% (n = 33) and 89% (n = 32), respectively. Five year survival rates with or without CD44v5 and CD44v6 expression were 71% versus 86% (log-rank test, P = 0.02) and 62% versus 81% (log-rank test, P = 0.001), respectively. For disease-free survival, expression of CD44v5, CD44v6 and CD44v7-8 showed a prognostic impact (log-rank test, P = 0.004, P = 0.0001 and P = 0.0001, respectively). However, multivariate analysis revealed that all investigated CD44 isoforms failed to be independent predictors of the patients outcome.

Signal-amplified colorimetric in situ hybridization for assessment of human papillomavirus infection in cervical lesions.

Detection and typing of human papillomavirus (HPV) infection may have a major impact in cervical-screening and follow-up. In this study various commercially available techniques for the detection of HPV were evaluated. HPV-status was determined in 86 samples of cervical cancer by PCR and direct sequencing, catalyzed signal amplified colorimetric DNA in situ hybridization (CSAC- ISH) (GenPoint system, DAKO), immunohistochemistry (IHC) and in 12 selected cases also by conventional, non-amplified ISH. Twenty-one samples of cervical intraepithelial neoplasias grade III (CIN III) were investigated by CSAC-ISH, conventional ISH and by IHC, in corresponding PAP smears HPV-detection and typing was performed by CSAC-ISH and Hybrid Capture test II (HC). In additional 20 PAP smears HPV typing was performed using HC and a novel immunocytochemical system for HPV detection and-typing. CSAC-ISH showed good correlation with PCR analysis in cervical cancers: In 87% of PCR positive cases, HPV infection was also detected by CSAC- ISH (66/76). HPV 16 was detected in 75% of PCR-positive cases (44/59), HPV 18 in 71% of PCR positive cases (5/7). CSAC-ISH detected HPV 31 in only 29% of PCR positive cases (2/7), and HPV 33 in 64% of PCR-positive cases (23/36). Nevertheless, CSAC-ISH- false negative cases for HPV 31 or 33 were nearly always combined infections with other HPV types, which were detectable by CSAC-ISH in most cases. CSAC-ISH revealed HPV infection in 20 of 21 HC-positive cervical smears, while in corresponding biopsies (CIN III) CSAC-ISH detected 100% of HPV infections. Conventional, non-amplified ISH showed significantly lower sensitivity compared with CSAC-ISH, and immunocyto- and -histochemistry were of very low sensitivity for detection of HPV. CSAC-ISH is an easy-to-handle method for detection and typing of cervical HPV infection, and shows sufficient sensitivity for clinical practice.

Kontraktile Fibroblasten (Myofibroblasten) in der Lamina propria der Hodenkanälchen vom Menschen

SummaryA special type of fibroblast is observed in the connective tissue of seminiferous tubules in human testes. These cells are characterized by a fibrillar system consisting of parallel arranged cytoplasmic filaments. The filaments have a mean diameter of 80 Å. Bundles of filaments run parallel to the surface of the flattened cells. The filaments insert in a dense, granular material which is connected with the cell membrane. This fibrillar system is thought to be contractile; the dense or more loose texture of the filaments within the bundles may correspond to the contracted or relaxed state of the cell. The specialized fibroblasts described are supposed to belong to the group of myofibroblasts (Gabbiani, Ryan and Majno, 1971).ZusammenfassungIn der Lamina propria menschlicher Hodenkanälchen wurden spezialisierte Fibroblasten beobachtet. Die Zellen sind durch Bündel parallel geordneter Plasmafilamente gekennzeichnet, deren Durchmesser rund 80 Å beträgt. Die Filamentbündel verlaufen parallel zur Oberfläche der lamellär ausgebreiteten Zellen und inserieren in elektronendichtem, granulärem Material, das der Innenseite der Zellmembran anliegt. Es wird angenommen, daß dieses Fibrillensystem kontraktil ist; die dichtere oder lockerere Vernetzung der Filamente innerhalb der Bündel würde dem kontrahierten oder erschlafften Zustand der Zellen entsprechen. Die beschriebenen spezialisierten Fibroblasten sollten der Gruppe der Myofibroblasten zugeordnet werden (Gabbiani, Ryan und Majno, 1971).

Is high-risk human papillomavirus infection associated with cervical intraepithelial neoplasia eliminated after conization by large-loop excision of the transformation zone?

OBJECTIVE To investigate whether high-risk HPV infection associated with cervical intraepithelial neoplasia (CIN) was successfully eliminated after electrosurgical conization by large-loop excision of the transformation zone (LLETZ). STUDY DESIGN 142 women, who were admitted for conization of CIN 1-3 were recruited into a prospective follow-up study of HPV infection, including cervical sampling for HPV DNA before, and then 3, 6 and 12 months after surgery. We examined whether there were any differences in the rate of HPV DNA positivity after LLETZ between specific risk groups, such as patients with primary (i.e. before surgical treatment) high-risk HPV infection, CIN of different grades, and positive margins. RESULTS We did not detect statistically significant differences between specific risk groups. According to the assay used (hybrid capture II) at the last follow-up visit 94% of primarily infected patients were completely free from infection with high-risk HPV types, while 6% had persisting HPV infection. CONCLUSIONS With a detection limit of 5000 genomes/ml HPV DNA the hybrid capture II results revealed, that after electrosurgical removal of CIN in 94% of patients testing positive for high-risk HPV DNA prior to surgery were negative 12 months post-surgery.

Tissue expression of CA 125 in benign and malignant lesions of ovary and fallopian tube: a comparison with CA 19-9 and CEA.

Sections of formalin-fixed and paraffin-embedded tissue specimens of 11 normal ovaries and tubes, 13 tubo-ovarian abscesses, 3 tubal carcinomas, and 115 ovarian tumors were investigated by immunohistochemistry. CA 125 and CA 19-9 were demonstrated with monoclonal antibodies, CEA with polyclonal antibodies. The tissue expression was visualized by the avidin-biotin method. In the germinal epithelium of all ovaries no tumor marker was confirmed. In 4 out of 11 tubes the epithelium was CA 125 positive, in 2 out of 11 cases CA 19-9 positive. Nine out of 13 tubo-ovarian abscesses were CA 125 and 5 out of 13 were CA 19-9 positive in their epithelium. Elevated serum levels of these markers might be due to expression via the epithelial cell of the inflamed tube. All normal and inflammatory adnexal tissues were CEA negative. In serous tumors and undifferentiated carcinomas, CA 125 was most frequently confirmed (85 and 70%, respectively). All mucinous tumors were CA 125 negative. The most frequently confirmed tumor marker was CA 19-9 (77%). In endometrioid tumors, CEA was most frequent (44%). In 42% of the borderline tumors and carcinomas only one marker was demonstrated, in 7% none. Here, immunohistochemistry may indicate the most adequate marker. Tumor marker expression was markedly heterogenous: tumor areas with strong, weak, and no reaction were adjacent. The tumor markers revealed no specificity for malignancy or disease.

Leiomyoma of the round ligament in a postmenopausal woman

OBJECTIVE Leiomyoma of the round ligament of the uterus is a rare condition. We present the first case of a postmenopausal, previously hysterectomised, woman who received combined hormonal replacement therapy and developed a leiomyoma of the right round ligament. METHOD In sections of the tumor, immunohistochemical reaction with monoclonal antibodies against smooth muscle actin, desmin, vimentin, estrogen receptor and progesterone receptor was performed. RESULTS The leiomyoma showed bizarre histologic appearance in absence of mitotic figures and degenerative changes. Leiomyocytes presented a positive immunohistochemical reaction with monoclonal antibodies against smooth muscle actin, desmin vimentin and progesterone receptor in absence of estrogen receptor. CONCLUSION Progestin as part of a combined hormonal replacement therapy may play an important role in promoting the development of leiomyoma in hormonal responsive tissue of the round ligament in postmenopausal women.

Evidence of androgen receptor expression in lichen sclerosus: an immunohistochemical study.

Objective: While topical androgen administration is widely used in the treatment of lichen sclerosus of the vulva, localization and level of expression of androgen receptor (AR) have not been described previously. Methods: Thirty-nine paraffin-embedded punch biopsies of patients with lichen sclerosus of the vulva were examined. Androgen receptor, estrogen receptor (ER), and progesterone receptor (PR) expression in lichen sclerosus and in normal vulvar skin were investigated by immunohistochemistry. Results: Five tissue specimens (12.8%) of lichen sclerosus showed nuclear staining with anti-AR in the parabasal cell layers of the epidermis. Median age of patients with positive nuclear staining for AR versus women without AR expression was 71 (range, 63-78) and 66.5 (range, 38-91) years, respectively. Estrogen receptor expression was present in only one patient. Nuclear staining reaction for PR expression was absent in all cases. Four of the five AR-positive women reported no complaints and therefore received no topical testosterone therapy. Conclusion: Our results suggest a lack of complaints in AR-positive lichen sclerosus patients. Our findings could justify a larger study comparing symptoms of patients with and without AR expression.

Preperitoneal lipoleiomyoma of the abdominal wall in a postmenopausal woman

Objective: Extrauterine leiomyomas are rare events. These tumors may be easily misdiagnosed as ovarian tumors at the clinical investigation. We present the first case of an otherwise healthy postmenopausal woman, hysterectomized 20 years ago, who developed a preperitoneal lipoleimoyoma in the 30-year-old scar of a Pfannenstiel incision. The patient received continuous hormone replacement therapy (HRT) for 5 years with 1.25 mg conjugated estrogen and 5 mg medrogeston per day. Methods: In sections of the tumor, immunohistochemical reactions with antibodies against actin, desmin, vimentin, estrogen and progesterone receptors and factor VIII related antigen was performed. Results: Histologic findings revealed cellular fascicles of spindle-shaped smooth muscle cells in a whorled arrangement. Mitotic figures were absent. Central degenerative changes and focal edema were observed. Between muscle fascicles, a significant amount of fat cells (20% of tumor volume) was visible. Leiomyocytes showed immunohistochemicaly positive reactions with actin, desmin, vimentin, and steroid hormone receptors. Based on these findings, the tumor was diagnosed as lipoleiomyoma. Conclusions: Origin of the tumor of smooth muscle cells of vessels located in the abdominal wall and development under influence of oral steroids seems most probable. HRT appears to promote the development of extrauterine leiomyomas in postmenopausal women.