Rosemarie Kühne-Heid

Screening for high‐grade cervical intra‐epithelial neoplasia and cancer by testing for high‐risk HPV, routine cytology or colposcopy

The validity of testing for high‐risk types of human papillomavirus (HPV) in cervical cancer prevention programs is undetermined. We compared the performance on primary screening of HPV DNA testing, cytology and colposcopy in detecting cervical intra‐epithelial neoplasia (CIN) grade 2 or 3 or cancer. A cohort of 4,761 women, median age 35 years, was screened by routine cytology, routine colposcopy and testing for high‐risk HPV by a PCR‐based method. Within an 8‐month period, women with abnormal findings on cytology or screening colposcopy or in whom high‐risk HPV types were detected were referred for colposcopy and biopsy. Women negative on all initial screening tests were followed by a second screening examination. To correct for work‐up bias, the true prevalence of CIN 2 or 3 or cancer was estimated by projection from histologically verified subgroups. Cervical biopsies were taken in 364 women (7.6%), of whom 114 (2.4%) showed CIN 2 (n = 34) or CIN 3 (n = 71) or cancer (n = 9). High‐risk HPV testing achieved bias‐corrected performance measures of 89.4% sensitivity, 93.9% specificity, 35.8% positive predictive value and 99.6% negative predictive value. Bias‐corrected rates of true‐ and false‐positives by high‐risk HPV testing compared to cytology (colposcopy) were about 4.5 (6.7) and 19.1 (7.4) times higher, respectively. The quality of routine cytology was controlled by computer‐assisted review, and the observed number of true‐positives more than doubled after adding automated review results. In middle‐aged women, testing for high‐risk HPV types, particularly when negative, may be used to increase the screening interval in programs for secondary prevention of cervical cancer. Int. J. Cancer 89:529–534, 2000.

Monocyte-chemo-attractant-protein-1 (MCP-1)-gene expression in cervical intra-epithelial neoplasias and cervical carcinomas

Chemokines play a central role in the chemotactic activation of immunological effector cells. One of the currently best characterized chemokines is the monocyte‐chemo‐attractant protein‐1 (MCP‐1), which is involved in the cross‐talk with cells of the monocyte‐macrophage lineage. Since macrophages and macrophage‐derived cytokines appear to be important in the transcriptional regulation of “high‐risk” types of human papillomaviruses (HPV), we monitored MCP‐1 expression by in situ hybridization (ISH) in histologically distinct stages of cervical intra‐epithelial neoplasms (CIN), cervical cancer and non‐HPV‐associated cases of erosive endocervicitis. Here, we demonstrate that high‐grade dysplasia (CIN III, n = 9) completely lacks both MCP‐1 expression and CD68+‐macrophage infiltration, while MCP‐1‐specific signals were occasionally detectable in one out of 5 CIN‐II and in one out of 3 CIN‐I lesions. Inspection of hyperplastic squamous epithelium adjacent to cervical carcinomas reveals high MCP‐1 expression and accumulation of infiltrating macrophages. In contrast, no macrophages could be detected in corresponding hyperplastic tissue areas surrounding CIN‐II and CIN‐III lesions, although MCP‐1 was found to be highly expressed. Finally, in agreement with our earlier in vitro data, invasive carcinomas of the cervix uteri showed MCP‐1‐specific hybridization signals and macrophage infiltration only in the stroma surrounding the carcinoma cells and in endothelial cells of capillaries, especially at the invasion front of the tumor, while the inner mass of the carcinomas was completely negative. On the other hand, ISH and histochemical evaluation of inflammatory, non‐HPV‐associated cases of erosive endocervicitis indicate strong MCP‐1 expression, which is regularly accompanied by chemotactic appearance of macrophages. These observations indicate that dysregulation of MCP‐1‐gene expression may represent an important step during HPV‐linked carcinogenesis, allowing the escape of virus‐positive cells from local immune response. Int. J. Cancer 82:6–11, 1999.

Identification of a new proliferation-associated protein NET-1/C4.8 characteristic for a subset of high-grade cervical intraepithelial neoplasia and cervical carcinomas

A large number of genes are known to be differentially expressed at distinct steps of carcinogenesis. By using a cell culture model system for cervical cancer, we had previously identified several genes that were more strongly expressed when comparing normal cervical epithelium with cervical intraepithelial neoplasia (CIN) and cervical cancer. In our study, we show that one of these genes, C4.8, is identical to NET‐1, which is a new member of the tetraspanin family of proteins. By generating a mouse polyclonal antiserum against the major extracellular domain of the protein, we could detect NET‐1/C4.8 expression both after ectopic expression of the gene in cell cultures and in cryostat sections of cervical biopsies. Moreover, immunohistochemic analyses of normal cervical epithelium, metaplasia, condyloma and CIN of different severity suggest that NET‐1/C4.8 expression is associated with neoplastic cell proliferation. Notably, expression of the protein throughout the entire epithelium is only evident for a subset of CIN3. The potential importance for this gene in cervical carcinogenesis is underlined by an invariably strong expression in all undifferentiated squamous cell cancers examined. This indicates that this gene may be of prognostic value.

Distribution of 14 high risk HPV types in cervical intraepithelial neoplasia detected by a non-radioactive general primer PCR mediated enzyme immunoassay.

AIM: To evaluate the presence of high risk human papillomaviruses (HPV) in cervical smears showing intraepithelial neoplasia (CIN). METHODS: The presence of 14 high risk HPV was evaluated in 114 cervical smears with CIN of different degrees, by comparing a non-radioactive polymerase chain reaction (PCR) enzyme immunoassay (EIA) with conventional PCR followed by radioactive Southern blot hybridisation. General primer PCR amplicons detecting low risk and high risk HPV were typed for 14 different high risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) by a non-radioactive PCR-EIA. Virus load of HPV 16 positive CIN was assessed using the semiquantitative PCR-EIA. RESULTS: Histological evaluation confirmed CIN I in 49 cases (mean age 29.0 years, range 17 to 52), CIN II in 31 cases (mean age 30.8 years, 18 to 54), and CIN III in 34 cases (mean age 31.1 years, 16 to 57). The non-radioactive PCR-EIA showed an overall agreement rate of 90% (kappa value 0.75) when compared with conventional general primer PCR followed by radioactive Southern blot hybridisation. High risk HPVs were detected in 47% of CIN I, 77% of CIN II, and 97% of CIN III (p < or = 0.02). HPV types 39, 51, 56, and 58 were found in CIN I exclusively (between 2% and 8%). HPV 16 and HPV 31 were detected in 12% and 2% of CIN I, 35% and 21% of CIN II, and 74% and 13% of CIN III, respectively (p < or = 0.03 and p < or = 0.04). The virus load estimated by the semiquantitative PCR-EIA of HPV 16 was similar in CIN I, CIN II, and CIN III. CONCLUSIONS: The PCR-EIA has high clinical sensitivity for detecting CIN II/III (90%). There was a significantly higher prevalence rate of HPV 16 and 31 in CIN III than in CIN I and II.

Value of laparoscopic evaluation of paraaortic and pelvic lymph nodes for treatment of cervical cancer

OBJECTIVE Laparoscopy was used to identify and localize suspicious lymph nodes in patients with cervical cancer. STUDY DESIGN Eighty-four patients with cervical cancer International Federation of Gynecology and Obstetrics stage IA2 to IV were staged by laparoscopic paraaortic and pelvic lymphadenectomy. The accuracy of laparoscopic assessment of lymph node status was compared with the histologic result. Positive lymph nodes were localized topographically by use of laparoscopy. RESULTS Sensitivity and specificity of laparoscopic evaluation for identifying positive paraaortic and pelvic lymph nodes was 92.3%. Combination of laparoscopic evaluation and frozen section helped to diagnose all patients with involved lymph nodes correctly. In 13 of 84 (15.4%) patients the result of lymph node assessment by laparoscopic evaluation and frozen section changed primary therapy. In two of these patients one positive lymph node was located in the lateral part of the cardinal ligament, and the hysterectomy was extended to be a more radical procedure. CONCLUSIONS Laparoscopic evaluation identified the lymph node status in patients with cervical cancer with high accuracy. Topographic localization showed that the lateral part of the cardinal ligament is involved early in lymph node spread.

Laparoscopic lymph node staging of cervical cancer in the 19th week of pregnancy

We present the case of a 39-year-old gravida I para 0 woman who underwent laparoscopic staging of lymph node involvement in cervical cancer in the 19th week of pregnancy. She had been diagnosed with adenosquamous carcinoma of the cervix, stage 1B 1, grade 2, with tumor involvement of the lymphovascular space and tumor involved resection margins via a cone biopsy in the 16th week of pregnancy. In order to decide whether it would be safe to proceed with the pregnancy, she was submitted to the laparoscopic exposure and removal of 18 parametric and pelvic lymph nodes. One positive lymph node was detected at the right internal iliac artery; therefore, an open radical hysterectomy with paraaortic lymphadenectomy was performed. This case shows that lymph node staging for cervical cancer can be done laparoscopically in the 2nd trimester. Information yielded during the course of this procedure can be crucial in deciding whether it is possible to preserve the pregnancy.

Detection of cancer‐related gene expression profiles in severe cervical neoplasia

The molecular signatures of 20 severe cervical intraepithelial neoplasia (CIN3) cases and 10 cervical squamous cell cancers were determined to define cancer‐related gene expression profiles. RNAs extracted from microdissected tissues were amplified by SMART technology and used as probes for hybridization of commercially available cDNA array filters comprising 1,176 cancer‐related genes. Ninety‐two differentially expressed genes were identified by comparison of pooled cDNA from CIN3 vs. cervical cancer. Heterogeneity in expression of this subset of genes was then analyzed for each biopsy using an algorithm for self‐organizing maps. For several gene clusters, the expression pattern for CIN3 differed significantly from that of cancer. Moreover, hierarchical clustering revealed significant differences in distribution of CIN and cancer. Several CIN cases were more strongly related to cancer, suggesting that gene expression profiling may be useful for subdividing pathologically indistinguishable precancers into different biologic entities. This approach also provides a basis for the identification of putative prognostic markers and for targeted molecular therapy.

A region on human chromosome 4 (q35.1-->qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis

Human papillomavirus (HPV) types 16 and 18 are known to play a major role in cervical carcinogenesis. Additional genetic alterations are required for the development and progression of cervical cancer. Previously, we showed that the introduction of an entire human chromosome 4 into HPV‐immortalized cells by microcell‐mediated chromosome transfer (MMCT) can induce senescence in cell hybrids. In the present study, we established eight new murine donor cell lines harboring different fragments of the human chromosome 4. These were tested for their ability to induce senescence by MMCT into HPV16‐immortalized keratinocytes (HPK II) and cervical carcinoma cells (HeLa). By exclusion, we could identify a region for a putative senescence gene or genes at 4q35.1→qter. Further evidence that this locus may be involved in cervical carcinogenesis was obtained by studying sections of high‐grade cervical intraepithelial neoplasias (CIN2/3) and cervical cancers from 87 women using a combination of interphase fluorescence in situ hybridization (I‐FISH) and microsatellite PCR. I‐FISH indicated copy number loss at 4q34→qter. Microsatellite analysis showed that loss of one or more alleles at chromosome 4 was more frequent in the cervical carcinomas than in the CINs. Loss of heterozygosity (LOH) affected four areas, D4S412 (4p16.3), D4S2394 (4q28.2), D4S3041 (4q32.3), and D4S408 (4q35.1), and was highest at D4S408. LOH at terminal 4q has been reported previously for cervical carcinomas and other human malignancies. This is the first report associating allelic loss at 4q34→qter with high‐grade intraepithelial neoplasia and cervical carcinoma, and the first experimental evidence that this locus or these loci can induce senescence in cervical carcinoma cells and HPV16‐immortalized cells.

Laparoskopische paraaortale und pelvine Lymphonodektomie

ZusammenfassungLaparoskopische Verfahren zur Entfernung pelviner und paraaortaler Lymphknoten gewinnen in der gynäkologischen Onkologie zunehmend an Bedeutung. In der Hand von Spezialisten gehören diese endoskopischen Verfahren bereits zum operativen Standardrepertoire beim Staging und der Behandlung von malignen Tumoren der Cervix uteri, des Endometriums, des Ovars und der Vulva. Die bisher veröffentlichten Daten unterstreichen folgende potentiellen Vorteile der laparoskopischen Lymphonodektomie: Die Kombination von Vergrößerungseffekt durch Videolaparoskopie und elektrochirurgischer Technik erlaubt subtiles anatomisches Operieren mit geringem Blutverlust und hoher Radikalität; eine verlängerte Operationsdauer wird durch einen kürzeren stationären Aufenthalt kompensiert und die intra- und postoperative Komplikationsrate nimmt mit zunehmender Erfahrung signifikant ab. Bisher fehlen prospektiv randomisierte Studien, die einen validen Vergleich zwischen laparoskopischer und konventioneller Lymphonodektomie erlauben. In künftigen Untersuchungen muß vor allem gezeigt werden, daß die Heilungsrate nach endoskopischen Verfahren, der durch konventionelle Techniken erreichten gleichwertig oder überlegen ist.

Trocar-site metastasis is not always due to laparoscopy.

The use of laparoscopic surgical techniques for the management of gynecologic malignancies has increased over the last years. Metastasis developing at the trocar insertion site is an emerging problem. We present the case of a 66-year-old woman with endometrial cancer who was diagnosed with an umbilical tumor after laparoscopically assisted vaginal hysterectomy (LAVH) and bilateral salpingoophorectomy. The interval between LAVH and diagnosis of the umbilical tumor was 13 months. The tumor was excised, and metastasis of endometrial cancer was histologically confirmed. Review of computer tomograms taken before LAVH showed a tumor in the umbilical area that had not been recognized before therapy. Therefore, tumor manifestation at the abdominal wall after laparoscopic surgery should not automatically be considered the result of iatrogenic spreading.