Proteomics in mammary cancer research.

During the past few years, the intensified detection of small (mammary) carcinomas causes an increase in the number of mammary cancers. Cancer of the mammary tissues has an almost individually unpredictable behavior and aggressiveness. Therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of mammary cancer, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications. In the following article we review proteomic techniques that are now commonly used in order to elucidate the role of proteins in breast cancer.

Expression of aromatase and estrogen sulfotransferase in preinvasive and invasive breast cancer.

PURPOSE: Intratumoral estradiol levels in postmenopausal women with breast cancer are thought to be mainly regulated by the aromatase-mediated conversion from androgens and estrogen sulfotransferase (EST)-mediated reduction of bioavailability. While in invasive breast cancer (IBC) the role of both enzymes has been extensively studied and has led to the use of aromatase inhibitors as a key therapeutic strategy, comparably little is still known about their role in the local regulation of estradiol in ductal carcinoma in situ (DCIS). METHODS: We have performed immunohistochemistry to investigate the expression of aromatase and sulfotransferase in custom-made breast cancer tissue arrays containing 96 samples of pure DCIS and in 104 tumor biopsies which contain both, DCIS and invasive components. RESULTS: We found that aromatase was equally detectable in epithelial components of both, DCIS and IBC (P = 0.884, Chi square test). However, stromal aromatase expression was significantly higher in IBC compared to adjacent DCIS components (P = 0.034, Chi square test). Whereas no significant difference was observed for epithelial aromatase expression in high versus non-high grade DCIS (P = 0.735 Chi square test), epithelial EST levels were found to be significantly down-regulated in high-grade DCIS compared to non-high grade cases (P = 0.042). CONCLUSION: We have demonstrated the presence of both aromatase and EST in malignant epithelium and adjacent stromal fibroblasts in DCIS. Lower stromal aromatase expression in preinvasive breast cancer and lower EST levels in high-grade DCIS suggest that the net effect of intratumoral estradiol (E2)-modulating enzymes results in lower local E2 levels in earlier stages of breast tumorigenesis.

Human papillomavirus DNA integration and messenger RNA transcription in cervical low- and high-risk squamous intraepithelial lesions in Austrian women.

The human papillomavirus (HPV) plays an important role in the progression of cervical carcinoma. High-risk (HR) HPV types have been mainly identified in cytologic high-grade squamous intraepithelial lesions (HSILs) and histologic invasive carcinoma of the cervix. We examined cervical swabs of patients with abnormal Papanicolaou (Pap) smears, diagnosed as low-grade squamous intraepithelial lesions (LSILs) including atypical squamous cells of uncertain significance or HSILs. Low-risk (LR) HPV and HR-HPV types were identified by the Digene Hybrid Capture II test. Two-dimensional (2D) gel electrophoresis was used to specify the physical state of HPV DNA sequences. Expression of E6/E7 messenger RNA (mRNA) transcripts was analyzed by reverse transcriptase-polymerase chain reaction. Histopathologic results were correlated to the patients' physical status and HPV DNA mRNA transcripts. Pap smears with HPV infections of LR and HR types were correlated to the degree of squamous intraepithelial lesions (SILs). Comparing the physical states of HPV DNA sequences with the expression of HPV E6/E7 mRNA transcripts, all types were identified only as extrachromosomal in benign cervical smears, cervical intraepithelial neoplasia (CIN) I and II. HPV16 showed all physical states in CIN III/carcinoma in situ (CIS), whereas HPV18 only existed in mixed and integrated forms. HPV31/33/52b/58 appeared in all stages of lesions most commonly in extrachromosomal form; in integrated form, they were present only in CIN III/CIS. Although integration of some HR-HPV types is not always necessary for progression of SILs, the above-mentioned method is useful to analyze the physical state of HPV DNA sequences and predict the progression of SILs.

Expression of aromatase and estrogen sulfotransferase in eutopic and ectopic endometrium: evidence for unbalanced estradiol production in endometriosis.

Endometriosis is an estrogen-dependent gynecological disease causing pelvic pain and infertility. Impaired estrogen metabolism is thought to play a pivotal role in the pathogenesis of the disease. While there is some information on factors involved in the synthesis of E2, information on E2-deactivating enzymes is still very limited. To elucidate the intracrinology of endometriotic tissues, the authors analyze the expression of aromatase and E2-inactivating estrogen sulfotransferase (EST) in paired biopsies obtained simultaneously from the endometrium and from endometrial lesions of each of 35 patients with peritoneal or ovarian endometriosis and in cycling endometria from 33 women without endometriosis. Protein localization was demonstrated by immunohistochemistry. Aromatase expression was found in endometriotic glands in 32 of 35 cases and was elevated in comparison to corresponding uterine endometria (25 of 35 cases, P = .021, chi(2) test). The difference was even more pronounced when uterine endometria from endometriosis patients were compared with that of healthy controls (8 of 33 cases, P < .001, chi(2) test). The EST levels were essentially unchanged. The elevated expression of aromatase in eutopic and ectopic endometrium from patients with endometriosis in the presence of comparable EST provides further evidence for unopposed local biosynthesis of estrogens in endometriosis.

Intratumoral IGF-I protein expression is selectively upregulated in breast cancer patients with BRCA1/2 mutations.

BRCA1/2 mutations predispose to early onset breast and ovarian cancers. The phenotypic expression of mutant alleles, however, is thought to be modified by factors that are also involved in the pathogenesis of sporadic breast cancer. One such protein is IGF-I, one of the strongest mitogens to breast cancer cells in vitro. We have utilized immunohistochemistry to compare the intratumoral IGF-I and IGF-I receptor (IGF-IR) protein expression in 57 BRCA1/2 mutation carriers and 102 matched breast cancer patients without a family history in a nested case-control study. BRCA1 silencing by siRNA was used to investigate the effect of BRCA mutations on IGF-I protein expression. IGF-I protein expression was detected in tumoral epithelium and surrounding stroma, and was significantly upregulated in tumors of BRCA mutation carriers when compared with matched sporadic tumors (epithelial: 87.7% vs 61.8%, P=0.001; stromal: 73.7% vs 34.3%, P<0.001). By contrast, IGF-IR protein expression was confined to malignant epithelium and was unchanged in mutation carriers (52.6% vs 39.2%, P=0.310). While in mutation carriers IGF-IR protein expression was significantly correlated with both epithelial (P=0.003) and stromal IGF-I (P=0.02), this association was less pronounced in sporadic breast cancer (P=0.02 respectively). siRNA-mediated downregulation of BRCA1 in primary human mammary gland cells triggered upregulation of endogenous intracellular IGF-I in vitro. The increased intratumoral IGF-I protein expression in BRCA mutation carriers suggests an involvement of the IGF-I/IGF-IR axis in the biological behavior of breast cancers in this population and could define a potential therapeutic target.

Malignant mixed Müllerian tumor with heterologous component arising in the fallopian tube--a case report.

Primary malignant mixed Müllerian tumors (MMMTs) of the fallopian tube are rarities in gynecologic oncology with only 26 cases of MMMTs with a heterologous component reported thus far. We report a case of FIGO Stage II primary MMMT of the fallopian tube with a heterologous tumor portion in an 80-year-old woman presenting with abdominal discomfort at the time of primary diagnosis. After performance of total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy follow-up examination three months postoperatively did not show signs of disease recurrence. The patient finally presented six months after the initial diagnosis with extensive intraabdominal metastasis and died several days thereafter. The present report supports the aggressive nature of these neoplasms. The efficacy of chemotherapy and radiation remains to be defined in future studies.

Interleukin-1alpha protein secretion in breast cancer is associated with poor differentiation and estrogen receptor alpha negativity.

While interleukins (IL)-1alpha and beta are thought to play an important role in malignant disease, little is still known about their expression in breast cancer. We have used reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) to analyze the expression of IL-1alpha and beta in breast cancer tissues, and compared their expression to estrogen receptor (ER) status and grading. In breast cancer cell lines, we found an inverse correlation between IL-1alpha and beta gene expression and differentiation, and only one highly invasive tumor cell line expressed IL-1alpha protein, while IL-beta was not detectable. Breast cancer tissue expressed variable amounts of IL-1alpha and beta messenger RNA, but consistently high levels of IL-1 type I receptor. IL-1alpha protein was detectable in malignant epithelium and adjacent stroma in 88% of cases. IL-1alpha expression was correlated with poor differentiation (P= 0.002; r= 0.469) and decreasing ERalpha expression (P= 0.004; r=-0.387). Stromal IL-1alpha was confined to areas with low or absent ERalpha protein expression in adjacent tumor epithelium (P= 0.001; r=-0.457). Taken together, we have demonstrated a functional IL-1 system in breast cancer and observed an inverse correlation between IL-1alpha and sex steroid receptor expression. We suggest that the expression of IL-1alpha in poorly differentiated, ERalpha-negative tumors contributes to their malignant phenotype.

Interleukin 1alpha and tissue-lytic matrix metalloproteinase-1 are elevated in ectopic endometrium of patients with endometriosis.

BACKGROUND: Matrix metalloproteinases (MMP) play an essential role in tissue remodelling and menstruation and appear to be regulated by cytokines such as interleukin-1alpha (IL-1alpha). In order to investigate their role in the pathogenesis of endometriosis, the aim of the present study was to compare the protein localization of matrix metalloproteinase-1 (MMP-1) and of its main stimulatory cytokine IL-1alpha in eutopic and dystopic endometrium of patients with endometriosis. METHODS: MMP-1 and IL-1alpha protein localization was analysed retrospectively in paired paraffin-embedded tissue biopsies obtained simultaneously from the endometrial cavity and from endometrial lesions of 37 patients with peritoneal or ovarian endometriosis and in cycling endometria from 37 women without endometriosis. Protein localization was demonstrated by immunohistochemistry; antibody specificity was confirmed by western blot analysis. RESULTS: MMP-1 and IL-1alpha protein staining in women suffering from endometriosis was significantly more pronounced in endometriotic lesions than in eutopic endometrium. This held true for both epithelial MMP-1 and IL-1alpha staining (P < 0.006 and P < 0.001), and for stromal MMP-1 and IL-1alpha staining (P < 0.001 and P < 0.001). Furthermore, stromal MMP-1 and IL-1alpha were significantly co-expressed in dystopic endometriotic tissue (P = 0.045). Endometrial MMP-1 and IL-1alpha protein expression pattern in eutopic endometrium from women suffering from endometriosis, however, did not differ significantly from the pattern seen in healthy women. CONCLUSIONS: The increased expression of both matrix-degrading MMP-1 and its major stimulatory cytokine IL-1alpha in endometriotic lesions and the selective co-expression in the stroma of endometriotic foci clearly suggests their involvement in the pathogenic mechanisms leading to local invasion and tissue destruction.

Presence of nanobacteria in psammoma bodies of ovarian cancer: evidence for pathogenetic role in intratumoral biomineralization.

AIMS: The presence of laminated, calcified extracellular debris known as psammoma bodies is a well-known histomorphological feature of ovarian adenocarcinomas and other human malignancies. Biomineralization has recently been found to be associated with a group of extremely small Gram-negative bacteria capable of precipitating calcium salts. The aim of the present study was to evaluate a possible pathogenic link between the development of psammoma bodies and nanobacteria infection. MATERIAL AND RESULTS: Immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyse nanobacterial protein and gene expression in eight psammona body-containing adenocarcinomas and in 10 malignant ovarian tumours without signs of biomineralization. Nanobacterial proteins were detected in eight out of eight (100%) psammoma-positive tumour samples. Conversely, none of the 10 psammoma-negative tissues (0%) was positive for nanobacterial antigens. Furthermore, nanobacterial mRNA was detectable in all of the four tissues (100%) that contained psammoma bodies, but was absent in all 10 ovarian cystadenocarcinomas (0%) that were psammoma negative. CONCLUSIONS: We found a 100% concordance between the expression of nanobacteria and the presence of psammoma bodies in malignant ovarian tumours. Several lines of evidence suggest the involvement of these organisms in the process of biomineralization. We therefore conclude that nanobacterial infection of malignant ovarian tissue contributes to mechanisms leading to the formation of calcified deposits known as psammoma bodies.

Predicting the clinical course of breast cancer patients undergoing trastuzumab-based therapy: an outlook.

Her-2/neu overexpressing breast cancer is associated with reduced overall survival, sex steroid receptor negativity and increased resistance to antihormonal therapy, and thus represents a subgroup with poor prognosis. The anti Her-2/neu receptor antibody trastuzumab (Herceptin), however, specifically targets this protein and provides a valuable addition to classical systemic therapies. Unfortunately, not all tumors that express Her-2/neu protein are also adequate candidates for trastuzumab therapy. Therefore, most clinicians now consider Her-2/neu oncoprotein overexpression and/or her-2/neu gene amplification a prerequisite for trastuzumab-based antineoplastic therapy. Nevertheless, due to the relatively low response rates that are observed even in this preselected patient cohort, better response predictors are clearly needed. Here, we review established parameters such as Her-2/neu immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and their ability to predict the clinical course of trastuzumab-treated breast cancer. We also evaluate promising parameters such as serum levels of the Her-2/neu extracellular domain (ECD) and the activation status of Her-2/neu oncoprotein (pHer-2/neu), and their use in the clinical setting. Finally, novel tumor-specific such as tumor M2-PK, IGF-IR and p53 are discussed and their potential to predict the efficacy of trastuzumab is assessed.

Expression of matrix metalloproteinases in patients with uterine smooth muscle tumors: an immunohistochemical analysis of MMP-1 and MMP-2 protein expression in leiomyoma, uterine smooth muscle tumor of uncertain malignant potential, and leiomyosarcoma.

OBJECTIVE: Matrix metalloproteinases (MMPs) have been suggested to play an important role in tumor invasion and metastasis. We compared the expression of MMP-1 and MMP-2 protein in patients with leiomyoma, uterine smooth muscle tumor of uncertain malignant potential (STUMP), and leiomyosarcoma (LMS). METHODS: MMP-1 and MMP-2 expression was investigated by immunohistochemistry from paraffin-embedded tissue in 26 patients with leiomyoma, in 24 patients with STUMP, and in 21 patients with LMS. RESULTS: MMP-1 was expressed in 92% of leiomyomas, in 83% of STUMP, and in 86% of LMS, whereas MMP-2 was expressed in 12% of leiomyomas, in 17% of STUMP, and in 48% of LMS. A statistically significant difference regarding the frequency of MMP-2 expression was observed between LMS and STUMP (P =.025) as well as between LMS and leiomyoma (P =.006), but not between STUMP and leiomyoma (P >.05). Likewise, the staining intensity did significantly differ between LMS and leiomyoma (P =.025), but no statistical significant difference was observed between LMS and STUMP (P >.05) and between STUMP and leiomyoma (P >.05). CONCLUSION: The stronger MMP-2 expression in patients with LMS compared with STUMP and leiomyoma indicates that this protein might be a marker for tumor invasion or metastasis in patients with uterine LMS. Furthermore, MMP-2 seems to be a useful immunohistochemical parameter to distinguish cases of smooth muscle tumors in which histologic features are ambiguous or borderline. Further studies including larger numbers of patients are necessary to establish MMP-2 as a routine marker for tumor invasion and progression.

MMP-1 and MMP-2 expression in uterine leiomyosarcoma and correlation with different clinicopathologic parameters.

OBJECTIVE: Matrix metalloproteinases (MMPs) have been suggested to play an important role in tumor invasion and metastasis because they degrade a wide range of components of the extracellular matrix. In the present study, we analyzed the expression of MMP-1 and MMP-2 proteins in patients with uterine leiomyosarcoma. METHODS: MMP-1 and MMP-2 expression was investigated by immunohistochemistry from paraffin-embedded tissue sections in 21 patients with uterine leiomyosarcoma (LMS). The immunohistochemical findings were correlated with different clinicopathologic characteristics of the patients. RESULTS: MMP-1 was expressed in 86% and MMP-2 was expressed in 48% of uterine LMS. There was a statistically significant positive correlation between vascular space involvement and MMP-2 expression (P =.05) and between age and MMP-2 expression, with patients over 50 years old having significantly more frequent MMP-2-positive tumors than patients younger than 50 years (P =.006). The relationship between MMP-2 expression and tumor stage and recurrence disease did not reach statistical significance. A trend towards prolonged disease-free survival was observed in women with MMP-2-negative LMS compared with patients with MMP-2-positive LMS (P =.09). Furthermore, a univariate analysis revealed that early tumor stage (P =.0001), age at diagnosis less than 50 years (P =.02), and the absence of vascular space involvement (P =.04) were associated with longer overall survival. CONCLUSION: The statistically significant positive correlation between MMP-2 expression and vascular space involvement as well as the prolonged disease-free survival rate in patients with MMP-2 negative uterine LMS suggest that MMP-2 plays an important role in tumor invasion and metastasis. Further clinical studies with larger numbers of cases need to be performed to verify these findings.

Her-2/neu-triggered intracellular tyrosine kinase activation: in vivo relevance of ligand-independent activation mechanisms and impact upon the efficacy of trastuzumab-based treatment.

Proteolytic cleavage of the Her-2/neu extracellular domain (ECD) has been shown to initiate receptor phosphorylation representing Her-2/neu activation in vitro. The present investigation was performed to evaluate the clinical relevance of ECD cleavage for Her-2/neu activation and the consequences of active intracellular Her-2/neu signalling reflected by tyrosine kinase phosphorylation in patients treated with the anti-Her-2/neu antibody trastuzumab. Sera from 62 patients receiving trastuzumab-based treatment for Her-2/neu overexpressing metastatic breast cancer were assessed for pretreatment ECD levels using an enzyme-linked immunosorbent assay. In parallel, Her-2/neu activation status of tumour specimens was assessed by immunohistochemistry using a Her-2/neu phosphorylation state specific antibody (PN2A) and correlated with the patients' ECD levels and clinical course of disease. Serum ECD levels were significantly higher in 15 (24%) patients with tumours exhibiting activated Her-2/neu as compared to those without detectable Her-2/neu phosphorylation (median 148.2 vs 28.5 ng ml(-1), P=0.010). Whereas response rate only showed a trend to be higher in patients with Her-2/neu-phosphorylated breast cancer (47 vs 34%, P=0.197), both uni- and multivariate analyses revealed that the median progression-free survival under trastuzumab-based treatment was significantly longer in patients with Her-2/neu-phosphorylated breast cancer-11.7 (95% CI 5.2-18.3) months-when compared to the progression-free survival of 4.5 (95% CI 3.4-5.6) months observed in patients with tumours lacking phosphorylated Her-2/neu (P=0.001). Proteolytic cleavage of the ECD represents a biologically relevant ligand-independent mechanism of Her-2/neu activation in vivo. The influence of Her-2/neu activation status upon the outcome of trastuzumab-based therapies merits further investigation in larger prospective trials.

Co-expression of ErbB-family members in human breast cancer: Her-2/neu is the preferred dimerization candidate in nodal-positive tumors.

Over-expression of members of the ErbB-receptor family has been associated with malignant transformation. The amplification of Her-2/neu in tumor tissue is now an established prognostic factor in breast cancer. In order to initiate signal transduction, ErbB-receptor monomers need to form homo- or heterodimers. The composition of these dimers is thought to influence both quality and quantity of downstream signaling pathways, and to determine the biological response. We have investigated the protein expression pattern of the four ErbB-receptors EGFR, Her-2/neu, Her-3 and Her-4, and correlated it with their putative ligands EGF, TGF-alpha and HRG in 74 women with invasive breast cancer. Using western blot-analysis on cell membrane isolates, we detected the co-expression of all four ErbB-family members in 79.7% of cases, and of all of the three investigated ligands in 82.4%. We did not observe a correlation between EGFR and Her-2/neu or Her-4 protein expression, EGFR and Her-3 (p = 0.005), and Her-3 and Her-4 (p = 0.05) were clearly co-expressed. The strongest overall correlation, was found between Her-2/neu and Her-3 (p < 0.001) and between Her-2/neu and Her-4 (p = 0.001). This was particularly true in nodal-positive tumors (p < 0.001 and p = 0.002) whereas in nodal-negative tumors the co-expression was either less significant (Her-2/neu and Her-3; p = 0.01) or not significant (Her-2/neu and Her-4). The co-expression of EGFR/Her-3 was associated with the expression of all ligands, whereas the Her-2/neu/Her-3 was correlated with HRG (p = 0.002), thereby indicating a functional relation between specific receptor-dimer combinations and putative ligands. Taken together, we have performed the first comprehensive survey of ErbB-system expression in breast cancer, and have demonstrated the presence of a co-regulated receptor/ligand system in vivo. We have further shown that Her-2/neu is the preferred co-expression partner in nodal-positive tumors and thus the most likely dimerization candidate in malignant breast tumors.

The detection of HPV DNA improves the recognition of cervical intraepithelial lesions.

UNLABELLED: Reasons that influence the efficacy of cervical cancer screening are failure to screen all women at risk, as well as inherent technical limitations of the conventional cervical smear. HPV DNA testing is a supplementary, objective test less independent on sampling failure. The aim of this study was to compare results of HPV DNA screening to cytological smears (CS) and histological diagnosis. From January 1995 to January 1999, cytological smears, cells for HPV DNA analysis and cervical biopsies were obtained from 280 women included in this study. STATISTICAL METHODS: Fisher's exact test (2x2 contingency tables, P<0.01), Pearson Chi-square, P< 0.05, Spearman's rank correlation R. Sixty patients (21.4%) tested positive for low-risk (LR-HPV), 227 (81.1%) positive for high-risk HPV (HR-HPV). The CS proved to be a strong predictor for the histological diagnosis, reaching a sensitivity of 93.4%, a specificity of 65.8% and a positive predictive value (PPV) of 77.4%. By combining cytology and HPV DNA testing, the sensitivity could be considerably enhanced (99.0%), though at a price of loss in specificity (30.1%). HPV DNA testing, available as a commercially standardized product, leads to a significant rise in sensitivity when used as an additional diagnostic tool to cytological screening methods and thus contributes to reduce the incidence of cervical carcinomas.

Interleukin-1 system and sex steroid receptor gene expression in human endometrial cancer.

OBJECTIVES: The interleukin-1 system is known to play a pivotal role in human physiology and reproduction. In the cycling endometrium, interleukin-1alpha activity is controlled by sex steroids and is confined to the perimenstrual phase, where it is involved in the events leading to tissue lysis and menstruation. Since local tissue degradation is also a feature of malignant tumors, our goal was to analyze the gene expression of interleukin-1alpha and other interleukin-1 family members and compare it with estrogen receptor alpha, estrogen receptor beta, and progesterone receptor mRNA expression in 27 endometrial carcinomas and 13 normal endometria. METHODS: Endometrial tumor tissues were obtained during hysterectomy for endometrial cancer, and normal endometrium was sampled in women undergoing surgical procedures for nonendometrial pathologies. Gene expression was analyzed by reverse transcription polymerase chain reaction. Protein expression was detected and localized by immunohistochemical staining. RESULTS: A strong gene expression of interleukin-1 type I receptor, estrogen recptor alpha, and progesterone receptor was detected in all tumor tissues and in the majority of benign endometrial tissues. However, in contrast to nonmalignant endometria, variable amounts of interleukin-1beta and interleukin-1 receptor antagonist mRNA were also detected in most of the tumor samples. Gene expression of interleukin-1alpha and estrogen receptor beta was considerably less frequent, with interleukin-1alpha being absent in all peri- and postmenopausal endometria and in all but one of the well-differentiated tumors. With decreasing differentiation interleukin-1alpha gene expression became more frequent. In these cases, interleukin-1alpha protein was detected predominantly in epithelial tumor cells of lower-grade tumors. CONCLUSION: We have demonstrated the presence of the interleukin-1 system in endometrial malignancies, and found a negative correlation between interleukin-1alpha and tumor differentiation. We hypothesize that the nonphysiological expression of interleukin-1alpha in less differentiated tumors might contribute to their invasiveness and malignant behavior.

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.

Prognostic parameters in carcinosarcomas of the uterus: a clinico-pathologic study.

BACKGROUND: Carcinosarcomas of the uterus are highly aggressive malignant neoplasms with early lymphatic and hematogenous spread. The most important prognostic factor in carcinosarcoma is the extent of the tumor at the time of diagnosis. The prognostic impact of other factors such as myometrial invasion, menopausal age, age, parity and adjuvant therapy is still being discussed controversially. MATERIALS AND METHODS: Nineteen patients with histologically proven carcinosarcoma were included in the analysis. The patients were staged according to a modification of the International Federation of Gynecology and Obstetrics (FIGO) staging system for endometrial cancer. For each patient, the histological material was reviewed by an experienced pathologist. Carcinosarcoma was defined histologically as any tumor of uterine origin composed of carcinomatous and sarcomatous components. RESULTS: The median follow-up time was 91 months (25% quartile, 47 months; 75% quartile, 145 months). The median overall survival of the 19 patients was 59 months, resulting in a 5-year overall survival rate of 43%. Three out of the nineteen (16%) patients demonstrated progressive disease while 6 out of 10 (32%) patients developed recurrent disease with a median disease free survival of 16 months (range 8-54). Eleven out of nineteen (58%) patients died of the disease. A univariate model revealed that early tumor stage (stage 1) (p<0.023), low myometrial invasion (p<0.017) and late onset of the menopause (p<0.050) were significantly associated with a lengthened overall survival in patients with carcinosarcoma. Age (p=0.34), parity (p=0.16) and adjuvant radiotherapy (p=0.45) did not influence overall survival of patients with carcinosarcoma. CONCLUSION: Early tumor stage, low myometrial invasion and late onset of the menopause are associated with a lengthened overall survival in patients with carcinosarcoma.

Oncogenic potential of c-erbB-2 and its association with c-K-ras in premalignant and malignant lesions of the human uterine endometrium.

The aim of this study was to detect activated c-K-ras by gene point mutation and to find c-erbB-2 gene amplification with p185 expression in association with the c-K-ras gene product p21 in the human endometrium. Specimens obtained from 25 normal, 31 hyperplastic and 72 malignant samples of the human endometrium were examined for point mutation in codons 12, 13 and 61 of the c-K-ras by direct sequencing and c-erbB-2 gene amplification with p185 and p21 expression by differential polymerase chain reaction (DPCR) and immunohistochemistry. Neither the normal endometrium nor endometrial hyperplasias were found to have mutations in the c-K-ras gene, although a double mutation of codons 12 and 13 as a single-point mutation was observed in one case of endometrioid carcinoma (2.8%). In each of two other cases of endometrioid carcinoma (2/72), two single-point mutations of codon 13 (5.6%) were shown. Using DPCR, we found c-erbB-2 to be amplified in 15 premalignant (48%) and 45 malignant (63%) samples. We noticed that nonamplification of the c-erbB-2 gene was associated with the absence of immunoreactivity. Our data indicate that, while c-erbB-2 plays a role in the early development of endometrioid carcinomas, c-K-ras gene activation by point mutation does not.