The role of fibroblast growth factor 2 in patients with uterine smooth muscle tumors: an immunohistochemical study.

OBJECTIVE: Fibroblast growth factor 2 (FGF-2) is considered to be a potent stimulator of angiogenesis and seems therefore to play an important role in the growth of tumors. We compared the immunohistochemical profile of FGF-2 in patients with uterine leiomyomas, smooth muscle tumors of uncertain malignant potential (STUMP) and leiomyosarcoma (LMS). Furthermore, we tried to clarify the prognostic role of FGF-2 in uterine leiomyosarcoma. STUDY DESIGN: FGF-2 expression was investigated by immunohistochemistry from paraffin-embedded tissue in 26 patients with leiomyoma, in 24 cases with STUMP and in 21 patients with LMS. The immunohistochemical profile of these 3 tumor entities was compared and regarding LMS correlated with different clinicopathologic parameters. RESULTS: FGF-2 was expressed in 85% of leiomyomas, in 88% of STUMP and in 57% of LMS. Significant differences regarding the frequency of FGF-2 expression were observed between leiomyoma and LMS as well as between STUMP and LMS (p<0.05). In uterine LMS FGF-2 expression was statistically more frequent in cases with high histological grade (p<0.05). Furthermore, FGF-2 positive tumors demonstrated a statistically significant higher rate of recurrence disease and tumor progression (p=0.005). Disease free as well as overall survival was significantly shortened in patients with FGF-2 positive compared to FGF-2 negative tumors (p<0.05). CONCLUSION: The significant correlation between FGF-2 expression and high histological grade indicates that FGF-2 might work as a negative predictive factor. Higher rates of recurrence disease as well as shortened disease free and overall survival among FGF-2 positive LMS support the potential role as prognosticator for poor clinical outcome.

Expression of p16 protein and epidermal growth factor receptor in patients with adenocarcinoma of the uterine cervix: an immunohistochemical analysis.

OBJECTIVE: The present study assessed the expression of p16 and epidermal growth factor receptor (EGFR) in patients with adenocarcinoma of the uterine cervix to determine their influence on prognosis and to evaluate a possible association between their expression and various clinicopathologic parameters. METHODS: p16 and EGFR expression was investigated by immunohistochemistry from paraffin-embedded tissue in 39 patients with adenocarcinoma of the uterine cervix. The immunohistochemical findings were correlated with different clinicopathologic parameters of the patients. RESULTS: p16 was expressed in 56% of the patients. A trend towards increased lymph vascular space invasion was observed in p16 positive tumors (p = 0.06). There was no statistically significant association between p16 expression and clinical stage, age, histology, tumor size, tumor grade, lymph node status and recurrence disease (p > 0.05). p16 expression did influence neither disease-free nor overall survival (p > 0.05). EGFR was expressed in 44% of the patients. There was no statistically significant correlation between EGFR expression and clinical stage, age, histology, tumor size, tumor grade, lymph vascular space invasion, lymph node status and recurrence disease (p > 0.05). EGFR expression did influence neither disease-free nor overall survival (p > 0.05). CONCLUSION: p16 and EGFR are frequently expressed in adenocarcinoma of the uterine cervix. Our study observed a trend towards increased lymph vascular space invasion in p16 positive tumors. Otherwise, the expression of the investigated parameters did not correlate with any clinicopathologic parameters and had no influence on overall and disease-free survival. So far, the investigation of p16 and EGFR is of limited use to assess patients' prognosis and guide clinical management.

Differential expression pattern of estrogen receptors, aromatase, and sulfotransferase in breast cancer tissue and corresponding lymph node metastases.

Patients with hormone receptor positive breast cancer who are treated with endocrine therapy generally have a good prognosis. However, resistance to hormonal therapy and progression occurs, and the reasons for this are manifold. It has been proposed that the local estrogenic environment has a role in the process of local invasion and progression. We have determined the expression pattern of estrogen receptor α, estrogen receptor ß, and the epithelial and stromal expression of the estrogen-metabolizing enzymes aromatase and sulfotransferase by immunohistochemistry in tissue arrays, containing 50 paraffin-embedded sets of tissues obtained from breast cancer and from corresponding metastatic axillary lymph nodes of the same patients. We have found statistically significant higher estrogen receptors α and ß expression in primary tumors than in corresponding lymph node metastases (p = 0.0004 and p = 0.003, respectively). Aromatase was also expressed more frequently in epithelial as well as in stromal cells of the malignant tumor when compared to according lymph node metastases (p = 0.08 and p = 0.12, respectively). While in lymph node metastases only estrogen receptor α and stromal aromatase expression were correlated (p = 0.01), significant associations were seen between the estrogen receptor ß and sromal aromatase, and epithelial sulfotransferase (p = 0.0006 and p = 0.03, respectively) in the primary tumor. We hypothesize that the decreased expression of local estrogens by aromatase, in combination with a decreased expression of estrogen receptors α and ß in lymphatic metastases, renders these metastases hormone insensitive and could contribute to the poor response to endocrine therapy that is often seen in nodal-positive tumors.

Oestrogen and progesterone receptor expression in patients with adenocarcinoma of the uterine cervix and correlation with various clinicopathological parameters.

OBJECTIVE: The expression of oestrogen and progesterone receptors in patients with adenocarcinoma of the uterine cervix was examined in order to determine their influence on prognosis and to evaluate the association between the steroid receptor expression and various clinicopathologic parameters. PATIENTS AND METHODS: Oestrogen and progesterone receptor expression was investigated by immunohistochemistry from paraffin-embedded tissue in 39 patients with adenocarcinoma of the uterine cervix. The immunohistochemical findings were correlated with various clinicopathological parameters of the patients. RESULTS: Oestrogen and progesterone receptors were expressed in 39% and 33% of the patients, respectively. The relationship between oestrogen and progesterone receptor expression and clinical stage, age, histology, tumour size, grade, lymph-vascular space invasion and lymph node status did not reach statistical significance (p>0.05). Neither oestrogen nor progesterone receptor expression significantly influenced disease-free and overall survival (p>0.05). CONCLUSION: Oestrogen and progesterone receptors were frequently expressed in adenocarcinoma of the uterine cervix. However, their expression did not correlate with clinicopathological parameters and had no influence on overall and disease-free survival. Thus, the investigation of steroid receptors adds little additional information to the clinical management and fails to play a prognostic role in cervical adenocarcinoma.

Beta-hCG/LH receptor (b-HCG/LH-R) expression is increased in invasive versus preinvasive breast cancer: implications for breast carcinogenesis?

INTRODUCTION: The role of the b-HCG/LH/LH-R system in breast cancer is conflicting. Whereas some reports suggest a protective effect of b-HCG on breast epithelium, vitro studies implicate a role of b-HCG/LH-R in the development and growth of breast tumors. MATERIAL AND METHODS: In order to further investigate a possible involvement of b-HCG/LH-R in breast carcinogenesis, immunofluorescence analyses of b-HCG/LH-R expression was performed on 70 preinvasive and adjacent invasive breast cancer specimen using tissue microarrays (TMAs). RESULTS: In 37 preinvasive samples available for further analysis, b-HCG/LH-R was found in 8/37 samples (21.6%; weak, intermediate and strong staining in 4/37 (10.8%), 2/37 (5.4%) and 2/37 (5.4%). In contrast, b-HCG/LH-R expression was observed in 19/27 (70.4%) adjacent invasive specimen with weak, moderate and strong immunostaining in 10/27 (37.0%), 6/27 (22.2%) and 3/27 (11.1%), respectively. This was statistically significant when compared to preinvasive components (P = 0.001, Chi Square Test). CONCLUSIONS: Based on the observation that b-HCG/LH-R was found to be selectively upregulated in invasive tumor components, we suggest that under certain circumstances, sensitivity of ductal cells to hormones that target b-HCG/LH-R could favour mammary carcinogenesis.

Incomplete surgical resection of ductal carcinomas in situ results in activation of ERBB2 in residual breast cancer cells.

ERBB2 amplification and consecutive overexpression is a predictor for poor prognosis in breast cancer patients. In addition, incomplete resection of ERBB2-overexpressing tumors leads to increased proliferation of residual breast cancer cells. While the local release of cytokines is thought to be responsible for the malignant behavior of remaining tumor tissue, the exact mechanism is still unknown. We have analyzed epidermal growth factor receptor (EGFR), activated (p)EGFR, and activated (p)ERBB2 protein expression in ERBB2-overexpressing and in non-ERBB2-overexpressing tumors from patients who underwent breast surgery and consecutive re-excision for involved margins, and compared expression levels by immunohistochemistry. While overall ERBB2 protein expression in the initial and the re-excised sample were comparable, we observed an increase in pERBB2 in ductal carcinomas in situ in both, ERBB2-overexpressing (16/21 vs 24/24; P=0.018, chi(2) test) and non-ERBB2-overexpressing tumors (3/28 vs 5/12; P=0.025, chi(2) test). pERBB2 was not increased in invasive tumors, regardless on whether the samples had been taken from a ERBB2-overexpressing (9/25 vs 6/17; P=0.261, chi(2) test) or a non-ERBB2-overexpressing tumor (1/27 vs 0/8; P=0.581, chi(2) test). EGFR expression was only detected in 1/47 ERBB2-overexpressing primary tumors and 2/48 non-ERBB2-overexpressing tumors, and was undetectable in re-excised specimen. Taken together, we have demonstrated an increase in ERBB2 receptor activation in incompletely resected preinvasive breast cancer. We hypothesize that receptor phosphorylation is caused by growth factor stimulation in response to intraoperative tissue damage, and perioperative inhibition of specific cytokines could become a promising therapeutic strategy.

beta-HCG/LH receptor (beta-HCG/LH-R) expression in eutopic endometrium and endometriotic implants: evidence for beta-HCG sensitivity of endometriosis.

BACKGROUND: Luteinizing hormone (LH) and human chorionic gonadotropin (HCG) target their receptor in gonadal and nongonadal cells to stimulate steroidogenesis and cell growth. The aim of the present study was to investigate the expression of HCG/LH-R in endometriosis to elucidate a possible impact of LH and HCG on this disease. MATERIALS AND METHODS: Analysis of HCG/LH-R protein expression in 23 paired samples of ectopic and eutopic tissue of cycling women with endometriosis and in endometrial samples from 22 healthy controls was conducted via immunofluorescence. HCG and HCG/LH-R gene expression in endometriotic lesions was confirmed by reverse-transcriptase polymerase chain reaction. RESULTS: In endometriotic implants, epithelial HCG/LH-R was found in 12/23 samples. No significant differences in HCG/LH-R levels were observed when compared with glands of uterine endometrium from the same patients or healthy controls. Messenger RNA transcripts for HCG were detected in all 12 samples, whereas HCG/LH-R mRNAs were observed in 10 of the 12 endometriotic lesions investigated. CONCLUSIONS: Although HCG/LH-R was not found to be selectively upregulated in endometriosis, the mere presence of HCG/LH-R in endometriotic tissue may suggest sensitivity of endometriosis to HCG and LH that target HCG/LH-R.

Differential gene expression profile in breast cancer-derived stromal fibroblasts.

BACKGROUND: Breast cancer is characterized by malignant transformation of epithelial cells, but stromal cells also play an important role in tumorigenesis. While tumor-derived fibroblasts display unique phenotypic properties, it is unclear whether they also represent are a specific subpopulation. MATERIALS AND METHODS: Stromal fibroblasts deriving from malignant tissue of 10 women with invasive breast cancer, and from normal breast tissue of 10 women with benign breast disorders, were subjected to differential complementary DNA Microarray Analysis by using a 2,400 gene cDNA array. Individual gene expression pattern were confirmed by RT-PCR. RESULTS: In a cDNA array that allows to analyze the differential gene expression of more than 2,400 genes, the mRNA expression of 135 genes were increased more than 2 fold in fibroblasts from malignant breast tumors. The majority of these genes encode tumor-promoting cytokines, transcription factors and cell-matrix associated proteins. The mRNA expression of 110 genes decreased to less than 0.5 fold. The remaining 2,155 genes were not significantly altered. RT-PCR performed on individual biopsies from breast cancer and normal breast tissues confirmed the validity of the pooled gene expression signature. CONCLUSION: Breast cancer-derived stromal fibroblasts show a distinctive gene expression pattern that differentiates them from normal breast stroma. Our observation of increased expression of tumor promotion-associated genes even in the absence of adjacent malignant epithelium suggests that tumor stroma is comprised of a fibroblastic subpopulation that provides for a microenvironment which supports tumor growth and invasion.

Type-specific antiviral antibodies to genital human papillomavirus types in mothers and newborns.

Type-specific antibodies to human papillomaviruses (HPVs) can be detected in most infected adult patients, and they have virus-neutralizing properties. However, there is a dearth of information on the seroprevalence of maternal and neonatal antibodies to HPV capsid antigens. Sera from 104 mothers, their newborns, and 3 twin pregnancies were analyzed by an enzyme-linked immunosorbent assay (ELISA) for the presence of specific IgG, IgM, and IgA antibodies to virus-like particles of HPV-6, -11, -16, -18, and -31. Maternal IgG positivity rates to HPV types 6, 11, 16, 18, and 31 were 23.1%, 2.9%, 8.7%, 5.8%, and 9.6%, respectively. Neonatal rates did not differ significantly, and individual IgG ELISA values of mothers and their infants and all paired twins showed a very high correlation. In contrast, nearly all IgM and IgA individual values in newborns were designated negative, whereas mothers' positivity rates ranged as high as 19.2%. Infants showed no HPV-related lesions at birth or at 4-year follow-up. Seven of 8 tested children lost IgG HPV antibodies in a follow-up examination. Similar anti-HPV IgG seropositivity in mothers and newborns and a lack of neonatal IgA and IgM together with twin and follow-up results indicate that neonatal IgG is not a sign of intrauterine HPV infection but, rather, maternofetal antibody transmission.

Differential spatial expression and activation pattern of EGFR and HER2 in human breast cancer.

Overexpression of HER family members is a well established prognostic factor and identifies potential targets for antibody-based receptor blocking strategies. While several studies have analyzed the expression of HER2 and other HER-family members in malignant tumors, considerably less is known about their expression and activation in non-involved breast tissue from breast cancer patients. We have therefore investigated the differential expression of EGFR, HER2, and their tyrosine-kinase activated forms (ptyr-1248 Her-2 and ptyr-845 EGFR) in 63 tumor specimen containing: a) malignant epithelium, b) in non-malignant tissue located at the peritumoral margin, and c) in uninvolved breast tissue obtained from tissue distant from the tumor. Using immunohistochemistry (IHC), we found significantly higher HER2 protein expression levels in malignant epithelium than in marginal and peripheral non-malignant epithelium (p=1.3 x 10(-10) Fisher's exact test). Epithelial EGFR expression did not differ between the three tissue types, but stromal EGFR protein was significantly more common in marginal and peripheral tissues when compared to tumor tissues (p=0.008, Fisher's exact test). When analyzing activated receptor forms, we found epithelial ptyr-1248 HER2 expression in one tumoral, one marginal and one peripheral sample. We did not observe ptyr-845 EGFR in any of the samples analyzed. We found a significant overall correlation between epithelial and stromal EGFR expression (r=0.442; p<0.0001; Spearman's Rho), and between stromal EGFR expression and normal tissue type (r=0.170; p<0.02; Spearman's Rho). Epithelial HER2 expression and normal tissue type (r=0.492; p<0.0001; Spearman's Rho) were inversely correlated. Taken together, we have observed a differential expression pattern of EGFR, HER2, and activated HER2 that is dependent on the spatial relation to a malignant tumor. Our findings of decreased intratumoral EGFR expression and the absence of activated EGFR suggests that, in contrast to HER2, EGFR inhibition might not be an ideal target for antibody therapy.

Gene profiling in Pap-cell smears of high-risk human papillomavirus-positive squamous cervical carcinoma.

OBJECTIVE: The purpose of the study was to investigate benign and malignant squamous cervical cells obtained by cervical swabs with regard to differentially expressed genes and gene expression profiling, in order to evaluate the biological behavior and clinical outcome of cervical malignancies. METHODS: Cervical squamous cells from six women with high-risk human papillomavirus positive [HR-HPV(+)] cervical carcinoma and from six HPV-negative women with normal ectocervical cells were analyzed by cDNA array. RESULTS: cDNA over-expression of several genes such as MET (c-met), Nm23-H1 (NME1), EGFR, KGFR, Nm23-H2 (NME2), ERBB2 (c-erbB-2), cyclin-dependent kinase inhibitor 4 (CDKN2A, p16INK4A), cytokeratin 8 (KRT8), KRAS (K-ras), FLT1, KGF (FGF7), BCL2-like 2 protein (BCL2L2), ERBB4, MYCN (N-myc), cyclin D1 (CCND1), KIT (c-kit), secreted phosphoprotein 1 (SPP1) and STAT1, was significant in cervical squamous cell carcinoma (CSCC). Gene expression was downregulated for 13 genes in CSCC, such as interleukin 1 alpha (IL1A), the transforming growth factor receptor beta superfamily (TGFbeta; TGFB), some members of the insulin-like growth factor binding proteins (IGFBPs) and the integrin family (ITGA6, ITGB1). CONCLUSION: This study was focused on the gene expression profiling of HR-HPV(-) and (+) cervical squamous cells and CSCC obtained by cytobrush. We observed gene expression patterns and signaling pathways that permit the investigator to distinguish between benign squamous cervical cells and CSCC with and without HPV infection.

Expression of the human MTA1 gene in breast cell lines and in breast cancer tissues.

MTA1 was reported as a metastasis-associated gene. Tumors with higher MTA1 mRNA level were shown to have higher rates of invasion and lymph node metastasis and tended to have higher rates of vascular involvement. The majority of invasive breast carcinomas were demonstrated to overexpress MTA1 compared to surrounding normal tissues. MTA1 was also found to be more expressed in metastatic breast cancer cell lines than in nonmetastatic ones. Originally we were interested in analyzing factors differently expressed in invasive and noninvasive breast cancer cell lines. Therefore we analyzed expression of MTA1 together with several other genes in correlation with cell invasiveness in 25 breast epithelial cell lines. Furthermore, we analyzed it in 90 primary breast tumor tissues and examined its correlation with expression of estrogen receptor, progesterone receptor, plasminogen activator inhibitor-1, E-cadherin, histopathological data, disease-free survival, and overall survival. Our results demonstrated that MTA1 expression was significantly higher in noninvasive cell lines than in invasive ones. It also correlated positively with expression of noninvasive factors and reverse correlated with invasive factors in both cell lines and tumor tissues.

Selective spatial upregulation of intratumoral stromal aromatase in breast cancer patients: evidence for imbalance of local estrogen metabolism.

The suppression of local estrogens levels is of key importance in the treatment of ER-positive breast cancer. Essentially all endocrine strategies act by either suppressing estrogen formation or competitively inhibiting receptor-binding in tumor cells. Nevertheless, little is still known about the local expression of aromatase and sulfotransferase which are the key modulators of intra-tumoral estrogen levels. We have performed immunohistochemostry to investigate the expression of aromatase and sulfotransferase in 42 samples obtained directly from malignant breast tumors, and compared it to biopsies obtained from uninvolved tissue in the vicinity of the invasion front, and to distant breast tissue. We found that aromatase was equally detectable in both tumor epithelial and stroma, but was mostly epithelial in non-malignant tissues (P=0.00008, Fisher's exact test). Also, aromatase protein expression was significantly more common in tumoral stroma when compared with peritumoral and distant breast stroma (P=0.00005, and P<0.00001 respectively). With the notable exception of cystosarcoma phylloides, sulfotransferase protein was detectable only in epithelial tissues, regardless of the location within the diseased breast. However, epithelial sulfotransferase was correlated with epithelial aromatase (r=0.35461, P=0.0009, Spearman's rho test) and with the epithelial ER status (r=0.29313, P=0.005). We have demonstrated a differential aromatase and sulfotransferase protein expression pattern that is dependent on the spatial relation to a malignant breast tumor. Our results indicate a net increase in intratumoral active estrogen levels through increased stromal aromatization, while physiological local inactivation by sulfotransferase activity remains essentially unchanged.

Tissue array-based expression of transglutaminase-2 in human breast and ovarian cancer.

Transglutaminase-2 is involved in the physiological regulation of cell growth, but has also been associated with a number of cancer-associated features such as cell adhesion, metastasis and extracellular matrix modulation. Despite its importance in tumor cell progression and survival, relatively little is known about its expression in human malignancies. We have therefore investigated the transglutaminase-2 expression pattern in breast and ovarian cancer by using tissue arrays which contained 57 invasive breast cancer biopsies and 62 ovarian cancers, and compared it to transglutaminase-2 protein levels in normal human tissues. By using immunohistochemistry, transglutaminase-2 protein was detected in 48 of 57 breast tumors (84%), with epithelial expression in 26 of 41 (63%) ductal invasive carcinomas and in all 6 (100%) lobular invasive carcinomas. Stromal transglutaminase-2 was present in 14 of 41 (34%) ductal subtypes and in 4 of 6 (67%) lobular subtypes, which is in sharp contrast to the infrequent expression in normal breast stroma (P<0.001, Mann-Whitney test) and somewhat also in normal breast epithelium (P = 0.065, Mann-Whitney test). In most other human tissues, transglutaminase-2 protein was less frequent and usually confined to either the epithelium or in adjacent stroma. In ovarian tumors, the protein was detected in 36 of the 62 cases (58%), and seen in all histological subtypes. Taken together, we have demonstrated increased transglutaminase-2 protein expression in both malignant breast epithelium and surrounding stroma, although its selective spatial expression pattern in normal tissues also indicates a physiological role in stromal-epithelial interactions.

Expression of thrombospondin 1 (TSP 1) in patients with uterine smooth muscle tumors: an immunohistochemical study.

OBJECTIVE: Angiogenesis is an essential component for tumor development regulated by both proangiogenic and antiangiogenic factors. Thrombospondin 1 (TSP 1) suppresses angiogenesis by inhibiting endothelial cell proliferation and inducing endothelial cell apoptosis. The aim of this study was to compare the expression of TSP 1 in cases with leiomyoma, uterine smooth muscle tumor of uncertain malignant potential (STUMP) and leiomyosarcoma (LMS). Furthermore, we evaluated the prognostic relevance of TSP 1 in uterine LMS. METHODS: TSP 1 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. Standard immunohistochemical techniques were used to study the expression of TSP 1 in 5-mum-thick tumor sections. TSP 1 expression was correlated with survival using the Kaplan-Meier method and log-rank test for univariate analysis. RESULTS: TSP 1 was expressed in 77% of leiomyomas, in 13% of STUMP and in 24% of LMS. A statistically significant difference regarding the frequency of TSP 1 expression was observed between leiomyoma and LMS (P < 0.05) as well as between leiomyoma and STUMP (P < 0.05), but not between LMS and STUMP (P > 0.05). Furthermore, a statistically significant correlation between vascular space involvement and TSP 1 expression was observed in patients with uterine LMS, with patients without vascular space involvement having more frequently TSP 1 positive tumors (P = 0.04). No statistically significant correlation between TSP 1 and clinical stage, age and recurrence disease could be detected (P > 0.05). CONCLUSIONS: We found that TSP 1 was more frequently expressed in leiomyoma compared to STUMP and LMS. Additionally, the statistically significant negative correlation between vascular space involvement and TSP 1 expression in patients with uterine LMS shows that TSP 1 might work as a predictive factor in patients with LMS. Further clinical studies are necessary to prove our results and to clarify the role of TSP 1 in uterine smooth muscle tumors.

Expression of KLF5 is a prognostic factor for disease-free survival and overall survival in patients with breast cancer.

PURPOSE: Kruppel-like factor (KLF5) is a cell growth mediator in various epithelial cells. Higher KLF5 increases cell growth rate and leads to transformed phenotypes. Because tumor cell proliferation is tightly associated with tumor progression, and consequently, with survival of cancer patients, we wanted to examine the prognostic value of KLF5 gene expression for patients with breast cancer. EXPERIMENTAL DESIGN: The gene expression levels of KLF5, ER, PR, HER2, and MKI67 were quantified in the tumor tissues of 90 patients with breast cancer and correlated with disease-free survival and overall survival of the patients. The correlations of gene expression between KLF5 and ER, PR, HER2, and MKI67 were analyzed. In addition, KLF5 expression was also compared with clinical data and age of patients. RESULTS: Statistically significant correlations were found between gene expression of KLF5 and both disease-free survival (univariate analysis) and overall survival (univariate and multivariate analysis). Patients with higher KLF5 expression had shorter disease-free survival and overall survival time, whereas patients with lower KLF5 expression had better survival. Moreover, KLF5 was also found to be positively correlated with HER2 and MKI67, and negatively correlated with age of the patients at diagnosis. CONCLUSION: The gene expression of KLF5 is directly correlated with cell proliferation in vivo and is a prognostic factor for patients with breast cancer. Patients with higher KLF5 expression have shorter disease-free survival and overall survival than patients with lower KLF5 expression. In addition, KLF5 has higher expression in patients ages 50 years old.

Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast.

The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-beta-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated beta-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with beta-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and up-regulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating in-hibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear beta-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the path-way could contribute to trophoblastic hyperplasia and local invasion.

Proteomic analysis in human breast cancer: identification of a characteristic protein expression profile of malignant breast epithelium.

Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.

Active (p)CrkL is overexpressed in human malignancies: potential role as a surrogate parameter for therapeutic tyrosine kinase inhibition.

CrkL is a nuclear adaptor and transcriptional activator in Bcr-Abl expressing cells and constitutes the major tyrosine phosphorylated protein in CML, but the expression and biological function of CrkL in other malignancies is largely unknown. Using immunohistochemistry, we have analyzed the protein expression of activated (p)CrkL in normal and malignant tissues. We then treated K562 leukemia cells with imatinib to analyze the effect of tyrosine kinase inhibition on CrkL activation. pCrkL expression was predominantly epithelial and detected in the majority of non-malignant prostate (79%), 49% of colon biopsies, 36% of skin biopsies, and 41% of samples obtained from normal brain. Protein expression was, however, considerably less frequent in normal breast (18%), lung (16%) and ovarian (12%) tissues. In contrast to their corresponding benign tissues, pCrkL expression was significantly more common in breast cancer samples (49%, p<0.0001; Fisher's exact test), lung carcinomas (55%, p=0.0002), lymphatic tissues (80% vs. 10%, p=0.012), skin cancer (67%, p=0.020), ovarian malignomas (50%, p<0.0001) and colon carcinomas (63%, p<0.03). By contrast, activated CrkL was significantly less frequent in prostate carcinoma samples when compared to corresponding non-malignant prostatic tissues (14% vs. 79%, p<0.0001). pCrkL expression was abrogated in K562 cells with the addition of the tyrosine kinase inhibitor imatinib, which indicates that phosphorylation of CrkL is mediated through targets of therapeutic TK inhibition. We hypothesize that pCrkL is selectively up-regulated in a number of malignant tumor entities and involved in malignant transformation. We further suggest that pCrkL might serve as a potential surrogate parameter for the efficacy of therapeutic TK inhibition.

Her-2/neu and EGFR tyrosine kinase activation predict the efficacy of trastuzumab-based therapy in patients with metastatic breast cancer.

Her-2/neu overexpression in human breast cancer leads to an aggressive biological behavior and poor prognosis. Although the anti-Her-2/neu antibody trastuzumab (Herceptin(R)) has become a valuable therapeutic option for patients with Her-2/neu-overexpressing breast cancer, many patients do not benefit from this therapy. To evaluate the effect of receptor activation on tumor response, we have investigated the phosphorylation status of Her-2/neu and EGFR in 46 Her-2/neu-overexpressing tumor samples from trastuzumab-treated metastatic breast cancer patients by immunohistochemistry. Activated (p)tyr-1248 Her-2/neu was detected in 9 of 46 breast cancers (20%), and activated (p)tyr-845 and (p)tyr-1173 EGFR were both present in 6 tumors (13%) while EGFR was present in 16 cases (35%). ptyr-1248 Her-2/neu showed a trend to correlate with increased response to trastuzumab (p = 0.063), while ptyr-845, ptyr-1173 EGFR and EGFR did not. The presence of ptyr-1248 Her-2/neu and ptyr-845 or ptyr-1173 EGFR, however, was a strong predictor of both response to trastuzumab-based treatment (OR = 8.0, p = 0.021 and OR = 8.0, p = 0.021) and clinical benefit (OR = 5.47, p = 0.041 and OR = 6.22, p = 0.028 multivariate logistic regression analysis). Furthermore, ptyr-845 EGFR and ptyr-1248 Her-2/neu were both independent predictors of progression-free survival (RR = 0.21, p = 0.01 and RR = 0.45, p = 0.026, multivariate analysis). Patients with ptyr-845 EGFR positive tumors also tended toward increased overall survival (RR = 0.17, p = 0.082). Taken together, we have demonstrated that the determination of activated EGFR improves the utility of ptyr-1248 Her-2/neu staining in predicting the clinical outcome of patients undergoing trastuzumab treatment. We hypothesize that the activation state of both Her-2/neu and EGFR are key determinants for trastuzumab efficacy.