Prognostic significance of methylated RASSF1A and PITX2 genes in blood- and bone marrow plasma of breast cancer patients.

Free circulating DNA is increased in the serum/plasma of cancer patients, and methylation of certain genes has been found to be characteristic for malignancy. Therefore, we investigated the prognostic value of two promising genes, PITX2 and RASSF1A, in peripheral blood-plasma (PB-P) and bone marrow plasma (BM-P) of breast cancer patients. Peripheral blood and bone marrow samples from patients with primary breast cancer were prospectively collected during primary surgery at the Department of Obstetrics and Gynecology in Innsbruck (n = 428) from June 2000 to December 2006. The study has been approved by the ethical committee of the Medical University of Innsbruck. Methylation analysis was performed using MethyLight, a methylation-specific quantitative PCR-method. In univariate survival analysis, methylated PITX2 in PB-P was found to be a significant indicator for poor overall survival (OAS) and distant disease-free survival (DDFS) (P = 0.001 and P = 0.023). Methylated RASSF1A in PB-P was also an indicator for poor OAS and DDFS (P = 0.001 and P = 0.004). RASSF1A had also significant prognostic potential when determined in BM-P (P = 0.016). In multivariate survival analysis methylated PITX2 and RASSF1A in PB-P remained as therapy-independent prognostic factors for OAS (P = 0.021, P < 0.001). For DDFS only RASSF1A in PB-P showed prognostic significance (P = 0.002). Methylated RASSF1A and PITX2 in PB-P appear to have promising potential as prognostic markers in clinical use.

Urinary neopterin does not reflect the local antitumor immune milieu in ovarian cancer.

The main objective of the present investigation was to study the urinary neopterin excretion in the context of the activation of the adaptive cellular immune system at the tumor site. For this purpose, we compared pre-treatment urinary neopterin levels measured in 92 ovarian cancer patients, with intratumoral levels of mRNA transcripts from factors either involved in the adaptive antitumor immune defense (CD3, IFN-γ, IRF-1, IRF-2, SOCS1 and iNOS) or immune tolerance (FoxP3). This study did not reveal an association between urinary neopterin and one of these investigated "on tumor site transcripts". From all the factors reflecting the magnitude of the local adaptive antitumor response, intratumoral IRF-1 expression above the edge of the 25th percentile was found to predict most reliably favorable progression-free (median 34 months vs. 10 months; p < 0.001) and overall (median 52 months vs. 16 months; p < 0.001) survival. In contrast, pre-treatment urinary neopterin excretion above 275 µmol/mol creatinine, which indicates an unspecific activation of the innate immune system, was associated with a very poor overall survival with a median of only 11 months when compared with a median overall survival of 40 months in patients with lower urinary neopterin excretion (p = 0.021). Interestingly, the considerable survival benefit in patients with high IRF-1-expressing cancers was completely abrogated as well for progression-free as for overall survival when urinary neopterin concentrations were found to be concomitantly elevated. These findings demonstrate that in ovarian carcinomas the unspecific "cancer-related inflammation" contributes to a significant subversion of the adaptive antitumor immune defense mounted at the tumor site.

Expression of coxsackie-adenovirus receptor is related to estrogen sensitivity in breast cancer.

This study analyzes the relationship between coxsackie-adenovirus receptor (CAR) expression (transmembrane and soluble isoforms) and hormone sensitivity in 95 breast cancers. Furthermore, prognostic significance of the expression of the various CAR isoforms was investigated. In addition, inducibility of CAR expression by estradiol and tamoxifen was assessed in various breast cancer cell lines. Expression of transmembrane CAR (hCAR) highly correlated with estrogen receptivity, but was independent of the expression of progesterone receptor (PR). Furthermore, hCAR expression was significantly higher in tumors with low-grade malignancy. However, no relationship between hCAR expression and tumor size, lymph node status, or survival was revealed. In the hormone receptor-positive breast cancer cell line T47-D expression of hCAR and its soluble isoforms was increased by treatment with estradiol and tamoxifen. In contrast, no induction of either CAR isoform was achieved in receptor-negative cell lines. Furthermore, enhancement of hCAR expression was significantly greater when cells were treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) than when treated with estradiol or tamoxifen. Moreover, sensitivity to TSA induction of hCAR was considerably greater in receptor-positive than in receptor-negative cell lines. No additive effect on CAR expression was found when TSA was combined with either estradiol or tamoxifen. In conclusion, the so far undescribed association between estrogen receptivity and the expression of hCAR in breast cancer seems to not only reflect a phenotype of low malignancy, but expression of hCAR may also be directly influenced by ER-specific ligands.

Methylated NEUROD1 promoter is a marker for chemosensitivity in breast cancer.

PURPOSE: Chemotherapy can be an integral component of the adjuvant management strategy for women with early stage breast cancer. To date, no tool is available to predict or monitor the efficacy of these therapies. The aim of this proof-of-principle study was to assess whether NEUROD1 DNA methylation is able to predict the response to neoadjuvant and adjuvant chemotherapy. EXPERIMENTAL DESIGN: Recently, we showed that NEUROD1 DNA is differentially methylated in neoplastic versus nonneoplastic breast tissue samples. In this study, we used MethyLight and analyzed NEUROD1 methylation in (a) 74 breast cancer tissue samples, (b) two independent sets of pretreatment core biopsies of 23 (training set) and 21 (test set) neoadjuvantly treated breast cancer patients, and (c) pretherapeutic and posttherapeutic serum samples from 107 breast cancer patients treated with adjuvant chemotherapy. RESULTS: High-grade tumors showed higher NEUROD1 methylation levels. Estrogen receptor-negative breast cancers with high NEUROD1 methylation were 10.8-fold more likely to respond with a complete pathologic response following neoadjuvant chemotherapy. Patients with positive serum pretreatment NEUROD1 methylation, which persisted after chemotherapy, indicated poor relapse-free and overall survival in univariate and multivariate analyses (relative risk for relapse, 6.2; 95% confidence interval, 1.6-24; P = 0.008, and relative risk for death, 14; 95% confidence interval, 1.6-120; P = 0.02). CONCLUSIONS: These data support the view that NEUROD1 methylation is a chemosensitivity marker in estrogen receptor-negative breast cancer.

Prognostic significance of TPA versus SCC-Ag, CEA and neopterin in carcinoma of the uterine cervix.

INTRODUCTION: Prognostic significance of squamous cell carcinoma antigen (SCC-Ag), tissue polypeptide antigen (TPA), carcinoembryonic antigen (CEA) and neopterin in cervical cancer patients was compared. MATERIALS AND METHODS: Pretreatment concentrations were determined in 138 women. RESULTS: Median age was 52 years, 85% squamous cell carcinomas, 15% adeno- or adenosquamous carcinomas were seen. In 36% Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage I, 24% stage II, 32% stage III and 8% stage IV was diagnosed. TPA was elevated in 22%, SCC in 68%, CEA in 42% and neopterin in 29%. These patients showed significantly worse overall survival in univariate analysis (p<0.001). TPA remained as independent prognostic factor in multivariate analysis. CONCLUSIONS: Elevation of TPA was associated with worse overall survival.

Favorable prognostic value of SOCS2 and IGF-I in breast cancer.

BACKGROUND: Suppressor of cytokine signaling (SOCS) proteins comprise a protein family, which has initially been described as STAT induced inhibitors of the Jak/Stat pathway. Recent in vivo and in vitro studies suggest that SOCS proteins are also implicated in cancer. The STAT5 induced IGF-I acts as an endocrine and para/autocrine growth and differentiation factor in mammary gland development. Whereas high levels of circulating IGF-I have been associated with increased cancer risk, the role of autocrine acting IGF-I is less clear. The present study is aimed to elucidate the clinicopathological features associated with SOCS1, SOCS2, SOCS3, CIS and IGF-I expression in breast cancer. METHODS: We determined the mRNA expression levels of SOCS1, SOCS2, SOCS3, CIS and IGF-I in 89 primary breast cancers by reverse transcriptase PCR. SOCS2 protein expression was further evaluated by immuno-blot and immunohistochemistry. RESULTS: SOCS2 expression inversely correlated with histopathological grade and ER positive tumors exhibited higher SOCS2 levels. Patients with high SOCS2 expression lived significantly longer (108.7 vs. 77.7 months; P = 0.015) and high SOCS2 expression proved to be an independent predictor for good prognosis (HR = 0.45, 95% CI 0.23 - 0.91, P = 0.026). In analogy to SOCS2, high IGF-I expression was an independent predictor for good prognosis in the entire patient cohort. In the subgroup of patients with lymph-node negative disease, high IGF-I was a strong predictor for favorable outcome in terms of overall survival and relapse free survival (HR = 0.075, 95% CI 0.014 - 0.388, P = 0.002). CONCLUSION: This is the first report on the favorable prognostic value of high SOCS2 expression in primary mammary carcinomas. Furthermore a strong association of high IGF-I expression levels with good prognosis was observed especially in lymph-node negative patients. Our results suggest that high expression of the STAT5 target genes SOCS2 and IGF-I is a feature of differentiated and less malignant tumors.

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies.

Prognostic significance of signal transducer and activator of transcription 1 activation in breast cancer.

PURPOSE: Signal transducers and activators of transcription (STATs)were shown to be activated in mammary carcinoma. Because different STAT factors are likely to have different functions in these tumors, an assessment of their individual role is mandatory. EXPERIMENTAL DESIGN: In this study we have separately determined activation of STAT1, STAT3, and STAT5 by measuring their DNA binding activity and tyrosine phosphorylation in breast cancer tissue samples. The predictive value of STAT activation on relapse-free and overall survival among women who received treatment for primary breast cancer was evaluated in a retrospective study. RESULTS: Survival analysis demonstrated that patients with high STAT1 activation have substantially longer overall and relapse-free survival, irrespective of whether STAT1 activation was determined by its DNA binding activity (P = 0.003 and 0.010, respectively) or by its tyrosine phosphorylation (P = 0.046 and 0.011, respectively). In accordance, Cox proportional hazard regression analysis revealed an enhanced hazard of death (hazard ratio, 3.77; P = 0.018) and relapse of disease (hazard ratio, 6.55; P = 0.013) for the group of women with low STAT1 activation. After adjusting for known prognostic variables (lymph node status, stage of disease, estrogen receptor status, and cathepsin D), STAT1 activation remained an independent prognostic value. Activation of STAT3 and STAT5 DNA binding did not significantly correlate with prognosis. CONCLUSION: Our study reveals a favorable and independent prognostic significance of STAT1 activation in mammary carcinoma, and is in accordance with the documented role of STAT1 in growth arrest, and in pro-apoptotic signaling pathways.

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

BACKGROUND: The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer. METHODOLOGY/PRINCIPAL FINDINGS: We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels. We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients. CONCLUSIONS/SIGNIFICANCE: We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.

The Role of Androgens in Normal and Malignant Breast Tissue.

Androgens, like estrogens, can be synthesized in the breast. As both active androgens and their corresponding receptors are present in breast tissue, we conclude that they play a role in breast physiology. This is supported by the fact that insufficient androgen production or sensitivity results in the development of gynecomastia. Complete androgen insensitivity due to receptor defects leads to normal female breast development in these XY women. While breast development is completely inhibited by male testosterone levels, partial but not total degradation of a developed breast by androgen treatment appears to be possible. Breast cancer in early stages seems to fulfill the prerequisites of androgen responsiveness. Androgen treatment of advanced breast cancer has shown similar effectiveness as anti-estrogen or estrogen-ablative therapy, but also considerable side effects. It has been speculated that the use of selective androgen modulators (SARMs), either alone or preferably in addition to anti-estrogens or aromatase inhibitors, may be a promising alternative to current therapy modalities in hormone-dependent breast cancer. In addition, future studies on the use of SARMs in prophylactic settings seem to be justified.

Modulation of CA-125 tumor marker shedding in ovarian cancer cells by erlotinib or cetuximab.

OBJECTIVE: The EGF receptor tyrosine kinase inhibitor erlotinib and the EGF receptor antibody cetuximab were investigated with respect to their antiproliferative effect in vitro and their influence on the synthesis and secretion of the tumor marker CA-125 in ovarian carcinoma and human peritoneal mesothelial (HPMC) cells. METHODS: Ovarian cancer cell lines and HPMC were cultured in vitro under the usual conditions. CA-125 surface expression was detected by a living cell radio-immunoassay. CA-125 concentration shed into supernatant medium was determined using a microparticle enzyme immunoassay. RESULTS: Proliferation was inhibited by erlotinib in a dose-dependent manner in 4/5 cell lines but in none of the HPMCs. Only the erlotinib-sensitive cell lines also responded by decreasing surface density of CA-125. Release of CA-125 into the supernatant medium was independent of its surface density and was increased by erlotinib treatment in all but HTB77 cancer cells but not in HPMCs. Very similar results were obtained when the EGFR antibody cetuximab was used in place of erlotinib. CONCLUSION: The results indicate that the effects observed were a consequence of EGFR inhibition. The influence exerted by erlotinib or cetuximab treatment on shedding and cell surface density of CA-125 leads us to conclude that CA-125 blood levels measured during therapy might not correctly indicate changes in tumor size.

Interferon-gamma expression is an independent prognostic factor in ovarian cancer.

BACKGROUND: Epithelial ovarian cancer prognosis is improved by the presence of intratumoral CD3 + T cells, which are known to produce interferon-gamma. We therefore speculated that interferon-gamma expression in ovarian cancer-infiltrating T-lymphocytes might cause better prognosis. PATIENTS AND METHODS: Reverse transcriptase polymerase chain reaction was performed to measure the expression of interferon-gamma and other related genes in normal ovaries (n = 19) and in ovarian cancer specimens (n = 99). Median follow-up of patients was 5.8 years. RESULTS: Interferon-gamma and CD-3 expression did not significantly differ in normal and malignant tissue. Patients with high levels of interferon-gamma expression had significantly longer progression-free and overall survival. Median time to progression was 10 and 29 months for patients with low and high interferon-gamma expression, respectively ( P = .039). Corresponding survival times were 29 and 44 months ( P < .032). Application of multivariate Cox regression analysis showed interferon-gamma expression to be an independent prognostic factor for progression-free and overall survival. CONCLUSION: Elevated interferon-gamma expression correlates with improved clinical outcome in patients with ovarian cancer.