Background

An altered expression of the activated leukocyte cell adhesion molecule (ALCAM) is associated with cancer progression in various cancer types. In some cancers ALCAM has a prognostic value or is predictive for the benefit of therapeutic interventions. To date there are no data on the role of ALCAM in cervical cancer available.

Methods

In this study, ALCAM expression was analysed by immunohistochemistry (IHC) in tissue samples of 233 patients with cervical cancer, among them 178 with complete follow-up information. In addition, soluble (s-)ALCAM was measured in sera of a subset of the included patients (n = 55) by enzyme-linked immunosorbent assay (ELISA).

Results

ALCAM overexpression was detected (immunoreactive score (IRS) 2-12) in 58.4% of the cervical cancer samples. The normal ectocervical or endocervical epithelium showed no ALCAM reactivity. In untreated patients, ALCAM overexpression in tumor tissue tended to be associated with shorter cancer-specific survival (CSS) and disease-free survival (DFS). Patients, whose tumor samples showed ALCAM overexpression receiving a cytotoxic therapy like radiotherapy or chemoradiation, however, had a favourable prognosis compared to those patients, whose cancers showed no or minimal ALCAM staining. This effect was particularly apparent in patients receiving chemoradiation where the CSS was significantly longer in patients with ALCAM-positive tumors (p = 0.038; cumulative incidence rates at 96 months 8%, 95% CI 0%-23%, and 26%, CI 3%-43% in ALCAM-positive and ALCAM-negative cases, respectively).

Median preoperative s-ALCAM concentration in sera from tumor patients was 27.6 ng/ml (range 17.5-55.1 ng/ml, mean 28.9 ng/ml), serum levels did not correlate with intratumoral ALCAM expression.

Conclusions

The data of our retrospective study suggest that the prognostic value of ALCAM expression in cervical carcinoma might be therapy-dependent, and that ALCAM might function as a predictive marker for the response to chemoradiation. This should be confirmed in further, prospective studies.

Introduction

With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Therefore, our aim was to identify molecular markers associated with the development of brain metastases in breast cancer.

Methods

Patterns of chromosomal aberrations in primary breast tumors and brain metastases were compared with array-comparative genetic hybridization (CGH). The most significant region was further characterized in more detail by microsatellite and gene-expression analysis, and finally, the possible target gene was screened for mutations.

Results

The array CGH results showed that brain metastases, in general, display similar chromosomal aberrations as do primary tumors, but with a notably higher frequency. Statistically significant differences were found at nine different chromosomal loci, with a gain and amplification of EGFR (7p11.2) and a loss of 10q22.3-qter being among the most significant aberrations in brain metastases (P < 0.01; false discovery rate (fdr) < 0.04). Allelic imbalance (AI) patterns at 10q were further verified in 77 unmatched primary tumors and 21 brain metastases. AI at PTEN loci was found significantly more often in brain metastases (52%) and primary tumors with a brain relapse (59%) compared with primary tumors from patients without relapse (18%; P = 0.003) or relapse other than brain tumors (12%; P = 0.006). Loss of PTEN was especially frequent in HER2-negative brain metastases (64%). Furthermore, PTEN mRNA expression was significantly downregulated in brain metastases compared with primary tumors, and PTEN mutations were frequently found in brain metastases.

Conclusions

These results demonstrate that brain metastases often show very complex genomic-aberration patterns, suggesting a potential role of PTEN and EGFR in brain metastasis formation.

Background

Current prognostic gene signatures for breast cancer mainly reflect proliferation status and have limited value in triple-negative (TNBC) cancers. The identification of prognostic signatures from TNBC cohorts was limited in the past due to small sample sizes.

Methodology/Principal Findings

We assembled all currently publically available TNBC gene expression datasets generated on Affymetrix gene chips. Inter-laboratory variation was minimized by filtering methods for both samples and genes. Supervised analysis was performed to identify prognostic signatures from 394 cases which were subsequently tested on an independent validation cohort (n = 261 cases).

Conclusions/Significance

Using two distinct false discovery rate thresholds, 25% and <3.5%, a larger (n = 264 probesets) and a smaller (n = 26 probesets) prognostic gene sets were identified and used as prognostic predictors. Most of these genes were positively associated with poor prognosis and correlated to metagenes for inflammation and angiogenesis. No correlation to other previously published prognostic signatures (recurrence score, genomic grade index, 70-gene signature, wound response signature, 7-gene immune response module, stroma derived prognostic predictor, and a medullary like signature) was observed. In multivariate analyses in the validation cohort the two signatures showed hazard ratios of 4.03 (95% confidence interval [CI] 1.71–9.48; P = 0.001) and 4.08 (95% CI 1.79–9.28; P = 0.001), respectively. The 10-year event-free survival was 70% for the good risk and 20% for the high risk group. The 26-gene signatures had modest predictive value (AUC = 0.588) to predict response to neoadjuvant chemotherapy, however, the combination of a B-cell metagene with the prognostic signatures increased its response predictive value. We identified a 264-gene prognostic signature for TNBC which is unrelated to previously known prognostic signatures.

Introduction

Current prognostic gene expression profiles for breast cancer mainly reflect proliferation status and are most useful in ER-positive cancers. Triple negative breast cancers (TNBC) are clinically heterogeneous and prognostic markers and biology-based therapies are needed to better treat this disease.

Methods

We assembled Affymetrix gene expression data for 579 TNBC and performed unsupervised analysis to define metagenes that distinguish molecular subsets within TNBC. We used n = 394 cases for discovery and n = 185 cases for validation. Sixteen metagenes emerged that identified basal-like, apocrine and claudin-low molecular subtypes, or reflected various non-neoplastic cell populations, including immune cells, blood, adipocytes, stroma, angiogenesis and inflammation within the cancer. The expressions of these metagenes were correlated with survival and multivariate analysis was performed, including routine clinical and pathological variables.

Results

Seventy-three percent of TNBC displayed basal-like molecular subtype that correlated with high histological grade and younger age. Survival of basal-like TNBC was not different from non basal-like TNBC. High expression of immune cell metagenes was associated with good and high expression of inflammation and angiogenesis-related metagenes were associated with poor prognosis. A ratio of high B-cell and low IL-8 metagenes identified 32% of TNBC with good prognosis (hazard ratio (HR) 0.37, 95% CI 0.22 to 0.61; P < 0.001) and was the only significant predictor in multivariate analysis including routine clinicopathological variables.

Conclusions

We describe a ratio of high B-cell presence and low IL-8 activity as a powerful new prognostic marker for TNBC. Inhibition of the IL-8 pathway also represents an attractive novel therapeutic target for this disease.

Introduction

Circulating tumor cells (CTCs) reflect aggressive tumor behavior by hematogenous tumor cell dissemination. The tissue inhibitor of metalloproteinase 1 (TIMP-1) plays a role in tissue invasion and is also involved in angiogenesis, abrogation of apoptosis and in chemoresistance. Carbonic anhydrase IX (CAIX) is a metalloenzyme involved in cell adhesion, growth and survival of tumor cells. The aim of the study was to investigate whether serum concentrations of TIMP-1 and CAIX are associated with the detection of CTC in metastatic breast cancer.

Methods

Blood was obtained in a prospective multicenter setting from 253 patients with metastatic breast cancer at the time of disease progression. Serum TIMP-1 and CAIX were determined using commercial ELISA-kits (Oncogene Science). CTC were detected with the CellSearch™ system (Veridex).

Results

Five or more CTCs were detected in 122 patients out of 245 evaluable patients (49.8%). Out of 253 metastatic patients 70 (28%) had serum TIMP-1 levels above 454 ng/mL. Serum CAIX was elevated above 506 ng/mL in 90 (35%) patients. Both serum markers had prognostic significance. Median progression free survival (PFS) was 7.2 months with elevated TIMP-1 vs. 11.4 months with non-elevated levels (p < 0.01). OS was 11.5 vs. 19.1 months (p < 0.01). Median PFS was 7.5 months with elevated CAIX vs. 11.7 months with non-elevated levels (p < 0.01), overall survival (OS) was 13.4 months vs. 19.1 months (p < 0.01). In patients with five or more CTCs, serum levels were above the cut-off for CAIX in 47% vs. 25% in those with less than five CTCs (p = 0.01). For TIMP-1, 37% patients with five or more CTCs had elevated serum levels and 17% of patients with less than five CTCs (p = 0.01). Including TIMP-1, CAIX, CTC and established prognostic factors in the multivariate analysis, the presence of CTCs, the therapy line and elevated CAIX remained independent predictors of OS.

Conclusions

Elevated serum levels of the invasion markers TIMP-1 and CAIX in metastatic breast cancer are prognostic markers and are associated with the presence of CTCs. Whether increased secretion of TIMP-1 and/or CAIX might directly contribute to tumor cell dissemination remains to be elucidated in further investigations.

Trial registration

Current Controlled Trials: ISRCTN59722891

Background

As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases.

Methods

The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo®3/7 Assay, respectively.

Results

Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008).

Conclusion

Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.

Summary

A better understanding of tumor biology has led to the development of a number of antibody-based targeted therapies in breast cancer. Several of these newer agents, such as trastuzumab and bevacizumab have demonstrated clinical activity and have improved the treatment of patients with metastatic breast cancer (MBC). Trastuzumab is a monoclonal antibody that binds to the extracellular domain of the HER2 receptor. The addition of trastuzumab to chemotherapy and also to endocrine therapy has enhanced efficacy of treatment. New antibody-based strategies directed against HER2 are under development. These new approaches include pertuzumab, an antibody with a different binding epitope that inhibits dimerization of HER2 with other members of the HER receptor family and TDM1, a trastuzumab-based antibody chemotherapeutic conjugate. Another approach to the treatment of solid tumors is inhibition of angiogenesis. The anti-VEGF antibody bevacizumab has been approved for treatment of MBC. Although the mechanism of action is still under investigation, bevacizumab is tested in other clinical settings such as adjuvant therapy, maintenance therapy, and in combination with both chemotherapy and other targeted agents. In this review, we will summarize the most important studies on trastuzumab and bevacizumab, and describe new antibodies currently under clinical development.

Introduction

MicroRNAs (miRs) are interesting new diagnostic targets that may provide important insights into the molecular pathogenesis of breast cancer. Here we evaluated, for the first time, the feasibility and clinical utility of circulating miRs as biomarkers for the detection and staging of breast cancer.

Methods

The relative concentrations of breast cancer-associated miR10b, miR34a, miR141 and miR155 were measured in the blood serum of 89 patients with primary breast cancer (M0, n = 59) and metastatic disease (M1, n = 30), and 29 healthy women by a TaqMan MicroRNA Assay.

Results

The relative concentrations of total RNA (P = 0.0001) and miR155 (P = 0.0001) in serum significantly discriminated M0-patients from healthy women, whereas miR10b (P = 0.005), miR34a (P = 0.001) and miR155 (P = 0.008) discriminated M1-patients from healthy controls. In breast cancer patients, the changes in the levels of total RNA (P = 0.0001), miR10b (P = 0.01), miR34a (P = 0.003) and miR155 (P = 0.002) correlated with the presence of overt metastases. Within the M0-cohort, patients at advanced tumor stages (pT3 to 4) had significantly more total RNA (P = 0.0001) and miR34a (P = 0.01) in their blood than patients at early tumor stages (pT1 to 2).

Conclusions

This pilot study provides first evidence that tumor-associated circulating miRs are elevated in the blood of breast cancer patients and associated with tumor progression.

Background

The integration of the non-cross-resistant chemotherapeutic agents capecitabine and vinorelbine into an intensified dose-dense sequential anthracycline- and taxane-containing regimen in high-risk early breast cancer (EBC) could improve efficacy, but this combination was not examined in this context so far.

Methods

Patients with stage II/IIIA EBC (four or more positive lymph nodes) received post-operative intensified dose-dense sequential epirubicin (150 mg/m² every 2 weeks) and paclitaxel (225 mg/m² every 2 weeks) with filgrastim and darbepoetin alfa, followed by capecitabine alone (dose levels 1 and 3) or with vinorelbine (dose levels 2 and 4). Capecitabine was given on days 1-14 every 21 days at 1000 or 1250 mg/m2 twice daily (dose levels 1/2 and 3/4, respectively). Vinorelbine 25 mg/m2 was given on days 1 and 8 of each 21-day course (dose levels 2 and 4).

Results

Fifty-one patients were treated. There was one dose-limiting toxicity (DLT) at dose level 1. At dose level 2 (capecitabine and vinorelbine), five of 10 patients experienced DLTs. Therefore evaluation of vinorelbine was abandoned and dose level 3 (capecitabine monotherapy) was expanded. Hand-foot syndrome and diarrhoea were dose limiting with capecitabine 1250 mg/m2 twice daily. At 35.2 months' median follow-up, the estimated 3-year relapse-free and overall survival rates were 82% and 91%, respectively.

Conclusions

Administration of capecitabine monotherapy after sequential dose-dense epirubicin and paclitaxel is feasible in node-positive EBC, while the combination of capecitabine and vinorelbine as used here caused more DLTs.

Trial registration

Current Controlled Trials ISRCTN38983527.

Immunocytochemical or molecular assays allow the detection of single disseminated tumor cells (DTCs) in the bone marrow (BM) or the peripheral blood in 10% to 60% of breast cancer patients without signs of metastasis. Results from recently reported studies suggest that circulating tumor cell (CTC) levels may serve as a prognostic marker and be used for early assessment of therapeutic response in patients with metastatic breast cancer. In early stage breast cancer, however, the impact of CTCs is less well established than that of DTCs in BM, where several clinical studies demonstrated that such cells are an independent prognostic factor at primary diagnosis. The characterization of DTCs/CTCs has already shed new light on the complex process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTCs should help to identify novel targets for biological therapies aimed to prevent metastatic relapse. In addition, understanding tumor 'dormancy' and identifying metastatic stem cells might result in the development of new therapeutic concepts.

Introduction

The aim of the study was to perform a comparative analysis of LOH (loss of heterozygosity) in primary tumors as well as peripheral blood and bone marrow (BM) of patients with breast cancer (BCa).

Methods

Performing PCR-based fluorescence microsatellite analysis and using a panel of seven polymorphic microsatellite markers, we compared the profiles of LOH in primary tumors, peripheral blood and BM plasma from patients with primary BCa (n = 40, stage M0) as well as tumor tissues and blood serum from metastatic BCa patients (n = 48, stage M1). During the course of systemic treatment blood samplings from 12 M0 and 16 M1 patients were at least once repeated.

Results

The overall incidences of LOH in tumor tissues, blood and BM samples were 27.5%, 9.0% and 5.0%, respectively. The marker D3S1255 was the only one in the panel that showed similar frequencies of LOH ranging from 19.0 to 24.5% in tumor, blood and BM samples. Both M0 blood serum and BM plasma samples displayed the same rate of 19.0%, whereas tumor and M1 serum showed a rate of 24.5% and 24.0%, respectively, at this locus. This marker also showed the highest frequency of LOH in serum and BM samples, whereas in tumor samples LOHs at the markers D13S218 (38%) and D17S855 (36%) were more frequent. Statistical analysis of the tumor samples showed that occurrence of LOH at the markers D3S1255 (P < 0.04), D9S171 (P < 0.05) and D17S855 (P < 0.03) correlated with undifferentiated nuclear grade. Additionally, significant associations of the number of LOH recorded at D17S250 with estrogen receptor (P < 0.02), progesterone receptor (P < 0.03) expression and high proliferation score (Ki-67 expression, P = 0.009) were observed. In blood serum samples a relationship between positive lymph node status and LOH at the marker D3S1255 was revealed (M0 stage, P = 0.05; M0+M1 stage, P = 0.004).

Conclusion

Our study demonstrates heterogeneous profiles and low rates of LOH, particularly on free DNA in BM and blood samples. However, the significant associations of LOH with some risk factors and the demonstrated possibility of monitoring free DNA in patients undergoing systemic therapy suggest that LOH analysis may be developed into a useful diagnostic tool.

In breast cancer patients, hematogenous tumor cell dissemination can be detected, even at the single cell level, by applying immunocytochemical and molecular assays. Various methods for the detection of circulating tumor cells in the peripheral blood have been described. Results from recently reported studies suggest that circulating tumor cell levels may serve as a prognostic marker and for the early assessment of therapeutic response in patients with metastatic breast cancer. However, in early-stage breast cancer, the impact of circulating tumor cells is less well established than the presence of disseminated tumor cells in bone marrow; several clinical studies have demonstrated that cells of the latter type are an independent prognostic factor at primary diagnosis. In this article we briefly summarize recent studies examining the presence of circulating tumor cells in the blood and discuss further clinical applications.

Early tumor cell dissemination at the single-cell level can be revealed in patients with breast cancer by using sensitive immunocytochemical and molecular assays. Recent clinical studies involving more than 4000 breast cancer patients demonstrated that the presence of disseminated tumor cells in bone marrow at primary diagnosis is an independent prognostic factor. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have recently been developed and some studies also suggest a potential clinical relevance of this measure. These findings provide the basis for the potential use of disseminated tumor cells in bone marrow or blood as markers for the early assessment of therapeutic response in prospective clinical trials.