EFEMP1 (Fibulin-3) belongs to the Fibulin gene family, a newly characterized family of 6 extracellular matrix (ECM) proteins that are localized at basal membranes, stroma and ECM fibers mediating cell to cell and cell to matrix communication. They are thought to provide organization and stabilization to ECM structures during organogenesis and vasculogenesis.1 While the potential role of Fibulin 1, 2, 4 and 5 in tumor development has been described in more detail,2–8 only little information has so far been available on the role of EFEMP1.9, 10 Klenotic et al.11 described a strong interaction of EFEMP1 with TIMP-3, the tissue inhibitor of metalloproteinases-3, an ECM-bound protein, which regulates matrix composition and affects tumor growth, invasion and angiogenesis. Recently, EFEMP1 was described to be an antagonist of angiogenesis.12 Among other results, Albig et al.12 showed that EFEMP1 prevents angiogenesis and vessel infiltration into bFGF-supplemented matrigel plugs implanted in genetically normal mice, and that EFEMP1 also decreases growth and blood vessel density in tumors produced by MCA102 fibrosarcoma cells implanted into syngeneic mice.
To characterize angiogenesis antagonist EFEMP1 in sporadic breast cancer and to correlate its expression with epigenetic alterations, we performed array-based RNA expression profiling, quantitative real-time PCR (QRT-PCR), immunohistochemistry, loss of heterozygosity (LOH) analysis, mutation screening and methylation studies in several breast cancer tissue panels. Because of the strong dependence of tumor growth and metastasis on angiogenesis, several components of the angiogenic pathway such as VEGF, VEGFR2 and HIF1A have been already analyzed for their prognostic and predictive impact in breast cancer.13 We therefore also aimed to evaluate the potential of EFEMP1 as a prognostic and/or predictive biomarker for this cancer entity.
Material and methods
Clinical tissue samples
The clinico-pathological characteristics of the tumors analyzed by microarray expression analysis, QRT-PCR, LOH analysis, mutation screening and methylation studies represent normal occurrence in clinical practice. Menopausal status: 81% post-, 19% pre- or perimenopausal; tumor size: 77% ≤ 2 cm, 23% > 2 cm; nodal status: 54% node-negative, 46% node-positive; Grade: 7% G1, 39% G2, 54% G3; histologic type: 78% ductal, 13% lobular, 9% others. For tissue microarray (TMA) analysis, malignant tumor samples were obtained from 203 clinically well characterized breast cancers (clinico-pathological characteristics given in Table I). Appropriate informed consent was obtained from all patients. The ethics committees of all participating centers approved the use of the tissues and the corresponding clinical data.
Table I. Clinico-Pathological Data of Tumor Samples Analyzed by TMA
Median age at diagnosis
58 years (range, 28–89)
Estrogen receptor status
Progesterone receptor status
Adjuvant systemic therapy
Human mammary epithelial cell line MCF-12A as well as breast cancer cell lines BT-20, MCF-7, SK-BR-3, T-47-D and ZR-75-1 were obtained from ATCC (Rockville, MD) and cultured under recommended conditions.
RNA amplification and expression analysis using Affymetrix microarray technology
Tumor material was snap frozen in liquid nitrogen immediately after surgery. Only samples with >70% tumor cells were selected for analysis. Isolation of total RNA was performed using TRIZOL reagent (Gibco-BRL, Glasgow, UK). cRNA was synthesized using 800–2000 ng purified RNA, linearly amplified, labeled with biotin and hybridized to the Human Genome U133A array (Affymetrix, UK) according to the recommendations of the manufacturer. Signal intensities were scanned using an Agilent Gene Array Scanner G2500A (Agilent Technologies, Waldbronn, Germany). Data analysis was performed using “Microarray Suite 5.0,” “MicroDB 3.0,” Data Mining Tool (Affymetrix) and BioConductor software (http://www.bioconductor.org/).
Quantitative real-time PCR
Cancer tissues were microdissected as described by Rhiem et al.14 Total RNA isolation was performed using TRIZOL (Gibco-BRL). Reverse transcription of RNA, PCR amplification and detection was performed using TaqMan EZ RT-PCR Core Reagents (Applied Biosystems, Weiterstadt, Germany) on a Sequence Detection System (ABI SDS 7700, PE Applied Biosystems) applying a standard one-step–protocol recommended by ABI. The sequences of the specific TaqMan primers and probe for EFEMP1 are given in Table II. The housekeeping gene GAPDH was used as a reference.
Table II. Primer Sequences Used in this Study
Bisulphite genomic sequenicng A2
Bisulphite genomic sequencing B2
5′-GAT GAA TGC AGA ACC TCA AGC-3′
5′-TCG TGG ATA ACA ACG GAA GC-3′
LOH analysis was carried out as described previously.15, 16 Primer sequences for the used microsatellite markers F2 and F3 are listed in Table II.
Genomic mutation screening was performed by DHPLC analysis (WAVE, Transgenomics, Omaha, NE) and exon sequencing with the adjacent intronic sequences using Big Dye chemistry (Perkin Elmer, Heidelberg, Germany) with separation of the fragments on an ABI capillary sequencer (ABI3100). Primer sequences for DHPLC analysis and exon sequencing as well as conditions for DHPLC analysis are given in Supporting Information 1.
Cells were seeded at a density of 1 to 3 × 105 cells/cm2 in a 6-well plate on day 0. The demethylating agent 5-Aza-2′-deoxycytidine (DAC) (Sigma-Aldrich, Steinheim, Germany) was added to a final concentration of 0.1–7.5 μM in fresh medium on day 1. Cells were harvested on day 4 for DNA and RNA extraction. Control cells were incubated without the addition of DAC. Restoration of EFEMP1 expression was analyzed applying real-time PCR using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany). Primer sequences are given in Table II. Gene expression was quantified by the comparative CT method, normalizing CT values to the housekeeping gene GAPDH and calculating relative expression values.16, 17
Bisulphite modification and bisulphite genomic sequencing
Genomic DNA was isolated using the DNA Extraction Mini Kit (Qiagen). Bisulphite reactions were performed using the CpGenome DNA Modification Kit according to the manufacturer's recommendations (Intergen, New York). For each bisulphite reaction, 0.5–3 μg of DNA was used. For bisulphite genomic sequencing-PCR reactions, 50 ng of bisulphite-treated DNA was used in a final reaction volume of 50 μL. Primer sequences are listed in Table II (PCR product A2, −739 to −962; PCR product B2, −868 to −1,132 relative to start codon). The amplified fragments were subcloned using the TOPO-TA cloning kit (Invitrogen, Karlsruhe, Germany). Inserts were sequenced using M13 primers.
A total of 203 tissue specimens of consecutive primary breast carcinomas were used for TMA construction. Median follow-up in patients still alive at the time of analysis was 92 months (range, 1–191). All clinico-pathological characteristics are given in Table I. Tumors had originally been fixed in neutral buffered formalin 4% and then paraffin embedded. TMAs were prepared using 2 peripheral and 1 central 1 mm2 tumor areas of each tumor.
Immunohistochemistry on tissue arrays
A specific polyclonal rabbit antibody against recombinant mouse Efemp1 protein was used for immunohistochemistry.18 Cross-reactivity of this antibody with human EFEMP1 was shown by western blot using recombinant human EFEMP119 and by specific immunostaining of human tissues (unpublished data). After dewaxing, endogenous peroxidase was blocked by 1% hydrogen peroxide (10 min). Antigen retrieval was performed using Protease XXIV (50 mg diluted in 15 mL of 0.1 M Tris/HCl, pH 8.0, 20 min at room temperature). Standard immunostaining used antibody goat anti-rabbit K 4002/4003 for EnVision-method (DAKO, Copenhagen, Denmark) on TechMate Horizon autostainer (DAKO) according to supplier's recommendations. Primary anti-Efemp1 polyclonal antibody was diluted 1:100 (antibody dilution and storage buffer, DAKO, F 3022) and incubated (room temperature, 2 × 25 min). Hematoxylin counterstaining was performed.
Interpretation of the immunohistochemistry results was performed by 2 independent scientists blinded to the corresponding clinicopathological data. Slides were evaluated using light microscopy and a semiquantitative immunoreactivity score (IRS). By recording the percentage of positive ECM staining (PP-value: 0 = negative, 1 = <10%, 2 = 10–50%, 3 = >50%) and staining intensity (SI-value: 0 = no, 1 = weak, 2 = moderate, 3 = strong) for each sample, IRS was calculated by multiplying PP with SI. If different scores were obtained for the central and the 2 peripheral areas of a tumor, a mean IRS value was calculated.
Statistical analysis of tissue microarray data
Statistical analysis was performed using the SPSS software version 14.0 (SPSS, Chicago, IL). Fisher's exact tests and χ2-tests were applied to analyze the correlation between clinico-pathological and immunohistochemical variables. All tests were performed two-sided. Disease-free and overall survival curves were calculated using the Kaplan-Meier method and compared by log-rank testing. Multivariate Cox proportional hazard models were used to define the potential prognostic significance of individual parameters. The significance level was set to 5%.
Analysis of EFEMP1 expression in primary breast tumors applying RNA microarray expression analysis
RNA microarray analysis was carried out in 27 macro-dissected primary breast cancer samples (>70% tumor cells) in comparison to 6 normal breast tissue samples. Clinical characteristics for the tumors represented normal occurrence in clinical practice and are given in the Material and Methods section “Clinical tissue samples.” For EFEMP1, down-regulation was observed in 59% (16/27) of cancer tissues with a 3.5-fold average change. Down-regulation was defined as an at least 2-fold reduction of expression compared with normal tissue.
Quantitative real-time PCR experiments in micro-dissected primary breast tumors
To confirm the results of the array analyzes, expression of EFEMP1 was also analyzed by quantitative real-time PCR in matched tumor/normal tissues of 18 micro-dissected primary breast cancer samples (>90% tumor cells). These analyzes revealed that 61% (11/18) of the tumors displayed down-regulation ranging between at least 50% and almost 100% compared with corresponding normal breast tissue (Fig. 1, black columns).
LOH and mutation analysis
To assess the allelic loss of the EFEMP1 gene locus, microsatellite markers F2 and F3 (Table II), located in close vicinity of the EFEMP1 locus, were used for LOH analysis. One hundred and one matched tumor/normal DNA sample pairs were analyzed with marker F2, 57 matched samples with marker F3. Allelic loss was observed in 32% (F2) and 21% (F3) of informative cases, respectively.
Twenty-one LOH positive tumors were subsequently screened for mutations in EFEMP1. Besides 3 known intronic polymorphisms (rs2277886, rs10496056, rs3748959 [dbSNP, NCBI]), only 1 deletion (1818 delT) (NM_004105) in the 3′ UTR in 1 of the tumors and 1 unknown missense mutation c.476A>C, p.Asp49Ala (NM_004105) in a second tumor were detected.
Analysis of epigenetic alterations by expression studies after treatment with demethylating agents in cell lines
The results of mutation analysis indicated that the major reason for the reduced gene expression is unlikely to be a result of LOH, associated with pathogenic point mutations on the remaining allele. We therefore analyzed whether epigenetic alterations may be the cause of the observed reduced expression. One nontumorous cell line (MCF-12A) and five breast cancer cell lines (BT-20, MCF-7, SK-BR-3, T-47 D, ZR-75-1) were treated with the demethylating agent DAC. Endogenous EFEMP1 expression levels in these cell lines before and after DAC treatment were assessed by quantitative real-time PCR. As shown in Figure 2a, the analyzed tumor cell lines displayed different levels of EFEMP1 expression before DAC treatment. Relative to the nontumorous cell line MCF-12A with an expression level set to 1.000, MCF-7 showed the lowest expression value (0.016) followed by BT-20 (0.177) and SK-BR-3 (0.290). In contrast, ZR-75-1 and T-47-D displayed higher expression with values of 2.037 and 7.127 relative to MCF-12A. Thus, similar to the above described expression results in native tumor tissues, 60% (3/5) of the tumor cell lines displayed a reduced expression compared with the nontumorous cell line.
After DAC treatment, the 2 cell lines with the lowest expression levels, MCF-7 and BT-20, displayed the most pronounced up-regulation with a 124-fold increase of expression in MCF-7 and a 19.4-fold increase in BT-20. In contrast, the nontumorous cell line MCF-12A together with tumor cell lines SK-BR-3 and T-47-D showed only moderate up-regulation with 8.9-, 6.7-, and 5.9-fold changes, whereas ZR-75-1 did not show any up-regulation.
To further investigate the influence of the EFEMP1 promoter methylation status on EFEMP1 expression in the analyzed cell lines, we applied bisulphite genomic sequencing in a genomic interval ranging between −739 and −1132 relative to the position of the start codon of EFEMP1. Per bisulphite-treated DNA sample, 16 clones with 25 CpG dinucleotides each were investigated. None of the 11 CpG dinucleotides located within interval −962 to −1132 displayed any methylation in any of the investigated cell lines. However, differential methylation within the different cell lines was observed for the 14 CpG dinucleotides within interval −739 to −963.
As expected, the nontumorous cell line MCF-12A displayed no methylation. The methylation status of tumor cell lines MCF-7 and BT-20 with the lowest expression levels showed the highest methylation levels with 91% and 76% of methylated CpGs (Fig. 2b). In contrast, cell lines ZR-75-1 and T47-D with high expression levels showed no significant methylation (6.7% and 0%). Thus, 40% (2/5) of the tumor cell lines displayed significant methylation in the promoter region of EFEMP1 which was correlated with a significant reduction of expression compared with the nontumorous cell line and a pronounced up-regulation after DAC treatment. The moderate increase of expression after DAC treatment in MCF-12A, SK-BR-3 and T-47-D may be explained by transgenic effects because of demethylation of other genes rather than by demethylation of the EFEMP1 promoter.
Methylation analysis of the putative EFEMP1 promoter in primary breast cancer samples
We next analyzed whether EFEMP1 promoter methylation also contributes to the reduction of EFEMP1 expression in primary breast cancer samples. Therefore, we performed methylation analysis by applying bisulphite genomic sequencing in the promoter region described before in 14 macro-dissected breast cancer samples with LOH at 1 allele, 4 micro-dissected tumor samples without LOH, and 8 micro-dissected normal breast tissues.
As described before, per bisulphite-treated DNA sample, 16 clones with 25 CpG dinucleotides (position −739 to 1,132, relative to position of start codon) each were investigated. Again, none of the 11 CpG dinucleotides located within interval −962 to −1,132 displayed any methylation neither in tumor samples nor in normal breast tissues. However, differential methylation was observed for the 14 CpG dinucleotides within interval −739 to −963 (Fig. 3). The endogenous methylation levels in the primary breast cancer and normal breast tissues analyzed are shown in Figure 4. Based on the expression and methylation results in the analyzed cell lines, a tissue was defined as methylation-positive, if at least 25% of the CpG dinucleotides were methylated (Table III, Fig. 4). Based on this definition, 57% (8/14) of the macro-dissected LOH-positive tumors (T1–T8) and 50% (2/4) of the micro-dissected LOH-negative tumors (T15, T16) were defined as methylation-positive with an average of 55% and 32.8% of methylated CpG dinucleotides, respectively (Table III). In contrast, none of the 8 normal breast tissues (N1–N8) was found to be methylation-positive, since their highest percentage of methylated CpG dinucleotides was 8.9% (average 3.4%) (Table III, Fig. 4).
Table III. Extent of Methylation Within Interval −739 to −969 of the 5′ Region of EFEMP1 in 18 Breast Tumor Tissues and 8 Normal Breast Tissues
Thus, these results strongly indicate that promoter methylation contributes to a major extent to the reduced EFEMP1 expression in the investigated sporadic breast cancer tissues.
EFEMP1 expression on protein level
After observing reduced EFEMP1 expression in sporadic breast tumors on the RNA-level, we investigated EFEMP1 expression on the protein level. A specific polyclonal rabbit antibody against recombinant mouse Efemp1 protein was used for immunohistochemistry (IHC).18 The amino acid sequences between human EFEMP1 and mouse Efemp1 are highly conserved (Supporting Information 2) and cross-reactivity of this antibody with human EFEMP1 was shown by western blot using recombinant human EFEMP119 and by specific immunostaining of human tissues (unpublished data).
Localization and expression of EFEMP1 in normal breast tissue
Localization of EFEMP1 was done by IHC, applying the mouse Efemp1 antibody described before. As shown in Figure 5a, EFEMP1 was detectable in normal breast tissues; faint staining was detected in myoepithelial and luminal cells of lobules and ducts, whereas intense staining was observed in the ECM as a linear pattern in the vicinity of the basal membrane of lobules (terminal duct-lobular-units) and as a diffuse pattern in the ECM of intralobular, interlobular and periductal stroma.
EFEMP1 expression in breast cancer tissues
IHC staining of 8 matched breast cancer/normal breast tissues revealed reduced or even abolished expression of EFEMP1 in the ECM in 62.5% (5/8) of the tumor samples compared with the ECM of normal breast tissues. Immunohistochemically, EFEMP1 was not detectable in the tumor cells themselves in most cases; faint tumor cell staining was only observed in few cases, thus, only ECM staining was evaluable by this method.
Tissue microarray analysis
After observing reduced EFEMP1 expression in 5 of 8 tumor samples as compared with matched normal breast tissues, we investigated a set of 203 clinically well characterized primary breast cancers using TMA analysis.
Strong EFEMP1 expression (IRS 6.5–9) was only seen in 9% of the tumor tissues, moderate expression (IRS 4.5–6.0) in 34%, whereas 57% of the tumors displayed weak or absent expression (IRS 0–4.0) (Fig. 5b).
Correlation of EFEMP1 protein expression with methylation status
EFEMP1 protein expression was then analyzed in selected tumor tissues with different methylation status (tumor 2 [T2] with 91.5% methylation; tumor 4 [T4] with 41.5% methylation; tumor 14 [T14] with 0% methylation, see also Fig. 4). Figure 6 displays the results of IHC staining. While the tumor with the highest methylation status, T2, showed the lowest protein expression (IRS 3.0), tumor T4 with 41.5% methylation displayed moderate expression (IRS 6.0), whereas the tumor without any methylation, T14, showed strong expression (IRS 9.0). Thus, these results strongly support the correlation between high methylation status and low levels of gene expression.
Correlation of EFEMP1-expression with patient outcome
To asses a potential clinical relevance of reduced EFEMP1 protein expression regarding patient outcome, stepwise Cox regression for multivariate survival modeling was performed for the above described 203 breast cancer samples. The following variables were included: tumor grade, steroid hormone receptor status, IRS for EFEMP1 protein expression, lymph node status, Her-2 status, tumor size and type of adjuvant systemic therapy.
We first analyzed the total cohort of 203 breast cancer samples, consisting of 17 node-negative and 186 node-positive cases. Looking at the total cohort, no obvious clinical relevance of EFEMP1 expression was seen. On the basis of assumed strong influence of EFEMP1 on tumor progression, we then analyzed the collective of the high-risk node-positive cases separately. This analysis revealed that next to tumor size and grade, EFEMP1 expression (applying an optimized IRS cut off value of 3.5) remained in the survival model as a relevant factor influencing disease-free and overall survival at borderline significance (DFS: p = 0.14; OS: p = 0.077).
Keeping in mind the fundamental differences between node-negative and node-positive patients regarding their primary therapy where particularly frequency and type of adjuvant systemic therapy may strongly influence their outcome results,20 we therefore investigated the relevance of EFEMP1 expression in patient subgroups with homogeneous adjuvant systemic therapy. Interestingly, this analysis revealed a significant correlation of low EFEMP1 expression with poor disease-free and overall survival (p = 0.037 and p = 0.032, respectively) in those node-positive patients who had received adjuvant anthracycline-containing chemotherapy (n = 31) (Fig. 7), whereas no significant impact was seen in node-positive patients treated either by cyclophosphamide-methotrexate-5-fluorouracil (CMF) chemotherapy (n = 49) (DFS: p = 0.605; OS: p = 0.934) or by adjuvant endocrine therapy (tamoxifen) alone (n = 106) (DFS: p = 0.735; OS: p = 0.275). While median disease-free survival in anthracycline-treated patients exceeded 10 years, if tumors showed high EFEMP1 expression, it was only 3.1 years in cases with low EFEMP1 expression (Fig. 7, Panel B). Median overall survival in these patients showed similar results with a median overall survival of more than 10 years for tumors with high EFEMP1 expression in contrast to only 4.5 years for those with low EFEMP1 expression (Fig. 7, Panel A).
In contrast to other members of the fibulin gene family,2–8 only little information has been available so far on the function of EFEMP1 (Fibulin 3) regarding tumor development and progression.1, 9–11 The recent description of EFEMP1 as an angiogenesis antagonist12 prompted us to further characterize EFEMP1 in several panels of sporadic breast carcinomas.
Our expression studies on RNA level in a total of 45 cancer tissues revealed down-regulation of EFEMP1 in ∼60% of the tumors and, thus, supporting our preliminary results on differential EFEMP1 expression in breast carcinomas by electronic northern approaches and CPA analysis21 as well as the results of Albig et al.12 On the basis of cDNA dot blot analysis, these authors had described down-regulation of EFEMP1 in 6 of 9 (67%) breast cancer tissues. On the protein level, we assessed EFEMP1 expression by immunohistochemical staining in a total of 211 primary breast cancers and found down-regulation in 57–62.5% of these tumor samples. Thus, we identified comparable levels of EFEMP1 loss both on RNA and protein level. Interestingly, there was no correlation between the amount of desmoplastic tumor stroma and intensity of EFEMP1 expression shown by immunohistochemistry.
Using DNA methylation analysis, we were then able to show that EFEMP1 down-regulation in primary breast tumors is very likely caused by hypermethylation of the EFEMP1 promoter. Bisulphite genomic sequencing of 14 macro-dissected and 4 micro-dissected breast cancer tissues revealed significant methylation with at least 25% of methylated CpG dinucleotides in 57% and 50% of the tumors, while none of the 8 investigated normal breast tissues was found to be significantly methylated. These observations are supported by the methylation and expression results in 5 breast cancer cell lines. Those cell lines with the lowest expression levels compared with a nontumorous cell line displayed the highest methylation levels. Moreover, after DAC treatment they showed the most pronounced up-regulation. In contrast, the 2 cell lines with high expression levels compared with 1 nontumorous cell line showed only low or even no methylation and therefore only moderate or no up-regulation after DAC treatment. These results strongly indicate that promoter methylation contributes to a major extent to the reduced EFEMP1 expression in the investigated sporadic breast cancer tissues. Furthermore, we were also able to prove the correlation between the EFEMP1 methylation status and its protein expression by applying IHC staining in tumor tissues of known methylation status (Fig. 6).
To our knowledge, these are the first data on altered EFEMP1 methylation in sporadic breast cancer. Our data on hypermethylation of EFEMP1 in sporadic breast carcinomas are also supported by very recent results of Yue et al.22 These authors reported on hypermethylation of the same 5′ regulatory region of EFEMP1 in lung cancer.
Angiogenesis is a physiological process involving the growth of new blood vessels from preexisting ones. Besides normal growth and development, this process is of great importance for growth, invasion and metastasis of malignant tumors, since it enables them to initiate recruitment of their own blood and nutrient supply by shifting the balance between specific pro- and antiangiogenic factors.13, 23 Most studies on the relationship of angiogenesis and patient outcome reveal a clear relationship between high levels of angiogenesis and poor prognosis.24 To evaluate the correlation between the expression of the angiogenesis antagonist EFEMP1 and patient outcome in sporadic breast carcinomas, we also investigated the correlation of clinicopathological variables to immunohistochemical EFEMP1 expression. By performing multivariate Cox regression analysis, we were indeed able to show that next to tumor size and grade, EFEMP1 expression had a clinically meaningful impact on disease-free and overall survival in a substantial cohort of 186 node-positive patients with a long-term median follow-up of 92 months. Moreover, taking adjuvant systemic therapy into account, we saw significant correlations only in those node-positive patients who had received anthracycline-containing adjuvant chemotherapy, but not in those treated by adjuvant CMF or endocrine therapy alone.
In our retrospective cohort, patients who had received anthracycline-containing adjuvant chemotherapy were also those patients with a clinically perceived high-risk of relapse. Thus, at present we cannot clearly distinguish between a potential predictive impact of EFEMP1 expression regarding response to anthracycline chemotherapy from a mere prognostic impact of this factor regarding survival in high-risk breast cancer. However, keeping the biological role of EFEMP1 in mind, it is not unreasonable to assume that its antiangiogenic properties may enhance an antiangiogenic chemotherapy effect which has, in fact, been attributed to anthracyclines.24, 25
In conclusion, the presented data provide evidence for a down-regulation because of promoter methylation of the angiogenesis antagonist EFEMP1 on RNA and on protein level in a substantial number of sporadic breast carcinomas, thus indicating EFEMP1 as a new candidate tumor suppressor gene in this cancer entity. Moreover, multivariate regression analysis revealed a correlation of reduced EFEMP1 expression with poor disease-free and overall survival in node-positive breast cancer. Taking adjuvant systemic therapy into account, the impact of EFEMP1 expression was most pronounced in patients with adjuvant anthracycline chemotherapy. Thus, these results point to a possible predictive value of EFEMP1 expression regarding anthracycline response which needs to be further validated in larger collectives of homogeneously treated breast cancer patients. In view of clinically emerging angiogenesis inhibitors, identification and characterization of components of the angiogenic pathway as specific prognostic as well as predictive markers is of great relevance for the success of this treatment option to overcome drug resistance and identify correct target populations.13 EFEMP1, with its antiangiogenic properties, may serve here as an important molecular marker for defining an adequate tumor-biology oriented therapeutic strategy.
The authors thank Mrs. R. Busch, Institute of Medical Statistics and Epidemiology (IMSE), Technical University, Munich, for her expert statistical analysis and Dr. Günter Kostka (deceased on January 19, 2006) for providing the Efemp1 antibody.