The Prevalence of CD146 Expression in Breast Cancer Subtypes and Its Relation to Outcome.

CD146, involved in epithelial-to-mesenchymal transition (EMT), might affect cancer aggressiveness. We here investigated the prevalence of CD146 expression in breast cancer subtypes, its relation to prognosis, the relation between CD146 and EMT and the outcome to tamoxifen. Primary breast cancer tissues from 1342 patients were available for this retrospective study and immunohistochemically stained for CD146. For survival analyses, pure prognosis was studied by only including lymph-node negative patients who did not receive (neo)adjuvant systemic treatment (n = 551). 11% of the tumors showed CD146 expression. CD146 expression was most prevalent in triple-negative cases (64%, p < 0.001). In univariable analysis, CD146 expression was a prognostic factor for both metastasis-free survival (MFS) (p = 0.020) and overall survival (OS) (p = 0.037), but not in multivariable analysis (including age, tumor size, grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67). No correlation between CD146 and EMT nor difference in outcome to first-line tamoxifen was seen. In this large series, our data showed that CD146 is present in primary breast cancer and is a pure prognostic factor for MFS and OS in breast cancer patients. We did not see an association between CD146 expression and EMT nor on outcome to tamoxifen.

The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer.

Breast tumors often display extreme genetic heterogeneity characterized by hundreds of gross chromosomal aberrations and tens of thousands of somatic mutations. Tumor evolution is thought to be ongoing and driven by multiple mutagenic processes. A major outstanding question is whether primary tumors have preexisting mutations for therapy resistance or whether additional DNA damage and mutagenesis are necessary. Drug resistance is a key measure of tumor evolvability. If a resistance mutation preexists at the time of primary tumor presentation, then the intended therapy is likely to fail. However, if resistance does not preexist, then ongoing mutational processes still have the potential to undermine therapeutic efficacy. The antiviral enzyme APOBEC3B (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B) preferentially deaminates DNA C-to-U, which results in signature C-to-T and C-to-G mutations commonly observed in breast tumors. We use clinical data and xenograft experiments to ask whether APOBEC3B contributes to ongoing breast tumor evolution and resistance to the selective estrogen receptor modulator, tamoxifen. First, APOBEC3B levels in primary estrogen receptor-positive (ER+) breast tumors inversely correlate with the clinical benefit of tamoxifen in the treatment of metastatic ER+ disease. Second, APOBEC3B depletion in an ER+ breast cancer cell line results in prolonged tamoxifen responses in murine xenograft experiments. Third, APOBEC3B overexpression accelerates the development of tamoxifen resistance in murine xenograft experiments by a mechanism that requires the enzyme's catalytic activity. These studies combine to indicate that APOBEC3B promotes drug resistance in breast cancer and that inhibiting APOBEC3B-dependent tumor evolvability may be an effective strategy to improve efficacies of targeted cancer therapies.

The 29.5 kb APOBEC3B Deletion Polymorphism Is Not Associated with Clinical Outcome of Breast Cancer.

Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90-1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69-1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71-1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may affect and therefore determine the prognostic value of APOBEC3B mRNA levels.

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen.

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens.

GATA3 mRNA expression, but not mutation, associates with longer progression-free survival in ER-positive breast cancer patients treated with first-line tamoxifen for recurrent disease.

In breast cancer, GATA3 mutations have been associated with a favorable prognosis and the response to neoadjuvant aromatase inhibitor treatment. Therefore, we investigated whether GATA3 mutations predict the outcome of tamoxifen treatment in the advanced setting. In a retrospective study consisting of 235 hormone-naive patients with ER-positive breast cancer who received tamoxifen as first-line treatment for recurrent disease, GATA3 mutations (in 14.0% of patients) did not significantly associate with either the overall response rate (ORR) or with the length of progression-free survival (PFS) after the start of tamoxifen therapy. Interestingly, among 148 patients for whom both mutation and mRNA expression data were available, GATA3 mutations associated with an increased expression of GATA3. However, only 23.7% of GATA3 high tumors had a mutation. Evaluation of the clinical significance of GATA3 mRNA revealed that it was associated with prolonged PFS, but not with the ORR, also in multivariate analysis. Thus, GATA3 mRNA expression, but not GATA3 mutation, is an independent predictor of prolonged PFS in ER-positive breast cancer patients who received first-line tamoxifen for recurrent disease. Besides GATA3 mutation, other mechanisms must exist that underlie increased GATA3 levels.

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds.

BACKGROUND: Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible. AIM: To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform. METHODS: Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples. RESULTS: Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified for various levels of significance (Pearson P<0.01, n = 1,432; P<0.05, n = 2,530; and P<0.10, n = 3,351 probes). Unsupervised hierarchical clustering of the 27 pairs using the resulting probes yielded 25, 21, and 19 correctly clustered pairs, respectively, compared to 1 pair when all probes were used. CONCLUSION: The proposed method enables comparison of gene expression profiles of FFPE and/or FF origin measured on the same platform.

Sensitivity to systemic therapy for metastatic breast cancer in CHEK2 1100delC mutation carriers.

PURPOSE: The role of CHEK2 in DNA repair by homologous recombination suggests that CHEK2-associated breast cancer (BC) patients might be more sensitive to chemotherapy inducing double-strand DNA breaks, but results hereon are lacking. We compared the sensitivity to first-line chemotherapy and endocrine therapy between CHEK2 1100delC and non-CHEK2 metastatic breast cancer (MBC) patients. METHODS: Sixty-two CHEK2 1100delC MBC patients were selected from three cohorts genotyped for CHEK2 1100delC (one non-BRCA1/2 cohort and two sporadic cohorts). Controls were 62 non-CHEK2 MBC patients, matched for age at and year of primary BC diagnosis, and year of metastatic disease. Objective response rate (complete and partial response) to, and progression-free survival (PFS) and overall survival (OS) after start of first-line chemotherapy and endocrine therapy were compared between CHEK2 and non-CHEK2 patients. RESULTS: Median age at BC diagnosis was 46 and 51 years at MBC diagnosis. First-line chemotherapy consisted of anthracycline-based chemotherapy (n = 73), taxanes (n = 16), CMF(-like) chemotherapy (n = 33) and taxane/anthracycline regimens (n = 2). CHEK2 and non-CHEK2 patients had a comparable objective response rate (44 vs. 52 %). Also, PFS and OS after start of chemotherapy were comparable between both patient groups (hazard ratio 0.91; 95 % confidence interval 0.63-1.30 and 1.03; 95 % CI 0.71-1.49, respectively). Thirty-six CHEK2 and 32 non-CHEK2 patients received first-line endocrine therapy (mainly tamoxifen) for MBC. No significant differences were observed in objective response rate to, and PFS and OS after start of endocrine therapy. CONCLUSION: No differential efficacy of chemotherapy and endocrine therapy given for MBC was observed in CHEK2 versus non-CHEK2 patients.

Elevated APOBEC3B correlates with poor outcomes for estrogen-receptor-positive breast cancers.

Recent observations connected DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. We addressed whether APOBEC3B is associated with breast cancer clinical outcomes. APOBEC3B messenger RNA (mRNA) levels were related in 1,491 primary breast cancers to disease-free (DFS), metastasis-free (MFS), and overall survival (OS). For independent validation, APOBEC3B mRNA expression was associated with patient outcome data in five additional cohorts (over 3,500 breast cancer cases). In univariate Cox regression analysis, increasing APOBEC3B expression as a continuous variable was associated with worse DFS, MFS, and OS (hazard ratio [HR] = 1.20, 1.21, and 1.24, respectively; all P < .001). Also, in untreated ER-positive (ER+), but not in ER-, lymph-node-negative patients, high APOBEC3B levels were associated with a poor DFS (continuous variable: HR = 1.29, P = .001; dichotomized at the median level, HR = 1.66, P = .0002). This implies that APOBEC3B is a marker of pure prognosis in ER + disease. These findings were confirmed in the analyses of five independent patient sets. In these analyses, APOBEC3B expression dichotomized at the median level was associated with adverse outcomes (METABRIC discovery and validation, 788 and 706 ER + cases, disease-specific survival (DSS), HR = 1.77 and HR = 1.77, respectively, both P < .001; Affymetrix dataset, 754 ER + cases, DFS, HR = 1.57, P = 2.46E-04; NKI295, 181 ER + cases, DFS, HR = 1.72, P = .054; and BIG 1-98, 1,219 ER + cases, breast-cancer-free interval (BCFI), HR = 1.42, P = 0.0079). APOBEC3B is a marker of pure prognosis and poor outcomes for ER + breast cancer, which strongly suggests that genetic aberrations induced by APOBEC3B contribute to breast cancer progression.

Evaluation of the ability of adjuvant tamoxifen-benefit gene signatures to predict outcome of hormone-naive estrogen receptor-positive breast cancer patients treated with tamoxifen in the advanced setting.

To identify molecular markers indicative of response to tamoxifen and easily implemented in the routine setting, we recently reported three gene signatures that could stratify post-menopausal tamoxifen-treated, estrogen receptor-positive (ER+) patients according to outcome in the adjuvant setting. Here, we evaluated the predictive potential of the total of 14 genes included in the 3 gene signatures using 2 hormone-naïve Dutch ER+ cohorts of a total of 285 recurrent breast cancer patients treated with first-line tamoxifen. mRNA levels were measured by reverse transcriptase quantitative PCR (RT-qPCR) and the length of progression-free survival (PFS) was used as the primary endpoint. A Mann-Whitney U test was used to select for differentially expressed genes between tumors of patients who showed or did not show progressive disease within 6 months after start of tamoxifen treatment. Cox univariate and multivariate regression analysis for PFS were used to further assess their (independent) predictive potential. Five (BCAR3, BCL2, ESR1, IGF1R, and NCOA1) of the 14 genes analyzed showed significantly higher mRNA levels in tumors of patients who showed no disease progression within 6 months. Only BCAR3, BCL2 and NAT1 were significantly associated with a favorable PFS in multivariate analysis that included the traditional predictive factors: age, dominant relapse site, disease-free interval, ER and progesterone receptor (PGR), and adjuvant chemotherapy. This study shows that BCAR3, BCL2 and NAT1 in particular exhibit predictive promise regarding the efficacy of tamoxifen treatment in recurrent disease, in addition to the previously shown favorable outcome in the adjuvant setting.

The CYP2C19*2 genotype predicts tamoxifen treatment outcome in advanced breast cancer patients.

AIMS: Tamoxifen is metabolized by cytochrome P450s, with an important role for CYP2D6. Recently, we demonstrated in 80 patients that CYP2C19*2 is associated with increased survival in breast cancer patients using tamoxifen. Here, we aimed to confirm this in a large group of 499 patients. MATERIALS & METHODS: A total of 499 estrogen receptor-positive primary breast tumor specimens of advanced disease patients treated with first-line tamoxifen were genotyped for CYP2C19*2 and *17 variant alleles, with primary end point time-to-treatment failure (TTF). Effects of CYP2C19, independent of treatment, were analyzed in 243 primary systematic untreated patients. RESULTS: CYP2C19*2 hetero- and homozygote patients combined showed significantly longer TTFs (hazard ratio [HR]: 0.72; 95% CI: 0.57-0.90; p = 0.004). In multivariate analysis, including CYP2D6*4 status, CYP2C19*2 remained independently associated with TTF (HR: 0.73; 95% CI: 0.58-0.91; p = 0.007). In untreated patients, the CYP2C19*17 allele was significantly associated with a longer disease-free interval (HR: 0.66; 95%CI: 0.46-0.95; p = 0.025). CONCLUSION: CYP2C19 genotyping is potentially important for tamoxifen therapy for advanced disease and for breast cancer prognosis.

MicroRNA-30c expression level is an independent predictor of clinical benefit of endocrine therapy in advanced estrogen receptor positive breast cancer.

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression and which have been implicated in cancer. We evaluated whether five candidate predictive miRNAs, derived from a pilot study in which 249 miRNAs were assayed, were associated with clinical benefit of tamoxifen therapy in advanced breast cancer. These five miRNAs were measured in an independent series of 246 estrogen receptor (ER)-positive primary breast tumors of patients who received tamoxifen for advanced disease by quantitative Real Time PCR. Univariate analysis showed that higher expression levels of hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 were significantly associated with benefit of tamoxifen treatment and with longer PFS (all P-values <0.01). In multivariate analysis, corrected for the traditional predictive factors, only hsa-miRNA-30c was an independent predictor (P-value <0.01). Finally, in an attempt to understand the biology connected to this miRNA, Global testing pathway analysis showed an association of hsa-miRNA-30c expression with HER and RAC1 signaling pathways. We identified hsa-miRNA-30c as an independent predictor for clinical benefit of tamoxifen therapy in patients with advanced breast cancer. Assessment of tumor levels and connected pathways could be helpful to improve treatment strategies.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response.

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.

Tumor tissue levels of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and outcome following adjuvant chemotherapy in premenopausal lymph node-positive breast cancer patients: A retrospective study.

BACKGROUND: We have previously demonstrated that high tumor tissue levels of TIMP-1 are associated with no or limited clinical benefit from chemotherapy with CMF and anthracyclines in metastatic breast cancer patients. Here, we extend our investigations to the adjuvant setting studying outcome after adjuvant chemotherapy in premenopausal lymph node-positive patients. We hypothesize that TIMP-1 high tumors are less sensitive to chemotherapy and accordingly that high tumor tissue levels are associated with shorter survival. METHODS: From our original retrospectively collected tumor samples we selected a group of 525 pre-menopausal lymph node-positive patients (adjuvant treatment: CMF, 324 patients; anthracycline-based, 99 patients; no adjuvant chemotherapy, 102 patients). TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. TIMP-1 was analyzed as a continuous variable and as a dichotomized one using the median TIMP-1 concentration as a cut point between high and low TIMP-1 groups. We analyzed the benefit of adjuvant CMF and anthracyclines in univariate and multivariable survival models; endpoints were disease-free (DFS) and overall survival (OS). RESULTS: In this selected cohort of high-risk patients, and in the subgroup of patients receiving no adjuvant therapy, TIMP-1 was not associated with prognosis. In the subgroup of patients treated with anthracyclines, when analyzed as a continuous variable we observed a tendency for increasing TIMP-1 levels to be associated with shorter DFS (multivariable analysis, HR 1.75, 95% CI 1.00-3.07, P = 0.05) and a significant association between increasing TIMP-1 and shorter OS in both univariate (HR 3.52, 95% CI 1.54-8.06, P = 0.003) and multivariable analyses (HR 4.19, 95% CI 1.67-10.51, P = 0.002). No statistically significant association between TIMP-1 and DFS was observed in the CMF-treated patients although high TIMP-1 was associated with shorter OS when analyzed as a dichotomized variable (HR 1.64, 95% CI 1.02-2.65, P = 0.04). CONCLUSION: In the subgroup of patients receiving adjuvant chemotherapy we found an association between shorter survival after treatment in TIMP-1 high patients compared with TIMP-1 low patients, especially in patients receiving anthracycline-based therapy. This suggests that high tumor tissue levels of TIMP-1 might be associated with reduced benefit from classical adjuvant chemotherapy. Our findings should be validated in larger prospective studies.

Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

CHEK2 1100delC and male breast cancer in the Netherlands.

Mutations in the breast cancer susceptibility genes BRCA1, BRCA2, and CHEK2 are known risk factors for female breast cancer. Mutations in BRCA1 and BRCA2 also are associated with male breast cancer (MBC). Similarly, it had been suggested in the original CHEK2 identification report that the CHEK2 1100delC mutation confers an increased risk for MBC. Here, we have evaluated the risk of CHEK2 1100delC for MBC by genotyping CHEK2 1100delC in 23 familial and 71 unselected Dutch MBC cases. None of the 23 familial MBC cases carried the CHEK2 1100delC mutation. In contrast, CHEK2 1100delC was present in 3 of the 71 (4.2%) unselected MBC cases, which was significantly more prevalent than the 1.1% Dutch population frequency assessed in 1,692 individuals (P = 0.05, OR = 4.1, 95% CI 1.2-14.3). Our data suggest that, in the Netherlands, CHEK2 1100delC is associated with an increased risk for MBC.

Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer.

PURPOSE: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. PATIENTS AND METHODS: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. RESULTS: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. CONCLUSION: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

Relevance of breast cancer antiestrogen resistance genes in human breast cancer progression and tamoxifen resistance.

PURPOSE: We have previously identified a set of breast cancer antiestrogen resistance (BCAR) genes causing estrogen independence and tamoxifen resistance in vitro using a functional genetic screen. Here, we explored whether these BCAR genes provide predictive value for tamoxifen resistance and prognostic information for tumor aggressiveness in breast cancer patients. PATIENTS AND METHODS: mRNA levels of 10 BCAR genes (AKT1, AKT2, BCAR1, BCAR3, EGFR, ERBB2, GRB7, SRC, TLE3, and TRERF1) were measured in estrogen receptor-positive breast tumors using quantitative reverse-transcriptase polymerase chain reaction. Normalized mRNA levels were evaluated for association with progression-free survival (PFS) in 242 patients receiving tamoxifen as first-line monotherapy for recurrent disease, and with distant metastasis-free survival (MFS) in 413 lymph node-negative (LNN) primary breast cancer patients who did not receive systemic adjuvant therapy. RESULTS: Concerning tamoxifen resistance, BCAR3, ERBB2, GRB7, and TLE3 mRNA levels were predictive for PFS, independent of traditional predictive factors. By combining GRB7 (or ERBB2) and TLE3 mRNA levels, patients could be classified in three subgroups with distinct PFS. For the evaluation of tumor aggressiveness, AKT2, EGFR, and TRERF1 mRNA levels were all significantly associated with MFS, independent of traditional prognostic factors. Using the combined AKT2 and EGFR mRNA status, four prognostic groups were identified with different MFS outcomes. CONCLUSION: The majority of BCAR genes, which were revealed to confer tamoxifen resistance and estrogen independence in vitro by functional screening, have clinical relevance, and associate with tamoxifen resistance and/or tumor aggressiveness in breast cancer patients.

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response.

PURPOSE: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. EXPERIMENTAL DESIGN: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. RESULTS: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. CONCLUSION: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

DNA hypermethylation of PITX2 is a marker of poor prognosis in untreated lymph node-negative hormone receptor-positive breast cancer patients.

BACKGROUND: In this study, we evaluated if PITX2 DNA methylation is a marker for disease recurrence in lymph node-negative (LNN), steroid hormone receptor-positive (HR+) breast cancer patients. In addition, we studied the association between PITX2 DNA methylation and PITX2 gene expression. PATIENTS AND METHODS: PITX2 DNA-methylation was measured in tumor tissue from 412 LNN/HR+ breast cancer patients who had not received any adjuvant systemic treatment. In addition, PITX2 DNA-methylation and mRNA expression was evaluated in 32 breast cancer cell lines. RESULTS: In univariate Cox regression analysis, DNA-methylation of PITX2 as a continuous variable was associated with early distant metastasis (HR = 1.71; P < 0.01) and poor overall survival (HR = 1.71; P < 0.01). In multivariate analysis together with the established prognostic factors age, tumor size and tumor grade, and steroid hormone receptor levels, both associations retained their significance (for MFS, HR = 1.74; P < 0.01; for OS, HR = 1.46; P = 0.02). In the breast cancer cell lines, PITX2 DNA methylation was inversely association with PITX2A and PITX2B mRNA expression (P < 0.01). CONCLUSIONS: Hypermethylation of PITX2 is, in cell lines, negatively associated with PITX2 mRNA expression and, in clinical specimens, positively associated with breast cancer disease progression.

Production of P-glycoprotein from the MDR1 upstream promoter is insufficient to affect the response to first-line chemotherapy in advanced breast cancer.

Multidrug resistance, the phenomenon by which cells treated with a drug become resistant to the cytotoxic effect of a variety of other structurally and functionally unrelated drugs, is often associated with the expression of P-glycoprotein, an efflux membrane pump coded by the MDR1 (ABCB1) gene. Transcription from MDR1 can start at 2 promoters: a well-characterized downstream promoter and an as yet uncharacterized upstream promoter (USP). We have previously determined that the USP is activated in some drug-resistant cell lines, in primary breast tumors and in metastatic epithelial cells isolated from the lymph nodes of breast cancer patients. In this study, we report the cloning and characterization of the MDR1 USP and studied its association with chemotherapy response in breast cancer patients. Deletion analysis indicated that a nearby endogenous retroviral long terminal repeat is not responsible for promoter activation, and that the region within the first 400 nucleotides upstream from the transcription start point contained all the elements necessary for promoter activity in drug-resistant cells. We identified an element recognized by the transcription factor NF-IL6 (activated upon interleukin-6 exposure) which is necessary for promoter activity in drug-resistant cells and plays a role in the activation of the promoter in response to interleukin-6 in breast cancer MCF-7 cells. Although transcripts from this promoter are associated with translating polyribosomes, their low abundance makes the amount of synthesized P-glycoprotein insufficient to affect the response to first-line chemotherapy in patients with advanced breast cancer.