COX2/PTGS2 Expression Is Predictive of Response to Neoadjuvant Celecoxib in HER2-negative Breast Cancer Patients.

BACKGROUND: The prognostic and predictive role of cyclo-oxygenase-2 (COX2) in breast cancer is still debated, and in particular, its role as a target of COX2 inhibitor (celecoxib) in neoadjuvant setting. MATERIALS AND METHODS: We analyzed a series of 156 breast cancer samples from patients of the COX2 inhibitor-treated arm included in the REMAGUS-02 randomized phase II trial. COX2 gene expression was assessed by reverse transcription and quantitative polymerase chain reaction using ribonucleic acid from frozen biopsies. Pathological complete response (pCR) was the surrogate end-point. RESULTS: Significantly higher rates of grade 3, and estrogen and progesterone receptor negativity were observed in tumors with the highest expression of COX2. pCR rates were significantly higher in COX2-overexpressing tumors in patients receiving celecoxib. The test for interaction between COX2 gene expression and the celecoxib effect was statistically significant (p<0.01), but was not retained in the multivariate analysis. CONCLUSION: COX2 overexpression is predictive of pCR in patients with celecoxib-treated tumors. The efficacy of celecoxib in breast cancer might be improved by quantification of COX2 gene expression.

PIK3R1 underexpression is an independent prognostic marker in breast cancer.

BACKGROUND: The present study focused on the prognostic roles of PIK3CA and PIK3R1 genes and additional PI3K pathway-associated genes in breast cancer. METHODS: The mutational and mRNA expression status of PIK3CA, PIK3R1 and AKT1, and expression status of other genes involved in the PI3K pathway (EGFR, PDK1, PTEN, AKT2, AKT3, GOLPH3, WEE1, P70S6K) were assessed in a series of 458 breast cancer samples. RESULTS: PIK3CA mutations were identified in 151 samples (33.0%) in exons 1, 2, 9 and 20. PIK3R1 mutations were found in 10 samples (2.2%) and underexpression in 283 samples (61.8%). AKT1 mutations were found in 15 samples (3.3%) and overexpression in 116 samples (25.3%). PIK3R1 underexpression tended to mutual exclusivity with PIK3CA mutations (p = 0.00097). PIK3CA mutations were associated with better metastasis-free survival and PIK3R1 underexpression was associated with poorer metastasis-free survival (p = 0.014 and p = 0.00028, respectively). By combining PIK3CA mutation and PIK3R1 expression status, four prognostic groups were identified with significantly different metastasis-free survival (p = 0.00046). On Cox multivariate regression analysis, the prognostic significance of PIK3R1 underexpression was confirmed in the total population (p = 0.0013) and in breast cancer subgroups. CONCLUSIONS: PIK3CA mutations and PIK3R1 underexpression show opposite effects on patient outcome and could become useful prognostic and predictive factors in breast cancer.

SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

BACKGROUND: Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. METHODS: This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. RESULTS: The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. CONCLUSION: These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

miRNA expression profiling of inflammatory breast cancer identifies a 5-miRNA signature predictive of breast tumor aggressiveness.

IBC (inflammatory breast cancer) is a rare but very aggressive form of breast cancer with a particular phenotype. The molecular mechanisms responsible for IBC remain largely unknown. In particular, genetic and epigenetic alterations specific to IBC remain to be identified. MicroRNAs, a class of small noncoding RNAs able to regulate gene expression, are deregulated in breast cancer and may therefore serve as tools for diagnosis and prediction. This study was designed to determine miRNA expression profiling (microRNAome) in IBC. Quantitative RT-PCR was used to determine expression levels of 804 miRNAs in a screening series of 12 IBC compared to 31 non-stage-matched non-IBC and 8 normal breast samples. The differentially expressed miRNAs were then validated in a series of 65 IBC and 95 non-IBC. From a set of 18 miRNAs of interest selected from the screening series, 13 were differentially expressed with statistical significance in the validation series of IBC compared to non-IBC. Among these, a 5-miRNA signature comprising miR-421, miR-486, miR-503, miR-720 and miR-1303 was shown to be predictive for IBC phenotype with an overall accuracy of 89%. Moreover, multivariate analysis showed that this signature was an independent predictor of poor Metastasis-Free Survival in non-IBC patients.

Identification of new candidate therapeutic target genes in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subgroup of breast cancer that is negative for estrogen and progesterone receptor and ERBB2 protein expression. It is characterized by its aggressive behavior and by the lack of targeted therapies. To identify new therapeutic targets in TNBC, we used real-time quantitative RT-PCR to analyze 63 TNBC samples in terms of their mRNA expression of 26 genes coding for the major proteins currently targeted by drugs used to treat other cancers or undergoing clinical trials in breast cancer. Six of the 26 genes tested (VEGFA, SRC, PARP1, PTK2, RAF1, and FGFR3) were significantly upregulated in 13% to 46% of the TNBCs. None of the 6 genes was specifically upregulated in the TNBCs compared with 3 other classical breast tumor subtypes. No association was observed between overexpression of these 6 genes (except for FGFR3) and PIK3CA mutation status. These results confirm the interest of targeting VEGFA and PARP1 in ongoing clinical trials in TNBC patients and also identify new target genes (SRC, PTK2, RAF1, and FGFR3). Clinical trials could be initiated easily with existing drugs. Our results also suggest that these target genes might serve as predictive biomarkers of the TNBC treatment response.

Identification of potential prognostic biomarkers for node-negative breast tumours by proteomic analysis: a multicentric 2004 national PHRC study.

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice.

Validation of tumor-associated macrophage ferritin light chain as a prognostic biomarker in node-negative breast cancer tumors: A multicentric 2004 national PHRC study.

Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. Large screening of different biological compartments, such as the proteome, by means of high throughput techniques may greatly help scientists to find such markers. The present retrospective multicentric study included 268 node-negative breast cancer patients. We used a proteomic approach of SELDI-TOF-MS screening to identify differentially expressed cytosolic proteins with prognostic impact. The screening cohort was composed of 198 patients. Seventy supplementary patients were included for validation. Immunohistochemistry (IHC) and immunoassay (IA) were run to confirm the prognostic role of the marker identified by SELDI-TOF-MS screening. IHC was also used to explore links between selected marker and epithelial-mesenchymal transition (EMT)-like, proliferation and macrophage markers. Ferritin light chain (FTL) was identified as an independent prognostic marker (HR = 1.30-95% CI: 1.10-1.50, p = 0.001). Validation step by means of IHC and IA confirmed the prognostic value of FTL level. CD68 IHC showed that FTL was stored in tumor-associated macrophages (TAM), which exhibit an M2-like phenotype. We report here, first, the validation of FTL as a breast tumor prognostic biomarker in node-negative patients, and second, the fact that FTL is stored in TAM.

Ki-67: level of evidence and methodological considerations for its role in the clinical management of breast cancer: analytical and critical review.

Clinicians can use biomarkers to guide therapeutic decisions in estrogen receptor positive (ER+) breast cancer. One such biomarker is cellular proliferation as evaluated by Ki-67. This biomarker has been extensively studied and is easily assayed by histopathologists but it is not currently accepted as a standard. This review focuses on its prognostic and predictive value, and on methodological considerations for its measurement and the cut-points used for treatment decision. Data describing study design, patients' characteristics, methods used and results were extracted from papers published between January 1990 and July 2010. In addition, the studies were assessed using the REMARK tool. Ki-67 is an independent prognostic factor for disease-free survival (HR 1.05-1.72) in multivariate analyses studies using samples from randomized clinical trials with secondary central analysis of the biomarker. The level of evidence (LOE) was judged to be I-B with the recently revised definition of Simon. However, standardization of the techniques and scoring methods are needed for the integration of this biomarker in everyday practice. Ki-67 was not found to be predictive for long-term follow-up after chemotherapy. Nevertheless, high KI-67 was found to be associated with immediate pathological complete response in the neoadjuvant setting, with an LOE of II-B. The REMARK score improved over time (with a range of 6-13/20 vs. 10-18/20, before and after 2005, respectively). KI-67 could be considered as a prognostic biomarker for therapeutic decision. It is assessed with a simple assay that could be standardized. However, international guidelines are needed for routine clinical use.

Importance of pre-analytical steps for transcriptome and RT-qPCR analyses in the context of the phase II randomised multicentre trial REMAGUS02 of neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial. METHODS: This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA. RESULTS: Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy. CONCLUSIONS: Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.

Expression analysis of mitotic spindle checkpoint genes in breast carcinoma: role of NDC80/HEC1 in early breast tumorigenicity, and a two-gene signature for aneuploidy.

BACKGROUND: Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer. METHODS: To obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples. RESULTS: The expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors. CONCLUSION: This study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.

Immunomarker studies of fine-needle cytopuncture cell blocks for tumor response prediction after preoperative chemotherapy and prognosis in operable nonmetastatic primary breast carcinoma.

Neo-adjuvant chemotherapy of breast cancer provides an opportunity to evaluate predictive factors at initial tumor biopsy. We evaluated these factors on cell blocks obtained by diagnostic fine-needle cytopuncture (FNC), with respect to tumor regression and outcome. A prospective study (1996-2003, median follow-up 82 months) involved 163 patients with breast carcinoma (T2 ≥ 3 cm, T3, T4 noninflammatory) diagnosed by means of FNC. Malignancy, cytologic grade, and the presence of lymphocytes were determined on cytologic smears. Ki67, estrogen receptor (ER), progesterone receptor (PgR), HER2, and p53 expression was assessed on cell blocks by means of immunohistochemistry. All the patients received anthracycline-based chemotherapy. A combined clinical and pathologic tumor regression score was calculated. Twelve cases (7.5%) showed a complete regression, 72 cases (44%) a partial regression and 79 cases (48.5%) no regression. Factors predictive of regression were high grade, presence of lymphocytes, pN0, high Ki67 expression, hormone receptor negativity, and the "triple negative" phenotype. In univariate analysis 5-year metastasis-free survival rate (MFS) correlated with cytologic grade, pN, ER, and p53 status, while overall survival (OS) correlated with cytologic grade, type of surgery, pN, and ER status. In multivariate analysis, MFS was significantly influenced by the regression score, Ki67, age, ER status, pN, HER2, and initial tumor size. Except for age, the same parameters correlated with OS. FNC with the cell block technique is a rapid, minimally invasive, reliable, and inexpensive method for analyzing predictive biomarkers, and may thus be useful in the management of breast cancer patients requiring neo-adjuvant chemotherapy.

CDO1 promoter methylation is a biomarker for outcome prediction of anthracycline treated, estrogen receptor-positive, lymph node-positive breast cancer patients.

BACKGROUND: Various biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy. METHODS: Quantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival. RESULTS: Of the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010). CONCLUSIONS: CDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.

DNA repair gene expression and risk of locoregional relapse in breast cancer patients.

PURPOSE: Radiation therapy appears to kill cells mainly by inducing DNA double-strand breaks. We investigated whether the DNA repair gene expression status might influence the risk of locoregional recurrence (LRR) in breast cancer patients. METHODS AND MATERIALS: We used a quantitative reverse transcriptase PCR-based approach to measure messenger RNA levels of 20 selected DNA repair genes in tumor samples from 97 breast cancer patients enrolled in a phase III trial (Centre René Huguenin cohort). Normalized mRNA levels were tested for an association with LRR-free survival (LRR-FS) and overall survival (OS). The findings were validated in comparison with those of an independent cohort (Netherlands Cancer Institute (NKI) cohort). Multivariate analysis encompassing known prognostic factors was used to assess the association between DNA repair gene expression and patient outcome. RESULTS: RAD51 was the only gene associated with LRR in both cohorts. With a median follow-up of 126 months in the CRH cohort, the 5-year LRR-FS and OS rates were 100% and 95% in the 61 patients with low RAD51 expression, compared with 70% and 69% in the 36 patients with high RAD51 expression, respectively (p < 0.001). RAD51 overexpression was associated with a higher risk of LRR (hazard ratio [HR], 12.83; 95% confidence interval [CI], 3.6-45.6) and death (HR, 4.10; 95% CI, 1.7-9.7). RAD51 overexpression was also significantly associated with shorter LRR-FS and OS in the NKI cohort. CONCLUSIONS: Overexpression of RAD51, a key component of the homologous DNA repair pathway, is associated with poor breast cancer outcome. This finding warrants prospective studies of RAD51 as a prognosticator and therapeutic target.

DNA methylation markers predict outcome in node-positive, estrogen receptor-positive breast cancer with adjuvant anthracycline-based chemotherapy.

PURPOSE: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy. EXPERIMENTAL DESIGN: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. RESULTS: DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone. CONCLUSIONS: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.

Identification of TACC1, NOV, and PTTG1 as new candidate genes associated with endocrine therapy resistance in breast cancer.

Cross-resistance to molecules used in endocrine therapy is among the main challenges in the treatment of estrogen receptor-alpha (ERalpha) positive breast cancer. In this study, we used two different cell models of resistance to anti-estrogens: MVLN/CL6.7 cells and VP229/VP267 cells selected after exposure to tamoxifen respectively in vitro and in vivo to characterize a phenotype rarely observed, i.e. acquisition of cross-resistance to the pure ER antagonist fulvestrant. As MVLN/CL6.7 cells and VP229/VP267 cell lines are original and valuable models of cross-resistance to tamoxifen and fulvestrant, we examined candidate genes using a RTQ-PCR strategy to identify new biomarkers of endocrine resistance. Out of the 26 candidate genes tested, 19 displayed deregulation of expression at the basal level in at least one of the two resistant cell lines. Eight genes (TACC1, NOV, PTTG1, MAD2L1, BAK1, TGFB2, BIRC5, and CCNE2) were significantly overexpressed in samples from ER-positive breast cancer patients who relapsed after tamoxifen treatment (n=24) compared with samples from patients who did not (n=24). Five genes (TACC1, NOV, PTTG1, BAK1, and TGFB2) were correlated with significantly shorter relapse-free survival (univariate analysis). Finally, we identified TACC1 and a three-gene expression signature (TACC1, NOV, and PTTG1) as independent prognostic markers (multivariate analysis). Aberrant mRNA and protein levels of TACC1, NOV, and PTTG1 were also observed under tamoxifen and/or fulvestrant exposure in resistant CL6.7 cells compared with their respective control MVLN cells. In conclusion, our data identify TACC1, NOV, and PTTG1 as promising new markers that could be used in the clinical management of ER-positive breast cancer patients.

Exemestane as neoadjuvant hormonotherapy for locally advanced breast cancer: results of a phase II trial.

BACKGROUND: Neoadjuvant hormonotherapy has recently been used for downstaging large or locally advanced (LA) breast cancer in postmenopausal women. PATIENTS AND METHODS: A phase II study was conducted in postmenopausal, hormone-receptor (HR) positive, T2-T4 patients, receiving 25 mg/day exemestane for 16 weeks. RESULTS: Among 42 patients, 57.1% underwent conservative surgery. The clinical objective response rate (ORR) was 73.3%, without progression. A pathological partial response was achieved in 16.7% of the patients. Exemestane significantly reduced the expression of Ki-67 and progesterone receptors (PgR) (p<0.001). A significant decrease in PgR was correlated with clinical ORR (p=0.028). The responders presented higher baseline PgR levels (p=0.017). No relationship was found between ORR and mRNA expression of aromatase or oestrogen receptors beta (ER-beta). CONCLUSION: Neoadjuvant exemestane provided satisfactory efficacy and safety profiles in LA breast cancer. The main biological effects consisted of a reduction in PgR expression for responders and a decrease in Ki-67 expression.

DNA-methylation of the homeodomain transcription factor PITX2 reliably predicts risk of distant disease recurrence in tamoxifen-treated, node-negative breast cancer patients--Technical and clinical validation in a multi-centre setting in collaboration with the European Organisation for Research and Treatment of Cancer (EORTC) PathoBiology group.

Our aim was to identify and validate DNA-methylation markers associated with very good outcome in node negative, hormone receptor positive breast cancer patients after adjuvant endocrine therapy which might allow identifying patients who could be spared the burden of adjuvant chemotherapy. Using a methylation microarray, we analysed 117 candidate genes in hormone receptor-positive tumours from 109 breast cancer patients treated by adjuvant tamoxifen. Results were validated in an independent cohort (n=236, 5 centres). Independent methodological validation was achieved by a real-time polymerase chain reaction (PCR)-based technique. DNA methylation of PITX2 showed the strongest correlation with distant recurrence. Its impact on patient outcome was validated in the independent cohort: 86% of patients with low PITX2 methylation were metastasis-free after 10 years, compared to 69% with elevated PITX2 methylation. Moreover, PITX2 methylation added significant independent information to established clinical factors. All clinical and technical findings were confirmed by quantitative DNA-methylation PCR. These results provide strong evidence that DNA-methylation analysis allows clinically relevant risk assessment in tamoxifen-treated primary breast cancer. Based on PITX2 methylation, about half of hormone receptor-positive, node-negative breast cancer patients receiving adjuvant tamoxifen monotherapy can be considered low-risk regarding development of distant recurrences and may thus be spared adjuvant chemotherapy. In addition, these low-risk postmenopausal patients seem to respond sufficiently well to tamoxifen so that they may not require up-front aromatase inhibitor therapy.