Baseline Radiomic Signature to Estimate Overall Survival in Patients With NSCLC.

INTRODUCTION: We aimed to define a baseline radiomic signature associated with overall survival (OS) using baseline computed tomography (CT) images obtained from patients with NSCLC treated with nivolumab or chemotherapy. METHODS: The radiomic signature was developed in patients with NSCLC treated with nivolumab in CheckMate-017, -026, and -063. Nivolumab-treated patients were pooled and randomized to training, calibration, or validation sets using a 2:1:1 ratio. From baseline CT images, volume of tumor lesions was semiautomatically segmented, and 38 radiomic variables depicting tumor phenotype were extracted. Association between the radiomic signature and OS was assessed in the nivolumab-treated (validation set) and chemotherapy-treated (test set) patients in these studies. RESULTS: A baseline radiomic signature was identified using CT images obtained from 758 patients. The radiomic signature used a combination of imaging variables (spatial correlation, tumor volume in the liver, and tumor volume in the mediastinal lymph nodes) to output a continuous value, ranging from 0 to 1 (from most to least favorable estimated OS). Given a threshold of 0.55, the sensitivity and specificity of the radiomic signature for predicting 3-month OS were 86% and 77.8%, respectively. The signature was identified in the training set of patients treated with nivolumab and was significantly associated (p < 0.0001) with OS in patients treated with nivolumab or chemotherapy. CONCLUSIONS: The radiomic signature provides an early readout of the anticipated OS in patients with NSCLC treated with nivolumab or chemotherapy. This could provide important prognostic information and may support risk stratification in clinical trials.

Prevention of Progression of Lipedema With Liposuction Using Tumescent Local Anesthesia: Results of an International Consensus Conference.

BACKGROUND: Lipedema is a chronic, progressive disorder of subcutaneous adipose tissue that usually affects the lower extremities of women. Also known as "two-body syndrome," the fat accumulations in lipedema are unsightly and painful. The disorder is well-known in Europe but is largely unrecognized and underdiagnosed in the United States. OBJECTIVE: To hold the First International Consensus Conference on Lipedema with the purpose of reviewing current European guidelines and the literature regarding the long-term benefits that have been reported to occur after lymph-sparing liposuction for lipedema using tumescent local anesthesia. METHODS: International experts on liposuction for lipedema were convened as part of the First International Congress on Lipedema in Vienna, Austria, June 9 to 10, 2017. RESULTS: Multiple studies from Germany have reported long-term benefits for as long as 8 years after liposuction for lipedema using tumescent local anesthesia. CONCLUSION: Lymph-sparing liposuction using tumescent local anesthesia is currently the only effective treatment for lipedema.

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC in in vitro and in vivo lung infection models using monoclonal antibodies.

Streptococcus pneumoniae isolates express up to three neuraminidases (sialidases), NanA, NanB and NanC, all of which cleave the terminal sialic acid of glycan-structures that decorate host cell surfaces. Most research has focused on the role of NanA with limited investigations evaluating the roles of all three neuraminidases in host-pathogen interactions. We generated two highly potent monoclonal antibodies (mAbs), one that blocks the enzymatic activity of NanA and one cross-neutralizing NanB and NanC. Total neuraminidase activity of clinical S. pneumoniae isolates could be inhibited by this mAb combination in enzymatic assays. To detect desialylation of cell surfaces by pneumococcal neuraminidases, primary human tracheal/bronchial mucocilial epithelial tissues were infected with S. pneumoniae and stained with peanut lectin. Simultaneous targeting of the neuraminidases was required to prevent desialylation, suggesting that inhibition of NanA alone is not sufficient to preserve terminal lung glycans. Importantly, we also found that all three neuraminidases increased the interaction of S. pneumoniae with human airway epithelial cells. Lectin-staining of lung tissues of mice pre-treated with mAbs before intranasal challenge with S. pneumoniae confirmed that both anti-NanA and anti-NanBC mAbs were required to effectively block desialylation of the respiratory epithelium in vivo. Despite this, no effect on survival, reduction in pulmonary bacterial load, or significant changes in cytokine responses were observed. This suggests that neuraminidases have no pivotal role in this murine pneumonia model that is induced by high bacterial challenge inocula and does not progress from colonization as it happens in the human host.

A retrospective analysis of cross-reacting cetuximab IgE antibody and its association with severe infusion reactions.

Severe infusion reactions (SIRs) at rates of 5% or less are known side effects of biological agents, including mAbs such as cetuximab. There are currently no prospectively validated risk factors to aid physicians in identifying patients who may be at risk of experiencing an SIR following administration of any of these drugs. A retrospective analysis of 545 banked serum or plasma samples from cancer patients participating in clinical trials of cetuximab was designed to evaluate whether the presence of pretreatment IgE antibodies against cetuximab, as determined by a commercially available assay system, is associated with SIRs during the initial cetuximab infusion. Patients with a positive test indicating the presence of pretreatment antibodies had a higher risk of experiencing an SIR; however, at the prespecified cutoff utilized in this analysis, the test has a relatively low-positive predictive value (0.577 [0.369-0.766]) and a negative predictive value of 0.961 (0.912-0.987) in an unselected patient population. Data collected in this large retrospective validation study support prior observations of an association between the presence of pretreatment IgE antibodies cross-reactive with cetuximab and SIRs. Further analysis of the test's ability to predict patients at risk of an SIR would be required before this assay could be used reliably in this patient population.

Differential gene expression patterns of EBV infected EBNA-3A positive and negative human B lymphocytes.

The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV-growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.

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.

Multicenter study using paraffin-embedded tumor tissue testing PITX2 DNA methylation as a marker for outcome prediction in tamoxifen-treated, node-negative breast cancer patients.

PURPOSE: We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor-positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. PATIENTS AND METHODS: Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor-positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). RESULTS: Reproducibility of the PCR assay in replicate measurements (r(s) > or = 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (r(s) = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). CONCLUSION: PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.

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.

The prognostic value of Stathmin-1, S100A2, and SYK proteins in ER-positive primary breast cancer patients treated with adjuvant tamoxifen monotherapy: an immunohistochemical study.

INTRODUCTION: We recently found that DNA methylation of S100A2, spleen tyrosine kinase (SYK), and Stathmin-1 (STMN1) correlates with response to tamoxifen therapy in metastatic breast cancer. In this retrospective study, we investigated immunohistochemically whether these three markers are predictors of relapse in early breast cancer (EBC) patients treated with adjuvant tamoxifen alone. METHODS: Immunohistochemical staining was performed for S100A2, SYK and STMN1 on a tissue microarray containing ER-positive invasive breast carcinomas from a study cohort of 215 operable breast cancer patients, who underwent radical local therapy and who were treated with adjuvant tamoxifen monotherapy. Cox regression was used to correlate staining intensity of the three markers with main endpoints in our study; disease-free survival (DFS), and disease-specific survival (DSS). RESULTS: In univariate analysis, only STMN1 staining intensity strongly correlated with DFS (P = 0.014) and DSS (P = 0.002). In the groups of low and high STMN1 intensity, DFS was 84% and 63%, and DSS was 89% and 70%. STMN1 retained its prognostic value for DFS (P = 0.002) and DSS (<0.001) in the multivariate model together with lymph node status. We found also a trend to better DFS in patients with low STMN1 intensity in both lymph node-positive (P = 0.001) and -negative patients (P = 0.065). As the tumour cells did not express S100A2 (except in one case) the potential prognostic value of this marker was not evaluated. CONCLUSIONS: Staining intensity of STMN1, but not SYK, predicted outcome in our collective of ER- positive tamoxifen treated EBC patients.

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.

Inhibition of vascular endothelial growth factor cotranslational translocation by the cyclopeptolide CAM741.

The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds.

The CR4 region of EBNA2 confers viability of Epstein-Barr virus-transformed B cells by CBF1-independent signalling.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene product is the key regulator of the latent genes of EBV and essential for EBV-mediated transformation of human primary B cells. Viral mutants were constructed carrying a deletion of the EBNA2 conserved region 4 (CR4). Primary resting B cells infected with the DeltaCR4-EBNA2 mutant virus were dramatically impaired for B cell transformation. Lymphoblastoid cell lines (LCLs) established with this mutant EBV revealed a prolonged population doubling time when cells were cultivated at low cell densities, which are not critical for wild-type-infected cells. Low-level spontaneous cell death occurred when the cells were cultivated at suboptimal cell densities. The phenotype of B cells and LCLs infected with the DeltaCR4-EBNA2 mutant virus indicated that the CR4 region of EBNA2 specifically contributes to the viability of the cells rather than affecting cell division rates.

Cellular target genes of Epstein-Barr virus nuclear antigen 2.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.

Epstein-Barr virus nuclear antigen 2 trans-activates the cellular antiapoptotic bfl-1 gene by a CBF1/RBPJ kappa-dependent pathway.

The human herpesvirus Epstein-Barr virus (EBV) establishes latency and promotes the long-term survival of its host B cell by targeting the molecular machinery controlling cell fate decisions. The cellular antiapoptotic bfl-1 gene confers protection from apoptosis under conditions of growth factor deprivation when expressed ectopically in an EBV-negative Burkitt's lymphoma-derived cell line (B. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000), and the EBV latent membrane protein 1 (LMP1) and its cellular functional homologue CD40 can both drive bfl-1 via an NF-kappaB-dependent enhancer element in the bfl-1 promoter (B. N. D'Souza, L. C. Edelstein, P. M. Pegman, S. M. Smith, S. T. Loughran, A. Clarke, A. Mehl, M. Rowe, C. Gélinas, and D. Walls, J. Virol. 78:1800-1816, 2004). Here we show that the EBV nuclear antigen 2 (EBNA2) also upregulates bfl-1. EBNA2 trans-activation of bfl-1 requires CBF1 (or RBP-J kappa), a nuclear component of the Notch signaling pathway, and there is an essential role for a core consensus CBF1-binding site on the bfl-1 promoter. trans-activation is dependent on the EBNA2-CBF1 interaction, is modulated by other EBV gene products known to interact with the CBF1 corepressor complex, and does not involve activation of NF-kappaB. bfl-1 expression is induced and maintained at high levels by the EBV growth program in a lymphoblastoid cell line, and withdrawal of either EBNA2 or LMP1 does not lead to a reduction in bfl-1 mRNA levels in this context, whereas the simultaneous loss of both EBV proteins results in a major decrease in bfl-1 expression. These findings are relevant to our understanding of EBV persistence, its role in malignant disease, and the B-cell developmental process.

Epstein-Barr virus nuclear antigen 2 induces FcRH5 expression through CBF1.

Fc-receptor homolog 5 (FcRH5) is a recently identified B-cell membrane protein of unknown function. In Burkitt lymphoma cell lines with chromosome 1q21 abnormalities, FcRH5 expression is deregulated, implicating FcRH5 in lymphomagenesis. Epstein-Barr virus infects and immortalizes B cells, and is implicated in the etiology of several tumors of B-cell origin. Overexpression of genes located on 1q21-25 has been proposed as a surrogate for Epstein-Barr virus in Burkitt lymphoma. We now report that Epstein-Barr virus nuclear antigen 2 (EBNA2) markedly induces the expression of the FcRH5 gene, encoded on chromosome 1q21. Induction occurred in the absence of other viral proteins and did not require de novo protein synthesis. EBNA2 lacks a DNA-binding domain and can target responsive genes through the host DNA binding protein CBF1. We show that induction of FcRH5 by EBNA2 is strictly CBF1 dependent, as it was abolished in CBF1-deficient cells. Accordingly, EBNA2 targeted CBF1 binding sites present in the FcRH5 promoter in vivo, as detected by chromatin immunoprecipitation. These results identify FcRH5 as a novel, direct target of EBNA2 that may contribute to the development of Epstein-Barr virus-associated tumors.

Differential DNA methylation of gene promoters in small B-cell lymphomas.

Improved care of patients with small B-cell lymphomas (SBCLs) is likely to result from the ongoing discovery of molecular markers that better define these malignant neoplasms. We identified multiple gene loci whose DNA methylation patterns differed between 3 types of SBCL: B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, and grades I and II follicular lymphoma. This analysis was performed using an oligonucleotide microarray that allowed determination of the DNA methylation status of 156 loci in 38 genes. Combined bisulfite restriction analysis and methylation-specific polymerase chain reaction were used to validate the differential methylation of 6 of these genes. By using non-Hodgkin lymphoma cell lines as models, these genes were examined further for methylation and gene expression relationships. This study illustrates nonrandom epigenetic alterations in SBCLs that seem to preferentially involve lymphomas of germinal center derivation.

Identifying DNA methylation biomarkers of cancer drug response.

In the last few years, DNA methylation has become one of the most studied gene regulation mechanisms in carcinogenesis as a result of the cumulative evidence produced by the scientific community. Moreover, advances in the technologies that allow detection of DNA methylation in a variety of analytes have opened the possibility of developing methylation-based tests. A number of studies have provided evidence that specific methylation changes can alter the response to different therapeutic agents in cancer and, therefore, be useful biomarkers. For example, the association of the methylation status of DNA repair genes such as MGMT and MLH1 illustrate the two main mechanisms of response to DNA damaging agents. Loss of methylation of MGMT, and the subsequent increase in gene expression, leads to a reduction in response to alkylating agents as a result of enhanced repair of drug-induced DNA damage. Conversely, the increase in methylation of MLH1 and its resulting loss of expression has been consistently observed in drug-resistant tumor cells. MLH1 encodes a mismatch repair enzyme activated in response to DNA damage; activation of MLH1 also induces apoptosis of tumor cells, and thus loss of its expression leads to resistance to DNA-damaging agents. Other methylation-regulated genes that could serve as biomarkers in cancer therapy include drug transporters, genes involved in microtubule formation and stability, and genes related to hormonal therapy response. These methylation markers have potential applications for disease prognosis, treatment response prediction, and the development of novel treatment strategies.

A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent.

CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.

Association of DNA methylation of phosphoserine aminotransferase with response to endocrine therapy in patients with recurrent breast cancer.

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor-positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor-positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor-positive patients with recurrent breast cancer.

DNA methylation markers - an opportunity to further individualize therapy in breast cancer?

Over the last few decades, a wealth of treatment options have become available for breast cancer. To specifically direct those therapies to patients with the highest need who will receive the greatest benefit, biomarkers are urgently needed. Two specific needs seem to be most pressing: first is the need for prognostic markers, which would determine which group of patients may recover without adjuvant chemotherapy. Second, predictive markers for specific treatments, such as different endocrine treatments, chemotherapies or targeted drugs, are expected to play a major role in the near future. Ideally, such markers should be strong single markers, or low-complexity marker panels containing only a few markers, to allow for easier assay development and improved reproducibility. The possibility to measure the marker(s) in formalin-fixed specimens would greatly facilitate integration into routine clinical practice. A common and early event in breast cancer is aberrant DNA methylation within gene regulatory regions, affecting a variety of genes with different functions. Data from recently published studies indicate that altered DNA methylation carries prognostic as well as predictive information in breast cancer. Together with the technical advantages of a DNA-based marker, DNA methylation may well constitute the ideal biomarker to further individualize breast cancer treatment. Here the recent literature is reviewed and the most interesting markers, which have the potential to significantly change breast cancer treatment and, therefore, warrant further systematic clinical validation, are highlighted.