Quantum Efficiency Measurement and Modeling of Silicon Sensors Optimized for Soft X-ray Detection.

Hybrid pixel detectors have become indispensable at synchrotron and X-ray free-electron laser facilities thanks to their large dynamic range, high frame rate, low noise, and large area. However, at energies below 3 keV, the detector performance is often limited because of the poor quantum efficiency of the sensor and the difficulty in achieving single-photon resolution due to the low signal-to-noise ratio. In this paper, we address the quantum efficiency of silicon sensors by refining the design of the entrance window, mainly by passivating the silicon surface and optimizing the dopant profile of the n+ region. We present the measurement of the quantum efficiency in the soft X-ray energy range for silicon sensors with several process variations in the fabrication of planar sensors with thin entrance windows. The quantum efficiency for 250 eV photons is increased from almost 0.5% for a standard sensor to up to 62% as a consequence of these developments, comparable to the quantum efficiency of backside-illuminated scientific CMOS sensors. Finally, we discuss the influence of the various process parameters on quantum efficiency and present a strategy for further improvement.

In vivo low-dose phase-contrast CT for quantification of functional and anatomical alterations in lungs of an experimental allergic airway disease mouse model.

Introduction: Synchrotron-based propagation-based imaging (PBI) is ideally suited for lung imaging and has successfully been applied in a variety of in vivo small animal studies. Virtually all these experiments were tailored to achieve extremely high spatial resolution close to the alveolar level while delivering high x-ray doses that would not permit longitudinal studies. However, the main rationale for performing lung imaging studies in vivo in small animal models is the ability to follow disease progression or monitor treatment response in the same animal over time. Thus, an in vivo imaging strategy should ideally allow performing longitudinal studies. Methods: Here, we demonstrate our findings of using PBI-based planar and CT imaging with two different detectors-MÖNCH 0.3 direct conversion detector and a complementary metal-oxide-semiconductor (CMOS) detector (Photonics Science)-in an Ovalbumin induced experimental allergic airway disease mouse model in comparison with healthy controls. The mice were imaged free breathing under isoflurane anesthesia. Results: At x-ray dose levels below those once used by commercial small animal CT devices at similar spatial resolutions, we were able to resolve structural changes at a pixel size down to 25 µm and demonstrate the reduction in elastic recoil in the asthmatic mice in cinematic planar x-ray imaging with a frame rate of up to 100 fps. Discussion: Thus, we believe that our approach will permit longitudinal small animal lung disease studies, closely following the mice over longer time spans.

Ptychographic Nanoscale Imaging of the Magnetoelectric Coupling in Freestanding BiFeO3.

Understanding the magnetic and ferroelectric ordering of magnetoelectric multiferroic materials at the nanoscale necessitates a versatile imaging method with high spatial resolution. Here, soft X-ray ptychography is employed to simultaneously image the ferroelectric and antiferromagnetic domains in an 80 nm thin freestanding film of the room-temperature multiferroic BiFeO3 (BFO). The antiferromagnetic spin cycloid of period 64 nm is resolved by reconstructing the corresponding resonant elastic X-ray scattering in real space and visualized together with mosaic-like ferroelectric domains in a linear dichroic contrast image at the Fe L3 edge. The measurements reveal a near perfect coupling between the antiferromagnetic and ferroelectric ordering by which the propagation direction of the spin cycloid is locked orthogonally to the ferroelectric polarization. In addition, the study evinces both a preference for in-plane propagation of the spin cycloid and changes of the ferroelectric polarization by 71° between multiferroic domains in the epitaxial strain-free, freestanding BFO film. The results provide a direct visualization of the strong magnetoelectric coupling in BFO and of its fine multiferroic domain structure, emphasizing the potential of ptychographic imaging for the study of multiferroics and non-collinear magnetic materials with soft X-rays.

Epigenomic Blood-Based Early Detection of Pancreatic Cancer Employing Cell-Free DNA.

BACKGROUND & AIMS: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma. METHODS: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs. RESULTS: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). CONCLUSIONS: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals.

The 5-Hydroxymethylcytosine Landscape of Prostate Cancer.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

The Cell Type-Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. SIGNIFICANCE: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265.

Characterization of Chromium Compensated GaAs Sensors with the Charge-Integrating JUNGFRAU Readout Chip by Means of a Highly Collimated Pencil Beam.

Chromium compensated GaAs or GaAs:Cr sensors provided by the Tomsk State University (Russia) were characterized using the low noise, charge integrating readout chip JUNGFRAU with a pixel pitch of 75 × 75 µm2 regarding its application as an X-ray detector at synchrotrons sources or FELs. Sensor properties such as dark current, resistivity, noise performance, spectral resolution capability and charge transport properties were measured and compared with results from a previous batch of GaAs:Cr sensors which were produced from wafers obtained from a different supplier. The properties of the sample from the later batch of sensors from 2017 show a resistivity of 1.69 × 109 Ω/cm, which is 47% higher compared to the previous batch from 2016. Moreover, its noise performance is 14% lower with a value of (101.65 ± 0.04) e- ENC and the resolution of a monochromatic 60 keV photo peak is significantly improved by 38% to a FWHM of 4.3%. Likely, this is due to improvements in charge collection, lower noise, and more homogeneous effective pixel size. In a previous work, a hole lifetime of 1.4 ns for GaAs:Cr sensors was determined for the sensors of the 2016 sensor batch, explaining the so-called "crater effect" which describes the occurrence of negative signals in the pixels around a pixel with a photon hit due to the missing hole contribution to the overall signal causing an incomplete signal induction. In this publication, the "crater effect" is further elaborated by measuring GaAs:Cr sensors using the sensors from 2017. The hole lifetime of these sensors was 2.5 ns. A focused photon beam was used to illuminate well defined positions along the pixels in order to corroborate the findings from the previous work and to further characterize the consequences of the "crater effect" on the detector operation.

A human tissue map of 5-hydroxymethylcytosines exhibits tissue specificity through gene and enhancer modulation.

DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers.

Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA.

Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort (n = 64) in comparison with a non-cancer cohort (n = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function (GATA4, GATA6, PROX1, ONECUT1, MEIS2), and cancer pathogenesis (YAP1, TEAD1, PROX1, IGF1). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of KRAS and inactivation of TP53. Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset, n = 79) and 0.92-0.94 (two independent test sets, n = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

Development of low-energy X-ray detectors using LGAD sensors.

Recent advances in segmented low-gain avalanche detectors (LGADs) make them promising for the position-sensitive detection of low-energy X-ray photons thanks to their internal gain. LGAD microstrip sensors fabricated by Fondazione Bruno Kessler have been investigated using X-rays with both charge-integrating and single-photon-counting readout chips developed at the Paul Scherrer Institut. In this work it is shown that the charge multiplication occurring in the sensor allows the detection of X-rays with improved signal-to-noise ratio in comparison with standard silicon sensors. The application in the tender X-ray energy range is demonstrated by the detection of the sulfur Kα and Kß lines (2.3 and 2.46 keV) in an energy-dispersive fluorescence spectrometer at the Swiss Light Source. Although further improvements in the segmentation and in the quantum efficiency at low energy are still necessary, this work paves the way for the development of single-photon-counting detectors in the soft X-ray energy range.

X-ray Fourier ptychography.

To a large extent, the performance of imaging systems is determined by their objectives, which affect properties as varied as collection efficiency, resolving power, and image distortions. Such limitations can be addressed by so-called aperture synthesis, a technique used, for instance, in radar, astronomy, and, increasingly, microscopy. Here, we apply such techniques to x-ray imaging and demonstrate how Fourier ptychography can be used at transmission x-ray microscopes to increase resolution, provide quantitative absorption and phase contrast, and allow for corrections of lens aberrations. We anticipate that such methods will find common and frequent applications, alleviating a number of limitations imposed by x-ray optical elements, offering an alternative approach to phase contrast imaging, and providing novel opportunities to mitigate radiation damage.

MÖNCH detector enables fast and low-dose free-propagation phase-contrast computed tomography of in situ mouse lungs.

Due to the complexity of the underlying pathomechanism, in vivo mouse lung-disease models continue to be of great importance in preclinical respiratory research. Longitudinal studies following the cause of a disease or evaluating treatment efficacy are of particular interest but challenging due to the small size of the mouse lung and the fast breathing rate. Synchrotron-based in-line phase-contrast computed tomography imaging has been successfully applied in lung research in various applications, but mostly at dose levels that forbid longitudinal in vivo studies. Here, the novel charge-integrating hybrid detector MÖNCH is presented, which enables imaging of mouse lungs at a pixel size of 25 µm, in less than 10 s and with an entrance dose of about 70 mGy, which therefore will allow longitudinal lung disease studies to be performed in mouse models.

Micrometer-resolution imaging using MÖNCH: towards G2-less grating interferometry.

MÖNCH is a 25 µm-pitch charge-integrating detector aimed at exploring the limits of current hybrid silicon detector technology. The small pixel size makes it ideal for high-resolution imaging. With an electronic noise of about 110 eV r.m.s., it opens new perspectives for many synchrotron applications where currently the detector is the limiting factor, e.g. inelastic X-ray scattering, Laue diffraction and soft X-ray or high-resolution color imaging. Due to the small pixel pitch, the charge cloud generated by absorbed X-rays is shared between neighboring pixels for most of the photons. Therefore, at low photon fluxes, interpolation algorithms can be applied to determine the absorption position of each photon with a resolution of the order of 1 µm. In this work, the characterization results of one of the MÖNCH prototypes are presented under low-flux conditions. A custom interpolation algorithm is described and applied to the data to obtain high-resolution images. Images obtained in grating interferometry experiments without the use of the absorption grating G2 are shown and discussed. Perspectives for the future developments of the MÖNCH detector are also presented.

PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor-positive breast cancer.

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.

The forkhead transcription factor FOXM1 promotes endocrine resistance and invasiveness in estrogen receptor-positive breast cancer by expansion of stem-like cancer cells.

INTRODUCTION: The forkhead transcription factor FOXM1 coordinates expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We previously showed that FOXM1 acts downstream of 14-3-3ζ signaling, the elevation of which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high has not been clearly defined yet. METHODS: We analyzed FOXM1 protein expression by immunohistochemistry in 501 ER-positive breast cancers. We also mapped genome-wide FOXM1, extracellular signal-regulated kinase 2 and ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. These binding profiles were integrated with gene expression data derived from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. We also modulated the levels of FOXM1 and newly discovered FOXM1-regulated genes and examined their impact on the cancer stem-like cell population and on cell invasiveness and resistance to endocrine treatments. RESULTS: FOXM1 protein expression was high in 20% of the tumors, which correlated with significantly reduced survival in these patients (P = 0.003 by logrank Mantel-Cox test). ChIP-seq analyses revealed that FOXM1 binding sites were enriched at the transcription start site of genes involved in cell-cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are associated with a poor prognosis in ERα-positive patients treated with tamoxifen. Integration of binding profiles with gene expression highlighted FOXM1 transcriptional networks controlling cell proliferation, stem cell properties, invasion and metastasis. Increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness. CONCLUSIONS: Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance. They highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.

Integration of molecular profiling and chemical imaging to elucidate fibroblast-microenvironment impact on cancer cell phenotype and endocrine resistance in breast cancer.

The tumor microenvironment is known to play a key role in altering the properties and behavior of nearby cancer cells. Its influence on resistance to endocrine therapy and cancer relapse, however, is poorly understood. Here we investigate the interaction of mammary fibroblasts and estrogen receptor-positive breast cancer cells in three-dimensional culture models in order to characterize gene expression, cellular changes, and the secreted protein factors involved in the cellular cross-talk. We show that fibroblasts, which are the predominant cell type found in the stroma adjacent to the cancer cells in a tumor, induce an epithelial-to-mesenchymal transition in the cancer cells, leading to hormone-independent growth, a more invasive phenotype, and resistance to endocrine therapy. Here, we applied a label-free chemical imaging modality, Fourier transform infrared (FT-IR) spectroscopic imaging, to identify cells that had transitioned to hormone-independent growth. Both the molecular and chemical profiles identified here were translated from cell culture to patient samples: a secreted protein signature was used to stratify patient populations based on gene expression and FT-IR was used to characterize breast tumor patient biopsies. Our findings underscore the role of mammary fibroblasts in promoting aggressiveness and endocrine therapy resistance in ER-positive breast cancers and highlight the utility of FT-IR for the further characterization of breast cancer samples.

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.

The detection and treatment of cancer has advanced significantly in the past several decades, with important improvements in our understanding of the fundamental molecular and genetic basis of the disease. Despite these advancements, drug-screening methodologies have remained essentially unchanged since the introduction of the in vitro human cell line screen in 1990. Although the existing methods provide information on the overall effects of compounds on cell viability, they are restricted by bulk measurements, large sample sizes, and lack capability to measure proliferation kinetics at the individual cell level. To truly understand the nature of cancer cell proliferation and to develop personalized adjuvant therapies, there is a need for new methodologies that provide quantitative information to monitor the effect of drugs on cell growth as well as morphological and phenotypic changes at the single cell level. Here we show that a quantitative phase imaging modality known as spatial light interference microscopy (SLIM) addresses these needs and provides additional advantages over existing proliferation assays. We demonstrate these capabilities through measurements on the effects of the hormone estradiol and the antiestrogen ICI182,780 (Faslodex) on the growth of MCF-7 breast cancer cells. Along with providing information on changes in the overall growth, SLIM provides additional biologically relevant information. For example, we find that exposure to estradiol results in rapidly growing cells with lower dry mass than the control population. Subsequently blocking the estrogen receptor with ICI results in slower growing cells, with lower dry masses than the control. This ability to measure changes in growth kinetics in response to environmental conditions provides new insight on growth regulation mechanisms. Our results establish the capabilities of SLIM as an advanced drug screening technology that provides information on changes in proliferation kinetics at the cellular level with greater sensitivity than any existing method.

14-3-3ζ as a predictor of early time to recurrence and distant metastasis in hormone receptor-positive and -negative breast cancers.

The 14-3-3ζ gene, on 8q22, is often amplified in breast cancer and encodes a survival factor that interacts with and stabilizes many key signaling proteins. We examined the relationship between the expression of 14-3-3ζ, estrogen receptor α (ERα), and other parameters ( tumor size, grade, nodal status, progesterone receptor, HER2, EGFR, and p53) in matched primary and recurrence tumor tissue and how these factors impact time to recurrence, properties of the recurred tumors, and site of metastasis. In this cohort of over 100 patients, median time to recurrence was 3 years (range 1-17 years). Our analyses of primary tumor microarray cores revealed that 14-3-3ζ status was significantly correlated with tumor grade, size, and ERα. Women with 14-3-3ζ-positive and ERα-negative tumors had the earliest time to recurrence (median 1 yr, p < 0.001, hazard ratio 2.89), while median time to recurrence was 7 years for 14-3-3ζ-negative and ER-positive tumors. Of recurred tumors, 70-75 % were positive for 14-3-3ζ, up from the 45 % positivity of primary tumors. High expression of 14-3-3ζ also correlated with site of recurrence and showed a propensity for distant metastases to lung and chest wall. Multifactor correlation regression analysis revealed 14-3-3ζ to be a non-redundant, independent variable that adds clinical strength in predicting risk for early recurrence in ER-positive and -negative breast cancers, providing information beyond that of all other clinical pathological features examined. Thus, high expression of 14-3-3ζ in the primary tumor was significantly associated with earlier time to recurrence and with distant metastasis. Furthermore, even when the primary breast cancers were negative-low for 14-3-3ζ, the majority acquired increased expression in the recurrence. The findings underscore the detrimental role played by 14-3-3ζ in tumor aggressiveness and suggest that reducing its expression or interfering with its actions might substantially improve the clinical outcome for breast cancer patients.