Sustained delivery of celecoxib from nanoparticles embedded in hydrogel injected into the biopsy cavity to prevent biopsy-induced breast cancer metastasis.

PURPOSE: We have previously reported that protracted Cyclooxygenase-2 (COX-2) activity in bone marrow-derived cells (BMDCs) infiltrating into biopsy wounds adjacent to the biopsy cavity of breast tumors in mice promotes M2-shift of macrophages and pro-metastatic changes in cancer cells, effects which were suppressed by oral administration of COX-2 inhibitors. Thus, local control of COX-2 activity in the biopsy wound may mitigate biopsy-induced pro-metastatic changes. METHODS: A combinatorial delivery system-thermosensitive biodegradable poly(lactic acid) hydrogel (PLA-gel) incorporating celecoxib-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Cx-NP/PLA-gel)-was injected into the biopsy cavity of Py230 murine breast tumors to achieve local control of COX-2 activity in the wound stroma. RESULTS: A single intra-biopsy cavity injection of PLA-gel loaded with rhodamine-encapsulated nanoparticles (NPs) showed sustained local delivery of rhodamine preferentially to infiltrating BMDCs with minimal to no rhodamine uptake by the reticuloendothelial organs in mice. Moreover, significant reductions in M2-like macrophage density, cancer cell epithelial-to-mesenchymal transition, and blood vessel density were observed in response to a single intra-biopsy cavity injection of Cx-NP/PLA-gel compared to PLA-gel loaded with NPs containing no payload. Accordingly, intra-biopsy cavity injection of Cx-NP/PLA-gel led to significantly fewer metastatic cells in the lungs than control-treated mice. CONCLUSION: This study provides evidence for the feasibility of sustained, local delivery of payload preferential to BMDCs in the wound stroma adjacent to the biopsy cavity using a combinatorial delivery system to reduce localized inflammation and effectively mitigate breast cancer cell dissemination.

Clinicopathologic Characteristics of a Single-institution Cohort of Ovarian Adult Granulosa Cell Tumors, With Biomarker and Therapeutic Implications Utilizing the Detection of Androgen, Estrogen, and Progesterone Hormone Receptor Expression by Immunohistochemistry.

Adult granulosa cell tumors (AGCTs) are rare ovarian tumors with generally good prognosis after surgical resection; however, they do have recurrence potential. Therapeutic and management options for recurrences are currently limited, and the need for expanded adjuvant therapies is increasingly recognized. Anti-hormonal therapy is being explored as an option, which relies on the detection and assessment of hormone receptor expression (androgen, estrogen, and progesterone receptors) as a biomarker and therapeutic target. Our study identifies several clinicopathologic characteristics with significant associations for recurrence of AGCT, which were younger age, higher stage, and larger tumor size. Our study also demonstrates that androgen receptor (AR) expression may be utilized as a potential biomarker for hormonal therapy and that detection of AR expression in AGCT by immunohistochemistry (IHC) varies depending on the antibody clone used for testing. AR was detected in 95% of samples tested with antibodies derived from clone AR27. This detection rate is much higher than previously reported.

Profibrotic COVID-19 subphenotype exhibits enhanced localized ER-dependent HSP47+ expression in cardiac myofibroblasts in situ.

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed 'profibrotic phenotype,' who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Selection of optimal quantile protein biomarkers based on cell-level immunohistochemistry data.

BACKGROUND: Protein biomarkers of cancer progression and response to therapy are increasingly important for improving personalized medicine. Advanced quantitative pathology platforms enable measurement of protein expression in tissues at the single-cell level. However, this rich quantitative cell-by-cell biomarker information is most often not exploited. Instead, it is reduced to a single mean across the cells of interest or converted into a simple proportion of binary biomarker-positive or -negative cells. RESULTS: We investigated the utility of retaining all quantitative information at the single-cell level by considering the values of the quantile function (inverse of the cumulative distribution function) estimated from a sample of cell signal intensity levels in a tumor tissue. An algorithm was developed for selecting optimal cutoffs for dichotomizing cell signal intensity distribution quantiles as predictors of continuous, categorical or survival outcomes. The proposed algorithm was used to select optimal quantile biomarkers of breast cancer progression based on cancer cells' cell signal intensity levels of nuclear protein Ki-67, Proliferating cell nuclear antigen, Programmed cell death 1 ligand 2, and Progesterone receptor. The performance of the resulting optimal quantile biomarkers was validated and compared to the standard cancer compartment mean signal intensity markers using an independent external validation cohort. For Ki-67, the optimal quantile biomarker was also compared to established biomarkers based on percentages of Ki67-positive cells. For proteins significantly associated with PFS in the external validation cohort, the optimal quantile biomarkers yielded either larger or similar effect size (hazard ratio for progression-free survival) as compared to cancer compartment mean signal intensity biomarkers. CONCLUSION: The optimal quantile protein biomarkers yield generally improved prognostic value as compared to the standard protein expression markers. The proposed methodology has a broad application to single-cell data from genomics, transcriptomics, proteomics, or metabolomics studies at the single cell level.

Quantile Index Biomarkers Based on Single-Cell Expression Data.

Current histocytometry methods enable single-cell quantification of biomolecules in tumor tissue sections by multiple detection technologies, including multiplex fluorescence-based immunohistochemistry or in situ hybridization. Quantitative pathology platforms can provide distributions of cellular signal intensity (CSI) levels of biomolecules across the entire cell populations of interest within the sampled tumor tissue. However, the heterogeneity of CSI levels is usually ignored, and the simple mean signal intensity value is considered a cancer biomarker. Here we consider the entire distribution of CSI expression levels of a given biomolecule in the cancer cell population as a predictor of clinical outcome. The proposed quantile index (QI) biomarker is defined as the weighted average of CSI distribution quantiles in individual tumors. The weight for each quantile is determined by fitting a functional regression model for a clinical outcome. That is, the weights are optimized so that the resulting QI has the highest power to predict a relevant clinical outcome. The proposed QI biomarkers were derived for proteins expressed in cancer cells of malignant breast tumors and demonstrated improved prognostic value compared with the standard mean signal intensity predictors. The R package Qindex implementing QI biomarkers has been developed. The proposed approach is not limited to immunohistochemistry data and can be based on any cell-level expressions of proteins or nucleic acids.

Individualized multi-omic pathway deviation scores using multiple factor analysis.

Malignant progression of normal tissue is typically driven by complex networks of somatic changes, including genetic mutations, copy number aberrations, epigenetic changes, and transcriptional reprogramming. To delineate aberrant multi-omic tumor features that correlate with clinical outcomes, we present a novel pathway-centric tool based on the multiple factor analysis framework called padma. Using a multi-omic consensus representation, padma quantifies and characterizes individualized pathway-specific multi-omic deviations and their underlying drivers, with respect to the sampled population. We demonstrate the utility of padma to correlate patient outcomes with complex genetic, epigenetic, and transcriptomic perturbations in clinically actionable pathways in breast and lung cancer.

Frequent upregulation of HER2 protein in hormone-receptor-positive HER2-negative breast cancer after short-term neoadjuvant endocrine therapy.

BACKGROUND: Endocrine resistant metastatic disease develops in ~ 20-25% of hormone-receptor-positive (HR+) breast cancer (BC) patients despite endocrine therapy (ET) use. Upregulation of HER family receptor tyrosine kinases (RTKs) represent escape mechanisms in response to ET in some HR+ tumors. Short-term neoadjuvant ET (NET) offers the opportunity to identify early endocrine escape mechanisms initiated in individual tumors. METHODS: This was a single arm, interventional phase II clinical trial evaluating 4 weeks (± 1 week) of NET in patients with early-stage HR+/HER2-negative (HER2-) BC. The primary objective was to assess NET-induced changes in HER1-4 proteins by immunohistochemistry (IHC) score. Protein upregulation was defined as an increase of ≥ 1 in IHC score following NET. RESULTS: Thirty-seven patients with cT1-T3, cN0, HR+/HER2- BC were enrolled. In 35 patients with evaluable tumor HER protein after NET, HER2 was upregulated in 48.6% (17/35; p = 0.025), with HER2-positive status (IHC 3+ or FISH-amplified) detected in three patients at surgery, who were recommended adjuvant trastuzumab-based therapy. Downregulation of HER3 and/or HER4 protein was detected in 54.2% of tumors, whereas HER1 protein remained low and unchanged in all cases. While no significant volumetric reduction was detected radiographically after short-term NET, significant reduction in tumor proliferation rates were observed. No significant associations were identified between any clinicopathologic covariates and changes in HER1-4 protein expression on multivariable analysis. CONCLUSION: Short-term NET frequently and preferentially upregulates HER2 over other HER family RTKs in early-stage HR+/HER2- BC and may be a promising strategy to identify tumors that utilize HER2 as an early endocrine escape pathway. CLINICAL TRIAL REGISTRY: Trial registration number: NCT03219476.

Sustained Inflammation of Breast Tumors after Needle Biopsy.

INTRODUCTION: Needle biopsy is essential for definitive diagnosis of breast malignancy. Significant histologic changes due to tissue damage have been reported in solid tumors. This study investigated the association between time from needle biopsy and inflammation in breast tumors. METHODS: A total of 73 stage I-II invasive breast cancer cases diagnosed by image-guided needle biopsy who had surgery as their first definitive treatment were retrospectively analyzed. Time from biopsy to surgical excision ranged from 8 to 252 days. Histological sections of surgically resected tumors with a visible needle tract were reviewed by histologic evaluation. Data were analyzed by McNemar's test for proportional differences, and the Benjamini-Hochberg procedure was used to assess the association between immune cell prevalence and clinical variables. RESULTS: Characteristic histology changes, including foreign body giant-cell reaction, synovial-cell metaplasia, desmoplastic repair changes, granulation tissue, fat necrosis, and inflammation, were frequently detected adjacent to the needle tract. Spatial comparison indicated that a higher proportion of cases had neutrophils, eosinophils, and macrophages adjacent to the needle tract than tumors distant from it. The presence of inflammatory cells adjacent to the needle tract was not associated with time from biopsy or subtype. Still, plasma cells were associated with residual carrier material from biopsy markers. CONCLUSION: Macrophages and eosinophils are highly abundant and retained adjacent to the needle tract regardless of time from the biopsy.

Quantification of spatial tumor heterogeneity in immunohistochemistry staining images.

MOTIVATION: Quantitative immunofluorescence is often used for immunohistochemistry quantification of proteins that serve as cancer biomarkers. Advanced image analysis systems for pathology allow capturing expression levels in each individual cell or subcellular compartment. However, only the mean signal intensity within the cancer tissue region of interest is usually considered as biomarker completely ignoring the issue of tumor heterogeneity. RESULTS: We propose using immunohistochemistry image-derived information on the spatial distribution of cellular signal intensity (CSI) of protein expression within the cancer cell population to quantify both mean expression level and tumor heterogeneity of CSI levels. We view CSI levels as marks in a marked point process of cancer cells in the tissue and define spatial indices based on conditional mean and conditional variance of the marked point process. The proposed methodology provides objective metrics of cell-to-cell heterogeneity in protein expressions that allow discriminating between different patterns of heterogeneity. The prognostic utility of new spatial indices is investigated and compared to the standard mean signal intensity biomarkers using the protein expressions in tissue microarrays incorporating tumor tissues from 1000+ breast cancer patients. AVAILABILITY AND IMPLEMENTATION: THE R CODE FOR COMPUTING THE PROPOSED SPATIAL INDICES IS INCLUDED AS SUPPLEMENTARY MATERIAL: . SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Theory, methods, and operational results of the Young Women's Health History Study: a study of young-onset breast cancer incidence in Black and White women.

PURPOSE: The etiology of young-onset breast cancer (BC) is poorly understood, despite its greater likelihood of being hormone receptor-negative with a worse prognosis and persistent racial and socioeconomic inequities. We conducted a population-based case-control study of BC among young Black and White women and here discuss the theory that informed our study, exposures collected, study methods, and operational results. METHODS: Cases were non-Hispanic Black (NHB) and White (NHW) women age 20-49 years with invasive BC in metropolitan Detroit and Los Angeles County SEER registries 2010-2015. Controls were identified through area-based sampling from the U.S. census and frequency matched to cases on study site, race, and age. An eco-social theory of health informed life-course exposures collected from in-person interviews, including socioeconomic, reproductive, and energy balance factors. Measured anthropometry, blood (or saliva), and among cases SEER tumor characteristics and tumor tissue (from a subset of cases) were also collected. RESULTS: Of 5,309 identified potentially eligible cases, 2,720 sampled participants were screened and 1,812 completed interviews (682 NHB, 1140 NHW; response rate (RR): 60%). Of 24,612 sampled control households 18,612 were rostered, 2,716 participants were sampled and screened, and 1,381 completed interviews (665 NHB, 716 NHW; RR: 53%). Ninety-nine% of participants completed the main interview, 82% provided blood or saliva (75% blood only), and SEER tumor characteristics (including ER, PR and HER2 status) were obtained from 96% of cases. CONCLUSIONS: Results from the successfully established YWHHS should expand our understanding of young-onset BC etiology overall and by tumor type and identify sources of racial and socioeconomic inequities in BC.

Prolonged Time from Diagnosis to Breast-Conserving Surgery is Associated with Upstaging in Hormone Receptor-Positive Invasive Ductal Breast Carcinoma.

BACKGROUND: Time to surgery (TTS) has been suggested to have an association with mortality in early-stage breast cancer. OBJECTIVE: This study aims to determine the association between TTS and preoperative disease progression in tumor size or nodal status among women diagnosed with clinical T1N0M0 ductal breast cancer. METHODS: Women diagnosed with clinical T1N0M0 ductal breast cancer who had breast-conserving surgery as their first definitive treatment between 2010 and 2016 (n = 90,405) were analyzed using the National Cancer Database. Separate multivariable logistic regression models for hormone receptor (HR)-positive and HR-negative patients, adjusted for clinical and demographic variables, were used to assess the relationship between TTS and upstaging of tumor size (T-upstaging) or nodal status (N-upstaging). RESULTS: T-upstaging occurred in 6.76% of HR-positive patients and 11.00% of HR-negative patients, while N-upstaging occurred in 12.69% and 10.75% of HR-positive and HR-negative patients, respectively. Among HR-positive patients, odds of T-upstaging were higher for 61-90 days TTS (odds ratio [OR] 1.18, 95% confidence interval [CI] 1.05-1.34) and ≥91 days TTS (OR 1.47, 95% CI 1.17-1.84) compared with ≤30 days TTS, and odds of N- upstaging were higher for ≥91 days TTS (OR 1.35, 95% CI 1.13-1.62). No association between TTS and either T- or N-upstaging was found among HR-negative patients. Other clinical and demographic variables, including grade, tumor location, and race/ethnicity, were associated with both T- and N-upstaging. CONCLUSION: TTS ≥61 and ≥91 days was a significant predictor of T- and N-upstaging, respectively, in HR-positive patients; however, TTS was not associated with upstaging in HR-negative breast cancer. Delays in surgery may contribute to measurable disease progression in T1N0M0 ductal breast cancer.

Control of CCND1 ubiquitylation by the catalytic SAGA subunit USP22 is essential for cell cycle progression through G1 in cancer cells.

Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.

Diffuse interstitial pneumonia-like/macrophage activation syndrome-like changes in patients with COVID-19 correlate with length of illness.

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

Exploring drivers of gene expression in the Cancer Genome Atlas.

Motivation: The Cancer Genome Atlas (TCGA) has greatly advanced cancer research by generating, curating and publicly releasing deeply measured molecular data from thousands of tumor samples. In particular, gene expression measures, both within and across cancer types, have been used to determine the genes and proteins that are active in tumor cells. Results: To more thoroughly investigate the behavior of gene expression in TCGA tumor samples, we introduce a statistical framework for partitioning the variation in gene expression due to a variety of molecular variables including somatic mutations, transcription factors (TFs), microRNAs, copy number alternations, methylation and germ-line genetic variation. As proof-of-principle, we identify and validate specific TFs that influence the expression of PTPN14 in breast cancer cells. Availability and implementation: We provide a freely available, user-friendly, browseable interactive web-based application for exploring the results of our transcriptome-wide analyses across 17 different cancers in TCGA at http://ls-shiny-prod.uwm.edu/edge_in_tcga. All TCGA Open Access tier data are available at the Broad Institute GDAC Firehose and were downloaded using the TCGA2STAT R package. TCGA Controlled Access tier data are available via controlled access through the Genomic Data Commons (GDC). R scripts used to download, format and analyze the data and produce the interactive R/Shiny web app have been made available on GitHub at https://github.com/andreamrau/EDGE-in-TCGA.

The p52 isoform of SHC1 is a key driver of breast cancer initiation.

BACKGROUND: SHC1 proteins (also called SHCA) exist in three functionally distinct isoforms (p46SHC, p52SHC, and p66SHC) that serve as intracellular adaptors for several key signaling pathways in breast cancer. Despite the broad evidence implicating SHC1 gene products as a central mediator of breast cancer, testing the isoform-specific roles of SHC1 proteins have been inaccessible due to the lack of isoform-specific inhibitors or gene knockout models. METHODS: Here, we addressed this issue by generating the first isoform-specific gene knockout models for p52SHC and p66SHC, using germline gene editing in the salt-sensitive rat strain. Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. Rats were dosed with 7,12-dimethylbenz(a)anthracene (DMBA) by oral gavage to induce mammary tumors, and progression of tumor development was followed for 15 weeks. At 15 weeks, tumors were excised and analyzed by RNA-seq to determine differences between tumors lacking p66SHC or p52SHC. RESULTS: Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. These data, combined with p52SHC being the predominant isoform that is upregulated in human and rat tumors, provide the first evidence that p52SHC is the oncogenic isoform of Shc1 gene products in breast cancer. Compared with WT tumors, 893 differentially expressed (DE; FDR < 0.05) genes were detected in p52SHC KO tumors compared with only 18 DE genes in the p66SHC KO tumors, further highlighting that p52SHC is the relevant SHC1 isoform in breast cancer. Finally, gene network analysis revealed that p52SHC KO disrupted multiple key pathways that have been previously implicated in breast cancer initiation and progression, including ESR1 and mTORC2/RICTOR. CONCLUSION: Collectively, these data demonstrate the p52SHC isoform is the key driver of DMBA-induced breast cancer while the expression of p66SHC and p46SHC are not enough to compensate.

Mapping genetic modifiers of radiation-induced cardiotoxicity to rat chromosome 3.

Radiation therapy is used in ~50% of cancer patients to reduce the risk of recurrence and in some cases improve survival. Despite these benefits, doses can be limited by toxicity in multiple organs, including the heart. The underlying causes and biomarkers of radiation-induced cardiotoxicity are currently unknown, prompting the need for experimental models with inherent differences in sensitivity and resistance to the development of radiation-induced cardiotoxicity. We have identified the parental SS (Dahl salt-sensitive/Mcwi) rat strain to be a highly-sensitized model of radiation-induced cardiotoxicity. In comparison, substitution of rat chromosome 3 from the resistant BN (Brown Norway) rat strain onto the SS background (SS-3BN consomic) significantly attenuated radiation-induced cardiotoxicity. SS-3BN rats had less radiation-induced cardiotoxicity than SS rats, as measured by survival, pleural and pericardial effusions, echocardiogram parameters, and histological damage. Mast cells, previously shown to have predominantly protective roles in radiation-induced cardiotoxicity, were increased in the more resistant SS-3BN hearts postradiation. RNA sequencing from SS and SS-3BN hearts at 1 wk postradiation revealed 5,098 differentially expressed candidate genes across the transcriptome and 350 differentially expressed genes on rat chromosome 3, which coincided with enrichment of multiple pathways, including mitochondrial dysfunction, sirtuin signaling, and ubiquitination. Upstream regulators of enriched pathways included the oxidative stress modulating transcription factor, Nrf2, which is located on rat chromosome 3. Nrf2 target genes were also differentially expressed in the SS vs. SS-3BN consomic hearts postradiation. Collectively, these data confirm the existence of heritable modifiers in radiation-induced cardiotoxicity and provide multiple biomarkers, pathways, and candidate genes for future analyses. NEW & NOTEWORTHY This novel study reveals that heritable genetic factors have the potential to modify normal tissue sensitivity to radiation. Gene variant(s) on rat chromosome 3 can contribute to enhanced cardiotoxicity displayed in the SS rats vs. the BN and SS-3BN consomic rats. Identifying genes that lead to understanding the mechanisms of radiation-induced cardiotoxicity represents a novel method to personalize radiation treatment, as well as predict the development of radiation-induced cardiotoxicity.

Neuronatin is a modifier of estrogen receptor-positive breast cancer incidence and outcome.

PURPOSE: Understanding the molecular mediators of breast cancer survival is critical for accurate disease prognosis and improving therapies. Here, we identified Neuronatin (NNAT) as a novel antiproliferative modifier of estrogen receptor-alpha (ER+) breast cancer. EXPERIMENTAL DESIGN: Genomic regions harboring breast cancer modifiers were identified by congenic mapping in a rat model of carcinogen-induced mammary cancer. Tumors from susceptible and resistant congenics were analyzed by RNAseq to identify candidate genes. Candidates were prioritized by correlation with outcome, using a consensus of three breast cancer patient cohorts. NNAT was transgenically expressed in ER+ breast cancer lines (T47D and ZR75), followed by transcriptomic and phenotypic characterization. RESULTS: We identified a region on rat chromosome 3 (142-178 Mb) that modified mammary tumor incidence. RNAseq of the mammary tumors narrowed the candidate list to three differentially expressed genes: NNAT, SLC35C2, and FAM210B. NNAT mRNA and protein also correlated with survival in human breast cancer patients. Quantitative immunohistochemistry of NNAT protein revealed an inverse correlation with survival in a univariate analysis of patients with invasive ER+ breast cancer (training cohort: n = 444, HR = 0.62, p = 0.031; validation cohort: n = 430, HR = 0.48, p = 0.004). NNAT also held up as an independent predictor of survival after multivariable adjustment (HR = 0.64, p = 0.038). NNAT significantly reduced proliferation and migration of ER+ breast cancer cells, which coincided with altered expression of multiple related pathways. CONCLUSIONS: Collectively, these data implicate NNAT as a novel mediator of cell proliferation and migration, which correlates with decreased tumorigenic potential and prolonged patient survival.

Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms.

Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan-Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0.98. Data-driven optimal cutpoints for outcome prediction by either platform were reciprocally applicable to the data derived by the alternate platform, identifying patients with low Nuc-pYStat5 at ~3.5-fold increased risk of disease progression. Our analyses identified two highly concordant fluorescence immunohistochemistry platforms that may serve as benchmarks for testing of other platforms, and low interoperator variability supports the implementation of objective tumor marker quantification in pathology laboratories.

Jak2-Stat5a/b Signaling Induces Epithelial-to-Mesenchymal Transition and Stem-Like Cell Properties in Prostate Cancer.

Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.