Differential Toxicity of Arsenic in Daphnia pulex Under Phosphorus and Food Limitation.

The on-going anthropogenic degradation of freshwater habitats has drastically altered the environmental supply of both nutrients and common pollutants. Most organisms living in these altered habitats experience interactive effects of various stressors that can initiate adjustments at multiple levels impacting their fitness. Hence, studies measuring response to a single environmental parameter fail to capture the complexities of the status quo. We tested both the individual and the interactive effect of arsenic (As) exposure, food quantity, and dietary phosphorus (P)-supply on six life-history traits (Juvenile Growth Rate; Adult Growth Rate; Age and Size at Maturity, Lifespan, and Fecundity) as surrogates for organismal fitness in the keystone aquatic grazer Daphnia pulex. We also tested the effect of food quantity and P-supply on somatic As accumulation in Daphnia. Our results indicated an influence of P-supply on neonatal growth and an influence of As and food quantity on growth and maintenance later in life. Maturation was strongly influenced by all three variables, with no reproduction observed in the presence of two or more environmental stressors. We found a strong interaction between As and dietary P, with increased P-supply intensifing the toxicity effect of As. No such effects were seen between As and food quantity, indicating a differential role of quantity versus quality on As toxicity. We found a nominal effect of diet on somatic As accumulation. The results from the present study emphasize the importance of considering such interactions between co-occurring environmental stressors and the dietary status of organisms, to better predict and manage impacts and risks associated with common environmental toxicants in highly vulnerable ecosystems. Environ Toxicol Chem 2024;00:1-13. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Does ROSE matter? Evaluation of final diagnostic, PD-L1 immunohistochemistry, and molecular testing yields of CT-guided lung biopsies performed before and after the onset of the COVID-19 pandemic.

INTRODUCTION: The onset of the COVID-19 pandemic, with urgent implementation of safety protocols limiting the number of on-site personnel, essentially terminated the use of rapid on-site evaluation (ROSE) for computed tomography (CT)--guided lung biopsies at our institution. The diminished use of ROSE during the pandemic prompted us to reevaluate the potential value of ROSE for CT-guided lung biopsies. MATERIALS AND METHODS: We retrospectively identified all CT-guided lung biopsies from 2017 to 2022. Associations between the use of ROSE, adequate diagnostic and ancillary testing (programmed death-ligand 1 immunohistochemistry and next-generation sequencing) outcomes, and other factors such as the number of passes performed and lesion size, were evaluated. RESULTS: Nine hundred twelve CT-guided lung biopsies were performed from 2017 to 2022; 171 (19%) utilized ROSE. The use of ROSE had been steadily decreasing prior to the pandemic but was essentially eliminated with the onset of the pandemic. By univariable analysis, the employment of ROSE was more likely to be associated with an adequate final diagnosis (odds ratio = 2.14, 95% confidence interval: [1.24-3.70], P = 0.006) and successful molecular testing (odds ratio = 2.16, 95% confidence interval: [1.11-4.21], P = 0.024). However, those associations were not present in multivariable analyses that incorporated the number of passes performed or lesion size. There were no differences in diagnostic adequacy or ancillary testing yields when comparing the periods 2017-2019 and 2020-2022, despite declining use of ROSE. CONCLUSIONS: If ROSE is not requested for CT-guided lung biopsies, proceduralists should err on the side of performing more, rather than fewer, passes, particularly for smaller lesions.

Enhancement of Radiation Therapy through Blockade of the Immune Checkpoint, V-domain Ig Suppressor of T Cell Activation (VISTA), in Melanoma and Adenocarcinoma Murine Models.

Radiation therapy (RT) has recently demonstrated promise at stimulating an enhanced immune response. The recent success of immunotherapies, such as checkpoint inhibitors, CART cells, and other immune modulators, affords new opportunities for combination with radiation. The aim of this study is to evaluate whether and to what extent blockade of VISTA, an immune checkpoint, can potentiate the tumor control ability of radiation therapy. Our study is novel in that it is the first comparison of two VISTA-blocking methods (antibody inhibition and genetic knockout) in combination with RT. VISTA was blocked either through genetic knockout (KO) or an inhibitory antibody and combined with RT in two syngeneic murine flank tumor models (B16 and MC38). Selected mRNA, immune cell infiltration, and tumor growth delay were used to assess the biological effects. When combined with a single 15Gy radiation dose, VISTA blockade via genetic knockout in the B16 model and via anti-VISTA antibodies in the MC38 model significantly improved survival compared to RT alone by an average of 5.5 days and 6.3 days, respectively (p < 0.05). The gene expression data suggest that the mechanism behind the enhanced tumor control is primarily a result of increased apoptosis and immune-mediated cytotoxicity. VISTA blockade significantly enhances the anti-tumor effect of a single dose of 15Gy radiation through increased expression and stimulation of cell-mediated apoptosis pathways. These results suggest that VISTA is a biologically relevant immune promoter that has the potential to enhance the efficacy of a large single radiation dose in a synergic manner.

Estrogen Therapy Induces Receptor-Dependent DNA Damage Enhanced by PARP Inhibition in ER+ Breast Cancer.

PURPOSE: Clinical evidence indicates that treatment with estrogens elicits anticancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor α (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains underused. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. EXPERIMENTAL DESIGN: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDX), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. RESULTS: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacologic suppression of the DNA damage response via PARP inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2-mutant and BRCA1/2-wild-type cell line and PDX models. CONCLUSIONS: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Alternating 17ß-Estradiol and Aromatase Inhibitor Therapies Is Efficacious in Postmenopausal Women with Advanced Endocrine-Resistant ER+ Breast Cancer.

PURPOSE: Strategies to implement estrogen therapy for advanced estrogen receptor-positive (ER+) breast cancer are underdeveloped. Preclinical data suggest that cycling treatment with 17ß-estradiol followed by estrogen deprivation can control tumor growth long-term. PATIENTS AND METHODS: Postmenopausal women with advanced ER+/HER2- breast cancer with recurrence or progression on ≥ 1 antiestrogen or aromatase inhibitor (AI)-based therapy were eligible. Patients received 17ß-estradiol (2 mg orally, three times a day) for 8 weeks followed by AI (physician's choice) for 16 weeks, alternating treatments on an 8-week/16-week schedule until disease progression. Patients then optionally received continuous single-agent treatment until a second instance of disease progression. Endpoints included 24-week clinical benefit and objective response per RECIST, and tumor genetic alterations. RESULTS: Of 19 evaluable patients, clinical benefit rate was 42.1% [95% confidence interval (CI), 23.1%-63.9%] and objective response rate (ORR) was 15.8% (95% CI, 5.7%-37.9%). One patient experienced a grade 3 adverse event related to 17ß-estradiol. Among patients who received continuous single-agent treatment until a second instance of disease progression, clinical benefit was observed in 5 of 12 (41.7%) cases. Tumor ER (ESR1) mutations were found by whole-exome profiling in 4 of 7 (57.1%) versus 2 of 9 (22.2%) patients who did versus did not experience clinical benefit from alternating 17ß-estradiol/AI therapy. The only two patients to experience objective responses to initial 17ß-estradiol had tumor ESR1 mutations. CONCLUSIONS: Alternating 17ß-estradiol/AI therapy may be a promising treatment for endocrine-refractory ER+ breast cancer, including following progression on CDK4/6 inhibitors or everolimus. Further study is warranted to determine whether the antitumor activity of 17ß-estradiol differs according to ESR1 mutation status.

Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer.

Purpose: Clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. Experimental Design: We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17ß-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. Results: Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2 -mutant and BRCA1 /2-wild-type cell line and PDX models. Conclusions: E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

Tumour, whole-blood, plasma and tissue concentrations of metformin in lung cancer patients.

AIM: Metformin is used for the management of type 2 diabetes mellitus (T2DM) and is being tested clinically as an anticancer agent. Metformin concentrations safely achievable in human solid tissues including tumours are unknown. This study was designed to determine metformin concentration in tissue compartments as a function of dose to inform rational dosing in preclinical models and interpretation of clinical results." METHODS: Subjects with solid tumours to be treated by resection and either (A) willingness to take metformin for 7-10 days before surgery or (B) taking metformin for T2DM were eligible. Whole blood, plasma, tumour, tumour-adjacent uninvolved tissue and subcutaneous adipose tissue were obtained for liquid chromatography with tandem mass spectrometry to measure metformin concentrations. RESULTS: All subjects had primary lung tumours. Metformin dose was significantly correlated with drug concentrations in all tissues analysed. Intersubject metformin concentrations varied by over two orders of magnitude. Metformin concentrations were significantly higher in tumour tissues and lower in adipose tissues compared to other tissues. Concentrations in blood and plasma were significantly correlated with concentrations in solid tissues. CONCLUSION: Metformin accumulates in cellular compartments. Concentrations observed in plasma, blood, lung and tumour tissues in subjects treated with US Food and Drug Administration-approved doses for T2DM are lower than those typically used in tissue culture studies. However, such tissue concentrations are in line with those found within cultured cells treated with supra-pharmacological doses of metformin. Given the large intersubject variability in metformin concentrations, it is imperative to determine whether there is an association between tissue metformin concentration and anticancer activity in humans.

In utero arsenic exposure and early childhood motor development in the New Hampshire Birth Cohort Study.

Introduction: High-level prenatal and childhood arsenic (As) exposure characteristic of several regions in Asia (e.g., Bangladesh), may impact motor function. However, the relationship between lower-level arsenic exposure (characteristic of other regions) and motor development is largely unstudied, despite the potential for deficient motor skills in childhood to have adverse long-term consequences. Thus, we sought to investigate the association between prenatal As exposure and motor function among 395 children in the New Hampshire Birth Cohort Study, a rural cohort from northern New England. Methods: Prenatal exposure was estimated by measuring maternal urine speciated As at 24-28 weeks of gestation using high-performance liquid chromatography (HPLC) inductively coupled plasma mass spectrometry (ICP-MS) and summing inorganic As, monomethylarsonic acid, and dimethylarsinic acid to obtain total urinary As (tAs). Motor function was assessed with the Bruininks-Oseretsky Test of Motor Proficiency, 2nd Edition (BOT-2) at a mean (SD) age of 5.5 (0.4) years. Results: Children who completed this exam were largely reported as white race (97%), born to married mothers (86%) with a college degree or higher (67%). The median (IQR) gestational urine tAs concentration was 4.0 (5.0) µg/L. Mean (SD) BOT-2 scores were 48.6 (8.4) for overall motor proficiency and 48.2 (9.6) for fine manual control [standard score = 50 (10)], and were 16.3 (5.1) for fine motor integration and 12.5 (4.1) for fine motor precision [standard score = 15 (5)]. We found evidence of a non-linear dose response relationship and used a change-point model to assess the association of tAs with overall motor proficiency and indices of fine motor integration, fine motor precision, and their composite, fine manual control, adjusted for age and sex. In models adjusted for potential confounders, each doubling of urine tAs decreased overall motor proficiency by -3.3 points (95% CI: -6.1, -0.4) for tAs concentrations greater than the change point of 9.5 µg/L and decreased fine motor integration by -4.3 points (95% CI: -8.0, -0.6) for tAs concentrations greater than the change point of 17.0 µg/L. Discussion: In summary, we found that levels of prenatal As exposure above an empirically-derived threshold (i.e., the change point) were associated with decrements in childhood motor development in a US population.

One-Sided Multidimensional Statistical Significance Testing: A New Method of Calculating the Statistical Significance of Spectra Used to Demonstrate Magnetic Nanoparticle Sensitivity.

Estimating statistical significance of the difference between two spectra or series is a fundamental statistical problem. Multivariate significance tests exist but the limitations preclude their use in many common cases; e.g., one-sided testing, unequal variance and when few repetitions are acquired all of which are required in magnetic spectroscopy of nanoparticle Brownian motion (MSB). We introduce a test, termed the T-S test, that is powerful and exact (exact type I error). It is flexible enough to be one- or two-sided and the one-sided version can specify arbitrary regions where each spectrum should be larger. The T-S test takes the-one or two-sided p-value at each frequency and combines them using Stouffer's method. We evaluated it using simulated spectra and measured MSB spectra. For the single-sided version, mean of the spectrum, A-T, was used as a reference; the T-S test is as powerful when the variance at each frequency is uniform and outperforms when the noise power is not uniform. For the two-sided version, the Hotelling T2 two-sided multivariate test was used as a reference; the two-sided T-S test is only slightly less powerful for large numbers of repetitions and outperforms rather dramatically for small numbers of repetitions. The T-S test was used to estimate the sensitivity of our current MSB spectrometer showing 1 nanogram sensitivity. Using eight repetitions the T-S test allowed 15 pM concentrations of mouse IL-6 to be identified while the mean of the spectra only identified 76 pM.

First-In-Human Study in Cancer Patients Establishing the Feasibility of Oxygen Measurements in Tumors Using Electron Paramagnetic Resonance With the OxyChip.

OBJECTIVE: The overall objective of this clinical study was to validate an implantable oxygen sensor, called the 'OxyChip', as a clinically feasible technology that would allow individualized tumor-oxygen assessments in cancer patients prior to and during hypoxia-modification interventions such as hyperoxygen breathing. METHODS: Patients with any solid tumor at ≤3-cm depth from the skin-surface scheduled to undergo surgical resection (with or without neoadjuvant therapy) were considered eligible for the study. The OxyChip was implanted in the tumor and subsequently removed during standard-of-care surgery. Partial pressure of oxygen (pO2) at the implant location was assessed using electron paramagnetic resonance (EPR) oximetry. RESULTS: Twenty-three cancer patients underwent OxyChip implantation in their tumors. Six patients received neoadjuvant therapy while the OxyChip was implanted. Median implant duration was 30 days (range 4-128 days). Forty-five successful oxygen measurements were made in 15 patients. Baseline pO2 values were variable with overall median 15.7 mmHg (range 0.6-73.1 mmHg); 33% of the values were below 10 mmHg. After hyperoxygenation, the overall median pO2 was 31.8 mmHg (range 1.5-144.6 mmHg). In 83% of the measurements, there was a statistically significant (p ≤ 0.05) response to hyperoxygenation. CONCLUSIONS: Measurement of baseline pO2 and response to hyperoxygenation using EPR oximetry with the OxyChip is clinically feasible in a variety of tumor types. Tumor oxygen at baseline differed significantly among patients. Although most tumors responded to a hyperoxygenation intervention, some were non-responders. These data demonstrated the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes.

Estimation of pO2 distribution in EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry, using oxygen-sensing implant such as OxyChip, is capable of measuring oxygen concentration in vivo - a critical tissue information required for successful medical treatment such as cancer, wound healing and diabetes. Typically, EPR oximetry produces one value of the oxygen concentration, expressed as pO2 at the site of implant. However, it is well recognized that in vivo one deals with a distribution of oxygen concentration and therefore reporting just one number is not representative_a long-standing critique of EPR oximetry. Indeed, when it comes to the assessment of radiation efficacy one should be guided not by the mean or median but the proportion of oxygenated cancer cells which can be estimated only when the whole oxygen distribution in the tumor is known. Although there is a handful of papers attempting estimation of the oxygen distribution they suffer from the problem of negative frequencies and no theoretical justification and no biomedical interpretation. The goal of this work is to suggest a novel method using the empirical Bayesian approach realized via nonlinear mixed modeling with a priori distribution of oxygen following a two-parameter lognormal distribution with parameters estimated from the multi-implant single component EPR scan. Unlike previous work, the result of our estimation is the distribution with positive values for the frequency and the associated pO2 value. Our algorithm based on nonlinear regression is illustrated with EPR measurements on OxyChips equilibrated with gas mixtures containing four values of pO2 and computation of the proportion of volume with pO2 greater than any given threshold. This approach may become crucial for application of the EPR oximetry in clinical setting when the sucsess of the treatment depends of the proportion of tissue oxygenated.

High estrogen receptor alpha activation confers resistance to estrogen deprivation and is required for therapeutic response to estrogen in breast cancer.

Estrogen receptor alpha (ER)-positive breast cancer is commonly treated with endocrine therapies, including antiestrogens that bind and inhibit ER activity, and aromatase inhibitors that suppress estrogen biosynthesis to inhibit estrogen-dependent ER activity. Paradoxically, treatment with estrogens such as 17b-estradiol can also be effective against ER+ breast cancer. Despite the known efficacy of estrogen therapy, the lack of a predictive biomarker of response and understanding of the mechanism of action have contributed to its limited clinical use. Herein, we demonstrate that ER overexpression confers resistance to estrogen deprivation through ER activation in human ER+ breast cancer cells and xenografts grown in mice. However, ER overexpression and the associated high levels of ER transcriptional activation converted 17b-estradiol from a growth-promoter to a growth-suppressor, offering a targetable therapeutic vulnerability and a potential means of identifying patients likely to benefit from estrogen therapy. Since ER+ breast cancer cells and tumors ultimately developed resistance to continuous estrogen deprivation or continuous 17b-estradiol treatment, we tested schedules of alternating treatments. Oscillation of ER activity through cycling of 17b-estradiol and estrogen deprivation provided long-term control of patient-derived xenografts, offering a novel endocrine-only strategy to manage ER+ breast cancer.

The impact of particulate electron paramagnetic resonance oxygen sensors on fluorodeoxyglucose imaging characteristics detected via positron emission tomography.

During a first-in-humans clinical trial investigating electron paramagnetic resonance tumor oximetry, a patient injected with the particulate oxygen sensor Printex ink was found to have unexpected fluorodeoxyglucose (FDG) uptake in a dermal nodule via positron emission tomography (PET). This nodule co-localized with the Printex ink injection; biopsy of the area, due to concern for malignancy, revealed findings consistent with ink and an associated inflammatory reaction. Investigations were subsequently performed to assess the impact of oxygen sensors on FDG-PET/CT imaging. A retrospective analysis of three clinical tumor oximetry trials involving two oxygen sensors (charcoal particulates and LiNc-BuO microcrystals) in 22 patients was performed to evaluate FDG imaging characteristics. The impact of clinically used oxygen sensors (carbon black, charcoal particulates, LiNc-BuO microcrystals) on FDG-PET/CT imaging after implantation in rat muscle (n = 12) was investigated. The retrospective review revealed no other patients with FDG avidity associated with particulate sensors. The preclinical investigation found no injected oxygen sensor whose mean standard uptake values differed significantly from sham injections. The risk of a false-positive FDG-PET/CT scan due to oxygen sensors appears low. However, in the right clinical context the potential exists that an associated inflammatory reaction may confound interpretation.

mNP hyperthermia and hypofractionated radiation activate similar immunogenetic and cytotoxic pathways.

OBJECTIVE: The goal of this study is to better understand the immunogenetic expression and related cytotoxic responses of moderate but clinically relevant doses of hypofractionated radiation (1x15 Gy and 3x8 Gy) and magnetic nanoparticle hyperthermia (mNPH, CEM43 30). METHODS: Genetic, protein, immunopathology and tumor growth delay assessments were used to determine the immune and cytotoxic responses following radiation and mNPH alone and in combination. Although the thermal dose used, 43 C°/30 min (CEM43 30), typically results in modest independent cytotoxicity, it has shown the ability to stimulate an immune response and enhance other cancer treatments. The radiation doses studied (15 Gy and 3x8 Gy) are commonly used in preclinical research and are effective in selected stereotactic and palliative treatment settings, however they are not commonly used as first-line primary tumor treatment regimens. RESULTS: Our RNA-based genetic results suggest that while many of the cytotoxic and immune gene and protein pathways for radiation and hyperthermia are similar, radiation, at the doses used, results in a more consistent and expansive anti-cancer immune/cytotoxic expression profile. These results were supported by immunohistochemistry based cytotoxic T-cell tumor infiltration and tumor growth delay studies. When used together radiation and hyperthermia led to greater immune and cytotoxic activity than either modality alone. CONCLUSION: This study clearly shows that modest, but commonly used hypofractionated radiation and hyperthermia doses share many important immune and cytotoxic pathways and that combining the treatments, as compared to either treatment alone, results in genetic and biological anti-cancer benefits.

AMPK Activation by Metformin Promotes Survival of Dormant ER+ Breast Cancer Cells.

PURPOSE: Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. EXPERIMENTAL DESIGN: Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. RESULTS: Dormant breast cancer cells exhibited upregulated 5' adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. CONCLUSIONS: AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

Approximations of the power functions for Wald, likelihood ratio, and score tests and their applications to linear and logistic regressions.

Traditionally, asymptotic tests are studied and applied under local alternative (Aivazian, et al., 1985). There exists a widespread opinion that the Wald, likelihood ratio, and score tests are asymptotically equivalent. We dispel this myth by showing that These tests have different statistical power in the presence of nuisance parameters. The local properties of the tests are described in terms of the first and second derivative evaluated at the null hypothesis. The comparison of the tests are illustrated with two popular regression models: linear regression with random predictor and logistic regression with binary covariate. We study the aberrant behavior of the tests when the distance between the null and alternative does not vanish with the sample size. We demonstrate that these tests have different asymptotic power. In particular, the score test is generally asymptotically biased but slightly superior for linear regression in a close neighborhood of the null. The power approximations are confirmed through simulations.

A Transcriptionally Definable Subgroup of Triple-Negative Breast and Ovarian Cancer Samples Shows Sensitivity to HSP90 Inhibition.

PURPOSE: We hypothesized that integrated analysis of cancer types from different lineages would reveal novel molecularly defined subgroups with unique therapeutic vulnerabilities. On the basis of the molecular similarities between subgroups of breast and ovarian cancers, we analyzed these cancers as a single cohort to test our hypothesis. EXPERIMENTAL DESIGN: Identification of transcriptional subgroups of cancers and drug sensitivity analyses were performed using mined data. Cell line sensitivity to Hsp90 inhibitors (Hsp90i) was tested in vitro. The ability of a transcriptional signature to predict Hsp90i sensitivity was validated using cell lines, and cell line- and patient-derived xenograft (PDX) models. Mechanisms of Hsp90i sensitivity were uncovered using immunoblot and RNAi. RESULTS: Transcriptomic analyses of breast and ovarian cancer cell lines uncovered two mixed subgroups comprised primarily of triple-negative breast and multiple ovarian cancer subtypes. Drug sensitivity analyses revealed that cells of one mixed subgroup are significantly more sensitive to Hsp90i compared with cells from all other cancer lineages evaluated. A gene expression classifier was generated that predicted Hsp90i sensitivity in vitro, and in cell line- and PDXs. Cells from the Hsp90i-sensitive subgroup underwent apoptosis mediated by Hsp90i-induced upregulation of the proapoptotic proteins Bim and PUMA. CONCLUSIONS: Our findings identify Hsp90i as a potential therapeutic strategy for a transcriptionally defined subgroup of ovarian and breast cancers. This study demonstrates that gene expression profiles may be useful to identify therapeutic vulnerabilities in tumor types with limited targetable genetic alterations, and to identify molecularly definable cancer subgroups that transcend lineage.

Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds.

Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.

Statistical determination of synergy based on Bliss definition of drugs independence.

Although synergy is a pillar of modern pharmacology, toxicology, and medicine, there is no consensus on its definition despite its nearly one hundred-year history. Moreover, methods for statistical determination of synergy that account for variation of response to treatment are underdeveloped and if exist are reduced to the traditional t-test, but do not comply with the normal distribution assumption. We offer statistical models for estimation of synergy using an established definition of Bliss drugs' independence. Although Bliss definition is well-known, it remains a theoretical concept and has never been applied for statistical determination of synergy with various forms of treatment outcome. We rigorously and consistently extend the Bliss definition to detect statistically significant synergy under various designs: (1) in vitro, when the outcome of a cell culture experiment with replicates is the proportion of surviving cells for a single dose or multiple doses, (2) dose-response methodology, (3) in vivo studies in organisms, when the outcome is a longitudinal measurement such as tumor volume, and (4) clinical studies, when the outcome of treatment is measured by survival. For each design, we developed a specific statistical model and demonstrated how to test for independence, synergy, and antagonism, and compute the associated p-value.

Effects of temperature, salinity, and sediment organic carbon on methylmercury bioaccumulation in an estuarine amphipod.

Mercury (Hg) is a global contaminant that poses a human health risk in its organic form, methylmercury (MeHg), through consumption of fish and fishery products. Bioaccumulation of Hg in the aquatic environment is controlled by a number of factors expected to be altered by climate change. We examined the individual and combined effects of temperature, sediment organic carbon, and salinity on the bioaccumulation of MeHg in an estuarine amphipod, Leptocheirus plumulosus, when exposed to sediment from two locations in the Gulf of Maine (Kittery and Bass Harbor) that contained different levels of MeHg and organic carbon. Higher temperatures and lower organic carbon levels individually increased uptake of MeHg by L. plumulosus as measured by the biota-sediment accumulation factor (BSAF), while the effect of salinity on BSAF differed by sediment source. Multi-factor statistical modeling using all data revealed a significant interaction between temperature and organic carbon for both sediments, in which increased temperature had a negative effect on BSAF at the lowest carbon levels and a positive effect at higher levels. Our results suggest that increased temperature and carbon loading, of a magnitude expected as a result from climate change, could be associated with a net decrease in amphipod BSAF of 50 to 71%, depending on sediment characteristics. While these are only first-order projections, our results indicate that the future fate of MeHg in marine food webs is likely to depend on a number of factors beyond Hg loading.