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In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), inhibition of the IL1β inflammatory pathway by canakinumab has been shown to significantly reduce lung cancer incidence and mortality. Here we performed molecular characterization of CANTOS patients who developed lung cancer during the study, including circulating tumor DNA (ctDNA) and soluble inflammatory biomarker analysis. Catalogue of Somatic Mutations in Cancer (COSMIC) database ctDNA mutations were detected in 65% (46/71) of the CANTOS patients with lung cancer, with 51% (36/71) having detectable ctDNA at the time point closest to lung cancer diagnosis and 43% (29/67) having detectable ctDNA at trial randomization. Mutations commonly found in lung cancer were observed with no evidence of enrichment in any mutation following canakinumab treatment. Median time to lung cancer diagnosis in patients with (n = 29) versus without (n = 38) detectable COSMIC ctDNA mutations at baseline was 407 days versus 837 days (P = 0.011). For serum inflammatory biomarker analysis, circulating levels of C-reactive protein (CRP), IL6, IL18, IL1 receptor antagonist, TNFα, leptin, adiponectin, fibrinogen, and plasminogen activator inhibitor-1 were determined. Patients with the highest level of baseline CRP or IL6, both downstream of IL1β signaling, trended toward a shorter time to lung cancer diagnosis. Other inflammation markers outside of the IL1β pathway at baseline did not trend with time to lung cancer diagnosis. These results provide further evidence for the importance of IL1β-mediated protumor inflammation in lung cancer and suggest canakinumab's effect may be mediated in part by delaying disease progression of diverse molecular subtypes of lung cancer. Significance: These findings suggest that targeting the IL1β inflammatory pathway might be critical in reducing tumor-promoting inflammation and lung cancer incidence.

Background For the advancement of cancer research, the collection of tissue specimens from drug‐resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post‐therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment‐resistant lung cancers. Methods Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non‐tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin‐fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD‐L1) clones. Next‐generation sequencing was performed on 13 specimens from 5 patients. Results Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD‐L1 IHC revealed heterogeneity within and between tumors. An AGK‐BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule‐associated protein‐like 4 to anaplastic lymphoma kinase (EML4‐ALK) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells. Conclusions Post‐therapy specimens demonstrated PD‐L1 heterogeneity and an acyl glycerol kinase to B‐rapidly accelerated fibrosarcoma (AGK‐BRAF) fusion in a patient with an EML4‐ALK–positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance., We collected high‐quality primary and metastatic lung cancer specimens from donors after death by rapid collection in the community. Protein analysis of this tissue revealed discordant programmed death ligand 1 (PD‐L1) results at different cancer sites from the same patient; genetic analysis identified anaplastic lymphoma kinase (ALK) and B‐rapidly accelerated fibrosarcoma (BRAF) gene fusions in a patient with a history of ALK inhibitor therapy and a clonal genetic origin of metastatic cancer cells.

IntroductionEconomic groups, such as microfinance or self-help groups are widely implemented in low-income and middle-income countries (LMICs). Women’s groups are voluntary groups, which aim to improve the well-being of members through activities, such as joint savings, credit, livelihoods development and/or health activities. Health interventions are increasingly added on to existing women’s economic groups as a public health intervention for women and their families. Here, we present the protocol for a mixed-methods systematic review we will conduct of the evidence on integrated economic and health interventions on women’s groups to assess whether and how they improve health-related knowledge, behaviour and outcomes in LMICs.Methods and analysisWe will search seven electronic databases for published literature, along with manual searches and consultation. The review will include (1) randomised trials and non-randomised quasiexperimental studies of intervention effects of integrated economic and health interventions delivered through women’s groups in LMICs, and (2) sibling studies that examine factors related to intervention content, context, implementation processes and costs. We will appraise risk of bias and study quality using standard tools. High and moderate quality studies will be grouped by health domain and synthesised without meta-analysis. Qualitative evidence will be thematically synthesised and integrated into the quantitative synthesis using a matrix approach.Ethics and disseminationThis protocol was reviewed and deemed exempt by the institutional review board at the American Institutes for Research. Findings will be shared through peer-reviewed publication and disseminated with programme implementers and policymakers engaged with women’s groups.PROSPERO registration numberCRD42020199998.

Background Traumatic hemothorax is a common injury that invites diagnostic and management strategy debates. Evidence-based management has been associated with improved care efficiency. However, the literature abounds with long-debated, re-emerging, and new questions. We aimed to consolidate up-to-date evidence on traumatic hemothoraces, focusing on clinical conundra debated in literature. Methods We conducted a scoping review of 21 clinical conundra in traumatic hemothorax diagnosis and management according to PRISMA-ScR guidelines. Experimental and observational studies evaluating patients (aged ≥18 years) with traumatic hemothoraces were identified through database searches (PubMed, EMBASE, Web of Science, Cochrane Library; database inception to Sep, 26 2020) and bibliography reviews of selected articles. Three reviewers screened and selected articles using standardized forms. Results We screened 1,440 articles for eligibility, of which 71 met criteria for synthesis. The review comprises 6 sections: (1) Presumptive antibiotics before tube thoracostomy; (2) Initial diagnostic and intervention decisions; (3) Chest tubes; (4) Retained hemothoraces; (5) Delayed hemothoraces; and (6) Chest tube removal). The 21 conundra across these sections follow the format of a question, our recommendation based on interpretation of available evidence, and succinct rationale. Rationale sections detail knowledge gaps and opportunities for future research. Conclusion Even practices engrained into surgical dogma, such as obtaining chest x-rays after inserting or removing chest tubes and mandating operation for patients who develop chest tube output above a certain threshold, deserve re-evaluation. Some knowledge gaps require rigorous future investigation; sound clinical judgment can likely supplement others.

Context The outcomes of specialty palliative care (PC) interventions for patients with hematologic malignancies (HMs) is under-investigated. Objectives We performed a systematic review to evaluate the effect of PC interventions on patient- and caregiver- reported outcomes and healthcare utilization among adults with HMs (leukemia, myeloma, and lymphoma). Methods From database inception through September 10, 2020, we systematically searched PubMed, CINAHL, Embase, Scopus, Web of Science, and Cochrane Reviews using terms representing HMs and PC. Eligible studies investigated adults aged 18 years and older, were published in the English language, and contained original, quantitative, or qualitative data related to patient- and/or caregiver-centered outcomes and healthcare utilization. Results We screened 5345 studies;16 met inclusion criteria and found that specialty PC led to improved symptom management, decreased likelihood of inpatient death, decreased healthcare utilization, decreased cost of healthcare, and improved caregiver-reported outcomes. Patients with HM have a high need for PC which, though increasing over time, is often provided late in the clinical disease course. Conclusions Specialty PC interventions improve healthcare outcomes for patients with HMs and should be implemented early and often. There remains a need for additional studies investigating PC use exclusively in patients with HMs.

Background: Reported 10-year survival rates in Caucasian, black, and Hispanic women with breast cancer are 80%, 66%, and 78%, respectively. One barrier to understanding disparities in these survival rates is the lack of data due to underrepresentation in clinical trials (including black, Hispanic, Native American, and Pacific Islander populations). US physicians at sites that treat minority patients with breast cancer identified lack of access to and financial burden of clinical trials as 2 key barriers to enrollment. Insufficient knowledge of genetic mutations specific to breast cancer is also a key barrier to identifying ideal treatment to extend survival in minorities. Patient-specific initiatives for the CompLEEment-1 study (CLEE011A2404; NCT02941926), a single-arm, international Phase 3b study of ribociclib (600 mg/d, 3 weeks on/1 week off) plus letrozole (2.5 mg/d) in adults with hormone receptor–positive, human epidermal growth factor–negative advanced breast cancer, were developed in the United States to increase the enrollment of diverse populations and better understand the disease and patient response to treatment. Methods: The enrollment initiatives included formation of a board of leading physicians and academics to advise on awareness initiatives and the cultural competency of tools used in clinical trials, a site selection process that brought the trial to the patient's clinic, a simplified expense reimbursement program to reduce patient economic burden, and partnerships with the Bridge Group and the National Black Church Initiative to bring clinical trial awareness and education to the African American church community. A tumor collection companion study (NCT03050398) was created to identify mechanisms of resistance among racial and ethnic groups. These initiatives aimed to increase the percentage of minorities from previous trials (eg, 5.7% in the MONALEESA-2 trial) and identify potential biological differences associated with race or ethnicity that might affect treatment response. Results: Enrollment in the CompLEEment-1 study is ongoing. Among the first 57 US patients enrolled, 8 (14.0%) were minorities (not identified as Caucasian or Asian), 32 (56.1%) were 30 miles outside of metropolitan areas. Of the open study sites with enrolled patients, 16 enrolled 1, 8 enrolled 2, and 5 enrolled ≥3 patients. Conclusions: Following discussions with physicians and assessments of patient feedback to identify reasons for underrepresentation of minority patient populations in clinical trials, patient-centered initiatives were developed for the CompLEEment-1 study in the United States to simultaneously reduce barriers to trial participation, reduce economic burden of enrolled patients, and identify biomarkers of therapeutic sensitivity and resistance. These initiatives resulted in an initial increase in the percentage of minorities enrolled to better reflect real-world populations. Citation Format: Small T, Marsano-Feeley J, Fernandez A, Grasso T, Feldman K, Petrone S, Bugazia N, Wong C, Meyer J, Schutta K, Purkayastha D, Saintil K, Spatz Caplan E, Waltman-Johnson K. Patient-centered initiatives for improving trial participation of diverse patient populations in the open-label phase 3b compLEEment-1 study of ribociclib plus letrozole in the treatment of HR+/HER2- advanced breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-10-07.

Introduction: TPI is a hallmark of cancer and plays a pivotal role in tumor initiation, development, progression, and invasiveness. High levels of tumor-promoting inflammatory cytokines e.g. IL-1β and IL-6 are associated with advanced malignancies and reduced survival. Selective IL-1β inhibitors like canakinumab (CAN) and gevokizumab (GEV) aim to target TPI within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In phase III CANTOS study, CAN significantly reduced lung cancer (LC) incidences and LC related mortalities in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in LC. We have utilized preclinical models to gain further insights into the mechanistic role of CAN and GEV in cancer development and progression. Methods: Preclinical efficacy of anti-human IL-1β antibodies, CAN and GEV was assessed using humanized mouse models of non-small cell lung cancer (NSCLC, H358), triple-negative breast cancer (TNBC, MDA-MB-231) and colorectal cancer (CRC, SW480) either as single agents or in combination with other immunomodulatory agents including PD-1 inhibitor pembrolizumab (PEM) and anti-VEGF therapy. A murine surrogate IL-1β blocking antibody clone 01BSUR was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as single agent or with PD-1 inhibitor and docetaxel (DOC). Tumor infiltrating lymphocytes (TIL) characterization was carried out by flow cytometry and IHC in syngeneic and humanized models, respectively. Results: The IL-1β inhibitors showed anti-tumor activity in both murine syngeneic and humanized models as single agents or with other agents. In the H358 NSCLC mouse model, CAN + PEM significantly inhibited tumor growth and was associated with >50% regression vs control. Decreased tumor growth was also observed with GEV treatment in the SW480 CRC mouse model, both alone and with anti-VEGF therapy. In GEV+anti-VEGF arm, a decrease in tolerogenic DC-10 immune populations and an increase in CD45+ immune cells and CD68+ myeloid cells were also observed. In the MDA-MB-231 TNBC mouse model, CAN single-agent showed a decrease in tumor growth vs control. In murine syngeneic 4T1 and LL-2 models, anti-IL-1β alone and anti-IL-1β + anti-PD-1 resulted in increased presence of CD8+ T cells and reduction of immunosuppressive myeloid-derived suppressor cells, neutrophils, and regulatory T cells in the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes were observed with combination of anti-IL-1β with DOC and anti-VEGF in murine syngeneic models. Conclusions: These data provide key insights into the biology behind IL-1β blockade currently being pursued in clinical trials with CAN and GEV. Additional preclinical studies to investigate mechanism of action of these agents and other novel combinations are planned. Citation Format: Pushpa Jayaraman, Vanessa Rodrik-Outmezguine, John Millholland, Neil O'Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff. Targeting tumor-promoting inflammation (TPI) via the IL-1βpathway for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5640.

Introduction: Inflammation is a hallmark of cancer wherein diverse immune cells exert either pro- or antitumor properties and affect therapeutic resistance (Ritter B et al, JEM 2019). High concentrations of tumor-promoting inflammatory cytokines such as IL-1β (Litmanovich A et al, Oncol Ther 2018) and IL-6 (Altundag O et al, J Clin Oncol 2005) correlate with advanced malignancies and are associated with reduced survival. A recent phase III clinical trial (CANTOS) demonstrated that canakinumab, a selective IL-1β inhibitor, could significantly reduce lung cancer incidences and lung cancer related mortalities as a secondary endpoint in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in lung cancer. Canakinumab and other selective IL-1β inhibitors such as gevokizumab aim to target tumor promoting inflammation (TPI) within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In an effort to gain further insights into the clinical mechanistic role of canakinumab and gevokizumab in cancer development and progression, we have utilized preclinical models to understand the immunological events following IL-1β blockade. Methods: Preclinical efficacy of anti-human IL-1β antibodies, canakinumab and gevokizumab, was assessed in humanized mouse models of NSCLC (H358) and CRC (SW480) respectively. Anti-mouse IL-1β blocking antibody was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as a single agent or in combination with PD-1 inhibitor and docetaxel. Tumor infiltrating lymphocytes (TIL) characterization was carried out either by flow cytometry or by IHC in syngeneic and humanized models. Results: Using both murine syngeneic and humanized mouse models, we demonstrate that IL-1β inhibition remodels the TME and slows tumor growth either alone or in combination with other agents. In the humanized H358 NSCLC mouse model, the combination of canakinumab and the anti-PD-1 monoclonal antibody, pembrolizumab, significantly slowed tumor growth by more than 50% vs control antibodies. Furthermore, canakinumab alone and in combination resulted in increased CD8 and CD3 positive TIL infiltration within the tumor. Decreased tumor growth was also observed with another IL-1β inhibitor, gevokizumab, in a humanized SW480 CRC mouse model, both alone and in combination with anti-VEGF therapy. In syngeneic 4T1 and LL-2 models, anti-IL-1β alone and in combination with anti-PD-1 resulted in increased infiltration of CD8+ cells along with the reduction of immunosuppressive myeloid derived suppressor cells (MDSCs), neutrophils and regulatory T cells (Tregs) into the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes to the tumor microenvironment were observed with the combination of anti-IL-1β with docetaxel. Additional combinations are being explored in other tumor models reflecting different indications. Conclusions: Our results highlight the role for IL-1β in tumor immunomodulation and that the pathophysiological role of the IL-1β pathway in innate immunity might have important consequences on T cell function and checkpoint blockade in cancer. These results support the ongoing clinical evaluation of IL-1β inhibitors such as canakinumab and gevokizumab and in combination with other therapeutic agents across several cancers. Citation Format: Pushpa Jayaraman, John Millholland, Neil O’Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Vanessa Rodrik-Outmezguine, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff . Targeting IL-1β pathway for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C103. doi:10.1158/1535-7163.TARG-19-C103

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR 1 -MR 4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR 4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR 4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5-6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.

Mammalian preimplantation embryo development is a complex process in which the exact timing and sequence of events are as essential as the accurate execution of the events themselves. Time-lapse microscopy (TLM) is an ideal tool to study this process since the ability to capture images over time provides a combination of morphological, dynamic and quantitative information about developmental events. Here, we systematically review the application of TLM in basic and clinical embryo research. We identified all relevant preimplantation embryo TLM studies published in English up to May 2012 using PubMed and Google Scholar. We then analysed the technical challenges involved in embryo TLM studies and how these challenges may be overcome with technological innovations. Finally, we reviewed the different types of TLM embryo studies, with a special focus on how TLM can benefit clinical assisted reproduction. Although new parameters predictive of embryo development potential may be discovered and used clinically to potentially increase the success rate of IVF, adopting TLM to routine clinical practice will require innovations in both optics and image analysis. Combined with such innovations, TLM may provide embryologists and clinicians with an important tool for making critical decisions in assisted reproduction. In this review, we perform a literature search of all published early embryo development studies that used time-lapse microscopy (TLM). From the literature, we discuss the benefits of TLM over traditional time-point analysis, as well as the technical difficulties and solutions involved in implementing TLM for embryo studies. We further discuss research that has successfully derived non-invasive markers that may increase the success rate of assisted reproductive technologies, primarily IVF. Most notably, we extend our discussion to highlight important considerations for the practical use of TLM in research and clinical settings.

Background: The evolution of BCR-ABL1 tyrosine kinase inhibitors (TKIs), from imatinib to newer agents such as nilotinib, has led to progressively lower levels of disease burden in patients with CML receiving TKI therapy. Currently, real-time quantitative PCR (RQ-PCR) is the most commonly used test for assessing MR in patients with CML and is able to detect BCR-ABL1 transcript levels down to MR4.0 to MR4.5 on the IS. It has been reported that dPCR can detect BCR-ABL1 with greater sensitivity and precision than RQ-PCR. In addition, unlike RQ-PCR, dPCR is able to perform absolute copy number quantification without the use of calibrators; thus, it should be free from calibrator biases. To date, validation of dPCR BCR-ABL1 assays with multiple control genes and standardization to the World Health Organization (WHO) IS reference material have not been reported. Standardization to the IS is critically important to enable appropriate assessment of patient response to therapy and to establish a standard of care. In this study, we describe the development, validation, and IS standardization of a sensitive BCR-ABL1 dPCR assay using 3 different control genes. We also tested the comparability of 3 different dPCR platforms. Methods: Four experiments were included in this study: (1) Three BCR-ABL1 dPCR assays were developed using ABL1, BCR, and GUS as control genes on the Bio-Rad dPCR platform. The ABL1 assay was standardized to the IS using the WHO Reference Panel. The BCR and GUS IS alignments are currently in process. All 3 assays were validated for linearity, precision, accuracy, limit of detection, and limit of blank, following in vitro diagnostics industry best practices and Clinical and Laboratory Standards Institute (CLSI) guidelines. (2) Multiple BCR-ABL1 and ABL1 primer/probe sets were tested on both RQ-PCR and dPCR for the selection of the best primer/probe design. (3) BCR-ABL1 and ABL1 quantifications were compared between 3 dPCR platforms: Bio-Rad QX200, RainDance RainDrop System, and Applied Biosystems QuantStudio3D. (4) Sensitivity of the BCR-ABL1 dPCR assay will be compared in CML PAXgene vs K2EDTA blood tube samples to assess the potential impact of blood-collection tube type on deep MR measurement in patients with CML. Results: (1) The BCR-ABL1/ABL1 dPCR assay WHO IS alignment resulted in a conversion factor of 1. Using the WHO IS-calibrated BCR-ABL1/ABL dPCR assay, MR in blood samples from patients with CML were quantified with sensitivity beyond MR4.5 to levels between MR5.0 and MR5.5 (Figure 1). Combining multiple dPCR wells, which enables analysis of thousands to millions of reactions together, improved the sensitivity and precision of the assay (Figure 2). (2) Evaluation of 10 BCR-ABL1 and 9 ABL1 primer/probe sets using the Bio-Rad dPCR platform resulted in < 2-fold differences in copy number detection compared with the 16- to 32-fold difference using RQ-PCR. This is likely due to dPCR measurement being an endpoint PCR reaction. (3) Less than 2-fold differences in both detected copy number and BCR-ABL1/ABL1 ratios were observed using 1 selected primer/probe set when a contrived sample panel representing key BCR-ABL1 levels from 10% to 0.0032% IS was tested across 3 different dPCR platforms, suggesting good cross-platform robustness of dPCR (Table). (4) The PAXgene vs K2EDTA blood tube comparison study is ongoing; data will be presented upon completion of the study. Conclusion: Three dPCR BCR-ABL1 assays were developed. The BCR-ABL1 dPCR assay demonstrated detection capability at levels below MR4.5, down to MR5.0 to MR5.5 in contrived samples from patients with CML. This increased sensitivity relative to RQ-PCR may aid future comparisons of deep MR rates across different CML therapies. dPCR assay performance is also more robust against primer/probe design changes than RQ-PCR, thus requiring less assay optimization. Our results demonstrate that dPCR offers robust BCR-ABL1 quantification on the WHO IS across commonly used control genes without requiring calibrators or extensive assay optimization. Disclosures Huang: Novartis: Employment. Salcius:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment, Equity Ownership. Wong:Novartis: Employment. Wang:Novartis: Employment, Equity Ownership.

Background Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo. Methodology/Principal Findings Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage. Conclusions/Significance We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo.

The centrosome duplicates once in S phase. To determine whether there is a block in centrosome reduplication, we used a cell fusion assay to compare the duplication potential of unduplicated G1 centrosomes and recently duplicated G2 centrosomes. By fusing cells in different cell cycle stages, we found that G2 centrosomes were unable to reduplicate in a cellular environment that supports centrosome duplication. Furthermore, G2 cytoplasm did not inhibit centrosome duplication in fused cells, indicating that the block to reduplication is intrinsic to the centrosomes rather than the cytoplasm. To test the underlying mechanism, we created mononucleate G1 cells with two centrosomes by fusing cells with enucleated cytoplasts. Both centrosomes duplicated, indicating that the block is not controlled by centrosome:nucleus ratio. We also found that human primary cells have tight control over centrosome number during prolonged S-phase arrest and that this control is partially abrogated in transformed cells. This suggests a link between the control of centrosome duplication and maintenance of genomic stability.

A novel protein in Caenorhabditis elegans, SAS-4, is a component of centrioles and is required for centriole duplication. Depletion of SAS-4 results in stunted centrioles and a smaller centrosome, suggesting a link to organelle size control.

Assessment of DNA damage is a vital part of oncology research and cancer drug development. In this study, we present a novel method using laser scanning cytometry (LSC) to automate a panel of DNA damage assays that are traditionally scored manually. The comet assay is one of the simplest and most widely-used methods to evaluate DNA damage. In order to fully automate this assay, we successfully developed the first algorithm that can reliably segment and quantify the amount of DNA present in the comet head versus the tail separately, providing a standardized and fully automated measurement of DNA damage. In the micronucleus (MN) assay, measurement of micronuclei formed from chromosome breakage and/or malsegregation provides an assessment of DNA damage and serves as a biomarker of genotoxicity and mutagenicity. Using simultaneous measurements by LSC of cellular morphology, nuclear and MN counts, precise DNA content, and specific molecular biomarker intensity, we developed a protocol to identify and quantify different cell sub-types within human buccal mucosa tissue consisting of basal cells, differentiated cells and bi-nucleated cells, as well as scoring cells which contain MN. The automated high-throughput generation of this data allows a high content analysis “cytome” assay of DNA damage, chromosome instability, cell death and the regenerative potential of human buccal mucosal tissue in response to exogenous and endogenous insults. The multiplexed DNA damage response assay combines DNA content measurement with assessment of ATM and H2AX phosphorylation. The integrated signal of each of the above markers allows correlation of the extent of DNA damage with the cell cycle phase. We employed LSC's automated imaging features to extend the dynamic range of measurable damage induced by various genotoxic agents, allowing identification and enumeration of DNA damage-reporting foci and analysis of expression and degree of overlap of these marker phospho-proteins in individual foci. Induction of cell senescence combined with reproductive cell death is the goal of chemotherapy and radiotherapy of solid tumors. The morphometric analytical capabilities of LSC were used to develop new biomarkers that reveal the degree (depth) of cell senescence based on senescence-characteristic morphological features. Combining these biomarkers with markers of DNA damage response, we assessed several factors that enhanced or attenuated senescence induced by anticancer drugs. In conclusion, we have developed a panel of LSC algorithms that allow previously unavailable automation and standardization of high-content, high-throughput DNA damage assays to be performed on a single platform. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-336. doi:10.1158/1538-7445.AM2011-LB-336

Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest. Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed. We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.