Detection of metabolic change in glioblastoma cells after radiotherapy using hyperpolarized 13 C-MRI.

Dynamic nuclear polarization (DNP) of 13 C-labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP-MRI chemical shift imaging with hyperpolarized [1-13 C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem-like cell-derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP-MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase-A expression in GBMJ1 following radiation exposure, consistent with DNP-MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP-MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients.

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

Improving Radiation Response in Glioblastoma Using ECO/siRNA Nanoparticles Targeting DNA Damage Repair.

Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.

Safety and Efficacy of an Enhanced Recovery Protocol After Hepatic Resection.

Enhanced recovery protocols (ERPs) have shown to improve outcomes in multiple specialties and were recently applied to hepatic resections. The objective of this study was to determine the safety and efficacy of ERP in hepatic resection. Between 2013-2017, 208 patients underwent hepatectomy. The ERP included early ambulation, early oral intake, and multimodal analgesia. Primary study end points were hospital length of stay (LOS) and overall morbidity; secondary end points were return of bowel function (ROBF), 30-day readmission, and 90-day mortality. Major hepatectomies were selected for separate analysis. Overall, pre-ERP (N = 99) and ERP (N = 109) were similar in demographics. ERP patients had earlier oral intake and ROBF with similar overall morbidity. Although median LOS was 5 days, 43% of ERP patients had LOS ≤4 days vs. 27% in the pre-ERP cohort (P = .02). 30-day readmission was similar (12%), and 90-day mortality was 2.8% vs. 3.0% (pre-ERP vs. ERP, P = .90). In major hepatectomies, pre-ERP (N = 41) and ERP (N = 33) demographics and operative characteristics were similar. ERP patients had earlier oral intake and ROBF with similar morbidity and mortality. There was no significant difference in median LOS; however, 36% of the major hepatectomy ERP patients had LOS ≤4 days vs. 17% of pre-ERP patients, P = .06. In conclusion, ERP can be safely implemented in hepatectomy, with earlier oral intake and ROBF, shorter LOS in some patients, and no increase in morbidity or mortality.

Reducing Interruptive Alert Burden Using Quality Improvement Methodology.

BACKGROUND: Increased adoption of electronic health records (EHR) with integrated clinical decision support (CDS) systems has reduced some sources of error but has led to unintended consequences including alert fatigue. The "pop-up" or interruptive alert is often employed as it requires providers to acknowledge receipt of an alert by taking an action despite the potential negative effects of workflow interruption. We noted a persistent upward trend of interruptive alerts at our institution and increasing requests for new interruptive alerts. OBJECTIVES: Using Institute for Healthcare Improvement (IHI) quality improvement (QI) methodology, the primary objective was to reduce the total volume of interruptive alerts received by providers. METHODS: We created an interactive dashboard for baseline alert data and to monitor frequency and outcomes of alerts as well as to prioritize interventions. A key driver diagram was developed with a specific aim to decrease the number of interruptive alerts from a baseline of 7,250 to 4,700 per week (35%) over 6 months. Interventions focused on the following key drivers: appropriate alert display within workflow, clear alert content, alert governance and standardization, user feedback regarding overrides, and respect for user knowledge. RESULTS: A total of 25 unique alerts accounted for 90% of the total interruptive alert volume. By focusing on these 25 alerts, we reduced interruptive alerts from 7,250 to 4,400 per week. CONCLUSION: Systematic and structured improvements to interruptive alerts can lead to overall reduced interruptive alert burden. Using QI methods to prioritize our interventions allowed us to maximize our impact. Further evaluation should be done on the effects of reduced interruptive alerts on patient care outcomes, usability heuristics on cognitive burden, and direct feedback mechanisms on alert utility.

Reliability of blood lactate as a measure of exercise intensity in different strains of mice during forced treadmill running.

Exercise has long been known to be beneficial to human health. Studies aimed at understanding the effects of exercise specifically focus on predetermined exercise intensities defined by measuring the aerobic capacity of each individual. Many disease models involving animal training often establish aerobic capacity by using the maximal lactate steady state (MLSS), a widely used method in humans that has frequently been used in rodent studies. The MLSS is defined as the highest exercise intensity at which blood lactate concentration remains constant and is roughly equivalent to 70-80% of maximal aerobic capacity. Due to our up-coming experiments investigating the effect of different exercise intensities in specific strains of tumor-bearing mice, the aim of the present study was to determine the MLSS in athymic nude (NCr nu/nu and NMRI), CDF1, and C3H mice by treadmill running at increasing speeds. However, despite thorough exercise acclimation and the use of different exercise protocols and aversive stimuli, less than half of the experiments across strains pointed towards an established MLSS. Moreover, gently prodding the mice during low to moderate intensity running caused a 30-121% (p<0.05) increase in blood lactate concentration compared to running without stimulation, further questioning the use of lactate as a measure of exercise intensity. Overall, MLSS is difficult to determine and large variations of blood lactate levels were observed depending on the exercise protocol, mice handling strategy and strain. This should be considered when planning experiments in mice using forced exercise protocols.

The Quiescent Metabolic Phenotype of Glioma Stem Cells.

INTRODUCTION: Glioblastoma (GBM) is the most common primary malignant brain tumor in humans and, even with aggressive treatment that includes surgical resection, radiation (IR), and chemotherapy administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem-like cells (GSLCs) exist, which are thought to be therapy resistant and are thus capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg Effect. There is no consensus on the metabolic characteristics of cancer stem cells. GSLCs have been shown to rely more heavily on oxidative phosphorylation, but there is also evidence that cancer stem cells can adapt their metabolism by fluctuating between energy pathways or acquiring intermediate metabolic phenotypes. We hypothesized that the metabolism of GSLCs differs from that of differentiated GBM tumor cell lines, and that the steady state metabolism would be differentially altered following radiation treatment. MATERIALS AND METHODS: We evaluated the oxygen consumption rate, extracellular acidification rate, and metabolic enzyme levels of GBM cell lines and GSLCs before and after irradiation using extracellular flux assays. We also measured absolute metabolite levels in these cells via mass spectroscopy with and without radiation treatment. RESULTS: GSLCs were found to be significantly more quiescent in comparison to adherent GBM cell lines, highlighted by lower glycolytic and maximal respiratory capacities as well as lower oxygen consumption and extracellular acidification rates. Analysis of individual metabolite concentrations revealed lower total metabolite concentrations overall but also elevated levels of metabolites in different energy pathways for GSLCs compared to GBM cell lines. Additionally, the metabolism of both GSLCs and GBM cell lines were found to be altered by IR. CONCLUSIONS: While there is not one metabolic alteration that distinguishes irradiated GSLC metabolism from that of GBM cell lines, therapies targeting more metabolically quiescent tumor cells and thus the resistant GSLC population may increase a cancer's sensitivity to radiotherapy.

Assessing the gene-disease association of 19 genes with the RASopathies using the ClinGen gene curation framework.

The RASopathies are a complex group of conditions regarding phenotype and genetic etiology. The ClinGen RASopathy Expert Panel (RAS EP) assessed published and other publicly available evidence supporting the association of 19 genes with RASopathy conditions. Using the semiquantitative literature curation method developed by the ClinGen Gene Curation Working Group, evidence for each gene was curated and scored for Noonan syndrome (NS), Costello syndrome, cardiofaciocutaneous syndrome, NS with multiple lentigines, and Noonan-like syndrome with loose anagen hair. The curated evidence supporting each gene-disease relationship was then discussed and approved by the ClinGen RASopathy Expert Panel. Each association's strength was classified as definitive, strong, moderate, limited, disputed, or no evidence. Eleven genes were classified as definitively associated with at least one RASopathy condition. Two genes classified as strong for association with at least one RASopathy condition while one gene was moderate and three were limited. The RAS EP also disputed the association of two genes for all RASopathy conditions. Overall, our results provide a greater understanding of the different gene-disease relationships within the RASopathies and can help in guiding and directing clinicians, patients, and researchers who are identifying variants in individuals with a suspected RASopathy.

Transgenerational Inheritance of Familial Lipomyelomeningocele.

Lipomyelomeningocele is a type of neural tube defect characterized by lipomatous tissue causing a defect in the vertebrae, infiltrating the dura, and tethering the spinal cord. Despite significant neurologic consequences, the underlying etiology remains poorly understood. We present a father and son with remarkably similar presentations of lipomyelomeningocele. Genetic testing did not reveal an underlying cause but whole exome sequencing identified variants in the ARHGAP29 and RADIL genes in the proband and his affected father. Genetic analyses of asymptomatic family members revealed several carriers of the ARHGAP29 or RADIL variants, but only the proband and his father carried both variants, suggesting a possible shared genetic mechanism. Rare cases of siblings affected with lipomyelomeningocele have suggested the possibility of autosomal recessive or germline mosaicism. We present the first documented cases of transgenerational lipomyelomeningocele with important implications for family counseling about the recurrence of lipomyelomeningocele.

Phacomatosis Pigmentokeratotica: A Mosaic RASopathy with Malignant Potential.

Phacomatosis pigmentokeratotica (PPK) is a rare epidermal nevus syndrome characterized by the co-occurrence of a nevus sebaceous arranged along the lines of Blaschko with a speckled lentiginous nevus (SLN). We report a novel KRAS mutation in a patient with a large nevus sebaceous and an SLN who subsequently developed a vaginal botryoid rhabdomyosarcoma, an association not previously reported in the literature. This case expands our knowledge of the genetic basis for phacomatosis, in which mutations in HRAS have been previously described, although this report provides evidence that activating mutations in KRAS or HRAS may cause PPK. This report confirms that PPK is a mosaic RASopathy with malignant potential and raises the question of whether screening for other RAS-associated malignancies should be performed for all children with PPK.

'In-Format' screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules.

Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an 'in-format' screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC50) when assessed in-format were identified. In contrast, the corresponding dAb monomers had ∼1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening naïve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.

Steric Clash in the SET Domain of Histone Methyltransferase NSD1 as a Cause of Sotos Syndrome and Its Genetic Heterogeneity in a Brazilian Cohort.

Most histone methyltransferases (HMTase) harbor a predicted Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain, which transfers a methyl group to a lysine residue in their substrates. Mutations of the SET domains were reported to cause intellectual disability syndromes such as Sotos, Weaver, or Kabuki syndromes. Sotos syndrome is an overgrowth syndrome with intellectual disability caused by haploinsufficiency of the nuclear receptor binding SET domain protein 1 (NSD1) gene, an HMTase at 5q35.2-35.3. Here, we analyzed NSD1 in 34 Brazilian Sotos patients and identified three novel and eight known mutations. Using protein modeling and bioinformatic approaches, we evaluated the effects of one novel (I2007F) and 21 previously reported missense mutations in the SET domain. For the I2007F mutation, we observed conformational change and loss of structural stability in Molecular Dynamics (MD) simulations which may lead to loss-of-function of the SET domain. For six mutations near the ligand-binding site we observed in simulations steric clashes with neighboring side chains near the substrate S-Adenosyl methionine (SAM) binding site, which may disrupt the enzymatic activity of NSD1. These results point to a structural mechanism underlying the pathology of the NSD1 missense mutations in the SET domain in Sotos syndrome. NSD1 mutations were identified in only 32% of the Brazilian Sotos patients in our study cohort suggesting other genes (including unknown disease genes) underlie the molecular etiology for the majority of these patients. Our studies also found NSD1 expression to be profound in human fetal brain and cerebellum, accounting for prenatal onset and hypoplasia of cerebellar vermis seen in Sotos syndrome.

In vivo biocompatibility of custom-fabricated apatite-wollastonite-mesenchymal stromal cell constructs.

We have used the additive manufacturing technology of selective laser sintering (SLS), together with post SLS heat treatment, to produce porous three dimensional scaffolds from the glass-ceramic apatite-wollastonite (A-W). The A-W scaffolds were custom-designed to incorporate a cylindrical central channel to increase cell penetration and medium flow to the center of the scaffolds under dynamic culture conditions during in vitro testing and subsequent in vivo implantation. The scaffolds were seeded with human bone marrow mesenchymal stromal cells (MSCs) and cultured in spinner flasks. Using confocal and scanning electron microscopy, we demonstrated that MSCs formed and maintained a confluent layer of viable cells on all surfaces of the A-W scaffolds during dynamic culture. MSC-seeded, with and without osteogenic pre-differentiation, and unseeded A-W scaffolds were implanted subcutaneously in MF1 nude mice where osteoid formation and tissue in-growth were observed following histological assessment. The results demonstrate that the in vivo biocompatibility and osteo-supportive capacity of A-W scaffolds can be enhanced by SLS-custom design, without the requirement for osteogenic pre-induction, to advance their potential as patient-specific bone replacement materials.

In vivo microscopy of microvessel oxygenation and network connections.

Abnormal or compromised microvascular function is a key component of various diseases. In vivo microscopy of microvessel function in preclinical models can be useful for the study of a disease state and effects of new treatments. Wide-field imaging of microvascular oxygenation via hemoglobin (Hb) saturation measurements has been applied in various applications alone and in combination with other measures of microvessel function, such as blood flow. However, most current combined imaging methods of microvessel function do not provide direct information on microvessel network connectivity or changes in connections and blood flow pathways. First-pass fluorescence (FPF) imaging of a systemically administered fluorescent contrast agent can be used to directly image blood flow pathways and connections relative to a local supplying arteriole in a quantitative manner through measurement of blood supply time (BST). Here, we demonstrate the utility of information produced by the combination of Hb saturation measurements via spectral imaging with BST measurements via FPF imaging for correlation of microvessel oxygenation with blood flow pathways and connections throughout a local network. Specifically, we show network pathway effects on oxygen transport in normal microvessels, dynamic changes associated with wound healing, and pathological effects of abnormal angiogenesis in tumor growth and development.

Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis.

BACKGROUND: Angiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model. METHODS: Human renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery. RESULTS: The window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis. CONCLUSIONS: Results show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed.

In vivo spectral and fluorescence microscopy comparison of microvascular function after treatment with OXi4503, Sunitinib and their combination in Caki-2 tumors.

Vascular targeting agents on their own have been shown to be insufficient for complete treatment of solid tumors, emphasizing the importance of studying the vascular effects of these drugs for their use with conventional therapies in the clinic. First-pass fluorescence imaging combined with hyperspectral imaging of hemoglobin saturation of microvessels in the murine dorsal window chamber model provides an easily implementable, low cost method to analyze tumor vascular response to these agents in real-time. In this study, the authors utilized these methods to spectroscopically demonstrate distinct vessel structure, blood flow and oxygenation changes in human Caki-2 renal cell carcinoma following treatment with OXi4503 alone, Sunitinib alone and both drugs together. We showed that treatment with OXi4503 plus Sunitinib destroyed existing tumor microvessels, inhibited blood vessel recovery and impaired Caki-2 tumor growth significantly more than either treatment alone.

Combination of spectral and fluorescence imaging microscopy for wide-field in vivo analysis of microvessel blood supply and oxygenation.

Hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence imaging of blood transit time were combined to analyze the oxygenation of and blood flow through microvessel networks. The combination imaging technique was demonstrated in a mouse dorsal window chamber model of a growing Caki-2 human renal cell carcinoma over time. Data from Hb saturation and blood supply time maps show the formation of arteriovenous malformations and shunting of blood directly from arteries to the tumor core and into veins in the periphery of the tumor. Images and data analysis show these malformations result in an oxygenated environment ideal for a tumor to proliferate.

Three-dimensional in vitro culture techniques for mesenchymal stem cells.

In recent years there has been a growing interest in culturing adherent cells using three-dimensional (3D) techniques, rather than more conventional 2D culture methods. This interest emerges from the realization that growing cells on plastic surfaces cannot truly re-create 3D in vivo conditions and therefore might be limiting the cells' potential. In addition, adult stem cells exist in specialized microenvironments, or niches, where the spatial organization of different niche elements (such as different cell types, extracellular matrix) contributes significantly to stem cell maintenance, which cannot be represented using 2D in vitro models. We have generated a range of different 3D approaches for the analysis of mesenchymal stem cells (MSCs) using both mono- and co-culture environments.

Comparative evaluation of differential laser-induced perturbation spectroscopy as a technique to discriminate emerging skin pathology.

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology. Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

Publication outcomes of phase II oncology clinical trials.

OBJECTIVES: Translation of evidence-based medicine into oncology practice depends on timely and full publication of clinical trials. We investigated publication outcomes of Phase II trial abstracts from the annual meetings of the American Society of Clinical Oncology (ASCO). METHODS: We searched the 1997, 1999, and 2001 ASCO annual meeting proceedings and identified all Phase II trials, excluding those that reported preliminary results. Literature search was performed using PubMed, EMBASE, and Google for corresponding publications in peer-reviewed journals. We attempted to contact authors of all unpublished trials. RESULTS: Only 60.8% of t he 559 trials identified were published, with a median time to publication of 41 months. At 5 years, 65.9%, 62.7%, and 57.0% of studies from 1997, 1999, and 2001, respectively, were published. Studies with larger samples were associated with a shorter time to publication, as were oral and poster presentations versus print only (P < 0.001). Common reasons for not publishing were uninteresting results, lack of time, and relocation of authors. Among abstracts reporting response rates, 37.7% showed different results in subsequent publications. Though not statistically significant, over the 5-year period, abstracts presented at later years had a lower rate of publication, longer time to publication, and a higher likelihood of showing a better tumor response. CONCLUSIONS: Almost half of Phase II trials presented at ASCO annual meetings within the last 10 years remain unpublished. Over one-third of published trials reported results different from those presented in abstracts. Like Phase I and III trials, Phase II trials often are unpublished.