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Bronchopulmonary dysplasia (BPD) is the most common serious morbidity of preterm birth. Short-term respiratory outcomes for infants with the most severe forms of BPD are highly variable. The mechanisms that explain this variability remain unknown and may be mediated by racial disparities.To determine the association of maternal race with death and length of hospital stay in a multicenter cohort of infants with severe BPD.This multicenter cohort study included preterm infants enrolled in the BPD Collaborative registry from January 1, 2015, to July 19, 2021, involving 8 BPD Collaborative centers located in the US. Included patients were born at less than 32 weeks' gestation, had a diagnosis of severe BPD as defined by the 2001 National Institutes of Health Consensus Criteria, and were born to Black or White mothers.Maternal race: Black vs White.Death and length of hospital stay.Among 834 registry infants (median [IQR] gestational age, 25 [24-27] weeks; 492 male infants [59%]) meeting inclusion criteria, the majority were born to White mothers (558 [67%]). Death was observed infrequently in the study cohort (32 [4%]), but Black maternal race was associated with an increased odds of death (adjusted odds ratio, 2.1; 95% CI, 1.2-3.5) after adjusting for center. Black maternal race was also significantly associated with length of hospital stay (adjusted between-group difference, 10 days; 95% CI, 3-17 days).In a multicenter severe BPD cohort, study results suggest that infants born to Black mothers had increased likelihood of death and increased length of hospital stay compared with infants born to White mothers. Prospective studies are needed to define the sociodemographic mechanisms underlying disparate health outcomes for Black infants with severe BPD.

OBJECTIVES To describe outpatient respiratory outcomes and center-level variability among children with severe bronchopulmonary dysplasia (BPD) who require tracheostomy and long-term mechanical ventilation. METHODS Retrospective cohort of subjects with severe BPD, born between 2016 and 2021, who received tracheostomy and were discharged on home ventilator support from 12 tertiary care centers participating in the BPD Collaborative Outpatient Registry. Timing of key respiratory events including time to tracheostomy placement, initial hospital discharge, first outpatient clinic visit, liberation from the ventilator, and decannulation were assessed using Kaplan-Meier analysis. Differences between centers for the timing of events were assessed via log-rank tests. RESULTS There were 155 patients who met inclusion criteria. Median age at the time of the study was 32 months. The median age of tracheostomy placement was 5 months (48 weeks’ postmenstrual age). The median ages of hospital discharge and first respiratory clinic visit were 10 months and 11 months of age, respectively. During the study period, 64% of the subjects were liberated from the ventilator at a median age of 27 months and 32% were decannulated at a median age of 49 months. The median ages for all key events differed significantly by center (P ≤ .001 for all events). CONCLUSIONS There is wide variability in the outpatient respiratory outcomes of ventilator-dependent infants and children with severe BPD. Further studies are needed to identify the factors that contribute to variability in practice among the different BPD outpatient centers, which may include inpatient practices.

INTRODUCTION Bronchopulmonary dysplasia (BPD) is a common respiratory sequelae of preterm birth, for which longitudinal outpatient data are limited. Our objective was to describe a geographically diverse outpatient cohort of former preterm infants followed in BPD-disease specific clinics. METHODS Seven BPD specialty clinics contributed data using standardized instruments to this retrospective cohort study. Inclusion criteria included preterm birth (

The coronavirus disease 2019 (COVID-19) pandemic, resulting from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused severe and widespread illness in adults, including pregnant women, while rarely infecting neonates. An incomplete understanding of disease pathogenesis and viral spread has resulted in evolving guidelines to reduce transmission from infected mothers to neonates. Fortunately, the risk of neonatal infection via perinatal/postnatal transmission is low when recommended precautions are followed. However, the psychosocial implications of these practices and racial/ethnic disparities highlighted by this pandemic must also be addressed when caring for mothers and their newborns. This review provides a comprehensive overview of neonatal-perinatal perspectives of COVID-19, ranging from the basic science of infection and recommendations for care of pregnant women and neonates to important psychosocial, ethical, and racial/ethnic topics emerging as a result of both the pandemic and the response of the healthcare community to the care of infected individuals.

To develop machine learning models predicting extubation failure in low birthweight neonates using large amounts of clinical data.Retrospective cohort study using MIMIC-III, a large single-center, open-source clinical dataset. Logistic regression and boosted-tree (XGBoost) models using demographics, medications, and vital sign and ventilatory data were developed to predict extubation failure, defined as reintubation within 7 days.1348 low birthweight (≤2500 g) neonates who received mechanical ventilation within the first 7 days were included, of which 350 (26%) failed a trial of extubation. The best-performing model was a boosted-tree model incorporating demographics, vital signs, ventilator parameters, and medications (AUROC 0.82). The most important features were birthweight, last FiOMachine learning models identified low birthweight ventilated neonates at risk for extubation failure. These models will need to be validated across multiple centers to determine generalizability of this tool.

Preterm infants and young children with bronchopulmonary dysplasia (BPD) are at increased risk for acute care utilization and chronic respiratory symptoms during early life. Identifying risk factors for respiratory morbidities in the outpatient setting could decrease the burden of care. We hypothesized that public insurance coverage was associated with higher acute care usage and respiratory symptoms in preterm infants and children with BPD after initial neonatal intensive care unit (NICU) discharge.Subjects were recruited from BPD clinics at 10 tertiary care centers in the United States between 2018 and 2021. Demographics and clinical characteristics were obtained through chart review. Surveys for clinical outcomes were administered to caregivers.Of the 470 subjects included in this study, 249 (53.0%) received employer-based insurance coverage and 221 (47.0%) received Medicaid as sole coverage at least once between 0 and 3 years of age. The Medicaid group was twice as likely to have sick visits (adjusted odd ratio [OR]: 2.06; p = 0.009) and emergency department visits (aOR: 2.09; p = 0.028), and three times more likely to be admitted for respiratory reasons (aOR: 3.04; p = 0.001) than those in the employer-based group. Additionally, those in the Medicaid group were more likely to have nighttime respiratory symptoms (aOR: 2.62; p = 0.004).Children with BPD who received Medicaid coverage were more likely to utilize acute care and have nighttime respiratory symptoms during the first 3 years of life. More comprehensive studies are needed to determine whether the use of Medicaid represents a barrier to accessing care, lower socioeconomic status, and/or a proxy for detrimental environmental exposures.

To establish consensus practices among a panel of national experts for the discharge of premature infants with bronchopulmonary dysplasia (BPD) from the hospital to home.We conducted a Delphi study that included US neonatologists and pediatric pulmonologists from the Bronchopulmonary Dysplasia Collaborative to establish consensus practices-defined as recommendations with at least 80% agreement-for infants with BPD being discharged from the hospital. Specifically, we evaluated recommendations for diagnostic tests to be completed around discharge, follow-up respiratory care, and family education.Thirty-one expert participants completed 3 rounds of surveys, with a 99% response rate (92 of 93). Consensus was established that infants with moderate-severe BPD (ie, those who remain on respiratory support at 36 weeks) and those discharged on oxygen should be targeted for in-person pulmonary follow-up within 1 month of hospital discharge. Specialized neonatal follow-up is an alternative for infants with mild BPD. Infants with moderate or severe BPD should have an echocardiogram performed after 36 weeks to screen for pulmonary hypertension. Infants with BPD warrant additional evaluations if they have growth restriction or poor growth, pulmonary hypertension, or tachypnea and if they are discharged to home on oxygen, diuretics, or nonoral feeds.This Delphi survey establishes expert consensus around best practices for follow-up respiratory management and routine evaluation for infants with BPD surrounding neonatal discharge. Areas of disagreement for which consensus was not established are discussed.

To test the hypothesis that daycare attendance among children with bronchopulmonary dysplasia (BPD) is associated with increased chronic respiratory symptoms and/or greater health care use for respiratory illnesses during the first 3 years of life.Daycare attendance and clinical outcomes were obtained via standardized instruments for 341 subjects recruited from 9 BPD specialty clinics in the US. All subjects were former infants born preterm (34 weeks) with BPD (71% severe) requiring outpatient follow-up between 0 and 3 years of age. Mixed logistic regression models were used to test for associations.Children with BPD attending daycare were more likely to have emergency department visits and systemic steroid usage. Children in daycare up to 3 years of age also were more likely to report trouble breathing, having activity limitations, and using rescue medications when compared with children not in daycare. More severe manifestations were found in children attending daycare between 6 and 12 months of chronological age.In this study, children born preterm with BPD who attend daycare were more likely to visit the emergency department, use systemic steroids, and have chronic respiratory symptoms compared with children not in daycare, indicating that daycare may be a potential modifiable risk factor to minimize respiratory morbidities in children with BPD during the preschool years.

Objective: To compare medications dispensed during the first two years in children born preterm and full-term. Study Design: Retrospective analysis of claims data from a commercial national managed care plan 2008–2019. 329 855 beneficiaries were enrolled from birth through two years, of which 25 408 (7.7%) were preterm (

Background The Accreditation Council for Graduate Medical Education has called for increased emphasis on reporting objective performance measures to trainees and programs. However, reporting of objective measures, including clinical volume, is largely omitted from training programs. Objective To use automated electronic medical systems at a tertiary pediatric care hospital to create a dashboard that reports objective trainee and program metrics, including clinical volume and diagnoses in a pediatrics residency. Methods We queried an enterprise data warehouse that aggregates data daily from multiple hospital systems to identify patient encounters during which senior pediatrics residents at Boston Children's Hospital had entered documentation over a 9-month period. From this query, we created a filterable dashboard to display clinical volume and diagnosis data by individual resident and in aggregate. Results A total of 44 of 45 senior residents (98%) in the program were included in analysis. We identified 12 198 patient encounters during which a senior pediatrics resident had entered documentation; these included a median of 332 inpatient encounters per resident, 122 emergency department encounters, and 84 outpatient encounters. The most common diagnoses stratified by clinical site were: inpatient – dehydration (median = 61); emergency department – long-term/current drug therapy (median = 16); and outpatient – encounter for immunization (median = 48). Conclusions We used electronic health record systems to generate performance dashboards for trainees in a pediatrics residency across different sites of care with reported volume by diagnosis. Our dashboards provide feedback to program leadership regarding individual and aggregate trainee experience and allow individual trainees to compare their clinical exposure to peers.

Economic evaluations performed alongside randomized controlled trials benefit from the protections against bias inherent in randomization. In this systematic review, we assessed the frequency and quality of economic assessments alongside randomized controlled trials of interventions in neonates published between 1990 and 2016. Over that period, 58 economic assessments were published, corresponding to approximately 2% of RCTs. We noted significant methodological limitations of these studies, including limitation of included costs to the health sector or payer rather than broader categories such as family or community expenditures (81%), short time horizon for cost measurement (less than one year in 60%), lack of reporting of uncertainty (26%), and infrequent analysis of costs and effects in a single metric (combined in 45%). Strategies for improving the quality and frequency of economic evaluations in neonatology are discussed, including selection of appropriate trials, funding, and peer review.

To describe the prevalence of apnea in otherwise healthy term newborns, identify attributable length of stay (LOS) and healthcare utilization (cost) of apnea, and measure hospital variation in attributable LOS and cost of apnea in this population.We conducted a secondary analysis of a national administrative dataset, the 2012 Kids' Inpatient Database, which included 3.4 million newborn discharges in the US. The birth hospitalizations of approximately 2.6 million otherwise healthy, full-term newborns were included for analysis. Attributable LOS and cost of apnea were calculated using multivariate analyses.Apnea was diagnosed in 1 in 1000 healthy full-term newborns. Multivariate analyses showed that newborns with apnea had 0.6 days longer LOS (P .001) and $483 greater costs (P .001) compared with healthy term newborns, per birth hospitalization. Newborns diagnosed with apnea plus hypoxia and/or bradycardia had 1.4 days longer LOS (P .001) and $653 greater costs (P .001). The attributable LOS and cost attributable to apnea varied between individual hospitals and differed by hospital region.Apnea is associated with higher LOS and cost in the newborn hospitalization, with variation in hospital practice. This suggests the need for better comprehension of the underlying physiology and standardization of practice in its management in the term newborn.

Background Observational studies suggest that asthma control improves after adenotonsillectomy, but longitudinal studies that correlate the effect of the procedure on the levels of biomarkers associated with airway inflammation are limited. Methods We conducted a longitudinal, observational study on pediatric patients, both with and without asthma, undergoing adenotonsillectomy. Asthma Control Test (ACT) scores and chitinase activity in the circulation were measured at time of surgery and at 6-month follow-up. Results 66 children with asthma and 64 control subjects were enrolled. Mean ACT scores improved by 3 points (p< 0.001) after 6 months. 85% of children with poorly-controlled asthma demonstrated an increase in ACT score of at least 3 points or a decrease in Emergency Department/Urgent Care visits, oral corticosteroid courses, or rescue short acting bronchodilator usage. Chitinase activity decreased significantly in asthmatics who improved (p< 0.01). Higher chitinase activity levels at baseline were associated with improved asthma control following surgery (p< 0.01). Conclusions In children with high pre-operative circulating chitinase activity levels, asthma control and healthcare utilization were significantly improved after adenotonsillecotmy. Chitinase activity decreased after surgery in children with improved control. This suggests that adenotonsillectomy modulates chitinase activity, affecting airway inflammation and improving airway disease.

Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mitogenic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit micromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with tamoxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5min, and dose-dependent, as low as 5μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH4.5. In addition, pretreatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism.

Poor prognosis in patients with later stage colorectal cancer (CRC) necessitates the search for new treatment strategies. Ceramide, because of its role in orchestrating death cascades in cancer cells, is a versatile alternative. Ceramide can be generated by exposure to chemotherapy or ionizing radiation, or it can be administered in the form of short-chain analogs (C6-ceramide). Because intracellular P-glycoprotein (P-gp) plays a role in catalyzing the conversion of ceramide to higher sphingolipids, we hypothesized that administration of P-gp antagonists with C6-ceramide would magnify cell death cascades. Human CRC cell lines were employed, HCT-15, HT-29, and LoVo. The addition of either tamoxifen, VX-710, verapamil, or cyclosporin A, antagonists of P-gp, enhanced C6-ceramide cytotoxicity in all cell lines. In depth studies with C6-ceramide and tamoxifen in LoVo cells showed the regimen induced PARP cleavage, caspase-dependent apoptosis, mitochondrial membrane permeabilization (MMP), and cell cycle arrest at G1 and G2. At the molecular level, the regimen, but not single agents, induced time-dependent upregulation of tumor suppressor protein p53; however, introduction of a p53 inhibitor staved neither MMP nor apoptosis. Nanoliposomal formulations of C6-ceramide and tamoxifen were also effective, yielding synergistic cell kill. We conclude that tamoxifen is a favorable adjuvant for enhancing C6-ceramide cytotoxicity in CRC, and demonstrates uniquely integrated effects. The high frequency of expression of P-gp in CRC presents an adventitious target for complementing ceramide-based therapies, a strategy that could hold promise for treatment of resistant disease.

Acid ceramidase (AC) occupies an important place in the control of cancer cell proliferation. We tested the influence of AC inhibition on the effects of PSC 833, a P-glycoprotein antagonist with potent ceramide-generating capacity, to determine whether AC could be a therapeutic target in pancreatic cancer.Ceramide metabolism was followed using (3)H-palmitate, and molecular species were determined by mass spectroscopy. Apoptosis was measured by DNA fragmentation, autophagy by acridine orange staining, and cell cycle was assessed by flow cytometry and RB phosphorylation. AC was measured in intact cells using fluorescent substrate.Exposure of human PANC-1 or MIA-PaCa-2 cells to PSC 833 promoted increases in de novo (dihydro)ceramides, (dihydro)glucosylceramides, and (dihydro)sphingomyelins, demarking ceramide generation and robust metabolism. Despite the multifold increases in (dihydro)ceramide levels, cells were refractory to PSC 833. However, PSC 833 produced a dose-dependent decrease in DNA synthesis and dose- and time-dependent decreases in RB phosphorylation, consistent with cell cycle arrest as demonstrated at G1. Cytostatic effects of PSC 833 were converted to cytotoxic end-point by acid ceramidase inhibition. Cytotoxicity was accompanied by formation of acridine orange-stained acidic vesicles and an increase in LC3 expression, indicative of autophagic response. Cell death was not reversed by preexposure to myriocin, which blocks PSC 833-induced ceramide generation.Although the role of ceramide in end-point cytotoxicity is unclear, our results suggest that acid ceramidase is a viable target in pancreatic cancer. We propose that AC inhibition will be effective in concert with other anticancer therapies.

Although ceramide has potent tumor suppressor properties, its use in cancer therapeutics has been limited; this is mainly due to rapid metabolism of ceramide by cancer cells, the inability to attain therapeutic levels when ceramide-generating agents are employed, and the inherent insolubility of ceramide as a therapeutic. Our approach circumvents these disadvantages and provides advantages over existing methodologies. For example, rather than promoting intracellular ceramide generation by application of exogenous drugs that activate de novo and sphingomyelinase pathways, we have chosen to administer ceramide exogenously in the form of short-chain C6-ceramide nanoliposomes; this overcomes solubility issues and improves uptake. In addition, nanoliposomes are efficient platforms for delivering other hydrophobic chemotherapeutics. The antiestrogen tamoxifen was selected for our studies because of its unusual “off-target” actions, which include inhibition of ceramide metabolism at glycosylation and hydrolysis. Blocking these junctures increases C6-ceramide intracellular residence time and diminishes production of sphingosine 1-phosphate, a mitogenic lipid derived via the action of ceramidase and sphingosine kinase. Hence, C6-ceramide and tamoxifen make up a versatile drug duo. Using in vitro models of human colon and breast cancer, acute mylogenous leukemia (AML), and melanoma, we show that simultaneous administration of nanoliposomal C6-ceramide and tamoxifen potently and synergistically decreased cell viability, compared to the single agents. The mix induced caspase-dependent apoptosis, cell cycle arrest at G1 and G2, upregulation of JNK, p38, and p53, and downregulation of Akt and survivin. In triple negative, hormone-insensitive breast cancer cells, C6-ceramide-tamoxifen induced cell cycle arrest, caspase-dependent apoptosis, and lysosomal and mitochondrial membrane permeability. Evaluation of single agents showed that tamoxifen elicited lysosomal membrane permeability and inhibited acid ceramidase. We have obtained similar results in in vitro models of human melanoma and AML. In an in vivo retroviral transduced murine AML model, the combination lowered leukemia burden in spleen and marrow. The combination also perpetuated synergistic increases in long-chain ceramides, likely compounding the overall ceramide effect. Of interest regarding in vivo utility, the major metabolites of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, were effective in combination with C6-ceramide. Our findings show that tamoxifen magnifies the antiproliferative effects of C6-ceramide via aggregate mechanisms. This proficient, ceramide-based nanoliposomal drug regimen may have potential as an effective anticancer therapeutic. Supported by NIGMS 77391; ABC's, Los Angeles; Fashion Footwear Association of New York Charitable Foundation. Citation Format: Samy A.F. Morad, Jonathan C. Levin, Noha Abdelmageed, Mark Kester, Sriram S. Shanmugavdlandy, Robert F. Paulson, Daniel W. Rosenberg, James P. Madigan, Myles C. Cabot. Tamoxifen amplifies antitumor impact of nanoformulated ceramide- pan efficacy in a myriad of tumor cell types. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2013-2027

Drug resistance in breast and other cancers results from multiple gene interactions. Ceramide, a key intermediate in the sphingolipid pathway, can act as a powerful tumor suppressor. The employ of ceramide-based agents as opposed to the administration of ceramide generators such as daunorubicin, could be a smart strategy for cancer therapy. Polychemotherapy remains the best option for treatment of breast cancer at the aggressive, metastatic stage. The aim of our study was to investigate the cytotoxic and antiproliferative effects of ceramide (short-chain C6-ceramide) administered in combination with tamoxifen, used here as an inhibitor of ceramide glycosylation. We previously demonstrated the cytotoxicity of this combination in MDA-MB-231 cells; however, little regarding mechanism was established. The present study employs nanoliposomal formulations of both agents, a strategy to enhance efficacy. Four triple-negative chemoresistant breast cancer cell lines, MDA-MB-231, MDA-MB-468, BT-20 and Hs578T were used. Cytotoxicity assays revealed that C6-ceramide/tamoxifen synergistically reduced viability, compared to single agents in all four cell lines. For example, in MDA-MB-468 cells, nanoliposomal C6-ceramide (2.5 µM), tamoxifen (5 µM), and the combination reduced cell viability to 70, 85, and 10% of control, respectively (96 hr). A hallmark of cancer, implicated in tumor growth as well as metastasis, is resistance to induction of apoptosis. Assessment of cellular apoptosis showed that C6-ceramide/tamoxifen treatment promoted DNA fragmentation in a dose-dependant manner, attaining a value that was 36% over control at 24 hr. Introduction of the pan-caspase inhibitor, z-VAD-fmk, completely reversed DNA fragmentation, showing that apoptosis was mediated by caspase activation. The aggressive and metastatic behavior of any tumor is dependent on the ability of the cells to proliferate. Interestingly, the lower concentrations of our drug combination (2.5 µM) induced cell cycle arrest at G1, the first growth phase. In summary, this approach has potential to circumvent limitations of ceramide-based therapies, such as rapid ceramide clearance due to metabolism, the inability to attain therapeutic levels when ceramide-generating agents are employed, and inherent insolubility that is encountered. These findings demonstrate the in vitro efficacy of combination C6-ceramide/tamoxifen nanoliposomal formulations for suppressing growth of breast cancer cells, and suggest that tamoxifen may be an effective adjuvant for enhancing ceramide-driven cell death cascades. Supported by NIGMS 77391 and ABC's, Los Angeles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3833. doi:1538-7445.AM2012-3833

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Even though the current treatment regimen has increased event-free survival to 70%, there still exists a poor prognosis for patients with unresectable primary tumors and those with clinically evident metastases. Insulin like growth factor-1 (IGF-1) and its receptor are thought to have a role in the pathogenesis, invasion, and distant metastasis of many human solid tumors. Our previous preclinical data using an IGF-1R antibody against xenograft models of OS demonstrated responses in four out of six OS xenografts suggesting this as a potential therapeutic target. It may be hypothesized that IGF-1R is selected for genomic amplification in some OS due to its proliferative and anti-apoptotic effects. Real time RT-PCR was performed on the primary patient samples, xenografts, and 143B, a standard cell line, to see the expression of IGF-1R. Only 3 of 20 patient samples, and 1 of 4 xenografts have shown higher level of expression of IGF-1R in reference to GAPDH. Genomic DNA was isolated from the same samples, and quantitative PCR was performed to detect gene copy numbers. 11 of 19 (58%)samples were shown to have more than 2 copies of IGF-1R (2 samples have >10 copies, 1 has 7, 2 have 5, 6 have 33), whereas 37% have 2 copies. Two OS xenografts, which respond to IGF-1R antibody in preclinical testing, showed increases in the copy number of IGF-1R. Fluorescence in situ hybridization (FISH) was used to detect the amplification of IGF-1R gene in both patient derived and xenograft OS cell lines. FISH was performed on metaphase slides of fixed cultured cells. Three BAC probes spanning the IGF-1R gene locus in 15q26.3 were produced by isolating BAC DNA, and labeling with AlexaFluor 488 by nick translation. Metaphase prep slides were then hybridized with IGF-1R probes and Texas Red-labeled chromosome 15 centromere probes, counter stained with DAPI, and viewed under fluorescence microscope. Copy number was counted in cells showing a full metaphase spread. Based on initial FISH results, it seems that in the majority of OS the IGF-1R gene is not amplified. This does not imply that IGF1R overamplification does not play a role in IGF-IR overexpression in OS: given the heterogeneity of these tumors and the limited sample size of the experiments thus far IGF-1R amplification may be a rare phenomenon in OS. As responses to IGF-1R are not observed in the majority, it remains possible that amplification correlates with therapeutic response to IGF-1R antibody. FISH on more patient cell lines and xenografts are underway, and even if no amplification is demonstrated, increased IGF-IR activity could still be explained by GOF mutation, or other mechanisms. Additional experiments including FISH in a larger pool of samples, comparing amplification results to clinical data and responsiveness of the cell lines to anti-IGF-IR therapy, and looking for other abnormalities in the IGF-I system, among others are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3195.