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BACKGROUND The US Preventive Services Task Force (USPSTF) includes multitarget stool DNA (mt-sDNA) testing as a colorectal cancer (CRC) screening option in average-risk individuals, but data on colonoscopy outcomes after positive mt-sDNA tests in community settings are needed. AIM The aim of this study was to investigate colonoscopy outcomes and quality following positive mt-sDNA in the population-based New Hampshire Colonoscopy Registry. METHODS We compared colonoscopy outcomes and quality between age-matched, sex-matched, and risk-matched patients from 30 endoscopy practices with and without a preceding positive mt-sDNA test. Main outcomes were colonoscopy findings of CRC, advanced noncancerous neoplasia, nonadvanced neoplasia, or normal examination. Quality measures included withdrawal time, bowel preparation quality, examination completion, and percentage of average-risk individuals with normal colonoscopies receiving a USPSTF-recommended 10 year rescreening interval. RESULTS Individuals with positive mt-sDNA tests (N=306, average age 67.0 y; 61.8% female) were significantly more likely than colonoscopy-only patients (N=918, 66.2 y; 61.8% female) to have CRC (1.3% vs. 0.4%) or advanced noncancerous neoplasia (27.1% vs. 8.2%) (P

Colorectal cancer is a growing burden in adults less than 50 years old. In 2018, the American Cancer Society published a guideline update recommending a reduction in the colorectal cancer screening start age for average-risk individuals from 50 to 45. Implementing these recommendations would have important implications for public health. However, the approximate number of people impacted by this change, the average-risk population ages 45–49, is not well-described in the literature. Here, we provide methodology to conservatively estimate the average-risk and screening-eligible population in the United States, including those who would be impacted by a lowered colorectal cancer screening start age. Using multiple data sources, we estimated the current average-risk population by subtracting individuals with symptomatic colorectal cancer, with a family history of colorectal cancer, and with inflammatory bowel disease and hereditary nonpolyposis colorectal cancer from the total population. Within this population, we estimated the number of screening-eligible individuals by subtracting those with previous colorectal cancer screening (45- to 49-year-old) or up to date with colorectal cancer screening (50- to 74-year-old). The total average-risk population is estimated between 102.1 and 106.5 million people, of whom 43.4–45.2 million people are eligible for colorectal cancer screening. Lowering the screening age would add roughly 19 million people to the average-risk population and increase the current number of screening-eligible individuals on immediate implementation by over 60% (from 27 to 44 million). Estimating the population size impacted by lowering the recommended colorectal cancer screening start age enables more accurate decision-making for policymakers and epidemiologists focused on cancer prevention.

Introduction Surveillance is essential to diagnose and more effectively treat hepatocellular carcinoma (HCC) in at-risk patients. However, the performance of currently recommended surveillance strategies is suboptimal, particularly for early-stage detection, and patient adherence remains low. Here, we establish the analytical performance of a novel liquid biopsy test to evaluate the presence of HCC. Methods The multi-target HCC blood test (mt-HBT) integrates results from three DNA methylation markers (HOXA1, TSPYL5, and B3GALT6), the protein biomarker α-fetoprotein (AFP), and patient sex. The methylation markers are quantified from cell-free DNA extracted from plasma, and AFP is measured from serum. We conducted analytical validation studies on the mt-HBT, including analytical sensitivity, linearity, cross-contamination, interference, analytical accuracy, and precision. Results The mt-HBT performance met all pre-specified analytical performance criteria. The test demonstrated high reproducibility, with ≥97% concordance relative to the expected results for six categories of surrogate samples across the test's dynamic range. Of 17 candidate interfering substances, none caused significant interference to biomarker quantitation, and no occurrences of sample-to-sample cross-contamination were observed. Conclusion These data demonstrate that the mt-HBT can produce consistent, reliable results for patients in the intended-use population, for whom surveillance is recommended.

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although biannual ultrasound surveillance with or without α-fetoprotein (AFP) testing is recommended for at-risk patients, sensitivity for early stage HCC, for which potentially curative treatments exist, is suboptimal. We conducted studies to establish the multitarget HCC blood test (mt-HBT) algorithm and cut-off values and to validate test performance in patients with chronic liver disease. METHODS Algorithm development and clinical validation studies were conducted with participants in an international, multicenter, case-control study. Study subjects had underlying cirrhosis or chronic hepatitis B virus; HCC cases were diagnosed per the American Association for the Study of Liver Diseases criteria and controls were matched for age and liver disease etiology. Whole blood and serum were shipped to a central laboratory and processed while blinded to case/control status. An algorithm was developed for the mt-HBT, which incorporates methylation biomarkers (HOXA1, TSPYL5, and B3GALT6), AFP, and sex. RESULTS In algorithm development, with 136 HCC cases (60% early stage) and 404 controls, the mt-HBT showed 72% sensitivity for early stage HCC at 88% specificity. Test performance was validated in an independent cohort of 156 HCC cases (50% early stage) and 245 controls, showing 88% overall sensitivity, 82% early stage sensitivity, and 87% specificity. Early stage sensitivity in clinical validation was significantly higher than AFP at 20 ng/mL or greater (40%; P < .0001) and GALAD (gender, age, Lens culinaris agglutinin-reactive AFP, AFP, and des-γ-carboxy-prothrombin score) of -0.63 or greater (71%; P = .03), although AFP and GALAD at these cut-off values had higher specificities (100% and 93%, respectively). CONCLUSIONS The mt-HBT may significantly improve early stage HCC detection for patients undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality. ClinicalTrials.gov number NCT03628651.

Background Readily available testing for SARS-CoV-2 is necessary to mitigate COVID-19 disease outbreaks. At-home collection kits, in which samples are self-collected without requiring a laboratory or clinic visit and sent to an external laboratory for testing, can provide convenient testing to those with barriers to access. They can prevent unnecessary exposure between patient and clinical staff, increase access for patients with disabilities or remote workers, and decrease burdens on health care resources, such as provider time and personal protective equipment. Exact Sciences developed an at-home collection kit for samples to be tested to detect SARS-CoV-2 that includes an Instructions for Use (IFU) document, which guides people without prior experience on collecting a nasal swab sample. Demonstrating successful sample collection and usability is critical to ensure that these samples meet the same high-quality sample collection standards as samples collected in clinics. Objective The aim of this study was to determine the usability of a SARS-CoV-2 at-home nasal swab sample collection kit. Methods A human factors usability study was conducted with 30 subjects without prior medical, laboratory, or health care training and without COVID-19 sample self-collection experience. Subjects were observed while they followed the IFU for the at-home sample collection portion of the SARS-CoV-2 test in a setting that simulated a home environment. IFU usability was further evaluated by requiring the subjects to complete a survey, answer comprehension questions, provide written feedback, and respond to questions from the observer about problems during use. Results All 30 subjects successfully completed the sample collection process, and all 30 samples were determined by reverse transcription–polymerase chain reaction (RT-PCR) testing to meet quality standards for SARS-CoV-2 testing. The subjects’ written feedback and comments revealed several recommendations to improve the IFU. Conclusions The study demonstrated the overall usability of an at-home SARS-CoV-2 collection kit. Various feedback mechanisms provided opportunities to improve the wording and graphics for some critical tasks, including placing the label correctly on the tube. A modified IFU was prepared based on study outcomes.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML. Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

// David K. Edwards V 1,* , David Tyler Sweeney 2,* , Hibery Ho 2 , Christopher A. Eide 2 , Angela Rofelty 2 , Anupriya Agarwal 2 , Selina Qiuying Liu 2 , Alexey V. Danilov 2 , Patrice Lee 3 , David Chantry 3 , Shannon K. McWeeney 4 , Brian J. Druker 2,5 , Jeffrey W. Tyner 1 , Stephen E. Spurgeon 2,** and Marc M. Loriaux 2,** 1 Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA 2 Division of Hematology & Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR, USA 3 Array BioPharma, Boulder, CO, USA 4 Department of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA 5 Howard Hughes Medical Institute, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA * These authors have contributed equally to this work ** These authors have contributed equally to this work Correspondence to: Marc M. Loriaux, email: // Keywords : colony-stimulating factor 1 receptor; chronic lymphocytic leukemia; tumor-associated macrophages; tumor microenvironment; small-molecule inhibitors Received: September 26, 2017 Accepted: March 01, 2018 Published: May 15, 2018 Abstract In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Background & Aims Hepatocellular carcinoma (HCC) can be treated effectively if detected at an early stage. Recommended surveillance strategies for at-risk patients include ultrasound with or without α-fetoprotein (AFP), but their sensitivity is suboptimal. We sought to develop a novel, blood-based biomarker panel with improved sensitivity for early-stage HCC detection. Methods In a multicenter, case-control study, we collected blood specimens from patients with HCC and age-matched controls with underlying liver disease but without HCC. Ten previously reported methylated DNA markers (MDMs) associated with HCC, methylated B3GALT6 (reference DNA marker), and 3 candidate proteins, including AFP, were assayed and analyzed by a logistic regression algorithm to predict HCC cases. The accuracy of the multi-target HCC panel was compared with that of other blood-based biomarkers for HCC detection. Results The study included 135 HCC cases and 302 controls. We identified a multi-target HCC panel of 3 MDMs (HOXA1, EMX1, and TSPYL5), B3GALT6 and 2 protein markers (AFP and AFP-L3) with a higher sensitivity (71%, 95% CI: 60–81%) at 90% specificity for early-stage HCC than the GALAD score (41%, 95% CI: 30–53%) or AFP ≥7.32 ng/mL (45%, 95% CI: 33–57%). The AUC for the multi-target HCC panel for detecting any stage HCC was 0.92 compared with 0.87 for the GALAD score and 0.81 for AFP alone. The panel performed equally well in important subgroups based on liver disease etiology, presence of cirrhosis, or sex. Conclusions We developed a novel, blood-based biomarker panel that demonstrates high sensitivity for early-stage HCC. These data support the potential for liquid biopsy detection of early-stage HCC to clinically benefit at-risk patients. This study was registered on ClinicalTrials.gov ( NCT03628651 ).

Secreted proteins in the bone marrow microenvironment play critical roles in acute myeloid leukemia (AML). Through an ex vivo functional screen of 94 cytokines, we identified that the pro-inflammatory cytokine interleukin-1 (IL-1) elicited profound expansion of myeloid progenitors in ∼67% of AML patients while suppressing the growth of normal progenitors. Levels of IL-1β and IL-1 receptors were increased in AML patients, and silencing of the IL-1 receptor led to significant suppression of clonogenicity and in vivo disease progression. IL-1 promoted AML cell growth by enhancing p38MAPK phosphorylation and promoting secretion of various other growth factors and inflammatory cytokines. Treatment with p38MAPK inhibitors reversed these effects and recovered normal CD34+ cells from IL-1-mediated growth suppression. These results highlight the importance of ex vivo functional screening to identify common and actionable extrinsic pathways in genetically heterogeneous malignancies and provide impetus for clinical development of IL-1/IL1R1/p38MAPK pathway-targeted therapies in AML.

Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise,This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise, Fgf2 -/- mice transplanted with retroviral BCR-ABL leukemia survive significantly longer than their +/+ counterparts when treated with TKI. Thus, inhibition of FGFR can modulate stromal function, reduce exosome secretion, and may be a therapeutic option to overcome resistance to TKIs. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., eLife digest Leukemias are cancers of white blood cells. The cells grow and divide rapidly, often because of mutations in proteins called kinases. Since the kinase mutations do not occur in healthy cells, they provide a good target for anti-leukemia drugs. Several such kinase inhibitors are effective at treating leukemia patients. However, most leukemia cells develop ways to resist the effects of the kinase inhibitors over time, leading to relapses of the disease. One way that leukemia cells resist kinase inhibitors is by taking advantage of signals coming from supportive cells, known as stromal cells, in the bone marrow. When patients are treated with kinase inhibitors, the bone marrow stromal cells produce more of a signaling protein called FGF2. The leukemia cells then use FGF2 to survive the effects of the kinase inhibitors. It was not clear how the FGF2 signal reaches the leukemia cells from the bone marrow stromal cells. Now, using biochemical techniques, Javidi-Sharifi, Martinez et al. show that bone marrow stromal cells package FGF2 into small compartments called exosomes. The stromal cells release the exosomes into the bone marrow, and the leukemia cells then engulf and internalize the exosomes. Leukemia cells that had taken up FGF2 in this way were better able to survive kinase inhibitor treatment than leukemia cells that had not. Javidi-Sharifi, Martinez et al. also observed that FGF2 also affects the bone marrow stromal cells themselves, causing them to grow faster, produce more FGF2 and release more exosomes. Blocking the effects of FGF2 on the stromal cells slowed their growth and caused fewer exosomes to be released. In addition, mice whose bone marrow stromal cells could not produce FGF2 survived leukemia for longer than mice whose stromal cells provided protective FGF2 in exosomes to leukemia cells. This suggests that taking advantage of drugs that prevent bone marrow stromal cells from releasing FGF2 in exosomes might improve treatments for leukemia. Further research will be needed to confirm whether this strategy would be effective in humans.

IntroductionPopulation-level screening has been shown to reduce the incidence and mortality of colorectal cancer (CRC). Unfortunately, adherence to screening recommendations among eligible US adults remains below national goals. A relatively new non-invasive screening modality, the Food and Drug Administration–approved multi-target stool DNA (mt-sDNA) assay (commercialised as Cologuard), which combines the detection of haemoglobin and DNA abnormalities, has been completed by more than 3 million individuals. Given mt-sDNA’s recent availability, the effectiveness of mt-sDNA screening with respect to CRC incidence and mortality reduction has not yet been established.Methods and analysisThrough an academic–industry collaboration, a prospective cohort study (Voyage) was designed with an initial enrolment target of 150 000 individuals with mt-sDNA ordered by their healthcare provider for CRC screening. Consented participants will be asked to complete a baseline questionnaire to collect sociodemographic and health information. Additional questionnaires will be provided after 1 year, and every 3 years thereafter, to collect data regarding CRC screening follow-up in order to estimate rates of CRC incidence and other health outcomes. Linkage to the National Death Index will be used to estimate mortality rates.Ethics and disseminationThe Voyage study will be conducted in accordance with international guidelines and local regulatory requirements and laws. Data will be stored and retained at Mayo Clinic. Only limited data elements required for research purposes will be transmitted between Mayo Clinic and Exact Sciences Laboratories. Results of the Voyage study will be disseminated through scientific presentations and publications.Trial registration numberNCT04124406.

To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5–conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.

Targeted therapy has proven to be successful in improving outcomes across multiple cancer types. However, many challenges still remain for implementation of this strategy in most patient cohorts, especially with the challenges of identifying the specific mutations or abnormalities in a heterogeneous tumor that are functionally significant. Previously, we developed a functional screening assay, RNAi-assisted protein target identification (RAPID) technology, which evaluates the viability of tumor cells after exposure to siRNAs against members of the tyrosine kinome and NRAS/KRAS. Here, we publish the comprehensive results of this screen for 332 primary AML patient samples. Data from these screening efforts have already helped identify previously unknown therapeutic targets, and will continue to provide insights into better treatment strategies for these patients.

Introduction: CPX-351 (Vyxeos) is a liposomal combination of cytarabine and daunorubicin at a synergistic 5:1 molar ratio. Recently, CPX-351 has been shown to be significantly more effective than the 7+3 standard of care chemotherapy in treating high-risk AML patients, including patients with the FLT3-ITD mutation. We previously treated primary patient samples with CPX-351 ex vivo and found that FLT3-ITD+ samples were significantly more sensitive to CPX-351 and showed enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in an activation of liposome uptake pathways, leading to increased sensitivity to CPX-351 and ultimately cell death. Furthermore, we examined the effect of combining CPX-351 with existing FLT3 inhibitors (e.g. quizartinib and midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains an activating FLT3 mutation) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We evaluated cell viability using a colorimetric assay and measured intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry. Additionally, we analyzed the synergy of exposing these cell lines to CPX-351 and FLT3 inhibitors in combination and at different dose schedules, followed by measuring cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351 and exhibited increased drug uptake compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These data provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351. This is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Citation Format: David K. Edwards, Nathalie Javidi-Sharifi, Angela Rofelty, Max Gordon, Riley Roth-Carter, Paul Tardi, Lawrence Mayer, Jeffrey W. Tyner. CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2017-1087

Introduction: CPX-351 (Vyxeos), a liposomal combination of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio, has recently been shown to be significantly more efficacious than the standard of care, 7+3 administration regimen of free (non-liposomal) cytarabine + daunorubicin in treating elderly high-risk AML patients, including patients with the FLT3-ITD mutation. We previously exposed AML blasts from primary patient samples to CPX-351 ex vivo and found that samples harboring FLT3-ITD were significantly more sensitive to CPX-351 and also demonstrated enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in upregulated liposome uptake pathways, leading to increased cell death and overall sensitivity to CPX-351. Furthermore, we wanted to examine the effect of combining CPX with existing FLT3 inhibitors (e.g. quizartinib or midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains mutant, activated FLT3) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We measured cell viability and intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry and using a fluorescent plate reader. Additionally, we performed synergy analyses on the cytotoxic activity in these cells, adding CPX-351 and FLT3 inhibitors in combination at different exposure sequences followed by measurement of cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351, and exhibited increased drug uptake, compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, indicating that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, in synergy studies we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These results provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351, which is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Disclosures Tardi: Jazz Pharmaceuticals: Employment. Mayer:Jazz Pharmaceuticals: Employment.

Background: Inflammatory cytokines secreted in the bone marrow microenvironment play important roles in modulating cell survival, proliferation, differentiation, and immune responses in cancer. Perhaps not surprisingly, there is also an association between chronic inflammation and tumor progression. We recently used an ex vivo functional screen of 94 cytokines to show that the pro-inflammatory cytokines IL1α and IL1β promoted the expansion of AML progenitors in 70% (40/60) of primary samples. We therefore hypothesized that inflammatory cytokines are crucial to clonal expansion and disease progression in AML and that therapeutic targeting of these pathways may circumvent disease heterogeneity. Here we provide in vitro and in vivo evidence that IL1-mediated signaling elicits profound expansion of leukemia progenitors in AML patients harboring various genetic mutations and promotes in vivo clonal expansion and disease progression in a murine AML model. Further, these effects are reversed by targeting IL1 signaling. Methods: We validated the role of IL1 signaling using a shRNA approach and in murine competitive repopulation and bone marrow transplantation models. We evaluated the influence of these cytokines on inflammatory markers using immunoblotting, flow cytometry, and Luminex assays, and assessed strategies to target these pathways using small-molecule inhibitors. Results: IL1 stimulation promoted a 3- to 20-fold increase in growth, survival, and clonogenic potential of AML CD34+ cells, while paradoxically suppressing growth of healthy CD34+ cells. To identify the influence of IL1 on in vivo clonal expansion of healthy and leukemic progenitors simultaneously, we established an in vivo murine competitive repopulation study utilizing TET2-null mice. IL1 treatment promoted clonal expansion of TET2-null myeloid cells over wild-type cells during 6 weeks of IL1β treatment. Consistent with this, both flow cytometry analysis and blood differential counts showed an increased percentage of granulocytes and reduced percentage of lymphocytes in IL1-treated mice. In this model, TET2-null monocytes have greater expression of IL1 than wild-type cells, suggesting IL1 promotes clonal growth of TET2-mutated early leukemic progenitors. Similarly, IL1β and IL1 receptors (IL1R1 and IL1RAP) were overexpressed in IL1-sensitive AML bone marrow and peripheral blood samples compared to nonsensitive AML samples and normal samples. Intracellular FACS showed that the majority of IL1β was secreted by monocytes and to some extent by myeloid progenitors. Accordingly, IL1-sensitive AML samples exhibited trends towards monocytic and myelomonocytic clinical features. Reduced survival of AML cells after monocyte depletion was rescued by IL1 treatment, suggesting that IL1 mediates paracrine regulation of AML cell growth. Silencing of the IL1 receptor, IL1R1, reduced the clonogenic potential of AML primary samples and oncogene (AML1-ETO9a, NRASG12D, and MLL-ENL)-transduced mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer than did recipients of wild-type marrow. We also found that IL1β increased phosphorylation of p38MAPK and MK2, as well as secretion of multiple downstream inflammatory cytokines (IL6, IL8, MCP1, MIP1α, and MIP1β) from CD34+ progenitors, in IL1-sensitive AML samples compared to IL1-nonsensitive progenitors. Conversely, treating AML cells with p38MAPK inhibitors such as doramapimod andralimetinib reduced the growth of AML cells by decreasing p38MAPK and MK2 phosphorylation and reducing secretion of inflammatory cytokines from AML progenitors. Clinical and demographic analyses suggest that AML patients are dependent on IL1 signaling irrespective of mutation status and clinical features. Targeting this unifying mechanism of IL1-mediated clonal expansion may thus have application across heterogeneous AML subtypes. Conclusion: We demonstrate that IL1 promotes in vitro and in vivo clonal expansion of leukemic cells and promotes disease progression in AML. As IL1 signaling is active across heterogeneous disease subtypes, AML patients may therefore benefit from drugs targeting IL1/p38MAPK signaling because of their potential to inhibit AML while enhancing normal hematopoiesis, a significant clinical advantage over traditional chemotherapy. Disclosures Agarwal: CTI BioPharma Corp: Research Funding.

Introduction The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. Methods A tissue microarray representing 248 clinically annotated stage I–II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). Results In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO ( p =0.017) and transcription factor GLI1 ( p =0.001), while SMO correlated with GLI1 ( p =0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression ( p =0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. Conclusions Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.

Introduction: CSF1R (M-CSFR) is a receptor tyrosine kinase that, upon binding with CSF1 ligand (M-CSF), activates downstream survival, proliferation, and differentiation pathways in myeloid lineage cells. Expression and activity of CSF1R has been shown to be important for sustaining tumor-associated macrophages (TAMs) in a variety of solid tumor types, and these TAMs have been shown to provide a supportive microenvironment that maintains tumor cell viability and growth. Monocyte and macrophage lineage cells have also been implicated in providing a supportive microenvironment in certain hematologic malignancies, such as nurse-like cells in CLL, but have not been extensively explored in AML. Using an ex vivo functional screening platform applied directly to primary specimens from AML patients, we have identified recurrent sensitivity to CSF1R inhibition or silencing due to a depletion of CSF1R-expressing monocytic-lineage cells in the AML microenvironment. Methods: To identify new therapies for AML patients, our lab has screened hundreds of AML patient specimens against panels of targeted small-molecule inhibitors or siRNA and evaluated the impact on cell viability. These panels have included two small-molecule inhibitors with exquisite specificity for CSF1R (GW-2580 and ARRY-382). We have also used a panel of recombinant growth factors and cytokines, including CSF1, to measure their impact on stimulating AML cell growth. Results: We found that 20-30% of AML specimens showed sensitivity to inhibition or silencing of CSF1R, with good concordance between CSF1R siRNA, GW-2580, and ARRY-382. Exhaustive analysis of the clinical, demographic, and genetic features of these patients failed to reveal correlation between CSF1R and any prominent disease subsets. Samples sensitive to CSF1R silencing or inhibition are significantly more likely than non-sensitive samples to respond to CSF1 growth stimulation, indicating a ligand-dependent mechanism of CSF1R signaling. Additionally, we examined CSF1R and CSF1 expression in discrete hematopoietic cell populations by flow cytometry in our specimens, both with and without CSF1R inhibition. Interestingly, we found that CSF1R was predominantly expressed a subpopulation of CD14-expressing monocytes, not tumor cells, and this population disappeared after exposure to the CSF1R inhibitor. Moreover, we identified a correlation between sensitivity to CSF1R inhibition and the presence of this CD14/CSF1R-expressing cell population. Conclusions: These results suggest that CD14/CSF1R-expressing cells support AML blasts through a ligand-dependent mechanism, and depleting these cells eliminates the supportive microenvironment, resulting in leukemia cell death. Therefore, we propose using CSF1R inhibitors, such as ARRY-382, as a promising new line of therapy to target AML by disrupting the tumor microenvironment. Disclosures Agarwal: CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Sage Bionetworks: Research Funding; Millipore: Patents & Royalties; Molecular MD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; McGraw Hill: Patents & Royalties; CTI Biosciences, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; AstraZeneca: Consultancy; Oregon Health and Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Novartis Pharamceuticals: Research Funding; ARIAD: Research Funding; Aptose Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding.

Background: Recent 'omics'-based approaches have revealed that acute myeloid leukemia (AML) has significant genetic heterogeneity, and that clonal evolution in AML occurs through stepwise acquisition of somatic mutations. However, extrinsic factors may also influence this process. Here we provide strong evidence that inflammatory cytokines secreted in the AML microenvironment play a critical role in clonal expansion and disease progression. Specifically, we show that the proinflammatory cytokine IL-1 promotes in vitro clonal expansion in a large subset of primary AML samples and in vivo disease progression in a murine AML model. Methods: We used several functional assays to determine which extrinsic cytokine-activated pathways are essential for AML cell survival. We analyzed 90 primary AML samples ex vivo in the absence and presence of dose gradients of 98 distinct cytokines. We used stromal cells conditioned media and a recombinant cytokine cocktail (IL-3, IL-6, SCF, GCSF, FLT3L) for positive controls. The distributions for the positive and negative control allowed us to derive rigorous empirical thresholds to define cytokine dependence for individual samples. We also performed siRNA screens targeting 188 cytokine and growth factor receptors, and measured 30 different cytokines secreted in the plasma of peripheral blood and bone marrow samples using a multiplex Luminex assay. Functionally relevant pathways were validated using shRNA and genetic mouse models. Results: Several of the 98 cytokines promoted the growth of primary AML cells, including IL-1, GM-CSF, G-CSF, TPO, CXCL-1, IL-3, M-CSF, MCPs, TNF-α, and BMP-4. Many of these are known to induce an inflammatory response and cluster with the growth response to IL-1, a master mediator of innate immunity and inflammation. IL-1α and IL-1β had the most profound effect on the clonal expansion of myeloid progenitors, leading to 3- to 20-fold growth increase in 70% (63/90) of primary AML samples. Consistent with our findings from the cytokine screen, treatment of CD34+ AML cells with IL-1 led to increases in cell growth, survival, and clonogenic potential. Paradoxically, IL-1 suppressed both growth and colony formation in normal CD34+ cells. In support of these ex vivo findings, IL-1β was overexpressed in IL-1-sensitive AML bone marrow and peripheral blood samples compared to non-sensitive AML samples and normal samples. Intracellular FACS showed that 80% of the total IL-1β is secreted by monocytes, and to some extent by myeloid progenitors and stromal cells, but not by B or T cells in the AML bone marrow microenvironment. Consistent with this, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. These results suggest that IL-1 secreted in the bone marrow microenvironment regulates AML cell growth in a paracrine manner. Silencing of the IL-1 receptor, IL1R1, reduced the viability of AML primary samples by 60-80% and led to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRASG12D and MLL-ENL) in mouse bone marrow. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer (39 days; range: 28-118) than did recipients of wild-type marrow (30 days; range: 27-61; p=0.012). Mechanistically, IL-1β regulates phosphorylation of p38MAPK, a downstream component of the IL-1 pathway, in AML progenitors and IL-1-sensitive AML samples exhibit more p38 phosphorylation as compared to normal cells. Conversely, knocking down IL1R1 or treating AML cells with p38MAPK inhibitors such as doramapimod reduced the growth of AML cells by decreasing p38MAPK phosphorylation. Conclusion: These results demonstrate a novel role for IL-1 and its receptor in promoting clonal growth and disease progression in a large subset of AML patients. Our findings suggest that AML patients may benefit from drugs targeting IL-1/IL1R1/p38MAPK signaling because of their potential to enhance normal hematopoiesis while inhibiting AML -- a significant clinical advantage over traditional chemotherapy, which kills both normal and leukemic cells.Since IL-1 and its receptors are not mutated in these patients, our data also highlight the importance of ex vivo functional screening for microenvironmental stimuli for the identification of novel therapeutic targets. Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sage Bionetworks: Research Funding; ARIAD: Research Funding; AstraZeneca: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding. Agarwal:CTI BioPharma: Research Funding.

Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients. Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive. Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity. Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. Disclosures Winski: Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

Background: Despite the great strides that have been made in the treatment of AML, resistance to treatment is common and signaling pathways influencing leukemic cellular proliferation have not been comprehensively defined. Cytokines and growth factors play important roles in cell survival, proliferation, differentiation, senesence, and immune response in both normal and cancer cells. Recent studies have demonstrated that certain inflammatory cytokines known to suppress normal hematopoiesis can have the opposite effect (i.e. enhancement of proliferation) on cell growth of AML cells. To test the importance of this mechanism, we systematically assessed the functional relevance of 98 cytokines and their receptors in primary human AML cells and identified interleukin-1 (IL-1) and its receptor as critical determinants in aberrant regulation of AML cell growth. Methods: To identify functionally important cytokine signaling pathways in AML pathogenesis we first quantified the proliferation of 50 primary AML patient samples in the presence of graded concentrations of 98 cytokines. We then employed a functional siRNA screen targeting 188 cytokine and growth factor receptors found to be highly expressed in 140 primary leukemia samples by gene expression analysis. Cytokine pathways of interest were functionally tested for their role as potential therapeutic targets utilizing primary patient samples and in vivo murine models. Results: We found that 40% of primary AML samples exhibited a 3- to 20-fold increase in cellular growth and a 2-fold decrease in apoptosis in the presence of IL-1α/β. Paradoxically, IL-1 suppressed the growth of normal CD34+ cells. Silencing of the IL-1 receptor, IL1R1, reduced the viability of these AML primary samples by 60-80%. Notably, most of the IL-1-sensitive AML samples exhibited monocytic and myelomonocytic features. To demonstrate the importance of IL-1 signaling in the survival of AML cells, we utilized IL1R1-/- mice and oncogene-induced leukemic cells in vitro and in vivo. We observed that IL1R1-/- mice and wild-type mice showed a normal distribution of stem and progenitor populations in their marrow by FACS analysis. Further, the absence of IL1R1 in murine bone marrow leads to a significant ablation of clonogenic potential (80% reduction) of oncogene-induced leukemic cells (AML1-ETO9a, NRASG12D and MLL-ENL) when compared to oncogene-induced leukemic cells from wild-type mice in a ligand-dependent manner. No difference in colony growth was observed with empty vector-transduced marrow cells. In a murine bone marrow transplantation model, recipients of IL1R1-/- marrow transduced with AML1-ETO9a/NRASG12D survived significantly longer for a median of 39 days (range: 28-118) compared to 30 days (range: 27-61) for recipients of wild-type marrow (p=0.012). Mice transplanted with IL1R1-/- marrow or wild-type marrow showed comparable white blood cell and platelet counts. However, histopathological analysis showed a significant reduction in myeloid infiltrates in liver and lungs as well as reduced marrow cellularity and reticulin fibrosis in IL1R1-/- leukemic mice as compared to wild-type leukemic mice. To exclude the possibility that this difference was related to differences in homing and engraftment of wild-type and IL1R1-/- bone marrow cells, we compared short-term homing and long-term engraftment of empty vector-transduced wild-type and IL1R1-/- marrow cells and found no significant differences. These results demonstrate an in vivo role of IL1-receptor in AML cell proliferation and progression. Mechanistically, exogenous IL-1 promotes the growth and survival of primary CD34+ AML cells and AML cell lines by increasing p38 MAPK phosphorylation. Conversely, knocking down IL1R1 or treating AML cells with p38 kinase inhibitors such as doramapimod (n=10; median IC50: 70 nM) reduced the growth of AML cells by decreasing phosphorylation of p38 kinase. No toxic effect of doramapimod was observed on normal cell growth, suggesting that targeting p38 kinase might be therapeutically beneficial for a subset of AML patients dependent on IL-1 signaling. Conclusion: These results demonstrate a novel in vitro and in vivo role for IL-1 and its receptor in promoting leukemic cellular growth and progression in a large subset of AML patients and warrant further investigation of this pathway as a therapeutic opportunity. Disclosures No relevant conflicts of interest to declare.

The gene KDM6A/UTX was recently identified as a putative tumor suppressor (van Haaften et al). The gene is frequently lost in a variety of malignancies but most commonly in esophageal squamous cell carcinomas (8%) and multiple myeloma (10%). The initial characterization of UTX abnormalities in multiple myeloma utilized a mixture of 58 cell lines and patient samples. To establish the frequency of UTX inactivation in multiple myeloma we studied the copy number profiling dataset (n=239) available from the Multiple Myeloma Genomics Initiative (MMGI) and our internal dataset of 68 human myeloma cell lines which were characterized by array-based CGH (Agilent 400k), whole exome sequencing, and mRNA sequencing. We identified homozygous deletions in 2.5% of patients and 26.5% of cell lines. There are no mutations in the available whole genome and exome sequencing data from 38 patients in the MMGI that partially overlaps the 239 copy number samples sequencing. However, in the cell lines another 14.3% of samples showed loss of UTX through a variety of mechanisms (missense, nonsense, frameshifts, single exon deletions and insertions of LINE elements) detected by exome and mRNA sequencing, bringing the total percentage of cell lines with UTX inactivation to 40.8%. The increased frequency of UTX inactivation in cell lines likely suggests that it is associated with a more aggressive disease or promotes the growth of the tumor outside of the normal bone marrow microenvironment. As part of the cell line characterization phase of the project we found that ARD has a homozygous deletion encompassing the entire UTX locus while its isogenic counterparts, ARP-1 and CAG, express wild-type UTX at a level comparable to other cell lines without UTX abnormalities. To ensure these isogenic cell lines represent the same tumor not different tumors from the same individual we cloned the immunoglobulin receptor rearrangements in all there lines, which confirmed these are derived from the same original tumor. To develop this model system we first generated single cell clones from each of the three cell lines. Six clones from each were randomly selected and their DNA content was determined by flow cytometry. In all cases the clones matched the parental population in ploidy (ARD 3.2N, ARP 3.3N, CAG 3.8N). We randomly select one subclone from each cell line to characterize in detail using spectral karyotyping, array-based CGH, whole exome sequencing, mRNA sequencing, and CHIP-seq for H3K4me3, H3K27ac, H3K27me1, H3K27me2, and H3K27me3. The detailed characterization of this isogenic model system will make it an excellent platform for future work aimed at understanding how UTX loss contributes to myelomagenesis. Citation Format: Kristi Allen, Venkata Yellapantula, Quan Zhao, David Edwards, Marta Chesi, Leif Bergsagel, John Carpten, Jonathan Keats. Characterization of an isogenic model system for KDM6A/UTX loss in multiple myeloma. [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 5209. doi:10.1158/1538-7445.AM2013-5209

Abstract 2920 Human myeloma cell lines (HMCL) provide both a discovery and validation platform to improve our understanding of the molecular pathogenesis of multiple myeloma. We have completed a project to characterize the underlying genetics of all commercially available HMCL with a primary goal of identifying appropriate model systems for findings from large scale patient studies like the multiple myeloma genomics initiative (MMGI). We first purchased all 33 commercially available HMCL from DSMZ, JCRB, ECACC, and ATCC. Subsequently each HMCL was thawed and cultured under strict parameters, which yielded cells for analysis, by Agilent 400k CGH, whole exome sequencing (Agilent 70Mb Exon+UTR), and mRNA sequencing. The combination of these three assays provides a detailed map of the genetic complexity underlying this deadly disease. For variant discovery, alignment was done using BWA followed by indel realignment, quality recalibration and duplicate removal. High quality calls were identified from the intersection of variants called by both Samtools and GATK. This identified a median of 32691 high confidence variants per sample with upper and lower quartile values of 34307.75 and 32241.25, respectively. To identify likely somatic mutations, we removed variants found in the 1000 genomes project and the NHLBI Exome Sequencing project. In addition, we removed variants present in dbSNP unless these mutations were also present in the COSMIC database. After these filtering steps a median of 702 potential mutations remained. From these lists we identified a median of 209.5 non-synonymous variants per sample and in genes which are typically expressed in the cohort, a median of 91 variants were found. Overall, these steps identified 2678 variants in 1978 genes. The primary goal was to identify appropriate models for novel findings from studies like the MMGI. For instance we identified HMCL with mutations in FAM46C and DIS3 among others. Secondarily, we focused on attempting to identify potential oncogenes and tumor suppressors through the integration of our three data types and data from published studies (Chapman et al. and Walker et al.). To identify potential oncogenes we focused on mutations that occurred at the same position in the genome or altered the same amino acid at a minimum. This identified 23 genes; including expected genes like KRAS (n=11) and, NRAS (n=7) but it also identified potentially activating mutations in IKBKB, SOX2, KDM4C, CD81, OSBP, NOTCH2, WDR92 and UBR2. To identify potential tumor suppressors we focused on genes that are typically expressed, which showed bi-allelic inactivation in two or more samples by either a homozygous deletion event, a deletion plus mutation, or two independent mutations. This identified 116 genes; including expected genes like TP53, CDKN2C, RB1, BIRC2/3, TRAF3, KDM6A, CDKN1B, FAM46C, and DIS3. Outside of the expected genes we identified recurrent inactivation in ANKRD11, ATP6AP1, ATXN1, BCL2L11, CDK8, RNF7, STS, TSPAN7, and TBL1XR1. These studies have highlighted the value in studying HMCL as most novel genes reported from recent studies were independently identified in this small cohort of samples. This is in large part because HMCL provide an unlimited DNA and RNA resource that allowed for multiple independent assays to be performed on each sample. Ultimately this study will provide the myeloma community with a detailed resource from which they can acquire appropriate model systems for their research goals from the various cell line repositories around the world. Disclosures: Keats: Tgen: Employment.

Background: Tyrosine kinase inhibitors (TKIs) are anticancer agents capable of specifically targeting tyrosine kinase receptors (TKR) often up-regulated in certain cancers. Sunitinib is a TKI acting on multiple TKRs including VEGFR, PDGFRβ, and FLT3. Sunitinib is approved for the treatment of advanced kidney and GIST cancers and is currently in clinical trials in other solid tumors, including NSCLC. We sought to identify in vitro microRNA (miRNA) biomarkers associated with sunitinib resistance (SR) in NSCLC cell lines. Methods: Sunitinib sensitivity was determined in seven NSCLC cell lines. Drug IC50s were determined using Alamar Blue proliferation assays. The sensitive NSCLC cell line H1703 was used to create SR cells by exposing three independent sets of cells to half the IC50 dose of sunitinib over several weeks. miRNA microarray profiling was performed on the three H1703 SR cell lines and the parent H1703 cells using the GenoExplorer miRNA Expression System (Sanger miRNA Registry version 14). Differences between the parent cells and the SR cells were identified using a t test with Benjamini-Hochberg correction and validated by qPCR. qPCR data were normalized to RNU6, and fold changes (FC) were computed against the parental H1703 line. A two-sided z-test was used to compute the p-values for the FC. To determine whether a miRNA can determine resistance across cell lines, a score was calculated using the following method. First, the FC were ternarized: significant increase (+1; FC >1 AND p-value < 0.05), significant decrease (−1; FC < 1 AND p-value < 0.05), non-significant FC(0). These values were then compared with the sunitinib IC50 of the cell lines: +1 on a resistant cell line or −1 on a sensitive cell line will be counted as one plus point, and otherwise as one minus point. A non-significant FC was not counted. Finally the score was computed as the total points divided by the number of cell lines. The “TKI resistance score” near “1” or “-1” suggests a good miRNA resistance indicator for the cell lines tested, Results: Three H1703 SR cell lines were successfully created with 2-7 fold increases in resistance as compared to the parental H1703 line. miRNA profiling identified up-regulation of miR-21, miR-23a, miR-23b, and miR-29 in the H1703 SR cells. TKI resistance scores based on qPCR data revealed miR-23b to be the best indicator of SR having a score of 1 when analyzing only the H1703 SR lines and a score of 0.67 when analyzing the H1703 SR lines together with the other six NSCLC lines. Conclusions: miRNA markers for SR exist in NSCLC cell lines as well as in H1703 cells with acquired SR. miR-23b appears to be the best potential biomarker for SR in NSCLC and if validated in clinical samples, may assist in enriching stratification for enrollment on NSCLC treatment clinical trials. Evaluation of miR-23b's mechanistic role in SR is ongoing. Supported by TGen Foundation, SHC Foundation, and the Flinn Foundation. 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 3048.

A randomized, placebo-controlled trial of human surfactant given intratracheally at birth (prophylactic) versus rescue admisistration after the onset of severe respiratory distress syndrome (RDS) was conducted among preterm infants born at 24 to 29 weeks of gestation. Singleton fetuses were randomly assigned to receive (1) placebo (air), (2) prophylactic surfactant treatment, or (3) rescue surfactant treatment; infants of multiple births received either (1) prophylactic or (2) rescue treatment. Of 282 potentially eligible fetuses, 246 infants received treatments at birth and 200 infants had RDS. Outcomes are presented both as an intention-to-treat analysis (including infants who met exclusion criteria at or atter birth) and as a full treatment protocol analysis for those infants with RDS and likely to benefit from surfactant. Preterm infants (mean 1.0 kg birth weight, 27 to 28 weeks of gestational age) randomly assigned to recelve prophylactic treatment received surfactant soon after birth; those assigned to receive rescue surfactant had instillation at a mean age of 220 minutes if the lecithin-sphingomyelin ratio was ≤2.0 and no phosphatidylglycerol was detected in either amniotic fluid or initial airway aspirate, oxygen requirements were a fraction of inspired oxygen of >0.5, and mean airway pressure was ≥7 cm H 2 O from 2 to 12 hours after birth. Up to four treatment doses (or air) were permitted within 48 hours; approximately 60% of surfactant-treated infants required two or more doses. Surfactant-treated infants had significantly less pulmonary interstitial emphysema than placebo-treated infant ( p =0.02), but there were no other significant differences in mortality rates or morbidity. Indexes of oxygenation and ventilation were improved in surfactant recipients during the first 24 hours. An intention-to-treat analysis found no significant differences between infants given placebo and surfactant-treated infants or between prophylactic- and rescue-treated infants; an improved total mortality rate ( p =0.002) was found among surfactant-treated infants in Helsinki but not in San Diego. Among infants with RDS, the total mortality rate was significantly improved ( p =0.004) with surfactant treatment but not the proportion alive and without bronchopulmonary dysplasia at 28 days ( p =0.052), or the proportion alive and without bronchopulmonary dysplasia at 38 weeks of postconceptional age ( p =0.18) to adjust for differeces in prematurity. Deaths caused by RDS or bronchopulmonary dysplasia were significantly reduced among surfactant reciplents ( p =0.0001). Neither among singletons nor among multiple-birth infants was there a selective advantage to prophylactic versus rescue treatment. Adverse effects (transient hypoxemia to 60% saturation) occurred in 1.5% of the infants treated with surfactant in the rescue group. Among preterm infants aged 24 to 29 weeks in this study, 19% did not have RDS. We conclude that surfactant treatment at 2 to 4 hours after birth (but after the onset of severe RDS) is as efficacious as treatment at birth.

We have compared the actual contribution of surfactant deficiency in respiratory distress syndrome to left-to-right shunting across a patent ductus arteriosus in premature infants with severe RDS by performing serial phospholipid analysis of tracheal aspirates and echocardiograms, and we have correlated these findings with the infants' clinical courses and management of ventilation. This analysis was combined with surgical or pharmacologic closure of PDA if a significant shunt existed. Qualitative surfactant abnormalities at birth and the subsequent maturation of phospholipids after birth were similar in all three groups of infants (Group 1, RDS alone; Group 2, RDS+PDA in infants weighing >1.2 kg; Group 3, RDS+PDA in infants ≤ 1.2 kg). The largest infants had isolated RDS (Group 1) and, after a brief period of stabilization after birth, had declining ventilatory and oxygen requirements. Infants in Group 2 had a gradual decrease in ventilatory and oxygen requirements which was accelerated markedly by cessation of PDA shunting. Infants in Group 3 had increasing ventilatory and oxygen requirements despite a maturing tracheal aspirate phospholipid pattern; their course, especially if they were asphyxiated, was characterized by early development of a significant left-to-right shunt and worsening clinical condition which improved following ablation of the shunt. The data suggest that severe RDS presents as a developmental spectrum and provides the justification for a controlled trial of very early closure of the PDA in the very low-birth-weight infant with severe RDS.

The course and complications of fifty-two infants with patent ductus arteriosus requiring closure were assessed prospectively. Twenty-six infants with a PDA received indomethacin for pharmacologic closure of the PDA, and 26 underwent ligation. The current study analyzes and compares the longitudinal follow-up with respect to somatic growth, neurologic function, psychomotor and mental development, and renal, ophthalmologic, and audiologic function in 21 infants in each group who entered the follow-up. No selective morbidity was attributable to PDA closure with indomethacin when compared to surgically treated infants.

Radiographic and clinical data were evaluated in 12 preterm infants with pneumopericardium complicating ventilator therapy of respiratory distress syndrome. Eight infants had massive or tension pneumopericardium, reflected by bradycardia, hypotension, and cyanosis of abrupt onset; cardiac size decreased dramatically but returned to approximately the former size after aspiration of the pneumopericardium. In puppies, pneumopericardium large enought to reduce heart size by 32 +/- 3% caused decreased mean arterial pressure (-22 +/- 7%) and right ventricular peak systolic pressure (-11 +/- 2%) and increased right ventricular diastolic and intrapericardial pressures. These findings suggest that pneumopericardium per se causes severe hemodynamic compromise. When it is large enought to reduce heart size, drastic circulatory impairment is produced and pericardiocentesis should be performed immediately.

I N 1 9 6 7, Northway and associates I described a form of chronic lung disease that they named bronchopulmonary dyplasia, basing their observations on newborn infants who received assisted ventilation for respiratory distress syndrome between 1962 and 1966. This disease appeared to progress through a series of four stages that were radiographically and pathologically distinct, terminating in chronic lung disease (Stage IV BPD). The radiographic appearance at each stage was characteristic, with Stage I appearing identical to uncomplicated RDS, Stage II being a period of pulmonary parenchymal opacity that cleared into a bubbly appearance (Stage III), which often ultimately became the rather distinctive pattern of chronic BPD (Stage IV).'-' Stage IV was defined chronologically as bubbly-appearing lungs persisting for 30 days or more. Subsequently this disease was observed by numerous investigators and described under a variety of names, including bronchopulmonary dysplasia, 3-1-~ pulmonary fibroplasia, 1~-1~ chronic pulmonary disease, TM respirator lung disease, ,~ and other descriptive terms? 9 -~ In most reports the diagnosis of BPD implies chronic disease, or Stage IV BPD. For most purposes, except when evolution of the condition is being discussed, this would appear to be a satisfactory convention. Since the initial descriptions of BPD, the disease appears to have changed, at least in certain of its radiographic manifestations. This article is a discussion of several recent observations, with particular emphasis on radiographic features and manifestations.

THE CHEST RADIOGRAPH is an essential modality in the diagnosis and monitoring of patients with bronchopulmonary dysplasia. 1 Some authors have questioned the degree of correlation of the radiographic staging of BPD with the pathologic staging of the diseaseY 3 Determining this correlation could facilitate attempts to establish prognosis, using changes in the chest radiograph as one of the clinical markers. The purpose of this study was to compare retrospectively the radiographic staging of BPD with the histopathologic staging of changes in the lungs of a series of patients with respiratory distress syndrome who died after receiving mechanically assisted ventilation and supplemental oxygen therapy.

The term hemifacial microsomia refers to unilateral defects in development of structures derived from the first and second branchial arches. Recently we evaluated three unrelated children who had a similar pattern of unilateral craniofacial defects that was associated with other structural abnormalities having a disruptive vascular pathogenesis. The clinical findings in these patients suggest that one cause of hemifacial microsomia is in utero interruption of blood flow.

We undertook a randomized, controlled trial to determine whether human surfactant administered endotracheally at birth to very premature infants (gestational age, 24 to 29 weeks) would prevent the respiratory distress syndrome or reduce its severity. Thirty-one treated infants (birth weight, 938 +/- 286 g) were compared in a blinded fashion with 29 control infants (birth weight, 964 +/- 174 g). The lecithin/sphingomyelin ratio was less than 2 in all infants, and phosphatidylglycerol was not present in amniotic fluid or tracheal fluids at birth, indicating a deficiency of surfactant in the lungs. The principal dependent variables were neonatal death, the incidence of bronchopulmonary dysplasia, and the infant's requirement for respiratory support (and its complications). The surfactant-treated group had significantly fewer deaths than the control group (16 percent vs. 52 percent, P less than 0.001), fewer cases of bronchopulmonary dysplasia (16 percent vs. 31 percent), and significantly fewer cases of pulmonary interstitial emphysema (P less than 0.001) and pneumothorax (P less than 0.02). Prophylactic treatment with human surfactant also substantially reduced the period of neonatal intensive care. We conclude that treatment with human surfactant offers promise for improving the survival of very premature infants with a surfactant deficiency and for reducing the pulmonary sequelae of the respiratory distress syndrome.

Idiopathic pulmonary hemosiderosis (IPH) is an uncommon but devastating disease in children [1]. The radiographic finding of a diffuse alveolar infiltrate [2], together with clinical findings of chronic, nonproductive cough, dyspnea, and irondeficiency anemia, strongly suggest IPH, but open-lung biopsy is usually necessary for definitive diagnosis [1 ]. We present a case of IPH in which MR imaging of pulmonary parenchymal hemosiderin showed a preferential T2 shortening by paramagnetic ferric iron similar to that occurring in evolving intracranial hematomas. This noninvasive diagnosis allowed initiation of therapy and stabilization of the patient’s cardiorespiratory status before lung biopsy. We suggest that in patients whose radiographic and clinical presentations suggest IPH, the MR findings permit sufficient diagnostic confidence to defer invasive maneuvers until the patient is clinically stable.

In 1967, Northway, Rosan, and Porter1 described a form of chronic lung disease that developed in certain infants who had received supplemental oxygenation and assisted ventilation for respiratory distress syndrome; they named this disease bronchopulmonary dysplasia to emphasize the pathological involvement of all lung tissues in the disease process. Subsequently, this disease was observed by numerous investigators and described under a variety of names, including bronchopulmonary dysplasia,2–10 pulmonary fibroplasia,11–13 respirator lung disease,14 chronic pulmonary disease,15 and other descriptive terms.16–22 This plethora of names is confusing and adds little to understanding of the disease. Further, despite minor theoretical objections,23 as Lynne Reid has remarked, the term bronchopulmonary dysplasia has much to commend it: it does not stipulate an etiology, and it presents the idea of a disturbance of growth.24

• Because investigation of bronchopulmonary dysplasia (BPD) has been hampered by imprecise methods for diagnosis and grading of severity, we evaluated new clinical and roentgenographic scoring systems in neonates with severe respiratory distress. The study population included 110 premature neonates who were admitted consecutively over a two-year period and who required mechanical ventilation. The clinical scoring system used measures of gas exchange, respiratory distress, and growth rate; roentgenographic scoring involved numerical assessment of features characteristic of BPD. A significant correlation was noted at 21 days of age between clinical and roentgenographic scores. In a linear stepwise multiple regression, we found that the best predictors of clinical score were birth weight (ie, degree of prematurity) and roentgenographic score. With further development and validation, BPD scoring should be helpful by improving our understanding of the epidemiology of this disease, providing a means for evaluation of treatment, and facilitating multicenter investigations. (AJDC1984;138:581-585)

Cytopathologic examinations of tracheobronchial aspirates from 108 infants sampled during mechanical ventilation demonstrated a well-defined progression of cytologic changes in bronchial epithelial cells that could be divided into three classes. Seventy percent of infants with respiratory distress syndrome who developed bronchopulmonary dysplasia had pulmonary effluent cytology designated Class III; no infants with RDS but without BPD had these cytologic findings. Additionally, a temporal progression of events involving polymorphonuclear leukocyte and macrophage populations occurred in the absence of infection; these events were associated with duration of assisted ventilation and oxygen exposure. The technique described provides a useful way to monitor the progression of lung injury and repair and offers a cytologic method to predict and diagnose the development of bronchopulmonary dysplasia.

A light and electroc microscopic study was carried out in 21 infants in whom the pathologic diagnosis of bronchopulmonary dysplasia had been made. All the infants except two had the respiratory distress syndrome at birth, and all 21 had been treated with respirator and oxygen therapy for various periods of time. The pathologic alterations observed in all the infants studied were primarily damage of the bronchial and bronchiolar ciliary apparatus and mucous membranes, severe necrotizing bronchiolitis, and marked bronchiolar and alveolar fibrosis. These changes were more pronounced in infants who survived the longest period of time. Such inflammatory and fibrotic changes are known to predispose to destruction of lung tissue, emphysema, and pulmonary hypertension. Six of these 21 infants developed symptoms and signs of cardiac atrial or ventricular stres, including cor pulmonale, prior to their demise. These infants were among those that survived the longest periods of time, had the longest exposure to supplemental oxygen, and showed histopathologically severe pulmonary fibrosis and emphysema.

• During a five-year period, 32 preterm infants weighing less than 2000 g were diagnosed as having postnatally acquired cytomegalovirus (CMV) infection in a neonatal intensive care unit. These CMV-infected infants were matched with 32 uninfected controls for gestational age, birth weight, and birth date; the two groups did not differ in Apgar scores or the incidence of respiratory distress syndrome and patent ductus arteriosus. Roentgenographic evidence of bronchopulmonary dysplasia (BPD) developed in 24 (75%) of 32 CMV-infected infants, an incidence significantly greater than that observed in control infants (12/32; 38%). Infants with acquired CMV infection required more respiratory support and longer hospitalization than uninfected controls. This association between acquired CMV infection in premature infants and the development of chronic lung disease provides further evidence that vigorous efforts to prevent CMV infection in hospitalized neonates is warranted. ( AJDC 1987;141:303-305)