SUMMARY Abnormalities in insulin/IGF-1 signaling are associated with infertility, but the molecular mechanisms are not well understood. Here we use liquid chromatography with electrospray ionization tandem mass spectrometry to show that the C. elegans insulin/FOXO pathway regulates the metabolism of locally acting lipid hormones called prostaglandins. C. elegans prostaglandins are synthesized without prostaglandin G/H synthase homologs, the targets of non-steroidal anti-inflammatory drugs. Our results support the model that insulin signaling promotes the conversion of oocyte polyunsaturated fatty acids (PUFAs) into F-series prostaglandins that guide sperm to the fertilization site. Reduction in insulin signaling activates DAF-16/FOXO, which represses the transcription of germline and intestinal genes required to deliver PUFAs to oocytes in lipoprotein complexes. Nutritional and neuroendocrine cues target this mechanism to control prostaglandin metabolism and reproductive output. Prostaglandins may be conserved sperm guidance factors and widespread downstream effectors of insulin actions that influence both reproductive and nonreproductive processes.

The mechanisms that guide motile sperm through the female reproductive tract to oocytes are not well understood. We have shown that Caenorhabditis elegans oocytes synthesize sperm guiding F-series prostaglandins from polyunsaturated fatty acid (PUFA) precursors provided in yolk lipoprotein complexes. Here we use genetics and electrospray ionization tandem mass spectrometry to partially delineate F-series prostaglandin metabolism pathways. We show that omega-6 and omega-3 PUFAs, including arachidonic and eicosapentaenoic acids, are converted into more than 10 structurally related F-series prostaglandins, which function collectively and largely redundantly to promote sperm guidance. Disruption of omega-3 PUFA synthesis triggers compensatory up-regulation of prostaglandins derived from omega-6 PUFAs. C. elegans F-series prostaglandin synthesis involves biochemical mechanisms distinct from those in mammalian cyclooxygenase-dependent pathways, yet PGF2α stereoisomers are still synthesized. A comparison of F-series prostaglandins in C. elegans and mouse tissues reveals shared features. Finally, we show that a conserved cytochrome P450 enzyme, whose human homolog is implicated in Bietti's Crystalline Dystrophy, negatively regulates prostaglandin synthesis. These results support the model that multiple cyclooxygenase-independent prostaglandins function together to promote sperm motility important for fertilization. This cyclooxygenase-independent pathway for F-series synthesis may be conserved.

Background:Treatment decisions in primary myelofibrosis (PMF) are guided by several prognostic systems based on disease-specific risk factors, including complete blood counts and cytogenetics. Patient specific comorbidities, e.g. non-hematopoietic organ dysfunction, are not incorporated into current prognostic models. Likewise, PMF risk stratification has not yet integrated large scale electronic health record (EHR) clinical data to refine these scoring systems. We have identified a PMF cohort within the Synthetic Derivative (SD), a de-identified, research-dedicated mirror of the EHR at Vanderbilt University Medical Center that contains 2.9 million individual records with 148 million ICD codes, and 125 million clinical notes. As a proof of concept, we leveraged the SD to develop a PMF cohort. We then aimed to identify novel patient specific comorbidities that may be associated with reduced overall survival (OS) in PMF via a phenome-wide association (PheWAS) study. Methods:We interrogated the SD for PMF via an algorithm that relied on ICD codes, natural language processing of physician notes, and medication history to identify high probability cases. Confirmation of PMF was based on strict hematologist review using 2016 WHO criteria. To this end, only patients with accessible hematopathology reports and cytogenetics, and more than 1 visit to the institution were enrolled. Patients with transformation to AML at presentation (e.g. within 30 days) were excluded. To evaluate each patient's overall comorbidity burden, we interrogated patient phecodes, which are grouped ICD9 codes shown to better mimic clinical phenotypes (PMID 20335276). Specifically, we extracted all ICD9 codes within 360 days of PMF diagnosis or referral and converted them to phecodes using the map available at https://phewascatalog.org/phecodes. PMF disease-related phecodes or codes that corresponded to DIPSS dependent variables were excluded. We identified 375 phecodes at PMF diagnosis, and conducted a PheWAS study to test the association of each phecode with survival. Survival was calculated as the interval between PMF diagnosis or referral and death or last follow-up (censor); patients who underwent hematopoietic stem cell transplant (HSCT) or transformed to AML were censored at that respective date. Survival from PMF diagnosis was estimated using the Kaplan-Meier method. We utilized the Cox proportional hazards model adjusted for the DIPSS predictors and evaluated the association of each comorbidity with the overall survival and reported those that are statistically significant after multiple testing adjustment (Bonferroni corrected P<0.00013). Results:We identified 193 cases of PMF from 1995-2016 that met strict pathology inclusion criteria. PMF median age of diagnosis was 59 (range 24-87), 42% were female, and 35 patients were referred greater than one year after diagnosis. Median OS was 39 months (range 1-265), with 23 patients developing AML and 40 patients treated with HSCT. Comorbidity analysis adjusted for DIPSS factors revealed five phecodes associated with reduced OS with Bonferroni correction (Figure 1); intracranial hemorrhage (HR 28.7; 95% CI 7-116; P=2.83E-06) invasive fungal infection (HR 41.1; 95% CI 7-235; P=2.90E-05), cerebral degeneration or hydrocephalus (HR 15.1; 95% CI 3-60;P=8.56E-05), encephalopathy or coma (HR 15; 95% CI 3-59; P=0.0001) and renal failure (HR 4; 95% CI 2-8; P=0.0001). Within the renal failure cohort, uric acid levels within 12 months of PMF diagnosis were elevated (N=18, mean 9.3 mg/dl) compared to the remaining PMF cases (N=132; mean 7.6 mg/dl) P=0.016. An additional 21 patient specific comorbidity patterns noted near Bonferroni cutoff (P<0.01), with highlights including pulmonary congestion (HR 6; 95% CI 1.7-21), pulmonary heart disease (HR 5.9; 95% CI 0.6-58), cardiomyopathy (HR 5; 95% CI 1.3-19), congestive heart failure (HR 4.3; 95% CI 1.3-14), and pneumonia (HR 3.3; 95% CI 1.4-7). Summary:We successfully leveraged our PMF cohort to identify potential high risk patient-specific comorbidities not included in current risk models. In addition, we illustrated the capacity to use these techniques to identify potential clinical intervention (e.g. chronic hyperuricemia and its impact on renal function). This study demonstrates the potential to refine prognostication and treatment decisions via EHR data to study large populations of rare myeloid disease. Disclosures Savona: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Patents & Royalties; AbbVie: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Background:Patients with clonal hematopoiesis (CH) in the absence of WHO-classified myeloid disease are of special interest given their increased prevalence with age, predisposition to morbid cardiovascular complications, and amplified risk of overt hematologic malignancy. Pts are often stratified by normal peripheral blood counts into clonal hematopoiesis of indeterminate potential (CHIP), or those with unexplained cytopenias as clonal cytopenias of undetermined significance (CCUS). However, less is known about pts with elevated counts and clonal hematopoiesis who do not fulfill WHO criteria for any myeloproliferative neoplasia (MPN). We leveraged Vanderbilt University Medical Center's unique biobank, BioVU, to identify the prevalence of JAK2V617Facross 48,000 pts to evaluate the clinical changes in progression from CH to overt myeloid disease. Methods:To develop a reference JAKV617Ftraining set, next generation sequencing via Illumina Trusight Myeloid Panel (NGS) was performed on BioVU samples (N=133) from pts with confirmed myeloproliferative malignancy. Of those pts, 78 harbored JAK2V617Fwith a range of variant allele frequencies (VAF). Matched samples in this training set (N=133) were also analyzed via Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAEX). SNP array JAK2V617Fvariant intensity was extracted (rs77375493; NM_004972.3(JAK2): c.1849G>T (p.Val617Phe). A regression model was built using NGS VAF as a dependent variable and MEGAEX intensity data as independent variable (r2=0.9931).Based on this model, we imputed JAK2V617FVAF for all 48,000 pts in our cohort. Pts with JAK2V617Fwere subdivided into: clinically confirmed myeloid disease, or JAK2V617Fwithout a diagnosis of MPN. Upon review of the EMR, the latter group was further dived into: 1) probable undiagnosedMPN, 2) CHIP, 3) CCUS, or 4) CH with associated elevated peripheral blood counts (CHAPbc). Only lab values after the date of JAK2V617Fdetection were included. Confirmed malignancy was defined by WHO classification of disease. Pts with evidence of possible WHO classified PV or ET with Hgb >18.5g/dl in men, >16.5g/dl in women, or PLT count >450k/mcl regardless of gender were classified as probable undiagnosedMPN. CHIP was defined as JAK2V617Fwithout abnormal counts across a patient's EMR lifetime, except when confounding events, e.g. trauma surgery or overt iron deficiency anemia, incorrectly skewed values. CCUS was defined as JAK2V617Fin the presence of unexplained cytopenias; hemoglobin (Hgb) <13.5g/dl men or <12g/dl women, leukocyte count (WBC) <3.9x10^3/uL or platelet (PLT) <135 x10^3/mcL. We classified pts with elevated blood counts who did not meet the WHO classification of MPN [e.g. WBC >10.7 regardless of gender, Hgb 18-18.5 g/dL in men or 16.0-16.5 g/dL in women with maximum Hgb no greater than 18.5g/dl in men and 16.5g/dl in women, or PLT count between 371-450k/mcl regardless of gender (and no values >450k/mcl)] as CHAPbc. Results:We identified 410 of 48,000 pts who harbored JAK2V617F(0.85% prevalence). Of those, 270/410 had clinically diagnosed hematologic malignancy including primary myelofibrosis (PMF) (79), ET (48), PV (43) and Ph-MPN NOS (29). MDS (29), AML (15), NHL (16), plasma cell dyscrasias (5), CML (3), other (3). There were 19/410 with insufficient clinical data to determine diagnosis. The remaining 121/410 JAK2V617Fpts did not have a related diagnosis. Figure 1a demonstrates imputed VAF differences in JAK2V617Fbetween known MPN vs. CH (including undiagnosed MPN) by age (mean VAF 0.44 vs. 0.17 respectively P<0.001). We identified several undiagnosed MPNs (22), which subdivided into polycythemia predominant (1), thrombocythemia predominant (16) or a combination of polycythemia and thrombocythemia (5). The remaining 99/410 cases were CH,with CHIP (55), CCUS (29), and CHAPbc (15). Ranges of blood counts appear to be a continuous variable among JAK2V617Fpts with CH or undiagnosed MPN (Fig 1b-1d). Summary: We used an unbiased approach to identify the prevalence of JAK2V617Facross all pts at a single institution. In this cohort, hematologic malignancy and CH did stratify by imputed VAF. Further, within JAK2V617FCH, CHAPbc may be differentiated from CHIP by clinical phenotype and further investigation will be required to determine its impact on patient outcomes. Disclosures Savona: Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; Selvita: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.