There are unresolved questions regarding the association between persistent leukocytosis and risk of thrombosis and disease evolution in polycythemia vera (PV), as much of the published literature on the topic does not appropriately use repeated-measures data or time-dependent modeling to answer these questions. To address this knowledge gap, we analyzed a retrospective database of 520 PV patients seen at 10 academic institutions across the United States. Taking hematologic laboratory data at ∼3-month intervals (or as available) for all patients for duration of follow-up, we used group-based trajectory modeling to identify latent clusters of patients who follow distinct trajectories with regard to their leukocyte, hematocrit, and platelet counts over time. We then tested the association between trajectory membership and hazard of 2 major outcomes: thrombosis and disease evolution to myelofibrosis, myelodysplastic syndrome, or acute myeloid leukemia. Controlling for relevant covariates, we found that persistently elevated leukocyte trajectories were not associated with the hazard of a thrombotic event (P = .4163), but were significantly associated with increased hazard of disease evolution in an ascending stepwise manner (overall P = .0002). In addition, we found that neither hematocrit nor platelet count was significantly associated with the hazard of thrombosis or disease evolution.

BackgroundEvasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo.MethodsTo investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2V617F–driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2V617F–driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis.ResultsWe found that JAK2V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2V617F-driven MPN.ConclusionLCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN.

Myeloproliferative neoplasms (MPN) are a class of hematological malignancies which result in the overproduction of myeloid lineage cells. These malignancies result in increased cytokine production and inflammation, which correlate with worsened symptom burden and prognosis. Other than bone marrow transplantation, there is no cure for myeloproliferative neoplasms. As such, treatments focus on reducing thrombotic risk, inflammation, and symptom burden. Because current pharmacological treatments carry significant side-effects, there is a need to explore low-risk therapies. One alternative is the Mediterranean diet, which is rich in anti-inflammatory foods, reduces inflammatory biomarkers, and beneficially alters the gut microbiome. Here, we performed a 15-week clinical trial of 28 individuals with MPN who were randomized to dietary counseling based on either a Mediterranean diet or standard U.S. Guidelines for Americans. Our primary objective was to determine if MPN patients were able to adopt a Mediterranean eating style when supported with dietician counseling. As exploratory endpoints, we investigated the impact of diet and inflammation on the gut microbiome. Using shotgun metagenomic sequencing, we found that microbiome diversity and composition were stable throughout the study duration in both cohorts. Furthermore, we discovered significant differences in the microbiomes between MPN subtypes, such as increased beta-dispersion in subjects with myelofibrosis. Lastly, we found several significant correlations between the abundances of multiple bacterial taxa and cytokine levels. Together, this study provides insight into the interaction between diet, inflammation, and the gut microbiome.

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Purpose: Chronic inflammation is integral to Myeloproliferative Neoplasm (MPN) pathogenesis. JAK inhibitors reduce cytokine levels, but not without significant side effects. Nutrition is a low-risk approach to reduce inflammation and ameliorate symptoms in MPN. We performed a randomized, parallel-arm study to determine the feasibility of an education-focused Mediterranean diet intervention among MPN patients. Experimental Design: We randomly assigned participants to either a Mediterranean diet or standard US Dietary Guidelines for Americans (USDA). Groups received equal but separate education with registered dietician counseling and written dietary resources. Patients were prospectively followed for feasibility, adherence, and symptom burden assessments. Biological samples were collected at four time points during the 15-week study to explore changes in inflammatory biomarkers and gut microbiome. Results: The Mediterranean diet was as easy to follow for MPN patients as the standard USDA diet. Over 80% of the patients in the Mediterranean diet group achieved a Mediterranean Diet Adherence Score of ≥8 throughout the entire active intervention period, whereas less than 50% of the USDA group achieved a score of ≥8 at any time point. Improvement in symptom burden was observed in both diet groups. No significant changes were observed in inflammatory cytokines. The diversity and composition of the gut microbiome remained stable throughout the duration of the intervention. Conclusions: With dietician counseling and written education MPN patients can adhere to a Mediterranean eating pattern. Diet interventions may be further developed as a component of MPN care, and potentially even be incorporated into the management of other chronic clonal hematologic conditions.

The Mediterranean diet, characterized by increased consumption of extra virgin olive oil (EVOO), nuts, legumes, vegetables, fruits, fish, and whole grain products, has proven to be beneficial in diseases where chronic subclinical inflammation plays a key role (Calder et al., 2011). For example, the PREDIMED (Prevención con Dieta Mediterránea) study demonstrated that a Mediterranean diet supplemented with EVOO or nuts reduced the incidence of major cardiovascular events (Estruch et. al, 2010). The Mediterranean diet's anti-inflammatory properties are attributed to its richness in phenolic compounds and main nutrients, such as: fiber, monounsaturated fatty acids, n-3 polyunsaturated fatty acids, vitamins C and E, and carotenoids (Casas et al., 2017). Overproduction of pro-inflammatory cytokines like IL-6 and TNF-α is a characteristic feature of Myeloproliferative Neoplasm (MPN) and correlates with high symptom burden and may also play a role in disease progression (Craver et al., 2018). Therefore, reduction of inflammation at the early stages of disease through low-risk interventions such as diet could lessen symptom burden and potentially blunt disease progression. To date, this nutrition trial will be the first to study the effects of the Mediterranean dietary pattern in MPN patients. To initially assess the feasibility of a Mediterranean diet intervention among MPN patients, we developed a prospective interventional proof-of-concept study of 30 MPN patients randomized (1:1) to either a Mediterranean diet supplemented with EVOO or the United States Dietary Guidelines for Americans (USDA). Inclusion and exclusion criteria are listed in Table 1 and study schematic is shown in Figure 1. The primary endpoint for this study is adherence to a Mediterranean diet with our diet curriculum. Exploratory endpoints include reduction in inflammatory biomarkers, reduction in symptom burden, and change in the gut microbiome. All participants meet once at the start of the intervention period (week 3) with a dietician for one-on-one counseling to educate the participant on the central components of the Mediterranean diet or the US Dietary guidelines and tailor the diet to meet each participant's medical needs and or cultural preferences. Participants are emailed 10 weekly installments of educational materials on their respective diet. At weeks 3 and 9, participants in the Mediterranean diet arm are given 750ml of EVOO and participants in the USDA arm receive a $10 grocery gift card. Six unannounced surveys and 24-hour food recalls are collected throughout the duration of the 15-week study. Conformity with the Mediterranean dietary pattern is assessed by the 14-item Mediterranean diet adherence score. Symptom burden is assessed using the MPN symptom assessment form (MPN-SAF). Four biological sample data points are collected during the 15-week study which includes collection of blood, stool, and urine. Complete blood count, Comprehensive Metabolic Panel, Lipid Panel, hsCRP are measured, and plasma is stored for cytokine analysis. Urine is used to quantify urine total polyphenol excretion. Stool samples are used to measure changes in the gut microbiome with diet. Since opening in October 2018, we have screened 44 potential participants. Four did not meet the inclusion criteria, 8 participants did not respond to initial surveys, and thus, did not progress to the intervention phase and were not randomized. Of the 32 participants who were randomized, 2 withdrew due to family illness. Eighteen participants have completed the 15-week study and 12 participants are currently in the active intervention phase. Demographics of the 30 participants who have completed this study or are currently receiving active intervention are shown in Table 2. In summary, this is a feasibility study to evaluate if MPN patients can adhere to a Mediterranean diet when given written curriculum and verbal counseling, and to explore whether a diet rich in anti-inflammatory properties can improve MPN symptoms. Geography was a limitation for this study, participants who were not in Southern California found it difficult to arrange travel for in-person visits. Patients who self-referred were more responsive and engaged than those recruited from clinic. Subsequent trials will test the impact of diet in a larger group of MPN patients, likely with a remotely administered intervention to obviate the need for travel. Disclosures Scherber: Incyte: Consultancy; Blueprint: Other: Ad board; Gilead: Consultancy. Mesa:Genotech: Research Funding; Celgene: Research Funding; Promedior: Research Funding; CTI Biopharma: Research Funding; Incyte: Research Funding; Samus: Research Funding; Novartis: Consultancy; La Jolla Pharma: Consultancy; AbbVie: Research Funding; Sierra Onc: Consultancy. Fleischman:incyte: Speakers Bureau.

Introduction A matched "tumor" and germline sample is required to unambiguously identify somatically acquired mutations in the blood or bone marrow of patients with hematologic malignancies or clonal hematopoiesis, particularly when variants of unknown significance (VUS) with a variant allele frequency (VAF) approximating 50% are encountered. Our goal was to identify sample sources for matched blood and germline samples to fit the following criteria: 1) non-invasive, 2) amenable to home collection by participants, and 3) stable at room temperature for an extended period. We hypothesized that saliva, comprised of mainly white blood cells as the DNA source, is a viable alternative to blood for identifying somatic hematopoietic mutations and that nail clippings provide adequate DNA devoid of contamination from blood cells to serve as a germline sample. To test this, a targeted NGS myeloid panel was performed on concurrently collected blood, saliva, and nail samples from patients with myeloproliferative neoplasms (MPN) or other clonal disorders. Methods Peripheral blood was collected in EDTA tubes, aliquoted into microcentrifuge tubes, and stored at -80°C until use. Saliva was collected using a DNA/RNA shield saliva collection kit (Zymo) and stored at -80°C until use. Fingernail or toenails were clipped into a plastic bag and stored at room temperature until use. For isolation of DNA from blood the DSP DNA Blood mini kit (QIAGEN) was used. For saliva and nails, the QIAmp DNA Investigator kit (QIAGEN) was used. After DNA quantification targeted NGS libraries were prepared from 100ng of DNA per sample using the ArcherDX VariantPlex Myeloid (SK0123) workflow. The resulting libraries were sequenced on an Illumina NextSeq500 instrument using v2 chemistry (Illumina, San Diego, CA), obtaining at least 4 millionreads per sample. The FASTQ data files were analyzed on ArcherDX Suite Analysis software (v.5.1.7) to identify SNPs, Indels, structural rearrangements, and Copy Number Variations. Results First, to test if nail clippings are a feasible source of germline DNA, we performed a targeted NGS myeloid panel on paired blood and nail from a patient with Polycythemia Vera (Patient #1). Clippings from approximately five nails was sufficient to yield 100ng of DNA. JAK2 V617F was detected at a low VAF in his nail sample (4% in nail vs 23% in blood), suggesting the presence of blood contamination. On subsequent samples, nail clippings were rinsed with water prior to DNA purification, and no MPN driver mutations were detected in washed nails (Patients #2-5). However, since processing of nails for DNA purification is time consuming and laborious, and the DNA yield from nails is quite limited, we are investigating alternative non-invasive sources for germline samples including nasal swabs. Sequencing of matched blood and saliva was performed from patients with all three MPN driver mutations, and in a patient with Clonal Cytopenia of Undetermined Significance (CCUS). All somatic mutations identified in the blood were also seen in the saliva, almost all had identical VAFs from both sample sources, even with VAFs as low as 3%. Conclusion Nail clippings are a feasible source for germline DNA in patients with hematologic malignancies and clonal hematopoiesis. Saliva is equivalent to peripheral blood for identifying somatic mutations in hematopoietic cells. These methods of sample collection are non-invasive, amenable to in home collection, and are temperature stable. Collection of saliva and nails could be easily utilized for remotely administered studies by mailing collection kits to participants, thus avoiding the cost and labor of a blood draw. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.