Abstract: Multiple myeloma (MM) is a genetically heterogeneous cancer of bone marrow plasma cells with variable outcome. To assess the prognostic relevance of clonal heterogeneity of TP53 copy number, we profiled tumors from 1777 newly diagnosed Myeloma XI trial patients with multiplex ligation-dependent probe amplification (MLPA). Subclonal TP53 deletions were independently associated with shorter overall survival, with a hazard ratio of 1.8 (95% confidence interval, 1.2-2.8; P = .01). Clonal, but not subclonal, TP53 deletions were associated with clinical markers of advanced disease, specifically lower platelet counts (P & lt; .001) and increased lactate dehydrogenase (P & lt; .001), as well as a higher frequency of features indicative of genomic instability, del(13q) (P = .002) or del(1p) (P = .006). Biallelic TP53 loss-of-function by mutation and deletion was rare (2.4%) and associated with advanced disease. We present a framework for identifying subclonal TP53 deletions by MLPA, to improve patient stratification in MM and tailor therapy, enabling management strategies.

Abstract: Background Myeloproliferative neoplasms (MPN) commonly result from mutations in genes encoding the kinase JAK2 or the multi-functional protein CALR. In preclinical studies, estrogen receptor alpha (ERα) modulation restores normal apoptosis in JAK2V617F hematopoietic progenitors (HSPCs). Use of selective ER modulators (SERM) such as tamoxifen may permit the molecular reduction of MPNs. Methods TAMARIN is a Trials Acceleration Programme, Phase II, multicentre, single arm A'herns design clinical trial assessing tamoxifen's safety and activity in reducing molecular markers of disease burden in MPN male patients aged ≥60 years and post-menopausal female patients with stable blood counts, no history of thrombosis and ≥20% mutated JAK2V617F, CALR 5bp insertion or CALR 52bp deletion. Based on tamoxifen's safety profile in ER+ breast cancer, an oral dose of 20 mg once daily was initially given and progressively escalated to 40 mg, in addition to standard cytoreductive therapy (excluding treatments known to lower allele burden eg interferon). Mutant allele burden was measured after 12 and 24 weeks (w) of treatment. The A'herns success criteria required the primary outcome ( & gt;50% reduction in allele burden at 24w) be observed in ≥3 patients (Barosi Leuk. 2015). Patient blood (baseline, 12 and 24w) samples were collected and CD34+ HSPCs were isolated in a subset for RNA-Seq, which was also performed on HEL and UKE-1 JAK2V617F-mutated human cell lines treated with tamoxifen/vehicle. Apoptosis and oxidative phosphorylation (OXPHOS) were measured in SERM-treated cell lines for confirmation. Results and Discussion 38 patients (37% essential thrombocythaemia (ET), 29% polycythaemia vera (PV), 16% primary myelofibrosis (PMF), 13% post-PV MF and 5% post-ET MF) were recruited over 112w. 33 patients completed ≥24w of tamoxifen treatment, 1 was untreated, 1 discontinued following an unprovoked thrombotic event and 3 discontinued due to toxicity. 4 patients achieved the primary outcome and 6 additional patients met the secondary outcome (≥25% reduction)(A-B). Responders included 4 JAK2V617F PV males, a JAK2V617F PMF female and ET patients of both genders carrying JAK2V617F, CALRdel52 or CALRins5 mutations. 4 patients remain on trial treatment beyond 48w as they are considered to be deriving clinical benefit. Two grade 3 adverse events unrelated to tamoxifen, as well as 1 superficial thrombophlebitis and 1 deep vein thrombosis (grade 2) occurred on study. HSPC transcriptome seggregates responders and non-responders perfectly at baseline (C), suggesting a potential predictive signature of response. Pathway analysis of differentially-expressed genes shows enrichment of myeloid differentiation and hormone-dependent transcriptional complex assembly in responders at baseline. In contrast, chromosome segregation, DNA replication, and chromosome condensation pathways are enriched in non-responders. Gene-set enrichment analysis (GSEA) reveals increased apoptosis and oxidative phosphorylation (OXPHOS) signatures in responders at baseline (D). Upregulated genes in responders are associated with H3K4me1 modification whilst genes upregulated in non-responders are associated with H3K9me3, suggesting the possibility that chromatin modifications account for tamoxifen sensitivity. 24w after treatment, OXPHOS and ROS pathways are downregulated in responder HSPCs (E) but upregulated in non-responders (F), suggesting striking differences in the metabolism of HSPCs in both groups and/or the eradication of sensitive HSPCs in responders. Reduced OXPHOS pathways and deregulated expression of unfolded protein response (UPR) genes were confirmed in HEL and UKE-1 cells. In fact, tamoxifen induces dose-dependent apoptosis in HEL and UKE-1 cells, where serum deprivation or UPR inducers sensitize resistant cells to tamoxifen-induced apoptosis, which is associated with decreased OXPHOS and energy (ATP) production. Conclusions These results demonstrate the safety and activity of tamoxifen in reducing mutant allele burden in a subset of MPN patients who could be prospectively identified based on their transcriptomic signature at baseline. Tamoxifen can induce apoptosis of human JAK2V617F or CALR mutated HSPCs through metabolic and transcriptional effects. These results advocate for future studies to test the effects of SERMs in MPN with careful consideration of thrombotic risk. Disclosures Harrison: Roche: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria; Shire: Honoraria, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Sierra Oncology: Honoraria; Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau. Mead:CTI: Consultancy; Gilead: Consultancy; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Abbvie: Consultancy. Knapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ewing:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. McMullin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Celgene: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees. Narayanan:Novartis: Other: Educational support to attend conferences; MSD: Speakers Bureau; Celgene: Other: Educational support to attend conferences; Alexion: Speakers Bureau; Takeda: Other: Educational support to attend conferences. Milojkovic:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Drummond:Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicine Corporation: Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Tamoxifen is a selective estrogen receptor modulator frequently used in estrogen receptor-positive breast cancer.

Abstract: Introduction Malignancy can be heralded by unprovoked venous thromboembolism (VTE) but also by arterial thrombosis. To date it is unknown whether this association is present also in myeloproliferative neoplasms (MPN), in which arterial thrombosis is more frequent that venous thrombosis and solid tumors are reported with an increased frequency. The MPN-K nested case-control study addressed the impact of cytoreductive drugs on the risk of developing second cancer in MPN patients (Barbui T et al, Leukemia 2019); here we re-examined the study database to evaluate the frequency and type of vascular complications in MPN patients with second cancer excluding leukemia and to establish whether arterial and venous thrombosis during follow-up after diagnosis of MPN could predict the occurrence of a second cancer. Patients and methods Cases were patients with second cancer diagnosed concurrently or subsequent to the diagnosis of MPN. Controls were MPN patients without second cancer. For each case with second cancer, up to 3 cancer-free controls were matched by each center for sex, age at MPN diagnosis, date of MPN diagnosis, and MPN disease duration. Each set consisting of one case and their matched-controls had a similar observational period (from MPN diagnosis until the index date of diagnosis for the second cancer). The study included 647 cases with second cancer (carcinoma, non-melanoma-skin cancers, hematological secondary cancer and melanoma). The most frequent category was carcinoma (n=426, 65.8%). Cases were comparable with the 1,234 matched controls for demographics, type of MPN, and exposure to potential confounders such as mutational profile, abnormal karyotype and cardiovascular risk factors. The thrombotic events of interest were ischemic stroke, transient ischemic attacks, acute coronary syndromes, peripheral arterial thrombosis, deep venous thrombosis (including thrombosis of cerebral and splanchnic veins) and pulmonary embolism. Thrombosis had to be concurrent with or in the 2 years before MPN diagnosis or occurring after MPN diagnosis. The cumulative incidence of either arterial or venous thrombosis from MPN diagnosis was estimated by the Kaplan-Meier method and was compared between cases and controls using the log-rank test. A conditional logistic regression model estimated the Odds Ratio (OR) with 95% Confidence Interval (CI) of second cancer associated with the occurrence of thrombosis before/at diagnosis of MPN and during follow-up. Other covariates were patient age, cardiovascular risk factors, the JAK2V617F mutation, and treatment during follow-up. Results Approximately 20% of either MPN cases or controls had thrombosis before MPN or at diagnosis (19.8% vs. 21.1%, respectively, p=0.462). After a median observation time from diagnosis of MPN to an index date of 4.5 years (interquartile range 1.5-8.2) in cases and 3.7 years (interquartile range 1.5-7.5) in controls, cases showed a percentage of thrombosis higher than in controls (75/647, 11.6% vs. 100/1234, 8.1%, respectively, p=0.013). Approximately one-third of thrombosis preceding cancer occurred in the 12 months before the diagnosis of second cancer (22/75, 29.3%). The excess of thrombosis in cases was due to a higher frequency of arterial thrombosis (6.2% vs. 3.7%, p=0.015), whereas no significant difference was found for venous thrombosis (5.4% vs. 4.3%, p=0.277). While the cumulative incidence of venous thrombosis over time was similar among cases and controls (p=0.864), the cumulative incidence of arterial thrombosis was higher in cases with second cancer (p=0.006) (Figure 1). The excess of arterial thrombosis after MPN diagnosis was limited to cases with carcinoma (6.8% vs 3.9%, p=0.027). In a multivariable model, arterial thrombosis during the follow-up was confirmed to be an independent predictor factor for carcinoma, with an odds ratio of 1.97 (95%CI 1.14-3.41, p=0.015). Conclusions. These findings reveal an association of arterial thrombosis with subsequent second cancer (namely carcinoma) in MPN patients. A possible biological plausibility for this link may be related to an underlying common pathogenic mechanism such as an aberrant inflammatory response consistently found in MPN. This observation may have practical implications and suggests careful clinical surveillance for early diagnosis of second cancer in MPN patients with arterial thrombosis during the follow-up. Disclosures Palandri: Novartis: Consultancy, Honoraria. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Bonifacio:Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Rumi:novartis: Honoraria, Research Funding. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Benevolo:Novartis Pharmaceuticals: Consultancy. McMullin:Italopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Daiko Sanyo: Membership on an entity's Board of Directors or advisory committees. Griesshammer:Novartis: Consultancy, Honoraria, Speakers Bureau. Vannucchi:CTI: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Abstract: European experts were convened to develop a definition of response to treatment in polycythemia vera (PV) and essential thrombocythemia (ET). Clinicohematologic (CH), molecular, and histologic response categories were selected. In ET, CH complete response (CR) was: platelet count less than or equal to 400 × 109/L, no disease-related symptoms, normal spleen size, and white blood cell count less than or equal to 10 × 109/L. Platelet count less than or equal to 600 × 109/L or a decrease greater than 50% was partial response (PR). In PV, CH-CR was: hematocrit less than 45% without phlebotomy, platelet count less than or equal to 400 × 109/L, white blood cell count less than or equal to 10 × 109/L, and no disease-related symptoms. A hematocrit less than 45% without phlebotomy or response in 3 or more of the other criteria was defined as PR. In both ET and in PV, molecular CR was a reduction of any molecular abnormality to undetectable levels. Molecular PR was defined as a reduction more than or equal to 50% in patients with less than 50% mutant allele burden, or a reduction more than or equal to 25% in patients with more than 50% mutant allele burden. Bone marrow histologic response in ET was judged on megakaryocyte hyperplasia while on cellularity and reticulin fibrosis in PV. The combined use of these response definitions should help standardize the design and reporting of clinical studies.

Abstract: Dysregulated nitric oxide (NO) homeostasis, a consequence of hemolysis, is a central feature of endothelial dysfunction (ED) in Sickle Cell disease (SCD). In addition to ED, scavenging of NO by free heme leads to increased cell adhesion and inflammation. Vascular inflammation and the production of superoxide may decrease BH4, an essential cofactor for NO production, thus creating an acquired BH4 deficiency. Restoring BH4 levels could potentially improve ED thereby favorably impacting complications of SCD. We assessed the safety and efficacy of 6R-BH4 on endothelial function in a Phase 2a, open-label, dose escalation study in SCD subjects using a non-invasive, operatorindependent technique of peripheral arterial tonometry (Endo-PAT; Itamar, Israel). Endo-PAT (PAT) scores were quantitatively determined as the ratio between the arterial pulse wave amplitude following a 5 min arterial occlusion in the forearm to the pre-occlusion value. A value of ≤1.67 represents an impaired response or endothelial dysfunction. Only patients with HbSS and HbSC disease and at least 15 years of age were enrolled. Patients were excluded if they: were on chronic hypertransfusion; had sickle cell crisis within 30 days of screening; had a history of bone marrow or stem cell transplant or were on hydroxyurea (HU) therapy during the 3 months prior to screening. Thirty-two African-American subjects, mean age 29 years (41% male) were sequentially treated for 4 weeks each with 6R-BH4 at 2.5, 5, 10, and 20 mg/kg/day at 12 US sites. Nine subjects discontinued therapy for various reasons including loss of follow up and pregnancy. Twenty-seven subjects had baseline PAT scores and the number of subjects with PAT scores varied at each treatment dose. There were no deaths and only one subject had a drug related adverse effect resulting in discontinuation. Overall, 6R-BH4 is safe and well-tolerated in subjects with SCD. The mean PAT scores for all participants at baseline was 1.58 ± 0.43 (mean ± SD). Mean PAT scores at baseline were 1.33 ± 0.17 in 18 patients with abnormal PAT scores and 2.09 ± 0.31 (p= 〈 0.001) in 9 patients with normal PAT scores. Mean PAT score for all subjects demonstrated significant improvement at 5mg/kg/day and 10mg/kg/day (dose, N, mean change +/− SD, mean % change and p value) (5 mg/kg/day, N=24, 1.79 ± 0.64, 22.4%, p= 0.042; 10 mg/kg/day, N=24, 1.95 ± 0.46, 28.2%, p=0.003). Eighteen of the 27 (67%) subjects who had abnormal PAT scores at baseline had statistically significant dose-dependent improvements over the 16 weeks of therapy with 6R-BH4 (2.5 mg/kg, N=15, 1.63 ± 0.37, 24.7%, p=0.012; 5mg/kg, N=14, 1.69 ± 0.56, 31.2%, p=0.025; 10mg/kg, N=15, 1.84 ± 0.47, 39.9%, p 〈 0.001; 20mg/kg, N=15, 2.01 ± 0.76, 56.6%, p=0.005). Consistent with the mechanism of action of 6R-BH4 subjects with normal Endo-PAT scores at baseline demonstrated no improvement with therapy. HbSS subjects appear to have more ED based on PAT scores compared with HbSC subjects, although the difference was not statistically significant 1.52 ± 0.45 vs 1.67 ± 0.39. More importantly, both HbSS and HbSC subjects demonstrated an improvement in mean change in endothelial function with increasing doses of 6R-BH4 with corresponding % mean changes from baseline being 48.8% and 15.5% respectively following 16 weeks of treatment. The majority of subjects in the study (17/27; 63%) were prescribed folic acid supplement by their physicians at baseline and throughout the study. Post hoc analysis demonstrated no difference in baseline PAT scores between subjects on folic acid supplementation and those not on it (1.60 ± 0.47 vs 1.55 ± 0.37). However, patients on folic acid demonstrated a better dose response to treatment with 6R-BH4 compared to patients not receiving folic acid (2.5 mg/kg: 1.72 ± 0.38 vs 1.69 ± 0.41; 5mg/kg: 1.93 ± 0.74 vs 1.56 ± 0.38; 10mg/kg: 1.89 ± 0.51 vs 2.06 ± 0.34; 20 mg/kg: 2.09 ± 0.73 vs 1.62 ± 0.34) In summary, 6R-BH4 is safe, well-tolerated and demonstrates a dose-dependent improvement in endothelial function in subjects with SCD. Best results were achieved in those with baseline endothelial dysfunction. Improvement in ED occurs regardless of genotype. Finally, patients receiving folic acid showed a better response to 6R-BH4 than those not receiving this supplement. These data provide further support for the development of 6R-BH4 as a treatment for sickle cell disease. 6R-BH4 is a potentially new effective modulator of NO for SCD patients who have ED.

Abstract: INTRODUCTION The incidence of secondary cancer (SC) in patients with myeloproliferative neoplasms (MPN) is high and comparable to that of thrombosis. However, the identification of patient subgroups that might be at increased susceptibility of developing SC has not been systematically addressed. We report here the results of an international case-control study (MPN-K) aimed at comparing the frequency of exposure to possible causes of SC in patients with classical MPN, polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). METHODS This European Leukaemia Network (ELN) study reports MPN patients from 28 sites of 5 European countries and Israel, diagnosed in the period from 2000 to 2016. Cases were MPN patients with concomitant diagnosis of a non-myeloid SC (n=15) or its presentation during the course of the disease (n=412). Controls were MPN patients cancer-free, matched to the paired case for sex, age (±5 years), date of MPN diagnosis (±5 years), and MPN disease duration (±6 years). A multivariable conditional logistic regression model was used to estimate the effect of selected variables on total SC risk and in different types of SC. RESULTS Among 1,259 MPN patients, there were 427 cases and 832 matched controls. Cases presented melanoma (n=20; 4.7%), non-melanoma skin cancer (n=69; 16.2% - basal/squamous cell carcinoma), non-skin solid cancer (n=290; 67.9%) including breast, ovary/uterus, colorectal, upper gastrointestinal, liver/pancreas, lung, prostate/urinary, other and lymphoproliferative diseases (n=48; 11.2%) including multiple myeloma, chronic lymphocytic leukemia, low and high grade B- and T-lymphoma. At diagnosis, there were slightly more patients with PV among SC cases (n= 152; 35.6%) than controls (n=256; 30.8%), while conversely there were slightly less ET patients among cases (n=196; 45.9%) than controls (n=426; 51.2%). Cases and controls presented similar proportion of MF diagnosis (n=79 cases, 18.5% and 150 controls, 18.0%). Driver mutations (JAK2 V617, EXON-12, CALR, MPL), non-driver mutations and abnormal karyotype were equally represented in cases and controls. Other variables such as cardiovascular risk factors, exposure to cancerogens, family history of cancer and chronic inflammatory diseases were reported with similar frequency in cases and controls. After MPN diagnosis, exposure to first and other lines of treatments until the index event, with Phlebotomy (n=193; 15.3%), Hydroxyurea (n=814; 64.7%), Anagrelide (n=14; 1.1%), Interferon (n=30; 2.4%), Pipobroman (n=8; 0.6%), Busulphan (n=13; 1.0%), Ruxolitinib (n=11; 0.9%), was similar in the two groups except for aspirin that was used less frequently (p=0.043) in cases (n=320; 74.9%) compared to controls (n=664; 79.9%). In particular, the lower use of aspirin was circumscribed to non-skin solid tumors. A multivariable analysis was carried out in all patients and stratified by different type of tumors (Table). In non-skin solid cancers, the time to exposure of the MPN disease 〉 5 years (OR=2.95; 95% CI 1.54-5.66, p=0.001) and the PV phenotype (OR=2.40, 95% CI 1.15-5.01, p=0.020) were more burdened by the incidence of events than the reference ET group. No difference in SC risk was found for MF patients compared to patients with ET. Interestingly, the independent protective role of aspirin retained its statistical significance only in non-skin SC. In non-melanoma skin cancer, multivariable analysis revealed that the presence of JAK2 mutation was less associated with SC (OR=0.32, 95% CI 0.13-0.81, p=0.016) and confirmed that exposure to HU and other cytotoxic agents was associated with a significantly higher risk of SC (OR=6.00, 95% CI 1.23-29.28, p=0.027 and OR=9.80, 95% CI 1.24-77.78, p=0.031, respectively). This finding was not seen in non-skin SC and in lymphoma. CONCLUSION The considered clinical and biological features, at MPN diagnosis, were not different in cases with SC and controls. During the course of the disease, three factors significantly and independently affected the risk of SC in these MPN patients: 1) patients with PV had a 77% higher risk than those with ET, 2) patients with MPN duration of more than 5 years had a twice higher risk than those with lower duration, 3) for the first time, we documented that in non-skin solid cancers, aspirin treatment reduced SC risk of 38%. Exposure to HU and other cytoreductive drugs was confirmed as a risk factor for non-melanoma skin cancer. Disclosures Palandri: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marchetti:Gilead: Consultancy; takeda: Speakers Bureau; amgen: Speakers Bureau; janssen: Speakers Bureau. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.