Diagnosis Of Essential Thrombocythemia Research Articles

Conventional Cytogenetic Analysis and Array CGH + SNP Identify Essential Thrombocythemia and Prefibrotic Primary Myelofibrosis Patients Who Are at Risk for Disease Progression.

The Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs) are a heterogeneous group of clonal hematopoietic malignancies that include polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (prePMF). In this study, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH + SNP) in patients with ET or prePMF to determine whether the combined analysis of both methodologies can identify patients who may be at a higher risk of disease progression. We performed a comprehensive genomic review on 169 patients with a clinical diagnosis of ET (154 patients) or prePMF (15 patients). Genomic alterations detected by CC or array-CGH + SNP were detected in 36% of patients. In patients who progressed, 68% had an abnormal genomic finding by either technology. There was a shorter progression-free survival (PFS) among patients who were cytogenetically abnormal or who were cytogenetically normal but had an abnormal aCGH + SNP result. Leveraging the ability to detect submicroscopic copy number alterations and regions of copy neutral-loss of heterozygosity, we identified a higher number of patients harboring genomic abnormalities than previously reported. These results underscore the importance of genomic analysis in prognostication and provide valuable information for clinical management and treatment decisions.

Polistemi Vera ve Esansiyel Trombositemi Tanısı Alan Hastalarda Elektrokardiyografik ve Ekokardiyografik Bulgularının Değerlendirilmesi

Background/Aims: Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative diseases. Along with the increase in myeloproliferative cell lines, they can cause various clinical outcomes such as arterial and venous thrombosis, pulmonary hypertension, and myocardial infarction. This study was designed to evaluate the cardiac effects of these diseases via electrocardiography and echocardiography. Methods: A total of 50 patients with a diagnosis of PV, 50 patients with a diagnosis of ET, and 50 healthy individuals as a control group were included in this study. Data on patient demographics were recorded in all subjects. All patients and control subjects had electrocardiography (ECG) recordings and routine transthoracic echocardiographic examination. Pulse wave velocity (PWV) was assessed with a Holter blood pressure device. Results: In total, 50 PV patients, 50 ET patients, and 50 control group were included in the study. The demographic characteristics of the PV, ET and control groups were similar. The PR interval was significantly shorter in control subjects than in PV and ET patients (p:0.007, p:0.024). Although the measured values were within normal limits, diastolic posterior wall thickness was significantly lower in the control group compared to PV and ET patients (p:0.019, p:0.009). PWV was significantly higher in ET patients compared to the control group (p:0.012). Conclusion: In this study, evaluating the effects of PV and ET on electrocardiography and transthoracic echocardiography; ECG parameters used to predict ventricular arrhythmias (QT, QTc, Tp-Te, Tp-Te/QT) and Pulmonary Artery Pressure showed no significant change, in opposition to existing literature. Nonetheless, similar to previous publications, PV and ET were found to negatively affect the diastolic function parameters on transthoracic echocardiography. While the aortic stiffness was significantly higher in ET patients compared to the control group, no significant difference was noted between PV patients and control subjects in terms of aortic stiffness.

An Approach to the Investigation of Thrombocytosis: Differentiating between Essential Thrombocythemia and Secondary Thrombocytosis.

Thrombocytosis is a common reason for referral to Hematology. Differentiating between secondary causes of thrombocytosis and essential thrombocythemia (ET) is often clinically challenging. A practical diagnostic approach to identify secondary thrombocytosis could reduce overinvestigation such as next generation sequencing (NGS) panel. All adult patients with thrombocytosis (≥450 × 109/L) who underwent molecular testing at a single tertiary care centre between January 1, 2018 and May 31, 2021 were evaluated. Clinical and laboratory variables were compared between patients with secondary thrombocytosis vs. ET. Clinical variables included smoking, thrombosis, splenectomy, active malignancy, chronic inflammatory disease, and iron deficiency anemia. Laboratory variables included complete blood count (CBC), ferritin, and myeloid mutations detected by NGS. The overall yield of molecular testing was 52.4%; 92.1% of which were mutations in JAK2, CALR, and/or MPL. Clinical factors predictive of ET included history of arterial thrombosis (p < 0.05); active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency were associated with secondary thrombocytosis (p < 0.05). A diagnosis of ET was associated with higher hemoglobin, mean corpuscular volume (MCV), red cell distribution width (RDW), and mean platelet volume (MPV), while secondary thrombocytosis was associated with higher body mass index, white blood cells, and neutrophils (p < 0.01). A practical approach to investigating patients with persistent thrombocytosis based on clinical characteristics such as active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency may assist in accurately identifying patients more likely to have secondary causes of thrombocytosis and reduce overinvestigation, particularly costly molecular testing.

Presence and Onset of Chronic Kidney Disease as a Factor Involved in the Poor Prognosis of Patients with Essential Thrombocythemia.

Chronic kidney disease (CKD) is an important risk factor for cardiovascular disease, thrombosis, and all-cause death. However, few studies have examined the association between CKD and the prognosis of patients with essential thrombocythemia (ET). We collected ET patients who met the WHO classification 2017 and performed a retrospective clinical study to clarify the association between the presence and onset of CKD and prognosis. Of 73 patients who met the diagnostic criteria, 21 (28.8%) had CKD at the time of ET diagnosis. The age of patients with CKD was significantly higher, and a high proportion of these patients had the JAK2V617F gene mutation. The presence of CKD was a risk factor for the prognosis (hazard ratio (HR): 3.750, 95% confidence interval (CI): 1.196-11.760, P=0.023), and the survival curve was significantly poorer. Furthermore, we analyzed patients without CKD at the time of ET diagnosis using the onset of CKD as a time-dependent variable and identified the onset of CKD as a risk factor for the prognosis (HR: 9.155, 95% CI: 1.542-54.370, P=0.005). In patients with renal hypofunction at the time of ET diagnosis or those with a reduction in the kidney function during follow-up, strict renal function monitoring at regular intervals is necessary.

Essential Thrombocythemia in Pregnancy: A Case Report

Essential thrombocythemia is a hematological problem that occurs during pregnancy. Diagnosis of essential thrombocythemia is difficult because it is a diagnosis of exclusion and there is a higher frequency of thrombocythemia due to other causes. A 26-year-old female patient with G1P0000 gestational weeks 30–31 came to the gynecology clinic after laboratory examination with thrombocyte count of 1,128,000 μL. There were no complaints of dizziness, nausea, vomiting, tingling, or fever. She was fatigue with compos mentis consciousness, with blood pressure 110/70 mmHg, heart rate 85 times/minute, respiratory rate 20 times/minute, and temperature 36.5 °C. Head to toe examination was within normal limits. An obstetric status examination was obtained. Fundal height: 3 fingers below the xiphoid process (31 cm, with estimation fetal weight by McD formula 2945 grams) and fetal heart rate 134 times/minute. Blood smear shows leukocytosis and thrombocythemia suspected myeloproliferative disorder (MPD), an essential thrombocythemia. She was tested for the JAK2V617F mutation but no mutation was detected. Doppler ultrasound test shows umbilical artery (RI: 0.69; PI: 0.95; S/D ratio: 2.76) and middle cerebral artery (RI: 0.74; PI: 1.48; S/D ratio 3.88). She was consulted to hematooncologist and was given aspirin 80 mg per day, Cal-95 1 tablet per day, and prenatal vitamins. Evaluation of patients with essential thrombocythemia includes a complete blood count, bone marrow biopsy, and genetic testing to evaluate gene mutations to obtain the appropriate diagnosis and therapy to prevent from its complication such as thrombosis during pregnancy, bleeding during pregnancy, gestational hypertension, preeclampsia, eclampsia, prematurity, placental abruption, intrauterine growth retardation and stillbirth.

Correlation between IPSET-t risk at diagnosis and subsequent hemorrhage in patients with essential thrombocythemia; a single institution experience.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by an increased risk of thrombotic and hemorrhagic events, that represent the leading causes of mortality and morbidity. Currently, while thrombotic risk is assessed through the IPSET-t and r-IPSET scores, there is no specific prognostic tool used to predict hemorrhagic risk in ET. The aim of the study was to define incidence and risk factors connected to hemorrhagic events by retrospectively analyzing 308 ET patients diagnosed between 1996 and 2022 at the Division of Hematology of Udine and treated according to the current international guidelines. According to molecular status, 193 patients (62.7%) were JAK2 mutated, 66 (21.4%) had a CALR mutation, 14 (4.5%) had a MPL mutation, 21 patients (6.8%) were "triple negative," and 14 patients (4.5%) were not evaluable. According to IPSET-t score, 49.7% patients were at high, 24.3% at intermediate, and 26.0% at low-risk, respectively. Twelve (3.9%) patients experienced bleeding at ET diagnosis, while 24 (7.8%) had at least one hemorrhagic event during follow-up at a median time of 103 months (range: 1-309). Forty hemorrhagic events were totally recorded and defined as minor in 22 cases, moderate in 11 cases, and severe in 7 cases. Cumulative incidence (CI) of hemorrhage at 10 and 20 years was 6.0% and 12.0%, respectively. A statistically significant correlation between hemorrhagic risk and IPSET-t score emerged: 10 years hemorrhage CI was 3.2% for low-risk, 2.9% for intermediate-risk, and 9.8% for high-risk patients, respectively (p=0.002). We found no correlation between hemorrhagic risk and gender or mutational status. Results of our study highlight the validity of IPSET-t score in predicting individual hemorrhagic risk among ET patients, suggesting a possible role of IPSET-t scoring system as a global evaluator for vascular events in ET patients.

Clinical and Disease Characteristics of Patients with Myelofibrosis and Essential Thrombocythemia Who Harbor a Calreticulin (CALR) Gene Mutation: Subanalysis of the MOST Study

Background: Calreticulin(CALR)gene mutations are the second most frequent driver of disease in myelofibrosis (MF) and essential thrombocythemia (ET). Compared with patients with JAK2 mutations, those with CALR mutations are considered to have lower-risk disease; however, prospective data on clinical characteristics and disease progression for patients with CALR mutations are limited. This study compared the disease characteristics and treatment patterns of patients with MF or ET who had CALR mutations vs those who had JAK2 mutations at enrollment in the Myelofibrosis and Essential Thrombocythemia Observational Study (MOST; NCT02953704). Methods: Patients enrolled in MOST were aged≥18 years with clinical diagnoses of MF or ET at clinical practices within the United States. Patients with MF included those with primary or secondary MF who were low risk (or intermediate-1 by age &amp;gt;65 years alone) per the Dynamic International Prognostic Scoring System (Passamonti F, et al. Blood. 2010;115:1703-1708). Patients in the ET cohort had a clinical diagnosis of ET and were ≥60 years or had history of thromboembolic events or were receiving ET-directed therapy at enrollment. Descriptive statistics were used to summarize the data. The median (range) follow-up time for CALR- and JAK2-positive patients was 52.8 (43-68) months and 56.6 (42-70) months, respectively. ET progression was defined as transformation to MF; MF progression was defined as worsening of objective clinical and laboratory assessments of disease. Results: A total of 881 patients ( CALR-positive, n=137 [16%]; JAK2-positive, n=750 [85%]; 6 patients had both CALR and JAK2 mutations) were included in this study. A summary of patient and disease characteristics are presented in Table 1 and Table 2. At enrollment, 25% and 75% of CALR-positive patients had a diagnosis of MF and ET, respectively, vs 16% and 84% of JAK2-positive patients. The majority of patients in both groups were women ( CALR-positive, 56%; JAK2-positive, 65%). CALR-positive patients were younger at enrollment and at diagnosis compared with JAK2-positive patients (median age at enrollment, 63.0 [24.0-89.0] vs 70.0 [26.0-93.0] years, P&amp;lt;0.001; median age at diagnosis, 59.3 [21.0-87.0] vs 66.3 [22.0-93.0] years, P&amp;lt;0.001). The majority of patients in this analysis (79% of CALR-positive, 90% of JAK2-positive) were receiving MPN-directed therapy at enrollment. The most common therapies in the CALR cohort were hydroxyurea (51%), interferon (12%), and anagrelide (8%); the common therapies among patients in the JAK2 cohort were hydroxyurea (66%), aspirin only (10%), and ruxolitinib (8%). Disease burden at enrollment was also assessed. The percentages of patients with white blood cell (WBC) count &amp;gt;10×10 9/L or platelet count &amp;gt;400×10 9/L were similar for both CALR- and JAK2-positive patients (13% vs 20% and 53% vs 51%, respectively). Percentages of patients with splenomegaly (SPM) were similar for CALR- vs JAK2-positive patients at enrollment (11.0% vs 13%). The mean MPN-SAF TSS was similar at baseline for CALR- and JAK2-positive patients (15.1 vs 16.5). The percentage of patients with MPN-SAF TSS &amp;gt;25 was 12% in the CALR-positive and 16% in JAK2-positive cohorts. Disease complications and progression were also assessed. Thrombotic and hemorrhagic events during the study period were infrequent in both CALR- and JAK2-positive patients (2% vs 4% and 1% vs 3%, respectively). Rates of disease progression were similar for CALR- and JAK2-positive patients, both for MF progression (50% vs 61%) and for ET progression to MF (2% vs 5%). Leukemic transformation occurred in 6 (4%) CALR-positive patients and 43 (6%) JAK2-positive patients. Deaths due to disease progression occurred in no CALR-positive patients and in 18 (2%) JAK2-positive patients. Conclusions: The results of this real-world analysis of the MOST study demonstrate that despite nearly 80% of CALR-positive patients receiving MPN-directed treatment, a substantial percentage of this group did not meet treatment goals, including control of symptom burden, WBC and platelet counts, and SPM. In addition, disease progression was observed in 50% of the patients with MF in the CALR-positive cohort. Further analysis of the MOST study may provide insight into optimal management of patients with CALR mutations.

A Real World, Single-Centre Study of Young Adult Patients with Philadelphia Negative Myeloproliferative Neoplasms

Classical ‘Philadelphia negative’ myeloproliferative neoplasms (MPNs) are clonal haematopoietic disorders and include essential thrombocythaemia (ET), polycythaemia vera (PV) and myelofibrosis (MF). MPNs have a heterogenous phenotype with an inherent risk of thromboembolic and haemorrhagic complications, and risk of blastic transformation. MPNs are commonly diagnosed in the 6 th decade or later, although up to 20% of patients are diagnosed &amp;lt; 40 years. Current risk stratification and treatment guidance, however, is frequently extrapolated from older cohorts. Here, we present a comprehensive retrospective analysis of 107 MPN adolescent and young adult (AYA) patients diagnosed &amp;lt;40 years, representing 19.6% of the total outpatient MPN cohort at University College London Hospital. Median follow up was 78 months (range (r), 1 - 507). Table 1 displays clinicopathological details of the entire cohort. Median age at diagnosis was 32 years (r,13-40). There was a female preponderance (66%); 57.9% of patients were Caucasian. Most patients had a diagnosis of ET (57.9%), with 24.3% PV and 9.3% MF, respectively. The most common driver mutation was JAK2 V617F, observed in 60.7%, followed by CALR (15.9%) and MPL (1.9%); 17% of patients were classified as triple negative (TN). Where data was available (n=27), median variant allelic frequency (VAF) for JAK2 V617F was 19.7% (r, 4-75%) and for CALR 43% (r, 5.7 - 53). 9/107 (8.4%) of patients had additional pathogenic gene abnormalities, most commonly variants of NRAS (22.2%), TET2 (22.2%) and ASXL1 (22.2%). We observed a high thrombosis rate of 19.6% (21/107); the majority had a diagnosis of ET (61.9%).. In 80% of such cases, this was the heralding event leading to the MPN diagnosis. Venous thrombosis was more frequent than arterial thrombosis (68% vs 33%). In particular, there was a disproportionate number of venous thromboses at unusual sites including cerebral venous sinus and splanchnic venous thromboses, representing 40% and 33% of total events, respectively. Only 3 (2.8%) patients were found to have a co-existent thrombophilia. Younger age at diagnosis appeared to associate with a higher rate of thrombosis (60% in patients aged &amp;lt;20, 18.2% in patients aged 20-30, and 17.2% in patients aged 31-40). Thrombotic events were most common in those with a JAK2V617 mutation (23.4%), followed by those who were TN (20.0%). Only 5.9% of CALR mutated cases had a thrombotic event. In terms of cardiovascular risk factors, 13.0% (14/107) and 6.5% (7/107) of patients had diagnoses of hypertension and type 2 diabetes, respectively. Haemorrhagic events were reported in 8.4% (9/107) of patients; 3.7% (4/107) had grade 3 or 4 bleeding complications, requiring transfusion and/or surgical intervention. All who experienced haemorrhagic events were on treatment with an anti-platelet or anti-coagulant agent. 4.7% (5/107) were diagnosed with acquired von Willebrand's disease, of whom one experienced a grade 3 haemorrhagic event. No patients transformed during follow up. 33 patients reported pregnancies, with 7/33 (21%) reporting antenatal or perinatal medical complications. Over 50% (55/107) of patients were commenced on cytoreduction. Most common agents employed were pegylated interferon (45.5%; 25/55) and hydroxycarbamide (43.6%%; 24/55). No patients required combination therapy. 92/107 (86%) patients were treated with an antiplatelet agent and 9/107 (8.4%) were on anti-coagulant treatment with the majority (88.8%) receiving warfarin rather than a DOAC. AYA MPN patients have unique characteristics and heterogeneous clinico-biological features differentiating them from older MPN cohorts. Our data highlights a female preponderance, higher incidence of venous thrombosis (particularly atypical sites), and lower incidence of arterial thrombosis. Whilst traditionally younger patients are considered to have ‘low risk’ disease, this real-world study highlights a significant thrombosis rate of ~ 20% which may have associated morbidity considering the relatively longer duration of the disease course and need for cytoreductive therapy. Specific AYA MPN directed risk stratification and therapeutic algorithms are required to inform holistic and age-appropriate patient management.

No Increased Incidence of Bleeding in Essential Thrombocythemia Patients with Extreme Thrombocytosis

CONCLUSIONS About 25% of essential thrombocythemia (ET) patients present with extreme thrombocytosis (ExT), defined as having a platelet count ≥1000 x 10^9/L. ExT patients may have an increased bleeding risk, at least in part associated with acquired von Willebrand disease. There are no specific guidelines for managing ExT patients, and many are started on cytoreduction, even if they are younger and otherwise at low thrombotic risk by the revised International Prognostic Score of Thrombosis for ET (R-IPSET). We analyzed the risk of bleeding and thrombosis in ExT vs. non-ExT ET patients to inform treatment decisions. METHODS : We retrospectively analyzed patients who received targeted gene sequencing at Massachusetts General Brigham / Dana-Farber Cancer Institute from 2014-2021 and met WHO 2016 criteria for ET. We performed medical record review and recorded information on treatment, diagnosis, blood counts, and von Willebrand antigen and activity levels. We abstracted the first major bleed, clinically relevant non-major bleed (CRNMB), and thrombotic event in a patient's disease course. Bleeding was categorized using the International Society on Thrombosis &amp; Haemostasis definitions.We determined the cumulative incidence rates of bleeding and thrombosis in ExT and non-ExT patients with death as a competing event and compared these rates using a Gray test. Risk factors for bleeding and thrombosis were evaluated using univariate and multivariate Fine Gray models. We identified 408 patients diagnosed with ET, of which 344 had a platelet count reported at diagnosis. There were 96 ExT patients and 248 non-ExT patients. Median follow-up was 11 and 6 years, and median platelet count at ET diagnosis was 1194 and 651, respectively. ExT patients, compared with non-ExT patients, were more likely to be younger (51 vs. 58 years old, p&amp;lt;.001), have the CALR mutation (49% vs. 23%, p&amp;lt;.001), have lower IPSET-thrombosis risk (65% vs. 49% very low or low risk, p&amp;lt;.001), and have lower von Willebrand activity levels at diagnosis or in disease course (60% vs. 94%, p&amp;lt;.001, Table 1). There were 7 (7%) major bleeds and 7 (7%) CRNMBs in the 96 ExT patients, and 11 (4%) major bleeds and 22 (9%) CRNMBs in the 248 non-ExT patients (p=.29 for major bleeding, p=.83 for CRNMB). 10 year cumulative incidence of major bleed and CRNMB was not different in ExT vs. non-ExT patients (4% vs. 5%, p=.68 for major bleed; 3% vs. 10%, p=.27 for CRNMB, Figure 1). Despite 65% of ExT patients in our sample categorized as very low or low risk for thrombosis, these patients were more likely to be treated with cytoreduction compared to non-ExT patients (63% vs. 50%, p=.21 for very low risk; 81% vs. 51%, p=.022 for low risk). However, there was no difference in the 10 year cumulative incidence of major bleeding and CRNMB in ExT patients who were treated vs. not treated with cytoreduction (6% vs. 0%, p=.13 for major bleed; 2% vs. 8%, p=.13 for CRNMB). There was also no difference in the cumulative incidence of bleeding in ExT and non-ExT patients who were not treated with cytoreduction (untreated or aspirin alone, 0% vs. 4%, p=.34 for major bleed; 8% vs. 9%, p=.75 for CRNMB). In univariate analysis, diabetes mellitus and thrombosis during disease course were significantly associated with increased combined major bleeding and CRNMB. In a multivariable analysis adjusting for these factors in addition to platelet count ≥1000 x 10^9/L and age at ET diagnosis, first von Willebrand factor activity, aspirin and cytoreduction during disease course, and JAK2 mutation status, diabetes mellitus (HR 3.45, 95% CI 1.31-8.72) and thrombosis during disease course (HR 2.92, 95% CI 1.41-6.03) remained as significant risk factors for bleeding. Cytoreduction was not significantly associated with a lower rate of bleeding in multivariable analysis (HR .42, 95% CI .09-3.06). 10 year cumulative incidence of thrombosis was not different in ExT vs. non-ExT patients (22% vs. 26%, p=.94). We found no difference in the cumulative incidence of bleeding and thrombosis in ET patients with ExT and importantly, no clear role for initiation of cytoreductive therapy in these patients who are otherwise low risk by R-IPSET. This confirms the current recommendations to initiate cytoreduction based on thrombotic risk and suggests that ExT alone should not be an indicator for cytoreduction.

Calr Variant Allele Frequency in Essential Thrombocythemia: Molecular Associations and Impact on Disease Phenotype and Outcome

Background. A mutation in calreticulin ( CALRm) is found in 30-35% of patients (pts) with Essential Thrombocythemia (ET). There are 2 main CALRm mutation types, Type 1/Type 1 like (T1) a 52bp deletion, and Type 2/Type 2 like (T2) a 5bp insertion. Other atypical mutations (T3) occur in a minority of pts. Recent study ( Guglielmelli P et al, BCJ 2023) in pts with myelofibrosis (MF) showed that higher CALR variant allele frequency (VAF) was associated with anemia at diagnosis and during FU, and to features of more advanced disease (CD34 + cell counts, ASXL1 mutation (mut), &amp;gt;2 mutated myeloid genes, and shorter leukocytosis-free survival. Aim. To evaluate whether the CALRm VAF in pts with ET was associated with predefined major clinical outcomes: evolution to MF, transformation to acute leukemia (AML), thrombosis, major bleeding, and survival (OS). Patients and Methods. Diagnosis of ET was strictly according to 2023 WHO and ICC to avoid mis-inclusion of prefibrotic MF. CALRm VAF was determined by capillary gel electrophoresis as the ratio (%) of areas under the curve of CALRm/ CALRm+ CALRwt. A panel of 45 myeloid neoplasm-associated genes was sequenced by NGS. Results. A total of 281 CALRm ET pts were identified from CRIMM (Florence, I), Quebec MPN Research Group centers' (Canada) and Mayo Clinic (Rochester, US) databases; 152 (54%), 101 (36%) and 28 (10%) were T1, T2 and T3 CALRm, respectively. Overall FU was 8.6y (0.3-39.4), median survival was not reached. Rate of death at 10y, 20y and 30y was 8%, 15% and 25%. MF transformation occurred in 50 pts (18%), AML in 2 (0.7%), 25 pts (8.9%) died. A major thrombotic event before or at ET diagnosis occurred in 17 (6.0 %) pts (14 arterial and 3 venous), whereas 28 pts (10.0%) had &amp;gt;1 major thrombosis during follow-up (53.6% arterial, 46.4% venous). Major bleeding occurred in 19 pts (6.8%; 16 at or before diagnosis). Ascertainment of correlation of CALRm VAF with predefined outcomes by continuous variable analysis (ROC curve) highlighted a significant correlation of VAF &amp;gt;60% with MF-free survival (MFS), with HR of 2.85 (95%CI, 1.4-5.6; P= .002. Fig. 1A). On the contrary, there was no impact of CALRm VAF as continuous variable on AML, thrombosis, bleeding and OS. We therefore used a threshold of &amp;gt;60% to compare main clinical and hematologic characteristics at diagnosis and outcomes during FU in pts categorized as CALR-high and CALR-low. Of all pts, 21 (7.5%) harbored a VAF &amp;gt;60%; of these, 52%, 19% and 29% were T1, T2, T3, respectively, compared to 54%, 37%, 8% of CALR-low (P= .008). We found no difference in age, gender, constitutional symptoms, splenomegaly, arterial and venous thrombosis at diagnosis and FU, IPSET revised risk stratification, and bleeding events. Hemoglobin was lower (12.8 g/dL (8.4-14.0) versus 13.6 (10.1-16.0); P=.02), while leukocytes and platelets did not differ. The proportion of pts with additional mutations in myeloid genes was significantly greater in CALR-high pts compared to CALR-low (66.7% vs 30.2%; P=0.01), as it was the proportion of pts with &amp;gt;2 mut myeloid genes (25% vs 11.6%, P=0.04). During FU, 52% of CALR-high pts (n=11) transformed to MF compared to 15% (n=39) of CALR-low (P&amp;lt;.0001); AML and death not differ. Anemia (&amp;lt;10g/dL) during the FU developed in 69% of CALR-high pts vs 18%, leukocytosis (&amp;gt;15x10 9/L) in 46% vs 8% and splenomegaly (greater than 5 cm from LCM) in 50% vs 12% (P&amp;lt;.0001 for all). The HR for anemia-free survival was 2.87 (95%CI, 1.3-6.3), for leukocytosis HR 3.71 (95%CI, 1.3-10.5) and splenomegaly HR 3.40 (95%CI, 1.2-9.2), all P&amp;lt;.01. MFS was significantly shorter in T1 (HR 2.0; P=0.04) or T3 (HR 2.7; P=0.03) using T2 as reference. Finally, we compared C ALR-mut, JAK2V617F mut and triple-negative (TN) ET pts. ROC analysis indicated a JAK2VAF &amp;gt;35% ( JAK2-high) as the best cutoff for shorter MFS. As in Fig. 1B, the MFS survival of CALR-high and JAK2-high was superimposable, and significantly shorter that in CALR/JAK2-low and TN pts. Conclusions. A CALR VAF &amp;gt;60% in pts with ET is associated with greater risk and shorter time to MF progression, development of anemia and splenomegaly. Similar findings in JAK2-high pts reinforce that that accumulation of mutated CALR and JAK2 alleles, evident since diagnosis, is detrimental for evolution to post-ET myelofibrosis. Longitudinal studies after diagnosis might provide further information about the kinetics of allele accumulation.

Quantification of JAK2 V167F in Patients with Myeloproliferative Neoplasms: Comparison of NGS and Digital PCR with Quantitative PCR

Introduction: JAK2 V617F is the most common mutation in the Philadelphia-negative myeloproliferative neoplasms (MPN), detected in 97% of polycythemia vera (PV), and approximately 55% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) cases. According to the WHO, JAK2 V617F positivity is a major criteria for PV, ET or PMF diagnosis. But JAK2 V617F quantification at diagnosis could be useful since a high allelic frequency (or VAF) has been associated with increased symptom burden in PV, risk of thrombotic events in ET and progression in PMF. The current gold standard technique for JAK2 V617F quantification is allele-specific quantitative PCR (qPCR). However, digital PCR (dPCR) is increasingly being employed since it permits absolute quantification of JAK2 V617F without the need for a standard curve. QPCR vs. dPCR comparisons have shown high conformance between the two methods. Moreover, next-generation sequencing technologies (NGS) are increasingly used for MPN diagnosis. The aim of this study was to evaluate the concordance between dPCR and NGS for JAK2 V617F quantification at diagnosis. Methods: JAK2 V617F was quantified in the same sample of genomic DNA, extracted from total leukocytes from peripheral blood or bone marrow at diagnosis, by digital PCR (dPCR) and by NGS. The Absolute Q™ Liquid Biopsy dPCR Assay (JAK2 12600) was used in the QuantStudio™ Absolute Q™ Digital PCR System (Applied Biosystems) with 25 ng DNA. This assay quantifies the number of JAK2 WT-VIC and JAK2 V617F-FAM positive signals in 20000 partitions, determined as copies JAK2 V617F/µl input sample, and expressed as percentage VAF, with a limit of detection of 0.1%. NGS was performed with the MiSeq (Illumina) platform and the targeted 30-gene panel Myeloid Solution™ (SOPHiA GENETICS) using 200 ng DNA. VAF was determined as the number of variant reads divided by the number of total reads (reported as a percentage), with a limit of detection of 2.5%. Statistical analyses were performed using R-commander version 2.8. P-values &amp;lt;0.05 were considered statistically significant. Results: The quantification of JAK2 V617F was carried out in a total of 52 patient samples with both techniques. Of the 52 patients, 5 had a diagnosis of ET and 47 had PV. The average VAF of JAK2 V617F using dPCR was 14.1% and 48.2%, and 17.1% and 47.6% using NGS for ET patients and PV patients, respectively. The concordance of positivity for JAK2 V617F between the two methods was 100%. With dPCR, the average VAF of JAK2 V617F was 44.62% (range 2.7%-100%). With NGS, the average VAF of JAK2 V617F was 44.95% (range 2.5%-96.5%), with an average coverage/read depth &amp;gt;3500 reads. At diagnosis, there was no difference in the mean VAF of JAK2 V617F determined using dPCR vs. NGS (44.62 vs. 44.95, p=0.792, paired Student t-test). There was an excellent correlation between the VAF of JAK2 V617F determined using dPCR vs. NGS (Pearson correlation coefficient 0.944, p&amp;lt;0.001; Figure 1). Only 7 samples had a deviation &amp;gt;10% in the VAF quantification. These determinations are indicated in orange in the graph and listed in the table . The cause of 2/7 deviations might have been a limited DNA volume/concentration available for dPCR. The high concordance of variant quantification by dPCR andNGS was maintained for JAK2 V617F with low VAFs (≤20%, mean 12.0% vs. 11.8%, respectively; p=0.840) and high VAFs (≥80%, mean 90.6% vs. 89.9%, respectively; p=0.858). Conclusions: Overall, excellent correlation was observed between the VAF of JAK2 V617F obtained using dPCR and NGS, showing that NGS is a robust quantification method. However, 7 samples had a difference &amp;gt;10% VAF, with a deviation &amp;gt;25% in 3 cases. Standardization of molecular techniques for JAK2 V617F quantification is important if the VAF is to be monitored during MPN patient follow-up as a biomarker for disease evolution and for response to treatment, with VAF reductions shown for pegylated interferon and ruxolitinib. DPCR might be better suited for follow-up quantification due to its higher sensitivity and lower price, while NGS at diagnosis, apart from detecting and quantifying JAK2 V617F at baseline, has the advantage of providing information on other gene mutations with prognostic value.

Conventional Cytogenetic Analysis and Array CGH+SNP Identify Essential Thrombocythemia (ET)/Prefibrotic Primary Myelofibrosis (pre-PMF) Patients Who Are at Risk for Disease Progression

ET and pre-PMF are myeloproliferative neoplasms (MPNs) that are each characterized by thrombocytosis but are difficult to distinguish by clinical and histopathological criteria. Moreover, each has the propensity to progress at variable rates to more advanced forms of the disease. The cause of progression to more overt forms of myelofibrosis (MF) or accelerated /blast phase (AP/BP) has yet to be fully understood, making prognostication difficult. To identify patients (pts) who are at risk for disease progression, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH+SNP) of 158 pts with a diagnosis of ET or pre-PMF from 2017 to 2022 at our institution. Among these pts, 58% were female (92 pts) and 42% were male (66 pts), and the median age was 61 years (range 13-92). Mutational analysis was performed in 155 pts, and JAK2 mutations were present in 63% of cases, CALR in 19%, and MPL in 7% of cases. The remaining 11% of pts were triple negative. An abnormal karyotype was observed in 15% (24/158), and the remaining 85% (134/158) were either normal or had non-diagnostic results due to failed or limited peripheral blood chromosome analysis. Of the 111 pts that had aCGH+SNP testing, 6% (7/111) had prior abnormalities as detected by conventional cytogenetic analyses, while abnormalities were found in 30% (33/111) with aCGH+SNP exclusively. In this cohort, 29% (46/158) of pts underwent disease progression. Among these cases, 65% (30/46) had progression to either MF or chronic myeloid leukemia or myelodysplasia, and 22% (10/46) progressed to AP/BP. In addition, 13% (6/46) evolved to polycythemia vera. Disease progression, irrespective of its form, was associated with genomic alterations detected by aCGH+SNP, cytogenetic analysis, or both at the time of first encounter. Of the 46 pts undergoing progression during their clinical course, 65% (30/46) had an abnormal genomic finding by either technology compared to 35% (16/46) who were normal (p&amp;lt;0.001). Strikingly, among 24 pts who were abnormal by classical cytogenetics, 79% (19/24) progressed to either AP/BP or MF compared to only 20% (26/125) who were cytogenetically normal (p&amp;lt;0.001). As shown in Figure 1, there was a shorter progression-free survival among pts who were cytogenetically abnormal as compared to normal pts (p&amp;lt;0.001). Among 134 pts with ET/pre-PMF who were normal or had non-diagnostic results by classical cytogenetic studies, aCGH+SNP testing was performed in 104 cases and additional genomic aberrations were detected in 32% (33/104). Within this group, 30% (10/33) progressed to MF with a trend towards a shorter progression-free survival. In those patients that progressed to MF, the most frequent abnormality was 9p CNLOH occurring in 6 pts. In the remaining 71 pts that were cytogenetically and aCGH+SNP normal, only 11% (8/71) had disease progression. Overall, the combination of both conventional cytogenetics and aCGH+SNP increased the sensitivity of detecting genomic abnormalities from 15% (cytogenetics alone) to 37% when incorporating aCGH+SNP. These data indicate that genomic alterations detected by conventional cytogenetic analyses or CGH+SNP array identify a subset of ET/pre-PMF pts that are at a higher risk of disease progression.

Sex-Specific Impact on Disease Outcome and Mutational Landscape in Essential Thrombocythemia

Background:Male sex has been confirmed as an independent risk factor on survival effect in patients withessential thrombocythemia by mutation-enhanced international prognostic scoring system for essential thrombocythemia (MIPSS-ET) (Tefferi A, et al., Br J Haematol. 2020). Females are more common in ET patients (Grinfeld, et al., N Engl J Med. 2018). Epigenetic alterations (EAs) are found in approximately 20% inET. However, the prognostic role of epigenetic alterations is still not established in this patient population. Sexual dimorphism significantly contributes to patient heterogeneity, and adopting a sex-informed perspective has emerged as a pioneering paradigm in the field of precision medicine. The objective of our study was to comprehensively elucidate the impact of sex diversity on essential thrombocythemia, encompassing disease genotype, phenotype, and clinical outcomes. Methods:In this study, we performed retrospective chart review of 556 consecutive patients aged 18 years or older with a diagnosis of essential thrombocythemia according to the 5 th World Health Organization classification of myeloid neoplasms (Khoury JD, et al., Leukemia. 2022). Recruitment periods for these patients were between January 2008 to June 2022. Overall survival (OS) from the time of diagnosis was estimated by Kaplan-Meier method with log-rank test. The effect of sex on clinical outcome, with OS, myelofibrosis-free survival, and thrombosis-free survival as the main endpoint, was analyzed in our cohort. Results:This study included 245 (44.1%) men and 311 (55.9%) women. The percentages of age&amp;gt;60 years at diagnosis were 26.1% for men and 21.2% for women (p=0.176). Patients in the women cohort had lower red blood cell levels (4.54 vs. 4.81, p&amp;lt;0.001), lower hemoglobin levels (135 g/L vs. 146 g/L, p&amp;lt;0.001), lower percentage of cardiovascular risk factor (21.8% vs. 39.3%, p&amp;lt;0.001), and lower percentage of abnormal karyotype (37.3% vs. 50.6%, p=0.009). The treatment distributions between men and women cohorts were comparable ( p&amp;gt;0.05). Sex biases were observed at the single-gene level with mutation in two genes enriched in males (SF3B1 p=0.008, ZRSR2 p=0.024). The ASXL1 gene mutation exhibited a discernible inclination towards a higher prevalence in males (p=0.098). Additionally, sex biases were observed in co-mutational pathways of founding genomic lesions (genes related to spliceosome mutation (SM), predominantly in men, p&amp;lt;0.001). Patients with SM had inferior OS and myelofibrosis-free survival than those without SM (p=2e-04, p=0.0078). Sex disparity was observed in the transcriptome signature of bone marrow CD34+ cells from ET patients. From results of GO analysis, we observed that up-regulated differentially expressed genes in male patients were significantly associated with the unfolded protein signaling pathway, as well as dysregulation of hematopoietic differentiation. Moreover, we found that lipid and atherosclerosis, and platelet activation pathways were enriched in male patients by KEGG analysis. Based on our analysis, we hypothesize that male ET patients exhibit enhanced bone marrow megakaryocyte production and platelet activation compared to female patients. Overall, Men had worse OS and thrombosis-free survival than women (both of median survival not reached; p=0.0081, p=0.019, respectively), but the myelofibrosis-free survival was not significantly different between men and women (p=0.52). Conclusions: The findings from our study indicate that the implementation of a sex-informed approach has the potential to enhance the precision of personalized decision making in individuals diagnosed with essential thrombocythemia. Therefore, it is imperative to incorporate this approach in the planning and execution of clinical trials in the future.

Sodium-Glucose Co-Transporter-2 Inhibitor Use in the Setting of Myeloproliferative Neoplasms: Impact on Hemoglobin/Hematocrit Levels and Thrombosis Risk

Background: Sodium-glucose co-transporter-2 inhibitors (SGLT-2I) are FDA-approved for diabetes mellitus, heart failure and chronic renal disease. Clinical trials on their anti-diabetic and cardiorenal protective effects showed an association with erythrocytosis. We have previously reported on the benign nature of SGLT-2I associated JAK2-unmutated erythrocytosis ( Blood 2021). Recently, we described the same phenomenon in the setting of a previously unrecognized myeloproliferative neoplasm (MPN), which led to unmasking of underlying MPN and thrombosis in 3 (33%) of 9 informative cases ( AJH 2023). The main objective of the current study was to assess the erythropoietic and thrombotic impact of SGLT-2I therapy in patients with a well-established MPN diagnosis, prior to treatment initiation. Methods: The Mayo Clinic MPN database was queried to identify patients prescribed SGLT2-I after diagnosis of essential thrombocythemia (ET), polycythemia vera (PV) or myelofibrosis (MF). Baseline Hgb/Hct at SGLT-2I initiation, peak Hgb/Hct, absolute change in Hgb/Hct levels, and thrombosis during treatment were recorded. Results: SGLT-2I use in essential thrombocythemia Eleven patients with ET (median age; 72 years, 55% males, 73% JAK2 mutated, 64% with thrombosis history) received SGLT-2I. Active treatments included aspirin ( n=10), cytoreductive therapy ( n=10) and systemic anticoagulation ( n=4). All 11 patients displayed increase in Hgb/Hct following SGLT-2I treatment. Baseline median (range) values for Hgb/Hct were 12.8 g/dL (9.5-14.9)/38.9% (30.3-44.6); median (range) peak Hgb/Hct levels during SGLT-2I therapy, were 14.5 g/dL (11-16.4)/44.4% (34.8-51.1), with median (range) increase in Hgb/Hct of 1.5 g/dL (1-4)/5.1% (2.6-13.8); 2 patients (18%), exceeded the proposed ICC Hgb/Hct diagnostic threshold for PV (Patient 7 and 8) (Figure 1). JAK2 mutation/VAF (p=0.54/ 0.20), or serum erythropoietin (Epo) (p=0.48) did not correlate with changes in Hgb/Hct levels. One patient experienced thrombosis, despite treatment with hydroxyurea and aspirin and Hgb/Hct levels below the threshold of concern (11.4 g/dL/37.7%). SGLT-2I use in polycythemia vera Nine patients with PV (median age 74 years, 66% males, JAK2 median VAF 14%, two patients with thrombosis history) received SGLT-2I. Concomitant treatments included aspirin ( n=9), cytoreductive therapy ( n=8), and systemic anticoagulation ( n=3). Phlebotomy intensity, defined as the number of phlebotomies, six months prior to and after SGLT-2I initiation, ranged from 0-3 and 0-12, respectively. Documented Hgb/Hct levels showed an increase in all 9 cases with baseline median (range) values for Hgb 14.1 g/dL (10.5-15.9) and Hct 42.7% (33.3-49.2) and peak median (range) values for Hgb/Hct, 15.4 g/dL (11.2-19.9)/46.7% (35.9-63.3). JAK2 VAF (p=0.85), and serum Epo (p=0.43) did not correlate with increase in Hct. Figure 2 highlights five patients (56%) with Hct exceeding 45% while on SGLT-2I; two patients (22%) had increased phlebotomy requirements; 6-month pre/post SGLT-2I phlebotomies for patient 5 and 9 were 3 and 6, and 0 and 12, respectively. Patient 9, experienced thrombosis in the context of uncontrolled Hgb/Hct level at 19.9 g/dl/63.3% and absence of cytoreductive therapy. SGLT-2I use in myelofibrosis Eight patients with myelofibrosis (primary 6, post-ET 2; median age 71 years; 50% males; 63% JAK2 mutated; MIPSS-70 2.0 int/high 6; on ruxolitinib 4),received SGLT-2I. Baseline median (range) values for Hgb/Hct were 10.6 g/dL (8.2-14.7)/27.5% (33.8-45.9); two (25%) patients displayed anemia with Hgb 8-10 g/dl. Peak median (range) values for Hgb/Hct were 11.5 g/dL (10.2-16.1)/35.3% (31.8-49.8), with median (range) baseline increase in Hgb/Hct of 1.6 g/dL (0.7-3.8)/4.3% (1-7.5). Patient 2 and 4 with baseline Hgb 8.2 g/dl and 9.4 g/dl, and not receiving ruxolitinib, had Hgb increase of 2 g/dl and 1 g/dl, respectively (Figure 3). One thrombotic event occurred during therapy with Hgb/Hct level of 11.6 g/dl/37.3%. Conclusion: The current study suggests that SGLT-2I therapy in MPN patients leads to increase in Hgb/Hct levels, regardless of disease subtype, and in some cases surpass the PV diagnosis and thrombosis risk thresholds. These observations mandate increased awareness to secure patient safety and diagnostic accuracy. The erythropoietic effect of SGLT-2I in MF suggests its potential use for MF-associated anemia.

Marrow Hypercellularity Does Not Exclude the Diagnosis of Essential Thrombocythemia (ET)

Background: The 2022 WHO criteria do not list bone marrow cellularity as a criterion for ET diagnosis (dx) 1, but a review discussing the International Consensus Classification criteria states that the marrow should be normocellular for age, with rare cases of mild hypercellularity 2. The ELN also cited normocellularity as a key factor in differentiating ET from polycythemia vera (PV) or prefibrotic myelofibrosis (MF), further suggesting that normocellularity is the expected standard in ET 3. However, we encountered a number of cases in our hematology practice with increased cellularity in bona fide ET patients (pts). Therefore, we decided to more systematically review the marrow cellularity and its prognostic impact. Methods: Medical records were queried for pts with ET per the ICD, versions 9-10. The dx of ET per WHO 2022 was confirmed by manual review. Cases lacking marrow biopsy were excluded. Structured query language was used for automated extraction of clinical, lab, and molecular findings. We utilized our previously described natural language processing pipeline to extract cellularity information from marrow reports 4. All hematopathology reports of hypercellular marrows were manually reviewed by 3 observers for confirmation and a randomly selected subset of the core and clot sections were reviewed for histomorphology and cellularity by 2 expert hematopathologists and 1 histomorphologist, blinded to pt age and clinical information prior to their review. Quantitative cellularity was determined independently and then congruently for consensus. Qualitative cellularity was defined using (100-age) ± 20% as the normal range with increases above this range considered hypercellular for age. Disease progression was defined as either progression to post-ET MF (PETMF) or direct transformation to acute myeloid leukemia. Transformation to PV was defined as pts with a JAK2 mutation for whom PV was thoroughly excluded at the time of ET dx but who developed absolute erythrocytosis and met the PV WHO diagnostic criteria later in their disease course. We performed univariable and multivariable analysis of progression, PV transformation, thrombosis and mortality risk using Cox proportional-hazards models. Overall (OS), progression-free (PFS), PV-free (PVFS) and thrombosis-free (TFS) survival were estimated using Kaplan-Meier methods. Results: 382 pts met WHO defined ET with marrow dx, of which 145 had pre-treatment diagnostic marrows available for review at Weill Cornell. In the other 237, the diagnostic biopsies had either been performed after treatment was initiated or only the report was available. Thirty-three (23%) were described as hypercellular and 112 (67%) were not. Review of 20 randomly selected marrows from the hypercellular group confirmed age-adjusted hypercellularity ranging from 30 to 80%. The hypercellular pts were significantly older at dx compared to those without hypercellularity (59 vs 50 yrs, p &amp;lt;0.001, Table 1). We identified no other significant differences in blood counts at dx, sex, driver mutation or thrombosis history between the groups. Univariate and multivariate analyses demonstrated no significant difference between the groups in PFS (HR 1.29, p=0.871), TFS (HR 1.39, p=0.671) or OS (HR 0.50, p=0.487) using age, thrombosis history and sex as covariates. A Cox model could not be generated for PVFS due to the low number of events. The 15-year PFS for pts with hypercellularity vs those without was 97% and 94% respectively ( p=0.56, Figure 1), 15-year PVFS was 100% and 67% respectively ( p=0.12), 15-year TFS was 79% and 87% respectively ( p=0.53) and 15-year OS was 84% and 81% respectively ( p=0.74). Conclusion: Our data to date suggests that hypercellularity is of importance diagnostically but not prognostically. Nearly a quarter of our pts with WHO ET had marrow that was hypercellular for age at dx. Pts with increased marrow cellularity tended to be older but were clinically indistinguishable from the remainder in terms of driver mutation, blood counts and thrombosis history. Univariate and multivariate analyses demonstrate that hypercellularity does not confer increased risk of progression, thrombosis or mortality. Thus, hypercellularity alone should not be used as a factor for excluding ET dx.

Low Thrombosis Risk CALR Mutations Confer Higher Risk of Essential Thrombocythemia Progression

Background: Patients (pts) with essential thrombocythemia (ET) face increased risk of thrombosis and the prospect of progression to myelofibrosis (MF) or acute leukemia. ET treatment recommendations from ELN 1 and NCCN 2 are largely governed by the revised IPSET-thrombosis score that identifies 4 risk categories from “very low” to “high” based on age, thrombosis history, and the presence of a JAK2 V617F mutation 3. Pts with CALR-mutated ET are known to have a lower risk of thrombosis than those with a JAK2 mutation, and for this reason, are often considered to have lower risk ET. Yet previous study of pts with clinically diagnosed ET found that CALR mutations may negatively impact progression risk 4. For this reason, we evaluated the impact of driver mutations on the risks of thromboembolic events, disease progression, and overall mortality for a cohort of pts with rigorously defined WHO ET treated at our institution. Methods: Medical records were queried for pts with the diagnosis (dx) of ET, as per ICD, code versions 9-10. The dx of ET and dx date were confirmed by manual review. All pts met the 2022 WHO criteria for ET, including a diagnostic marrow in all cases. Confounding diagnoses, including pre-fibrotic MF, polycythemia vera, or presumed ET in pts lacking a marrow biopsy, were strictly excluded by thorough review of the rich data available. Structured query language supplemented by manual review was used to extract clinical, laboratory, molecular, and marrow findings and treatment course. Overall (OS), MF-free (MFS), and thrombosis-free (TFS) survival were estimated using Kaplan-Meier methods. Univariable and multivariable analysis of post-ET MF (PETMF), thrombosis and mortality risk were performed using Cox proportional-hazards models using IPSET-survival 5 and revised IPSET-thrombosis variables as covariates. Results: The query returned 751 pts but only 338 met our strict criteria for ET with diagnostic marrows and had annotated driver mutations. Of these, 216 (64%) were positive for JAK2 V617F, 85 (25%) CALR, 19 (6%) MPL, and 18 (5%) triple negative (TN). We identified no significant differences among the driver mutation cohorts in dx age, sex, or median follow up duration. Semi-quantitative features of the diagnostic marrow biopsies, including age-adjusted cellularity and reticulin fibrosis score, were not significantly different among the groups. Red cell parameters at dx were mildly higher in the JAK2 pts (HCT, HGB, RBC all with p&amp;lt;0.001), WBC count was highest in TN pts ( p=0.012), and PLT count was not significantly different ( p=0.064). 20-year TFS for JAK2 was 71%, CALR 100%, MPL 90% and TN 83% ( p=0.0027, Figure 1); 20-year MFS for JAK2 was 87%, CALR 48%, MPL 65% and TN 94% ( p=0.00053, Figure 2); 20-year OS for JAK2 was 76%, CALR 86%, MPL 89% and TN 90% ( p=0.66). Multivariable analysis showed that CALR was associated with a significantly higher risk of PETMF as compared to JAK2 (HR 11.52, CI 1.88-70.51, p=0.008), independent of age, sex, WBC, and thrombosis history. Conversely, CALR was associated with a lower risk of thrombosis (HR 0.08, CI 0.01-0.58, p=0.013) when adjusting for sex and revised-IPSET thrombosis parameters. Discussion: We found that although pts with CALR-mutated ET have lower thrombosis risk, they experience higher risk of progression to MF. Current risk models for thrombosis yield treatment recommendations well suited to reducing thrombosis risk 3. With this success, pts are expected to live with ET for decades, thereby exposing them to other ET risks, most notably that of malignant disease progression. These risks are of particular concern for younger pts who suffer the greatest relative risk 6. It is important that thrombosis risk and fibrosis risk are not conflated as our data suggest that pts with low or very low risk CALR-mutated ET can be at higher risk of progression. Our findings reinforce the need for long-term data to guide therapy for ET based not only on the near-term thrombotic risk, but also on the long-term risk of progression.

Interpretable Artificial Intelligence (AI) Differentiates Prefibrotic Primary Myelofibrosis (prePMF) from Essential Thrombocythemia (ET): A Multi-Center Study of a New Clinical Decision Support Tool

Introduction Overlapping clinical, molecular, and histopathological characteristics pose challenges in differentiating prePMF from ET. The median overall survival, however, significantly differs between prePMF and ET (11.9 vs 22.2 years, Jeryczynski, 2017). The difference in survival highlights the need to distinguish between these two myeloproliferative neoplasms (MPNs) to select disease-specific therapeutic options. This area of unmet need often requires expert assessment at high-volume academic institutions to render a definitive diagnosis. Our aim in this study is to develop and validate a biologically-motivated AI algorithm to rapidly, accurately, and inexpensively diagnose prePMF and ET directly from diagnostic bone marrow (BM) biopsy digital whole-slide images (WSI). Methods Patients with a clinical/histopathological diagnosis of prePMF or ET as determined by the International Consensus Classification of Myeloid Neoplasms were identified at the University of Florence, Italy (Florence) between 06/2007 and 05/2023 and Moffitt Cancer Center, Tampa, FL (Moffitt) between 01/2013 and 01/2022. Diagnostic H&amp;E-stained BM biopsy slides were digitized using Aperio AT2 slide scanners (Leica Biosystems, Deer Park, IL) at each institution . The training cohort comprised of 200 (100 prePMF / 100 ET) patients from Florence, and the external test cohort entailed 26 (6 prePMF / 20 ET) patients from Moffitt. In total, the resultant model was trained on 32,226 patient-derived WSI. Our chosen pretrained neural network, RetCCL, was previously trained on 32,000 diagnostic WSIs to potentially represent a histologically-informed model (Wang, 2023). BM WSI were tessellated into representative image tiles extracted at 10x magnification (302 microns per image dimensions) for model training. Finally, a prediction upon each patient's WSI was calculated by attention-based multiple instance learning, which is a method that automatically assigns a numeric weight to an image portion representing its relative importance to the classification task. Model performance was assessed utilizing the area under the receiver operator curve (AUC). The cutoff threshold for diagnosis classification was determined by maximizing Youden's Index. For qualitative assessment, attention scores were plotted as a heatmap across the BM WSI and reviewed for morphological features by an expert hematopathologist. Custom scripts were written using our open-source AI framework, Slideflow (Dolezal, 2021). Model development was performed on the Minerva High Performance Computer at Mount Sinai Hospital. Evaluation time upon a single WSI was estimated using a consumer-grade computer with an NVIDIA RTX 3080 graphics processing unit. Results Within the training cohort, 5-fold cross validation resulted in a mean AUC of 0.90 and standard deviation of 0.04. A final locked model re-trained on the entire training cohort resulted in an AUC of 0.90 upon evaluation of the test cohort ( Figure 1). We optimized the classification threshold to balance sensitivity and specificity; the final diagnostic classification accuracy on the test cohort was 92.3% with a sensitivity and specificity for prePMF diagnosis of 66.6% and 100%, respectively. Upon review of the slides with highest prediction value per class, attention heatmaps highlighted the model's reliance on areas of cellular marrow without reliance on image artifacts or background ( Figure 2). Using affordable consumer-grade hardware, evaluation upon a previously unseen WSI was completed in approximately 6.1 seconds (4.9 for preprocessing and 1.2 for evaluation). Conclusion We developed a novel AI model with high accuracy for distinguishing between prePMF and ET in distinct clinical cohorts. To our knowledge, this study represents the largest image-based AI study within MPNs with external validation. Our proposed model may assist clinicians in appropriately identifying patient cohorts who would benefit from disease-specific therapies or enrollment onto clinical trials. We imagine that a potential high-speed, low-cost algorithm may reliably distinguish prePMF from ET patients with high specificity which can be democratized to the MPN clinical community in routine practice and drive clinical trial accrual for biologically rational novel therapeutics.

Near-early T-cell precursor acute lymphoblastic leukemia following a diagnosis of essential thrombocythemia.

Near-early T-cell precursor acute lymphoblastic leukemia following a diagnosis of essential thrombocythemia.

Abdominal aortic calcification in patients newly diagnosed with essential thrombocythemia.

Although atherosclerosis is likely to be involved in the development of arterial thrombotic events in patients with essential thrombocythemia (ET), abdominal aortic calcification (AAC) has rarely been investigated. We evaluated the prevalence and clinical relevance of AAC at the time of ET diagnosis. This retrospective study included patients newly diagnosed with ET who underwent abdominal computed tomography (CT) at the time of diagnosis between January 2002 and December 2021 at Chungnam National University Hospital, Daejeon, Korea. CT images were reviewed and an aortic calcification score was assigned. Of the 94 patients (median age, 62 yr; range, 18‒90 yr), AAC was detected in 62 (66.0%). AAC was most commonly mild (33.0%), followed by moderate (22.7%) and severe (5.3%). Old age [odds ratio (OR), 34.37; 95% confidence interval (CI), 12.32‒95.91; P＜0.001] was an independent risk factor for AAC. The patients with AAC had a higher WBC count (11.8±4.7 vs. 9.7±2.9×109/L, P=0.017), higher neutrophil-to-lymphocyte ratio (4.3±2.7 vs. 3.1±1.5, P=0.039), and higher JAK2V617F positivity (81.5% vs. 58.8%, P=0.020) compared to those without AAC. AAC was an independent risk factor for arterial thrombotic vascular events that occurred before or at diagnosis of ET (OR, 4.12; 95% CI, 1.11‒15.85; P=0.034). AAC is common in patients with ET and is associated with arterial thrombotic events.

JAK2V617F mutation and circulating extracellular vesicles in essential thrombocythemia.

The clinical course of essential thrombocythemia (ET) is complicated with thrombosis which significantly impacts patients' mortality. Studies have identified JAK2V617F mutation as an independent risk factor for thrombosis. Circulating extracellular vesicles (EVs) were evaluated in several studies regarding myeloproliferative neoplasms and thrombosis as potential biomarkers. The present study investigates the relationship between JAK2V617F mutation and EVs levels in 119 ET patients. Our analyses revealed that JAK2V617F-positive patients are at a significantly increased risk of thrombosis within five years before the ET diagnosis (hazard ratio [95% CI]: 11.9 [1.7-83.7], P = 0.013), and that JAK2V617F mutation is an independent risk factor for thrombosis at ET diagnosis or during the follow-up (hazard ratio [95% CI]: 3.56 [1.47-8.62], P = 0.005). ET patients have higher levels of platelet-EVs, erythrocyte-EVs and procoagulant activity of EVs than the healthy population. Absolute and relative counts of platelet-EVs are increased in the presence of JAK2V617F mutation (P = 0.018, P = 0.024, respectively). In conclusion, our results support the role of JAK2V617F mutation in the pathogenesis of thrombosis in essential thrombocythemia through enhancing platelet activation.