Balliu, Manjola

Background: The JAK2 46/1, also referred to as the GGCC, haplotype, is represented by 4 main SNPs spanning a region of 250kb in chr9 including the JAK2 gene, that are in complete linkage disequilibrium. The GGCC haplo was found to confer susceptibility to JAK2 mutated myeloproliferative neoplasms (MPN) (Jones AV, 2009; Olcaydu D, 2009; Kilpivaara O, 2009) while association with JAK2-wildtype MPN remains controversial (Pardanani A, 2010; Jones AV, 2010; Guglielmelli P, 2010; Trifa A, 2016). The GGCC haplo was also found enriched in MPN pts with splancnic vein thrombosis (SVT) (Smalberg JH, 2011), and to correlate with higher JAK2V617F variant allele frequency (VAF) (Alvarez-Larran, A, 2012). Nullizigosity for GGCC haplo was associated with reduced survival (OS) in pts with myelofibrosis (MF) (Tefferi A, 2019), but no information on the impact of GGCC haplo status with phenotype and outcome in PV is available. Aim: To assess the clinical correlates and the prognostic significance of GGCC haplo status in a large cohort of pts with PV. Pts and Methods: A total of 399 PV pts, all JAK2V617F mutated, diagnosis revised according to 2022 ICC criteria, were genotyped for 46/1 haplotype by PCR allele discrimination with rs12343867 (with the “C” allele standing for 46/1 haplotype). Results: A total of 301 pts (75.4%) had the C allele, of which 143 (47.5%) were CC homo and 158 (52.5%) CT hetero; 98 (24.6%) were nullizygous for GGCC (TT). Since preliminary analysis disclosed no significant difference between CT and TT haplotype for all variables considered, homozygous CC pts were compared to TT+CT. The CC haplotype was associated with higher leukocytes (median 11.3x10e9/L vs 9.6, p<.001), higher hemoglobin (18.4 g/dL vs 17.5, p<.001), lower platelets (441x10e9/L vs 514, p=.002) more aquagenic pruritus (51.8% vs 33.2%, p=.001), and higher JAK2VAF (60.8+-24.8% vs 36.8+-19.7%, p<.001) at diagnosis. Pts with a VAF >50% were 69% in CC versus 24.1% in TT+CT pts (p<.001). ASXL1 mutations were enriched among CC pts (20.3% vs 9.4%, p=0.04), otherwise no difference regarding additional myeloid mutations was noted. All above differences remained significant when considering CC versus TT and CT separately. More pts with CC haplotype suffered from venous thrombosis (VT) during the FU (16.1% vs 4.3%, p<.001), no difference was noted for arterial thrombosis nor for splancnic vein thrombosis. VT-FS by Kaplan Meyer was significantly shorter (p<.001) in CC pts; the rate of VT at 5, 10, 15y after diagnosis, was 5.5%, 12%, 20.2% for CC versus 2.6%, 3.4%, 6.4% for TT+CT, respectively (all p<.001). In multivariable analysis, including JAK2VAF >50%, age >60y, history of VT, WBC >15x10e9/L, only CC haplo remained significant (HR 3.6, 95%CI 1.4-9.5, p=.009). Progression to post-PV MF occurred in 81 pts (20.3%), significantly more among CC (n=45, 31.5%) than TT+CT (n=36; 14.1%; p<.001). Myelofibrosis-FS was significantly shorter in CC vs other genotypes (p=.02). However, in multivariable analysis, only BM fibrosis G1 remained significant (HR 3.2, 1.7-5.9; p<.001). Evolution to blast-phase occurred in 13 pts (3.2%), 9 (6.3%) with CC haplo vs 4 TT+CT (1.6%; p=.01). During the follow-up, 102 pts (25.5%) died, 50 (35%) CC and 52 TT+CT (20.3%; p=.001). In multivariable analysis, including MIPSS-PV variables (Tefferi A 2020), the CC haplo remained significant (HR 1.8, 1.04-3.2, p=.03) together with age (HR 3.0, 1.6-5.7; p<.001), WBC (HR 2.6, 1.3-4.8; p=.004), and thrombosis history (HR 1.9, 1.0-3.7; p=.03). Conclusions. Homozygosity for the 46/1 or CC haplotype contributes to the phenotype diversity of PV, in part possibly mediated by associated higher JAK2V67F VAF, and predicts for incident VT and shorter OS. Supported by AIRC, 21267; MIUR,2022F4WMR3; IHM, PNRR-MAD-2022-12376695

Abstract Among 281 patients with essential thrombocythemia and calreticulin (CALR) mutation, we found a variant allele frequency of ≥60% to be associated with significantly shortened myelofibrosis-free survival, mostly apparent with CALR type-1 and CALR type-indeterminate mutations.

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), >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 >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 >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 >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 >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 >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<.0001); AML and death not differ. Anemia (<10g/dL) during the FU developed in 69% of CALR-high pts vs 18%, leukocytosis (>15x10 9/L) in 46% vs 8% and splenomegaly (greater than 5 cm from LCM) in 50% vs 12% (P<.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<.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 >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 >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.