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Megakaryocytic activation and fibrotic evolution of MPN
M-ACT and age (P=1.00), Hgb level (P=0.43), MPL muta- tions (P=1.00) and A/V thrombosis (P=0.42; Table 1).
Similarly to what happened in the PV cohort, when we correlated M-ACT status with PFS, we found that patients with early/prefibrotic PMF and with M-ACT had a signif- icant lower PFS than those without M-ACT (Table 1; Figure 2 panel B, for PFS: median PFS for M-ACT positive patients 44 months vs. median PFS for M-ACT negative patients 77 months, P<0.0001, HR 3.17, 95% CI: 2.27- 4.44). Moreover, male sex, CALR type 1 mutations, WBC count >11x109/L, presence of palpable splenomegaly, PLT≥600x109/L and LDH ≥250 U/L had a significant corre- lation with a worse PFS (P=0.0187, P<0.0001, P<0.0001, P<0.0001, P<0.0001 and P=0.0025, respectively, Online Supplementary Figure 2S). Conversely, age (P=0.8831), major bleeding (P=0.7244), JAK2 V617F allele burden≥ 50% (P=0.3459), Hgb level (P=0.5234), MPL mutations (P=0.2268) and A/V thrombosis (P=0.2003) did not show significant correlation with PFS.
Multivariate analysis of PFS, including M-ACT status, CALR status, WBC count, sex, LDH serous level, splenomegaly, and platelet count, showed that the pres- ence of M-ACT and CALR type 1 mutation, WBC count >11x109/L and male sex were the significant predictors (for M-ACT status, P<0.0001, HR 2.1510, 95% CI: 1.5598- 2.9661; for CALR status, P=0.0285, HR 1.446, 95% CI: 1.0395-2.0124; for WBC count, P=0.0211, HR 1.5425, 95% CI: 1.0673-2.2294; for sex, P=0.0074, HR 1.5024, 95% CI: 1.1153-2.0240; Table 4).
In the non-PV MPN cohort, we also analyzed a small subgroup of 23 ET patients. We did not find M-ACT in any of the ET BM biopsies performed at the time of the diagnosis. The ET patients had a better PFS in comparison to patients with early/prefibrotic PMF either with M-ACT (Online Supplementary Figure S3; P<0.0001) and without M- ACT (Online Supplementary Figure S3; P<0.0001).
Interestingly, we found that the incidence of M-ACT among triple-negative patients in the non-PV cohort was significantly lower in respect to patients with a driver gene mutation (21 of 85 [24.7%] triple negative patients vs. 90 of 222 [40.5%]; P=0.0115), and that triple-negative patients with M-ACT also had a significant lower PFS than those without M-ACT (median PFS for M-ACT positive triple-negative patients 56 months vs. median PFS for M-ACT negative triple-negative patients 79 months, P<0.0023, HR 2.76, 95% CI: 1.44-5.32; data not shown).
Discussion
For about two decades, one of the most important prob- lems in the treatment of patients with MPN has been the identification of biological and non-biological factors that could represent a determinant key to the prediction of prognosis. Accordingly, several prognostic scores have succeeded over time, mainly based on clinical, hematolog- ical and molecular parameters, in identifying the fraction of MPN patients that could have a high risk of developing a leukemic transformation or a bone marrow fibrotic fail- ure. However, none of these models take the morpholog- ical parameters into factual consideration, while these parameters play an important role in the diagnostic phase.
In this retrospective and single-center study, we propose a novel morphological parameter, defined as M-ACT, as a new possible predictive marker of fibrotic evolution
among Philadelphia-negative MPN. Furthermore, this new parameter seems to be useful to supplement WHO 2017 classification criteria in the differential diagnosis of the MPN subtype between ET and early/prefibrotic PMF.
In our study, carried out on a large cohort of MPN BM biopsies at diagnosis, extensive evidence support this statement. In fact, in univariate analysis M-ACT correlates with relevant MPN clinical and hematologic parameters (see Table 1 and 2), such as palpable splenomegaly, WBC or PLT count, and LDH levels, but also with molecular profiles defined by the JAK2 V617F allele burden and CALR mutations (especially the CALR type 1 mutation).
In PV patients the PFS was influenced at the multivariate analysis by the JAK2 V617F allele burden >50%, as already reported;26 in early/prefibrotic PMF patients’ PFS was influenced by the presence of the CALR type 1 muta- tion, WBC count >11x109/L and male sex, in agreement with previous reports.27
Moreover, patients with M-ACT had a significant corre- lation with a worse PFS and with an overt-myelofibrotic BM failure, in both PV and early/prefibrotic PMF (P<0.0001). This last result is also confirmed at multivari- ate analysis. Interestingly, PV and early/prefibrotic PMF patients with this parameter showed a rapid clinical pro- gression before the end of the 5-year follow-up, suggest- ing that M-ACT could be an early predictive marker capa- ble of precociously identifying patients that need a closer follow-up.
Numerous scientific papers have highlighted that in the evolution towards myelofibrosis of MPN, a central role seems to be played by MK. Patients with MPN and fibrot- ic evolution showed a significantly increased number of MK with an abnormal nuclear/cytoplasmic ratio and a reduced polyploid state, often organized in clusters.27,28 Experiments using in vitro cultures of CD34+ hematopoiet- ic stem cells of patients with fibrotic MPN have shown that MK expand excessively, are immature and show delayed apoptosis owing to increased expression of the anti-apoptotic factor BCL-XL.29 Moreover, mice with a MK-specific deficiency of the transcription factor–encod- ing gene GATA1 show elevated numbers of immature MK in the BM and an increased and pathologic neutrophil emperipolesis that may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenviron- ment of in vivo experiments.14,16 Finally, MK from individ- uals with PMF secrete increased levels of the fibrotic cytokines such as TGF-b, compared to MK from healthy individuals, and the extracellular matrix (ECM) microenvi- ronment, especially the fibronectin component, is able to sustain progenitor cell proliferation and megakaryopoiesis in a TPO-independent manner.16,30,31 These pro-fibrotic cytokines would presumably act mainly in the microenvi- ronment near to those MK clusters which are, in turn, their main producers. Furthermore, the criteria defining the megakaryocytic activation could represent the mor- phological counterpart of what is postulated by in vitro and in vivo studies regarding the role of MK in the BM fibrotic evolution of patients with MPN.
Recent evidence has suggested that treating patients with early-stage MF may lead to better outcomes with a less severe splenomegaly, a lower incidence of cytopenia, and less-severe BM fibrosis. However, the argument is debated, especially considerating the adverse events of the JAK2 inhibitor treatment (ruxolitinib). M-ACT parameter,
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