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Megakaryocytic activation and fibrotic evolution of MPN
study group, which enabled survival estimation at the time of diagnosis primarily employing five clinical and hemato- logic parameters;5 this model was further revised as the Dynamic International Prognostic Scoring System (DIPSS) and then as DIPSS-plus score.6,7 The above, nonetheless, applied to already-established myelofibrosis (both PMF and post-PV/ET MF) only, determining survival from the time of disease progression/transformation to death with- out considering the heterogeneous disease history before the appearance of BM changes.8
On the basis of advances in MPN molecular profiling, in order to improve the prognostic prediction in PMF patients, novel models included JAK2, CALR, and MPL mutation status in addition to the IPSS parameters.8 Moreover, novel insights were provided by in-depth analy- sis of genomic subsets with different clinical outcomes.9 Recent publications have introduced new risk models for PMF, namely MIPSS70 (mutation-enhanced international prognostic scoring system for transplant-age patients),10 MIPSS70+ version 2.0 (karyotype-enhanced MIPSS70) and GIPSS (genetically-inspired prognostic scoring system).11,12 Similar risk models have been recently introduced for both ET and PV under the name of MIPSS-ET and MIPSS-PV, highlighting the prognostic contribution of spliceosome gene mutations.13 However, all these predictive models do not consider morphological and phenotypical features, except BM fibrosis grade in the MIPPS70 model.
In this study we evaluated a new morphological param- eter, defined by the coexistence of emperipolesis of megakaryocytes (MK) (i.e., the presence of an intact cell within the cytoplasm of another cell), MK clustering and peri-MK fibrosis in BM biopsy, which was named megakaryocytic activation (M-ACT). Larocca et al. in 2015 demonstrated that extensive BM emperipolesis associated to BM fibrosis was present in patients affected by gray platelet syndrome, with up to 65% MK containing two of four leukocytes engulfed within the cytoplasm;14 a similar phenomenon has been described either in BM patients with PMF,15 and in the BM of animal models of myelofi- brosis.16,17
We demonstrated that M-ACT is a useful morphological parameter in forecasting both PV and early/prefibrotic PMF to myelofibrosis progression and could also help in the dif- ferential diagnosis between ET and early/prefibrotic PMF.18
Methods
Patients' features
Formalin-fixed, paraffin-embedded BM biopsy specimens, obtained from the posterior superior iliac spine,19 were available in our Institute of Pathology for 460 patients clinically diagnosed with a MPN and followed at our Institute of Hematology (Fondazione Policlinico Universitario “A. Gemelli”, IRCCS) from January 2005 to October 2019. The study was carried out in accor- dance with the Declaration of Helsinki and the consent for retro- spective analysis of all clinical data, according to the Ethical Committee of the Università Cattolica del Sacro Cuore School of Medicine, and obtained by all the patients at the hospital admis- sion. Patients were clinically followed-up over the observation time by one single team physician (VDS and ER as senior mem- bers).
All 286 cases were sorted until October 2019, according to three inclusion criteria: clinical diagnosis of either PV or non-PV MPN, first BM biopsy at diagnosis for non-PV cohort and within 0-24
months from the clinical diagnosis for PV cohort and no grade 2-3 BM fibrosis. Accordingly, patients with diagnosis of overt PMF or secondary myelofibrosis were excluded. Furthermore, BM biop- sies were revised by two skilled pathologists (LMR and MM) and categorized according to the WHO 2017 criteria (PV, ET, early/pre- fibrotic PMF).
Clinical and hematological data (according to WHO 2017 crite- ria) were collected in order to trace lactose dehydrogenase (LDH) increase (i.e., LDH serum levels ≥250 UI/L), palpable splenomegaly, leukocytosis (i.e., white blood cell [WBC] count ≥ 11×109/L), high hemoglobin (Hg) level (i.e., Hgb >16 g/dL for women and Hgb > 16.5 g/dL for men) and thrombocytosis (i.e., PLT ≥ 450×109/L) for each patient at diagnosis. We also verified the occurrence of arterial/venous (A/V) thrombotic events and/or major bleeding events during the clinical course (until October 2019) for each case. Thrombotic and bleeding events were defined as previously described.20 JAK2 V617F mutation and allele burden analysis, CALR exon 9 mutations and MPL exon 10 mutations were performed as previously described.20 Progression to second- ary myelofibrosis was defined from the patient chart review and based on the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) consensus criteria.21
The main clinical, hematological and molecular characteristics of the 286 patients are shown in Table 1 for the PV cohort (64 patients), in Table 2 and the Online Supplementary Table S1 for non- PV cohort (including 199 early/prefibrotic PMF patients [Table 2] and 23 ET patients [Online Supplementary Table S1]).
Bone marrow biopsy analysis and megakaryocytic activation histological parameters
All biopsy specimens had a suitable length (at least 1.5-2 cm) in order to obtain at least ten partially preserved intertrabecular areas, since subcortical medullary lacunae are less cellular than deep ones (especially in the elderly) and since focal pathologies can have a deep localization.22 After collection, each biopsy spec- imen was kept in a properly-labeled clean container filled with 10% natural buffered formalin at pH 7.6 for 12 hours for fixation, was then decalcified with a Decalcifier II solution (Leica Biosystems, Milan, Italy) for 1 hour at room temperature, then fixed with 10% natural buffered formalin at pH 7.6 for 2 hours and finally embedded in paraffin. Sections (3-5 mm thick) were cut from each block for staining with hematoxylin and eosin (H&E) and Gordon&Sweet’s silver staining to evaluate morphological features and fibrosis.23,24 The specimens were concurrently exam- ined and reviewed by two pathologists experienced in BM biopsy interpretation (LML and MM), who were blinded toward the patients’ characteristics and survival. Cases with disagreement were discussed using a multiheaded microscope until agreement was achieved. The agreement indices (Cohen’s K) between the two pathologists were very good: k=0.83 and k=0.85 for PV group and for non-PV group, respectively.
In the definition of M-ACT the following parameters were examined in detail (as shown in Figure 1): (i) MK emperipolesis, (ii) MK clustering and (iii) peri-MK fibrosis: i) MK emperipolesis was defined as the presence of one or more leukocyte or a precursor of hematopoiesis within the cytoplasm of at least 30% MK in the specimen; ii) MK clustering was defined as an aggregation of three or more megakaryocytes in close contact with each other and at least 25% of MK distributed in clusters in the specimen; iii) peri- MK fibrosis was defined as the arrangement of collagen fibers around the perimeter of the vast majority of MK, underlining their primary role in the genesis of fibrosis.
M-ACT positive patients showed the contemporary presence of all three parameters and M-ACT was evaluated only on the first BM biopsy at diagnosis and before any treatment.
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