Academic literature on the topic 'Myeloproliferative neoplasms, prognosis, outcome, myelofibrosis'

Abstract Substantial progress has been made in our understanding of the pathogenetic basis of myeloproliferative neoplasms. The discovery of mutations in JAK2 over a decade ago heralded a new age for patient care as a consequence of improved diagnosis and the development of therapeutic JAK inhibitors. The more recent identification of mutations in calreticulin brought with it a sense of completeness, with most patients with myeloproliferative neoplasm now having a biological basis for their excessive myeloproliferation. We are also beginning to understand the processes that lead to acquisition of somatic mutations and the factors that influence subsequent clonal expansion and emergence of disease. Extended genomic profiling has established a multitude of additional acquired mutations, particularly prevalent in myelofibrosis, where their presence carries prognostic implications. A major goal is to integrate genetic, clinical, and laboratory features to identify patients who share disease biology and clinical outcome, such that therapies, both existing and novel, can be better targeted.

Abstract Substantial progress has been made in our understanding of the pathogenetic basis of myeloproliferative neoplasms. The discovery of mutations in JAK2 over a decade ago heralded a new age for patient care as a consequence of improved diagnosis and the development of therapeutic JAK inhibitors. The more recent identification of mutations in calreticulin brought with it a sense of completeness, with most patients with myeloproliferative neoplasm now having a biological basis for their excessive myeloproliferation. We are also beginning to understand the processes that lead to acquisition of somatic mutations and the factors that influence subsequent clonal expansion and emergence of disease. Extended genomic profiling has established a multitude of additional acquired mutations, particularly prevalent in myelofibrosis, where their presence carries prognostic implications. A major goal is to integrate genetic, clinical, and laboratory features to identify patients who share disease biology and clinical outcome, such that therapies, both existing and novel, can be better targeted.

Myelofibrosis (MF) is subtype of myeloproliferative neoplasm (MPN) characterized by a relatively poor prognosis in patients. Understanding the factors that drive MF pathogenesis is crucial to identifying novel therapeutic approaches with the potential to improve patient care. Driver mutations in three main genes (janus kinase 2 (JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL)) are recurrently mutated in MPN and are sufficient to engender MPN using animal models. Interestingly, animal studies have shown that the underlying molecular mutation and the acquisition of additional genetic lesions is associated with MF outcome and transition from early stage MPN such as essential thrombocythemia (ET) and polycythemia vera (PV) to secondary MF. In this issue, we review murine models that have contributed to a better characterization of MF pathobiology and identification of new therapeutic opportunities in MPN.

Primary myelofibrosis (PMF) has the least favorable prognosis of the Philadelphia chromosome–negative myeloproliferative neoplasms, which also include essential thrombocythemia (ET) and polycythemia vera (PV). However, clinical presentations and outcomes of PMF vary widely, with median overall survival ranging from years to decades. Given the heterogeneity of PMF, there has been considerable effort to develop discriminatory prognostic models to help with management decisions, particularly for the consideration of hematopoietic stem cell transplantation in patients at higher risk. Although earlier models incorporated only clinical features in risk stratification, contemporary models increasingly use molecular and cytogenetic features, leading to more comprehensive prognostication. This article reviews the most widely adopted prognostic models used for PMF, including the International Prognostic Scoring System (IPSS), dynamic IPSS (DIPSS)/DIPSS+, mutation-enhanced IPSS for transplant-age patients (MIPSS70)/MIPSS70+/MIPSS70+ version 2.0, genetically inspired prognostic scoring system, and Myelofibrosis Secondary to PV and ET-Prognostic Model in patients with post-ET/PV myelofibrosis. We also discuss newly emerging prognostic models and provide a practical approach to risk stratification in patients with PMF and post-ET/PV myelofibrosis.

Abstract Introduction Myeloproliferative Neoplasms can shorten the life of patients and affect severely their quality of life as a result of constitutional symptoms mediated by cytokines and from massive splenomegaly characteristic of these diseases. The objective measurement of symptoms in Myelofibrosis is essential for the presence of general constitutional symptoms as fatigue and is considered adverse prognostic value for survival. The systematic measurement of symptoms in Myelofibrosis is essential for assessing the outcome of treatment with JAK-2 inhibitors. Material and Methods To assess the impact on quality of life in patients with Myeloproliferative Neoplasms treated with the inhibitor Ruxolitinib, there was applied the questionnaire Myelofibrosis Symptom Assessment Form (Mesa et al. Leukemia Research. 2009: 33; 1199-1203) to 16 patients included in the Compassionate Use of Ruxolitinib Program: 8 men and 8 women were included with a median age of 63 years; 4 patients with primary Myelofibrosis, 3 with Myelofibrosis post Polycitemia vera and 9 with Myelofibrosis post essential Thrombocythemia. The median patient follow-up was 9 months. The patients started with a dose of 20 mg Ruxolitinib orally every 12 hours. Results The questionnaire was administered to all patients at each visit and the results of the ratings of the patients were presented as median of each answer at baseline, 3, 6 and 12 months of follow-up (table 1). By comparing the differences of each parameter measured between the start and at 3, 6 and 12 months were statistically significant for all variables (P <0.001, CI: 95%). The most significant changes were observed when comparing data at baseline and follow-up to the maximum time, however were obvious from the first quarter of observation on fatigue and symptoms dependent splenomegaly in most general symptoms, which actually disappeared completely after 3 months of treatment and not relapsed during the track and in the perception of the quality of life of patients. The variables with lower proportional change recorded were general activity, mood, walking tolerance and everyday work. Discussion and conclusions The treatment of Myeloproliferative Neoplasms with Ruxolitinib represents a major advance in the management of malignant Myeloproliferative and achieved excellent improvement in overall constitutional symptom control, splenomegaly and the general perception of the quality of life of these patients. The objective measurement of symptoms of Myelofibrosis is essential, since the presence of general constitutional symptoms as fatigue is considered adverse prognostic value for survival and systematic measurement of symptoms in Myelofibrosis is essential for assessing the outcome of treatment with inhibitors JAK-2. Disclosures: No relevant conflicts of interest to declare.

Primary myelofibrosis (PMF) is a BCR-ABL1 negative myeloproliferative neoplasm characterized by clonal proliferation of myeloid cells. This leads to reactive bone marrow fibrosis, ultimately resulting in progressive marrow failure, hepatosplenomegaly, and extramedullary hematopoiesis. PMF is considered the most aggressive of the BCR-ABL1 negative myeloproliferative neoplasms with the least favorable prognosis. Constitutional symptoms are common, which can impact an individual’s quality of life and leukemic transformation remains an important cause of death in PMF patients. The development of the Janus kinase 2 (JAK2) inhibitors have provided a good option for management of PMF-related symptoms. Unfortunately, these agents have not been shown to improve overall survival or significantly alter the course of disease. Allogenic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment option in PMF. However, allo-HSCT is associated with significant treatment-related morbidity and mortality and has historically been reserved for younger, high-risk patients. This review examines patient, disease, and transplant-specific factors which may impact transplant-related outcomes in PMF. Through the vast improvements in donor selection, conditioning regimens, and post-transplant care, allo-HSCT may provide a safe and effective curative option for a broader range of PMF patients in the future.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are divided in three major groups: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The 2016 WHO classification incorporates also prefibrotic PMF (pre-PMF) and overt PMF. This study aimed to discriminate the clinical features, genetic alterations, and outcomes in patients with prefibrotic, overt PMF, and secondary MF (SMF). This study included 229 patients with diagnosed myelofibrosis (MF). Among 229 patients, 67 (29%), 122 (53%), and 40 (18%) were confirmed as SMF, overt PMF, and pre-PMF, respectively. The JAK2 V617F mutation was differentially distributed in SMF and PMF, contradictory to CALR and MPL mutations. Regarding nondriver mutations, the occurrence of ASXL1 mutations differed between PMF and SMF or pre-PMF. The three-year overall survival was 91.5%, 85.3%, and 94.8% in SMF, overt PMF, and pre-PMF groups. Various scoring systems could discriminate the overall survival in PMF but not in SMF and pre-PMF. Still, clinical features including anemia and thrombocytopenia were poor prognostic factors throughout the myelofibrosis, whereas mutations contributed differently. Molecular grouping by wild-type SF3B1 and SRSF2/RUNX1/U2AF1/ASXL1/TP53 mutations showed inferior progression-free survival (PFS) in PMF, SMF, and pre-PMF. We determined the clinical and genetic features related to poor prognosis in myelofibrosis.

Abstract Introduction: Although myeloproliferative neoplasms (MPN) are driven by three mutually exclusive driver mutations (JAK2, CALR and MPL), targeted deep sequencing studies identified multiple additional somatic mutations potentially impacting MPN evolution. Presence of a high molecular risk (HMR: ASXL1, EZH2, SRSF2 and IDH1/2) or a TP53 mutations has been associated with adverse prognosis. However, to date, the effect of clonal evolution (CEv) on MPN patients' outcome has not been evaluated, as most of the studies assessed mutational-based prognosis stratification from single baseline molecular genotyping. The objective of our study was to describe the clinical and molecular characteristics of patients with CEv in a large cohort of MPN patients and analyze its impact on patients' outcome. Methods: A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. From this large retrospective cohort, we included in this study 446 patients who had at least 2 molecular analyses during the chronic phase of MPN, performed at diagnosis and/or during follow-up using next generation sequencing (NGS), targeting a panel of 36 genes involved in myeloid malignancies. Significant variants were retained with a sensitivity of 1%. CEv was defined as the acquisition of a new additional non-driver mutation between baseline and subsequent NGS evaluation. Statistical analyses were performed using the STATA software (STATA 17.0 for Mac Corporation, College Station, TX). Results: The median age at MPN diagnosis in our whole cohort was 51 years [IQR 41 - 60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with polycythemia vera, essential thrombocythemia and primary myelofibrosis (MF) respectively. With a median interval of 1.6 years [IQR 1.0 - 2.8] between the first and the second NGS analysis in the whole cohort, CEv occurred in 128 patients (29%). Patients with CEv were significantly older compared to patients without CEv (n=318) (p=0.03). MPN diagnosis, the type of driver mutation and complete blood counts at MPN diagnosis did not differ between the 2 groups. Eighty-one (63%) and 198 (62%) patients with or without CEv respectively had at least one additional non-driver mutation at baseline NGS (p=0.59), while the rate of HMR (n=25 (20%) versus n=79 (25%)) or TP53 (n=7 (5%) versus n=20 (6%)) mutations at baseline NGS did not differ between the 2 groups. Thirty six out of 128 (28%) of patients with CEv had more than 1 acquired mutation. Most recurrently acquired mutations involved the epigenetic regulators TET2 and DNMT3A that were mutated in respectively 33% and 25% of patients with CEv (Figure 1A). Moreover, 38% of CEv patients acquired HMR (ASXL1 (14%), EZH2 (6%), SRSF2 (3%), IDH1/2 (2%)) or TP53 (13%) mutations. After a median follow up of 10.8 years [IQR 6.6 - 17.2] in the whole cohort representing a total of 5635 patient years, 32 (7%) patients died, and 11 (2.5%) and 11 (2.5%) patients with at least 2 NGS performed during MPN chronic phase transformed respectively into secondary MF or myelodysplastic syndrome / acute myeloid leukemia (MDS/AML). Interestingly, CEv (HR 11.27, 95%CI [5.09; 24.96], p&lt;0.001) (Figure 1B), age at MPN diagnosis (HR 1.11, 95%CI [1.07; 1.15], p&lt;0.001) and the presence of HMR mutations at baseline NGS (HR 4.48, 95%CI [2.05; 9.77], p &lt;0.001) independently adversely impacted OS in a COX regression multivariate analysis. CEv also independently adversely impacted MDS/AML free survival (HR 13.15, 95%CI [3.88; 44.47], p&lt;0.001) and secondary MF free survival (HR 21.13, 95%CI [6.18; 72.20], p&lt;0.001) in a COX regression multivariate analysis. Conclusion: Our study on a large retrospective clinically and biologically annotated real-life cohort of MPN patients with long-term follow up shows that CEv independently adversely impacts OS, MDS/AML and secondary MF free survivals. CEv occurred in a clinically relevant proportion of MPN patients (28%) and was associated with patients' age. Acquired mutations mainly involved epigenetic regulators, HMR and TP53 genes. These results suggest that serial molecular monitoring using NGS could be routinely implemented in MPN patients follow up, to assess more accurately disease evolution and potentially update therapeutic management. Figure 1 Figure 1. Disclosures Raffoux: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ABBVIE: Consultancy; ASTELLAS: Consultancy. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Benajiba: Gilead: Research Funding; Pfizer: Research Funding.

Abstract Background: Myeloproliferative neoplasms (MPNs) are a group of disorders of clonal hematopoiesis and include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Cardiovascular disease, including atrial fibrillation (AF), heart failure (HF), pulmonary hypertension (PH), and atherosclerotic heart disease, is an underappreciated cause of morbidity and mortality in MPNs. Additionally, MPNs harbor mutations commonly seen in clonal hematopoiesis of indeterminate potential (CHIP) which has been shown to be associated with increased cardiovascular risk. PH has been associated with MPN and MPN-associated PH is classified as group 5 PH according to the World Health Organization. Although studies have shown PH to occur in between 3.8% to 58% of patients with PMF and has been associated with decreased overall survival in PV, its impact on cardiovascular prognosis, including cardiovascular death (CV death), in patients with MPN has been understudied. Our study aims to investigate the prognostic implication of PH in a high risk MPN population, those with cardiovascular disease. Methods: Our study is a single center retrospective cohort study. Using an institutional database, we identified 197 patients with MPN (ET, PV or PMF) who had cardiovascular disease (AF, hospitalization for HF or acute coronary syndrome after MPN diagnosis) and had undergone right heart catheterization (RHC) or transthoracic echocardiography (TTE). PH was defined as either a mean pulmonary artery pressure ≥ 20 mmHg on RHC or estimated right ventricular systolic pressure (RVSP) ≥ 50 mmHg on TTE. Our primary outcome was CV death (death due to pulmonary embolism, myocardial infarction, stroke, arrhythmia, sudden death or HF). Other outcomes of interest included all-cause mortality, HF hospitalization, VTE and arterial thrombosis. Continuous variables were compared using Student's t-test, categorical variables and outcomes were compared with the Fisher exact test. We used a Fine-Gray model to determine the effect of PH on CV death using other causes of death as competing events. To investigate the effect of PH on our outcomes we also used a multivariable logistic regression model using age, sex, MPN type, MPN treatment, arterial and venous thrombosis prior to and after MPN diagnosis, diabetes, prior HF diagnosis, HF hospitalization after MPN diagnosis, AF, hyperlipidemia, leukemic or secondary myelofibrosis transformation, and diabetes as co-variables. Results: There were 92 patients with PH and 105 patients without PH. Median age at MPN diagnosis was higher in PH vs non-PH patients (73 vs 71, p = 0.038). The median time to diagnosis of PH from MPN diagnosis was 69 months (IQR 43, 113). Patients with PH had higher rates of prior HF (54% vs 32%, p = 0.001) and hypertension (83% vs 64%, p = 0.004), but lower rates of AF (64% vs 79%, p = 0.026) compared with patients without PH. MPN types and driver mutations were not statistically significant between PH and non-PH groups. More patients with PH had leukemic or secondary myelofibrosis transformation compared with patients without PH (23% vs 11%, p = 0.037). Patients with PH had higher rates of CV death (35% vs 9%, p &lt; 0.0001), all-cause death (58% vs 37%, p = 0.004), HF hospitalization (73% vs 36%, p &lt; 0.0001) and VTE (25% vs 12%, p = 0.027) but not arterial thrombosis (39% vs 41%, p = 0.88). Cumulative incidence of CV death was higher in PH compared with non-PH patients (Figure 1A, Fine-Gray HR 5.53, 95% CI 2.58-11.85). After multivariable regression, PH was associated with higher odds of CV death (Figure 1B, aOR 6.1, 95% CI 2.2-19.0), VTE (aOR 3.3, 95% CI 2.0-25.5), HF hospitalization (aOR 3.9, 95% CI 1.6-10.2) but not all-cause death (aOR 1.5, 95% CI 0.7-3.2) or arterial thrombosis (aOR 0.9, 95% CI 0.4-1.9), Figure 1B. Conclusions: In patients with MPN and cardiovascular diseases, PH is associated with worse outcomes including CV death, VTE, HF hospitalization and leukemic/secondary myelofibrosis transformation compared with patients without PH. More investigation is needed but our results suggests early screening for PH may be beneficial in this patient population. Figure 1 Figure 1. Disclosures Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rosovsky: Janssen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Inari: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hobbs: Bayer: Research Funding; Incyte Corporation: Research Funding; Merck: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie.: Consultancy.

Abstract The application of genomic techniques, including cytogenetics and DNA sequencing, to decipher the molecular landscape of patients with myeloproliferative neoplasms (MPNs) has radically modified diagnostic approach and management through improved risk stratification. Three driver mutated genes (JAK2, MPL, CALR) are variably harbored by &gt;80% of patients and associated with clinical characteristics, as well as major disease-related complications and different survival outcomes. Therefore, JAK2 V617F mutation is included in the revised International Prognosis Score of Thrombosis for Essential Thrombocythemia score for prediction of thrombosis in patients with essential thrombocythemia and prefibrotic primary myelofibrosis, while a CALR type 1 mutated genotype constitutes a favorable variable for survival in patients with myelofibrosis (MF). Novel, integrated clinical and cytogenetic/mutation scores (Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients with Primary Myelofibrosis [MIPSS70/v2], genetically inspired prognostic scoring system [GIPSS], Myelofibrosis Secondary to PV and ET- Prognostic Model [MYSEC-PM]) have been devised that guide selection of stem cell transplantation candidates with MF or help predict the risk associated with the transplant procedure (Myelofibrosis Transplant Scoring System), with greater performance compared with conventional scores based on hematologic and clinical variables only. On the other hand, several clinical needs remain unmet despite the great amount of molecular information available nowadays. These include the prediction of evolution to acute leukemia in a clinically actionable time frame, the identification of patients most likely to derive durable benefits from target agents, in primis JAK inhibitors, and, conversely, the significance of molecular responses that develop in patients receiving interferon or some novel agents. Here, we discuss briefly the significance and the role of genomic analysis for prognostication in patients with MPNs from a clinician's point of view, with the intent to provide how-to-use hints.

Les néoplasies myéloprolifératives (NMP), non BCR-ABL1, regroupent principalement la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose primitive (MFP).Ces pathologies partagent, dans des proportions variables, une mutation commune, la mutation JAK2 V617F. La protéine JAK2 mutée a une activité tyrosine kinase constitutive, impliquée dans le développement de la maladie. Cette mutation, seule, n’explique pas l’hétérogénéité phénotypique au sein des NMP. L’avènement des techniques de séquençage haut débit a permis de mieux appréhender la physiopathologie. Notre travail avait pour objectif l’identification de mutations additionnelles au sein de deux cohortes suivies au long cours en lien avec un risque d’aggravation de la maladie, l’une regroupant des patients en phase chronique (TE et PV JAK2 V617F), la seconde regroupant des patients avec une myélofibrose traitée par interféron. A l’instar d’autres travaux récents, nous avons montré que le nombre de mutations et la présence de mutations additionnelles sont associés à l’évolution de la maladie, voire à la réponse au traitement.Parmi les mutations identifiées, certaines pourraient influencer l’épissage. La deuxième partie de ce travail a donc consisté à étudier l’épissage alternatif en fonction des mutations présentes, et en particulier la mutation JAK2 (V617F) et de manière globale dans les TE. Un saut de l’exon 14 de JAK2 a été décrit chez des patients NMP présentant, ou non, la mutation JAK2 V617F. Cette mutation du gène JAK2 est prédite pour altérer un site de fixation de la protéine SRSF6 régulatrice de l’épissage. Nous observons que le saut de l’exon 14 est un événement peu fréquent chez les patients, modulé en partie par l’expression des protéines SR. L’analyse transcriptomique montre une grande hétérogénéité entre les patients en termes d’expression et d’épissage, ce qui ne nous a pas permis de mettre en évidence de profil caractéristique. Ces résultats soulignent l’importance de l’identification des mutations additionnelles au diagnostic et au cours du suivi.Nous avons pu, en outre, identifier quelques transcrits alternatifs associés à la présence de ces mutations. Le rôle fonctionnel de ces variants reste à définir
Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are a group of Philadelphia-negative myeloproliferative neoplasm (MPN). These diseases share a common mutation, JAK2 V617F, in varying proportions. The mutated JAK2 protein has a constitutive tyrosine kinase activity, implicated in the physiopathology of MPN. This mutation alone does not explain the phenotypic heterogeneity within MPN.High throughput sequencing techniques helped understanding the physiopathology. This work aimed to identify additional mutations in two patient cohorts related to the aggravation risk of the disease. The first one consisted of patients in chronic phase (JAK2 V617F ET and PV), the second consisted in patients with myelofibrosis treated with interferon. Like other studies, we have shown that the number of mutations and the presence of additional mutations are associated with disease progression or with response to treatment. Some identified mutations could influence splicing. The second part of this work aimed at studying the putative impact of the JAK2 V617F mutation, on alternative splicing (AS).We also analyzed global AS profiles in ET. JAK2 exon 14 skipping has been described in NMP patients with or without the JAK2 V617F mutation.This mutation was predicted to alter the binding site of the SRSF6 splice-regulating protein. We observed that exon 14 skipping was an uncommon event in patients, in part related to SR protein expression. In addition, our transcriptomic-wide analysis showed a great heterogeneity between the patients with respect to both gene expression and splicing. This prevented us from identifying any characteristic profile. These results underscore the importance of identifying additional mutations at diagnosis and during follow-up. We have also been able to uncover some alternative transcripts associated with the presence of these mutations.The functional role of these variants remains to be defined

The classic chronic myeloproliferative neoplasms (MPNs) comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), originate from de-regulated clonal proliferation of hematopoietic stem cells and are associated with overproduction of mature blood cells. MPN are characterized by a high molecular complexity. Although JAK2 mutations have been shown to be the phenotypic drivers in MPN, there is evidence of clonality and mutational events preceding the acquisition of JAK2V617F. An increasing number of mutations in genes distinct from JAK2 have been recently identified in patients with MPN. Mutations can be divided in: “phenotypic driver” (JAK2, MPL and CALR) since the expression of the mutated gene in cell lines caused cytokine independent growth and in animal models phenotypes closely resembling a myeloproliferative disease; “subclonal mutations” that usually occur in hematopoietic cell subclones of variable size, often but not invariably together with one of the phenotypic driver mutations. Although different studies have found statistically significant correlations between the presence (or allelic burden) of certain molecular abnormalities and clinical phenotype in MPN, particularly in myelofibrosis, these novel molecular information still have modest influence on risk stratification and treatment decisions. This project focused on identifying novel disease-associated genetic alterations in MPNs, addressing their relevance for disease progression and leukemia transformation. Elucidation of these issues would facilitate the understanding of genotype-phenotype correlations, in order to explain the molecular puzzle underlying the pathogenesis of MPNs, as well as refining risk stratification and improving therapeutic management of the patients. In the first set of experiments we studied two independent cohorts of patients with PMF, in order to clarify the prognostic relevance of newly described mutations, including TET2, CBL, IDH1 or IDH2, ASXL1, EZH2, DNMT3A and SRSF2. The first cohort of 483 patients, from multiple centres in Europe, studied within one year of diagnosis, was needful to select out which one of these mutations predicted worse outcome. Overall and leukemia-free survivals (OS and LFS) were inter-independently predicted by ASXL1, SRSF2 or EZH2 mutations and ASXL1, SRSF2 and IDH1/2 mutations, respectively. We classified the entire patient cohort into those who displayed at least one (“molecularly high-risk”, HMR) or (“molecularly low risk”, LMR) none of the four mutations and we found that pts at LMR had better outcome respect to pts in HMR category. The correlation between the mutational status and the disease outcome was significant in terms of both OS and LFS. We then found that also the “number” of prognostically detrimental mutated genes had impact on outcome. Patients with 2 or more HMR mutations had significantly reduced overall and leukemia-free survival compared no only to unmutated but also to those presenting only one mutation in HMR genes. These results were validated both in learning and validation cohorts. In the second part of my work I focused my attention on the very recent discovery of CALR mutations in MPNs in order to clarify different aspects regarding the identification and the impact of these mutations on clinics. Based on the common feature of CALR mutations to generate the same frame at C-terminus of the protein, we developed a polyclonal antibody against a 17 peptide residue in the variant C-terminus of mutated calreticulin, that proved able to label selectively bone marrow sections from CALR mutated patients, distinguishing them from normal subjects and ET and PMF patients harboring the JAK2 or MPL mutations. The immunostaining was almost entirely confined to cells of the megakaryocyte lineage, while myeloid and erythroid cells showed fainter labelling. So we demonstrated that the use of mutation specific antibody may be useful for molecular classification of ET and PMF patients and also for studying different aspects of hematopoiesis, and particularly megakaryocytopoiesis, in patients harboring CALR mutations. In a second moment we investigated the prevalence, characteristics and clinical and laboratory features associated with CALR mutations and the prognostic relevance of mutated CALR in large series of ET and PMF patients, in multicentre studies, and we demonstrated that CALR-mutant ET and CALR-mutant PMF have a relatively indolent clinical course compared with the respective JAK2-mutant and MPL-mutant disorders. On the contrary, PMF with non-mutated JAK2, CALR, or MPL (triple negative, TN) has a poor prognosis with a particularly high risk of leukemic transformation. Also the prognostic advantage of CALR mutation in PMF regards only patients harboring type 1/type 1-like mutation, while the survival of those harboring type 2/type 2-like mutation does not differ from JAK2V617F and MPLW515 mutated ones, maybe due to the molecular complexity of subclonal mutations. The interesting results that we obtained in the study on primary myelofibrosis, in terms of better OS of CALR mutated patients compared with JAK2+, MPL+ and TN pts, and the association of high molecular risk signature with negative outcome in PMF patients, prompt us at defining the role for outcome prediction of these mutational status in secondary myelofibrosis (sMF). We evaluated Post-Polycythemia Vera and Post-Essential Thrombocythemia Myelofibrosis (PPV-MF and PET-MF) patients (from 4 Italian centres) at time of diagnosis and we reported that in sMF there is not a strong impact of prognostically detrimental mutated genes on outcome that we conversely demonstrated in PMF patients, even if ASXL1 and SRSF2 still negative prognostic factor in sMF. We can suppose that in the secondary form the mutational profiling is much complex, probably due to the acquisition of different mutations, at different point during the disease progression. In our study on ET and PMF triple negative resulted the category with the worst prognosis, they present a higher risk of developing anemia and thrombocytopenia, suffer from reduced overall survival compared to other genotypes, particularly to CALR type1/type1like mutations, and may be at higher risk of leukemic transformation. Therefore, in the last part of my work, I focused my attention on an extensive molecular characterization of 28 PMF resulted triple negative in our cohort of 396 PMF patients. We evaluated 18 genes known to be prognostically relevant in previous studies (EZH2, ASXL1, IDH1/2, SRSF2, TP53, TET2, RUNX1, CBL, NRAS, KRAS, DNMT3A, SF3B1, IKZF1, NFE2, SH2B3, U2AF1). We observed that among triple negative patients there was a significantly higher proportion of patients clustered in the high molecular risk (both with at least 1 and >2 HMR mutations), compared with JAK2V617F, CALR Type1/1-like and CALR Type2-type2-like mutated patients. We could observe that patients displaying more than 2 mutations had the worst outcome, both in terms of overall and leukemia-free survival, but without reaching statistical significance. So we can conclude that the outcome of triple-negative PMF is markedly worse than JAK2, CALR or MPL mutant PMF, the dismal outcome could be explained by the high number of subclonal mutations displayed in these patients. Overall, these findings show that the molecular landscape of MPN is even more complex and that mutational profiling, in particular in PMF, is needful for refine current risk stratification models and highly relevant to clinical decision-making as regards diagnostic approach and prognostication.

Clinically chronic myeloproliferative neoplasms are biphasic or triphasic diseases that are usually diagnosed in the initial ‘chronic’, ‘indolent’, or ‘stable’ phase and then spontaneously evolve after some years into an advanced phase. In patients with chronic myeloid leukaemia, the advanced phase can sometimes be subdivided into an earlier accelerated phase and a later blast phase resembling acute leukaemia. Patients with essential thrombocythaemia and polycythaemia vera transform initially to myelofibrosis and then to acute myeloid leukaemia (AML). Rarely they transform directly from the initial indolent phase to AML; patients with primary myelofibrosis also transform directly to AML. Although much is known about the molecular biology and genomic landscape of the initial phases, the molecular basis of disease progression and transformation remains obscure. The clinical management of transformed disease is generally difficult, and most patients tend to have a poor prognosis. This chapter reviews what is known of the mechanisms underlying the transformation of these myeloproliferative neoplasms from a chronic phase to frank leukaemia and also discusses the current and future treatment strategies.

The BCR-ABL-negative myeloproliferative neoplasms (MPN) collectively represent a diverse assortment of clonal myeloid malignancies with variability in both genotypic and phenotypic presentation. MPN subtypes including essential thrombocythaemia (ET), polycythaemia vera (PV), myelofibrosis (MF), systemic mast cell disorders have been best studied in terms of symptom profiles and heterogeneously incur disease burdens that impact treatment options, overall quality of life, and survival. Recent treatment strategies have aimed to incorporate disease burden assessment into the selection of therapeutic interventions. Patient-reported outcome (PRO) tools have been developed to objectively assess the MPN disease burden in both clinical and trial formats. Their development supports recent FDA efforts to promote PROs in supporting labelling claims for novel targeted agents. This chapter discusses the spectrum of symptoms inherent to MPN disorders, available risk scoring tools and PRO options, and the emerging role of patient outcomes as trial endpoints.

The latest version of the World Health Organization guidelines focuses mainly on the genetic and cytogenetic features of hematologic neoplasms as predictors of diagnostic, treatment decision, prognostic outcome, and for treatment monitoring in hematological malignancies. There are different techniques to identify these abnormalities. Live cells are needed for chromosome preparation. The Hematological malignancies include myeloid and lymphoid neoplasms. The myeloid neoplasms include Myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemias. The Lymphoid neoplasms include acute and chronic lymphocytic leukemias, plasma cell neoplasms, myeloma, hodgkin, and non-hodgkin lymphomas. The first chromosomal abnormality discovered in connection with cancer is the Philadelphia chromosome, which is an abnormal chromosome 22, formed due to the translocation between chromosomes 9 and 22. The presence of this abnormal chromosome confirms the diagnosis of “CML”. After that, hundreds of chromosomal abnormalities have been identified in hematological malignancies in different parts of the world. In AML, specific abnormalities were identified as having a good prognosis, intermediate prognosis, and poor prognosis. In other hematological malignancies also there some specific chromosome abnormalities are associated with prognostication. Now a day’s clinicians depend mainly on genetic abnormalities for the proper treatment management of hematological malignancies, so the study of chromosomal abnormalities is essential.