Unclassifiable Myeloproliferative Neoplasms Research Articles

Morphologic and Genetic Features of Unclassifiable Chronic Myeloid Neoplasms: Five-Year Experience of a Canadian Provincial Referral Center

Introduction: The challenge of diagnosing and definitively classifying myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) remains a conundrum in the field of hematology. While the World Health Organization has outlined diagnostic criteria for various categories, there remain cases that elude specific classification, falling under the umbrella of MPN, unclassifiable (MPN-U) and MDS/MPN, unclassifiable (MDS/MPN-U). In this study, we aimed to compare the morphological and genetic profiles of cases classified as MPN-U or MDS/MPN-U against more definitively classified cases (including chronic myelomonocytic leukemia (CMML), essential thrombocythemia (ET), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and primary myelofibrosis (PMF), including prefibrotic PMF (pre-PMF)). Methods: Morphologically unclassifiable myeloid neoplasm cases referred for pathologist review to our laboratory in the largest tertiary care center in British Columbia, Canada were identified through pathology database query over a five-year period. Bone marrow materials were reviewed and reanalyzed with standardized collection of qualitative and semiquantitative data by two hematopathologists. Morphological parameters studied included the myeloid to erythroid ratio, bone marrow cellularity, lineage dysplasia, megakaryocyte histotopography, bone marrow blast frequency, fibrotic changes, and bone marrow vascularity, among others. Clinical data, cytogenetic findings, and results of a targeted sequencing panel assessing a set of approximately 50 genes recurrently mutated in myeloid malignancies were collected by electronic medical record review. A cohort of contemporarily collected myeloid neoplasms with definitive pathologic diagnoses was reviewed for comparison. Statistical analyses were performed using python3. Results: The cohort comprised 29 cases classified as MPN-U, 12 cases of MDS/MPN-U, and 5 cases each of ET, PMF, pre-PMF, CMML, and MDS/MPN-RS-T. MPN-U cases were further subcategorized based on the apparent reason for difficulty in classification, including: overlapping features with another MPN; late stage/overt fibrosis; early stage without fully-developed morphologic features; and coexisting atypical morphologic findings. MPN-U with overlapping features had highly variable reticulin fibrosis, while, as expected, late stage entities had a high degree of reticulin fibrosis and early stage entities showed less overt fibrosis (Figure, Left, demonstrating select morphologic features of evaluated chronic myeloid neoplasms). MPN-U as a whole appeared to show less prominent fibrosis-associated findings than MDS/MPN-U and to, in general, show a greater degree of neovascularization, similar to changes observed in cases with PMF. Splicing factor and epigenetic modifier mutations appeared to be overrepresented in MDS/MPN-U relative to MPN-U, notably excluding MPN-U with atypical morphologic features (Figure, Right, demonstrating the distribution of pathogenic variants identified in evaluated myeloid neoplasms; results are indicated as presence (1) or absence (0) of a variant for each gene). Notably, no CALR mutations were identified in the MDS/MPN-U category or early stage / atypical morphology MPN-U, though they were enriched in overtly fibrotic MPN-U entities. Discussion/Conclusion: In this cohort we observed, as expected, significant overlap in cases classified as MPN-U and MDS/MPN-U with better delineated entities, though patterns of morphologic change and mutational spectra suggest potential directions for further investigation of additional criteria that may guide accurate classification of these neoplasms. These findings reflect the heterogeneity of MPNs described in the literature and the complexity of their genetic underpinnings, particularly in the context of concurrent dyshematopoiesis; suggest enhanced bone marrow vascularization may be an informative differential diagnostic criterion in evaluation of these neoplasms, though further study is required; and emphasize the need for broader genetic testing and perhaps further subtyping of MPN-U cases for a more nuanced analytical approaches for these disorders.

Classification of Philadelphia-Negative MPN As Low Risk and High Risk MPN Based on Peripheral Blood Values and Molecular Genetics Only

Background: Philadelphia-negative myeloproliferative neoplasms (Ph -MPN) are characterized by overproduction of differentiated hematopoietic cells mediated in the majority of cases by mutations in JAK2, MPL or CALR (MPN driver genes). Ph - MPN include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and MPN, unclassifiable (MPN-U) which differ in clinical presentation, prognosis, fibrosis and leukemic transformation rate as well as therapeutic options. However, WHO based classification requires experienced pathologists, in particular to diagnose prefibrotic PMF as high risk MPN, and thus remains observer dependent. Aim: Observer independent classification of Ph - MPN solely based on molecular genetics and peripheral blood (PB) parameters. Patients and Methods: We analyzed 267 Ph - MPN cases diagnosed as ET (n=76), PV (n=74), PMF (n=68) or MPN-U (n=49) following the WHO4R classification including only patients with an MPN driver mutation (median age: 68 years [22-91]; female: 41%; median follow-up: 5 years). All samples were analyzed by cytomorphology and whole genome sequencing (median coverage 100x). Mutation status of 20 genes associated with myeloid malignancies were analyzed in detail. Results: Genomic landscape of the MPN cohort showed mutations in JAK2 in 222 (83%) (n=132: single mutation; n=90: either mutation and copy neutral loss of heterozygosity or mutation and 9p gain), in CALR in 36 (14%), and in MPL in 9 (3%) patients. The median number of mutations per patient was two (range: 1-6). Within the cohort, 141 patients (53%) harbored MPN driver mutations only, while in 126 (47%) one to 5 additional mutations were found. The most frequent additional mutations were ASXL1 (n=50), TET2 (n=49; thereof 15 biallelic), SRSF2 (n=26), DNMT3A (n=18), U2AF1 (n=11) and TP53 (n=9; thereof 3 biallelic). Regarding PB parameters, high WBC (≥11×10 9/ L) were detected in 127 patients (48%), high PLT (≥450×10 9/ L) in 116 (43%) and anemia (HB <♂12 or ♀11 g/dL) in 50 patients (19%). We then performed Cox regression analyses for overall survival (OS) including only genetic abnormalities and PB values. In univariate analyses, biallelic TET2 inactivation ( TET2bi: hazard ratio/HR: 3.789), RUNX1 (n=4; HR: 13.479), ASXL1 (HR: 1.886), SRSF2 (HR: 3.218) mutations as well as high PLT (HR: 0.440), high WBC (HR: 1.998) and PB blasts >1% (n=57; HR: 1.904) showed significant associations with OS (for all p<0.02), while no effect was observed for MPN driver mutations, DNMT3A and monoallelic TET2 mutations. In addition, anemia showed a trend towards poor prognosis (HR: 1.595; p=0.079). Following this, we determined high and low risk MPN. In the absence of thrombocytosis, high risk MPN was defined if any of the four following risk factors were present: i) mutation in ASXL1 or SRSF2 or RUNX1 or TET2bi, ii) PB blasts >1%, iii) high WBC or iv) anemia. In the presence of thrombocytosis, at least two of these risk factors were required (Figure 1). Survival analysis revealed a significantly worse median OS of high risk (n=137; 51%) compared to low risk MPN (n=130; 49%) (8 years vs. not reached; p<0.001). This proposed novel stratification reflected prognosis better than WHO entities (median OS: MPN-U: 7 years; MF: 8 years; PV/ ET not reached; overall p=0.001) (Figure 1A). All 76 ET cases (100%) and 44/74 PV cases (59%) were classified as low risk MPN, while 63/68 PMF cases (93%) and 44/49 MPN-U cases (90%) were classified as high risk MPN (Figure 1B). Within low risk and high risk MPN, no significant OS differences were observed between the WHO entities ( p=0.114 and p=0.355, respectively). Conclusions: JAK2-pathway mutated Ph - MPN can be categorized into low and high risk MPN based on molecular genetics and PB values only. The presence of these objectively assessable risk factors in a patient with ET or PV warrants for a profound reevaluation of bone marrow histology (eventually by a reference pathologist for MPN) and clinical course before a high risk MPN (including prefibrotic PMF) can be ruled out. Whether the predictive value of low risk MPN based on molecular genetics and PB values only could be sufficient to omit BM biopsy in selected patients, needs to be evaluated in further prospective studies.

Labor market affiliation of patients with myeloproliferative neoplasms: a population-based matched cohort study

Background Patients with myeloproliferative neoplasms (MPNs) suffer from substantial symptoms and risk of debilitating complications, yet observational data on their labor market affiliation are scarce. Material and methods We conducted a descriptive cohort study using data from Danish nationwide registries, including patients diagnosed with MPN in 2010-2016. Each patient was matched with up to ten comparators without MPN on age, sex, level of education, and region of residence. We assessed pre- and post-diagnosis labor market affiliation, defined as working, unemployed, or receiving sickness benefit, disability pension, retirement pension, or other health-related benefits. Labor market affiliation was assessed weekly from two years pre-diagnosis until death, emigration, or 31 December 2018. For patients and comparators, we reported percentage point (pp) changes in labor market affiliation cross-sectionally from week −104 pre-diagnosis to week 104 post-diagnosis. Results The study included 3,342 patients with MPN and 32,737 comparators. From two years pre-diagnosis until two years post-diagnosis, a larger reduction in the proportion working was observed among patients than comparators (essential thrombocythemia: 10.2 [95% CI: 6.3–14.1] vs. 6.8 [95% CI: 5.5–8.0] pp; polycythemia vera: 9.6 [95% CI: 5.9–13.2] vs. 7.4 [95% CI: 6.2–8.7] pp; myelofibrosis: 8.1 [95% CI: 3.0–13.2] vs. 5.8 [95% CI: 4.2–7.5] pp; and unclassifiable MPN: 8.0 [95% CI: 3.0–13.0] vs. 7.4 [95% CI: 5.7–9.1] pp). Correspondingly, an increase in the proportion of patients receiving sickness benefits including other health-related benefits was evident around the time of diagnosis. Conclusion Overall, we found that Danish patients with essential thrombocythemia, polycythemia vera, myelofibrosis, and unclassifiable MPN had slightly impaired labor market affiliation compared with a population of the same age and sex. From two years pre-diagnosis to two years post-diagnosis, we observed a larger reduction in the proportion of patients with MPN working and a greater proportion receiving sickness benefits compared with matched individuals.

Differential diagnosis of thrombocytosis: a case study

The article presents a clinical case of an unclassifiable myeloproliferative neoplasm in a 65-year old patient. Platelet count is not a criteria for differential diagnosis of reactive and tumor processes. A typical course of the disease is associated with the persistence of symptoms of microcirculation disorder against the background of previous asymptomic thrombocytosis for several years. The diagnosis of an unclassifiable myeloproliferative neoplasm is established by a combination of clinical, laboratory, instrumental, molecular genetic and morphological methods of research.

GENETIC CHIMERISM IN MYELOPROLIFERATIVE NEOPLASMS: CLINICAL CASE OF COEXISTENCE OF JAK2V617F MUTATION AND BCR-ABL TRANSLOCATION

Essentialy, the diagnostics feature of myeloproliferative neoplasms (MPNs) is a genetic mutations. While bcr-abl translocation belongs to CML, Jak2V617F mutation is commonly associated with other MPNs. The recent decade has been clarified a data when a human blood presents bcr-abl translocation and Jak2V617F mutation both. This clinical case reports of bcr-abl translocation and Jak2V617F mutation when they were detected simultaneously by polymerase chain reaction (PCR) in a patient with myeloproliferative neoplasm, unclassifiable (MPN-U).

Molecular Profiles and Clinical Features of Myeloproliferative Neoplasms Unclassifiable (MPN-U) with and without JAK2/CALR/MPL Mutations

Introduction: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic neoplasms and include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), etc., each with specific diagnostic criteria. Patients who display features compatible with MPN but do not meet the diagnostic criteria for a specific entity are commonly assigned a diagnosis of MPN, unclassifiable (MPN-U). JAK2, CALR, and MPL are well-known driver mutations in PV, ET, and PMF. With the advancement and application of next-generation sequencing (NGS), the molecular profiles of the above 3 well-defined MPNs have been extensively studied. However, the molecular profile of MPN-U remains ill-defined. In this study, we aimed to describe the clinical and molecular features of MPN-U in our institution and then compare these features between patients with and without JAK2, CALR, or MPL mutations to better understand the clinicopathologic characteristics and prognosis of MPN-U. Method: The patients with a diagnosis of MPN-U and available NGS test results at our cancer center were identified and divided into two groups: cohort A) MPN-U with mutations in JAK2, CALR, or MPL, and cohort B) triple-negative. The patients' survival data, clinical parameters, bone marrow findings, and cytogenetics were collected for comparison. Baseline characteristics and somatic mutation distributions were compared using the chi-square test (categorical variables) and Wilcoxon rank sum test (continuous variables). Overall survival (OS) was calculated using the Kaplan-Meier method and compared using the log-rank test. A Cox proportional-hazards model was used to evaluate the association between OS and clinical factors. Results: Out of 8285 patients in our institutional clinical molecular database which houses NGS, we identified and confirmed MPN-U diagnosis in 53 patients. Among this group, 39 patients harbored JAK2 (n=36), CALR (n=1), MPL (n=1), or JAK2 and CALR co-mutations (n=1) and were assigned to cohort A. The remaining 14 patients were triple-negative and assigned to cohort B. The triple-negative patients in cohort B had higher median WBC count (18.5 x 10 9/L vs 9.98 x 10 9/L, p=0.04) and bone marrow cellularity (81% vs 64%, p=0.02) (Table 1). The mutational profile of cohort B was enriched for CUX1, ETV6, NRAS, SETBP1, and SRSF2 mutations. ASXL1 and SETBP1 were the most frequently mutated genes in the triple-negative group, and were often co-mutated with ETV6, NRAS, and/or SRSF2 (Figure 1). TET2 mutations appeared more common in cohort A, but this finding was not statistically significant. There were no significant differences in the other parameters, including gender, bone marrow blast percentage, karyotype, hemoglobin concentration, platelet count, and bone marrow fibrosis. Kaplan-Meier analysis demonstrated that the median OS time in cohorts A and B was not reached, and survival differences were not statistically significant (p-value 0.23). We used a Cox proportional-hazards model to evaluate the association between OS and clinical factors. Via backward elimination, we uncovered that high WBC count and low hemoglobin concentration were significantly associated with worse outcome (hazard ratios of 1.04 and 1.33, respectively, and p-values of 0.007 and 0.01). Conclusion: In our small cohort, the patients with MPN-U who were triple-negative for JAK2,CALR, and MPL mutations had more frequent CUX1, ETV6, NRAS, SETBP1, and SRSF2 mutations. ASXL1 and SETBP1 were the most frequently mutated genes in the triple-negative group, and were often co-mutated with ETV6, NRAS, and/or SRSF2. In addition, triple-negative patients had higher WBC counts and higher bone marrow cellularity. A Cox proportional-hazards model identified high WBC count and low hemoglobin concentration as being significantly associated with a worse outcome. Kaplan-Meier analysis did not reveal a difference in OS between triple-negative and JAK2, CALR, or MPL mutated MPN-U. A larger cohort is needed to evaluate the survival outcome of these two distinct groups. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical, Molecular, and Histopathological Correlates in a Series of 24 Consecutive Cases of Suspected Myeloproliferative Neoplasms with a JAK2V617F Variant Allele Frequency ≤1%

Background: JAK2V617F is detected in >95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F variant allele frequency (VAF) is associated with different clinical characteristics and outcome. Highly sensitive molecular assays led to detect JAK2V617F mutation also in healthy subjects, associated with a higher risk of coronary heart disease (N Engl J Med. 2017;377(2):111-121) in the context of CHIP. To date, comprehensive data on suspected MPN patients with a JAK2V617F VAF≤1% are lacking. Aim and methods: The aim was to analyze clinical, molecular, and histopathological correlates in a series of suspected MPN patients (pts) with a JAK2V617F VAF≤1% (from 2017 to 2022) at our Center. JAK2V617F VAF was measured in granulocytes by highly sensitive RTQ-PCR. Conventional karyotype and target next-generation sequencing (NGS) analysis were performed (complete data will be presented at the meeting). Bone marrow (BM) biopsies were reviewed by two independent expert pathologists, R.S. and U.G., the latter included as an author in the International Consensus Classification (Blood 2022; 2022015850). Results: 21 males and 3 females (M/F=7:1), with a median age of 58 y (range, 22-81) were included. Median JAK2V617F VAF was 0.2% (0.1-1). In the suspicion of MPN, complete diagnostic work-up was carried out in pts presenting with erythrocytosis (n=15), thrombocytosis (n=4), history of atypical thrombosis (n=3), splenomegaly (n=1) and leukocytosis (n=1), (Fig.1). In pts with erythrocytosis, median (range) Hb and Hct values were 17 g/dL (15.8-18.5) and 51.5% (50.4-54.7), whereas leukocytes (6.6 x 109/L, 5-11) and platelet (258 x 109/L, 172-440) were within normal range. EPO level (10 mUI/mL; 1.8-12.5) was subnormal in 3 pts. Abnormal karyotype was documented in one case (45,X,-Y[7]/46,XY[13]). Additional mutations were documented in 3 pts: HBB T124N, SH2B3 S186I and cKIT D816V. In pts with thrombocytosis, platelet range was 460-1450 x 109/L; Hb, Hct and leucocytes values were normal. MPL W515K and CALR L367Tfs*46 additional mutations were in one and two pts, respectively. Considering pts with atypical thrombosis, one had a history of arterial events while the others experienced cerebral venous sinus and portal vein thrombosis. Thrombophilia screenings were negative. Karyotype was normal in two pts, whereas one had 45,X,-Y[3]/46,XY[20]. Additional CBL p.D460dup was detected in one case. The subjects investigated for splenomegaly/leukocytosis had normal karyotype, no additional mutations. Morphological features consistent with PV were documented in 4 pts (Fig. 2, A-B). In one of these cases, multifocal dense infiltrates of CD117+/CD25+ mast cells were detected, fulfilling the diagnostic criteria for systemic mastocytosis with associated hematological neoplasm. In the other cases, microscopic examination showed age-adjusted normal or focally increased cellularity, regular or mildly expanded erythroid lineage with no/only minor increase in granulocytic lineage and a higher number of megakaryocytes with PV-like features (i.e. polymorphic forms without atypia and no dense cluster formation) (Fig. 1, C-D). Neither reticulin fibrosis nor excess blasts were detected and these pts had a final diagnosis of MPN unclassifiable (MPN-U), with a PV-like morphology. In pts with thrombocytosis histology was consistent with ET in three cases and overt-PMF in one. Among ET, one pts harbored MPLW515K with grade 1 reticulin fibrosis (Fig. 2, E-F), whereas the overt PMF had CALR type 1 additional mutation. BM biopsies of pts presenting with atypical thrombosis, leukocytosis and mild splenomegaly showed non-specific MPN-like features (including focally increased cellularity, mild expansion of erythroid or granulocytic lineages, higher number of megakaryocytes with a TE-like or a PV-like morphology) and were diagnosed as MPN-U. Conclusions: This study highlights the importance of using highly sensitive assays to detect JAK2V617F, as well as the need to search for CALR and MPL mutations, particularly in pts with thrombocytosis. Moreover, in pts presenting with erythrocytosis and JAK2V617F positivity with VAF≤1%, histopathological diagnostic clues to MPN may be subtle as in the early phase of the disease, emphasizing the need to integrate histopathology with clinical and genetic data in order to address patients to proper clinical management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Distinct Clinico-Molecular Arterial and Venous Thrombotic Scoring Systems for MPN Patients Risk Stratification

Arterial and venous thrombotic events are the major complications of myeloproliferative neoplasms (MPN), responsible for a high rate of morbidity and mortality. Risk stratification and prediction of these events remain challenging clinical questions in MPN. We investigated clinical and molecular risk factors of arterial or venous thrombotic events within a cohort of 1055 patients with MPN followed in our MPN reference center from 2011 to 2021. Using a univariate and multivariate Cox regression model, we first defined independent factors associated to arterial or venous thrombotic events, and then developed predictive risk scores for arterial thrombosis on one hand and venous thrombosis on the other hand. The discrimination power of each score was compared to the currently recommended European Leukemia Net (ELN) and IPSET scores. Our cohort included 494 (46.82%) patients with essential thrombocythemia (ET), 362 (34.31%) with polycythemia vera (PV), 161 (15.26%) with myelofibrosis (MF), 9 (0.85%) with MDS/MPN and 29 (2.75%) with unclassified MPN. 115 patients (10.90%) and 146 patients (13.84%) presented arterial or venous thrombotic events respectively, prior to and/or at the time of MPN diagnosis. After a median follow-up of 9.45 years, 72 patients and 79 patients developed 75 arterial thrombotic events (0.75% patients-year) and 97 venous thrombotic events (0.97 % patients-year), respectively. The incidence rate for arterial and venous thrombotic events during follow up were 0.69% and 0.95% patients-year respectively in patients with PV, 0.75% and 0.78% for ET patients and 0.43% and 1.59% for MF patient. Acute myocardial infarction (32%), stroke (26.67%) and transient ischemic attack (24%), splanchnic thrombosis (37.11%) and deep vein thrombosis (27.83%) were the main arterial and venous thrombotic events during follow-up, respectively. Prior arterial thrombotic events (HR 2.03, 95%CI[1.09;3.58], p=0.024), age >60 years at diagnosis (HR 2.29, 95%CI[1.34;3.91], p =0.002), cardiovascular risk factors (HR 1.86, 95%CI[1.09;3.46], p=0.023) and presence of TET2 or DNMT3A additional mutations (HR 1.74, 95%CI[1.08;2.80], p=0.022) were independently associated with arterial thrombotic events in multivariate analysis. An arterial thrombosis risk score, based on these four factors, defined low and high-risk patients (p=0.0001) (Figure 1). The performance of our arterial risk score was superior to ELN-risk score (ROC curves AUC: 0.68 versus 0.58; p=0.0008), across all MPN subtypes. Interestingly, cytoreductive treatment with interferon alpha (HR 0.45, 95%CI[0.24;0.83], p=0.011) or ruxolitinib (HR 0.049, 95%CI[0.006;0.35], p=0.003) was associated with decreased arterial thrombotic events, while hydroxyurea treatment was not (HR 1.27, 95%CI[0.73;2.20], p=0.381). Prior venous thrombotic events (HR 2.48, 95%CI[1.42;4.34], p=0.001) and presence of a JAK2V617Fdriver mutation (HR 2.66, 95%CI[1.47;4.86], p=0.001) were independently associated with venous thrombotic events in multivariate analysis. A venous thrombosis risk score, based on these two factors, defined low and high risk patients (p=0.035) (Figure 2). The discrimination potential of our venous risk score was similar to ELN-risk score (ROC curves AUC: 0.52 versus 0.50, p=0.67). As opposed to arterial events, all cytoreductive treatments; hydroxyurea (HR 0.43, 95% CI[0.28;0.68], p=0.001), interferon alpha (HR 0.15, 95% IC [0.07;0.31], p=0.0001) or ruxolitinib (HR 0.48, 95% CI[0.23;0.99], p=0.049) were associated with decreased venous thrombotic events. Acute myeloid leukemia/myelodysplastic syndrome transformation free survival was shorter in patients who developed arterial thrombotic events than in those who harbored venous thrombosis (p=0.034). No difference was observed for secondary myelofibrosis transformation free survival or overall survival. Our study offers a comprehensive landscape of MPN associated thrombosis across a large clinically and molecularly annotated long-term follow-up MPN patients' cohort. Our results pinpoint arterial and venous thrombosis clinico-molecular differences and define specific risk scores for each unique entity, hence improving patients' stratification. If further validated on external cohorts, our findings warrant consideration of arterial and venous thrombosis separately for future scoring systems and preventive treatment recommendations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Thrombosis with Non-Proliferative Complete Blood Count Indicative of Underlying Myeloproliferative Neoplasm, Sythrom, a Study on Behalf of the FIM Group

Introduction Thrombosis is the main cause of morbidity in myeloproliferative neoplasms (MPN) and can be the mode of revelation of these malignant diseases. Most of them are characterized by specific driver mutations such as JAK2 V617F, CALR or MPL. An idiopathic or atypical thrombotic event justifies looking for MPN by searching these MPN-related mutations, even in the absence of myeloproliferative features on the complete blood count (CBC). This is especially crucial in case of splanchnic and cerebral thromboses, but not only. In this context the diagnosis of MPN and the choice of treatment are made difficult by the absence of increased blood counts. Objectives - To constitute an observational cohort of patients (pts) presenting an arterial or venous thrombotic event leading to a suspicion of MPN without signs of myeloproliferation in CBC. - To study the characteristics of pts at diagnosis, the rate of recurrence of thrombosis and the rate of hematological progression. Patients and method Inclusion criteria were as follows: i)-diagnosis of an arterial or deep venous thrombotic event, ii)-CBC not suggestive of polycythemia vera (PV), essential thrombocythemia (ET) or myelofibrosis (MF) (CBC below WHO 2016 thresholds) but iii)-detection of a molecular abnormality (JAK2, CALR or MPL driver mutations) and/or an abnormality on bone marrow trephine in favor of MPN and/or spontaneous progenitor growth. Data were collected retrospectively and prospectively. The study is registered on ClinicalTrial.gov (NCT04539678). Results Between 05/2019 and 06/2022, the participation of 31 centers in France and Switzerland allowed the collection of data from 216 pts. The analysis is presented for 208 pts after exclusion of incomplete non-evaluable cases (n=5) or those not meeting inclusion criteria (n=3). The M/F sex ratio was 1.3 with a median age of 55 years (22-91) at the date of molecular testing. Inaugural thrombosis was venous (80%) or arterial (20%) and involved either splanchnic (50%), cerebral (24%), other locations (23%) or multiple territories (3%). Median CBC values were hemoglobin 14 g/dL, hematocrit 43%, platelets 298x109/L and leukocytes 7,1 x109/L. Pts had either a driver mutation of JAK2 V617F (n=201), CALR (n=2) or MPL (n=2), or none of these but spontaneous progenitor growth (n=3). According to WHO criteria, the diagnoses retained were MPN in 91 (44%) cases (including 26 PV, 4 MF and 61 unclassifiable MPN), while 38 pts (18%) lacked MPN criteria and 79 (38%) were not assessable (no trephine). A clinical follow-up of at least 3 months was available in 197 pts, with a median of 61 months (range 3 months - 36 years). Recurrent thrombosis occurred for 42 pts (21%, respectively 24% of initially arterial and 19% of initially venous). Recurrence was splanchnic for 13 pts (31%). The median delay of recurrence was 37 months (12 days - 18 years) after the thrombotic event that led to a suspicion of MPN. Recurrence occurred more often in pts with initially non-splanchnic thrombosis (30% vs. 13% p=0.004). Thrombosis recurrences were not significantly different between pts classified as MPN and those without MPN criteria (18% vs. 30% p=0.1). Hematological progression was observed for 27 pts with a median delay of 31 months (1 - 105) since thrombosis. According to WHO MPN criteria, they were 9 PV, 14 TE and 4 MF. Pts with thrombotic recurrence more frequently presented hematological progression or died (31 month progression-free survival [PFS] 85% vs 94% p=0.008). PFS was significantly reduced in cases of initially non-splanchnic thrombosis (86% vs 97% at 31 months p<0.0001). An explanation could be that pts with splanchnic thrombosis were more frequently treated with cytoreductive agents (CRA) (71% vs 41% p=0.03). Analysis of the effect of antithrombotic therapy is ongoing. Conclusion Thrombosis cases with a non-proliferative CBC but with MPN driver mutations leading to MPN suspicion constitute a heterogeneous group of pts, with many cases of non-splanchnic involvement. The latter pts show more frequent thrombotic recurrences and hematological progression than pts with splanchnic involvement. The role of cytoreduction and antithrombotic therapies needs to be explored in this context.

Healthcare resource utilization in patients with myeloproliferative neoplasms: A Danish nationwide matched cohort study.

Few studies have assessed healthcare resource utilization (HRU) in patients with Philadelphia-negative myeloproliferative neoplasms (MPN) using a matched cohort design. Further, no detailed assessment of HRU in the years preceding an MPN diagnosis exists. We conducted a registry-based nationwide Danish cohort study, including patients with essential thrombocythemia, polycythemia vera, myelofibrosis, and unclassifiable MPN diagnosed between January 2010 and December 2016. HRU data were summarized annually from 2 years before MPN diagnosis until emigration, death, or end of study (December 2017). We included 3342 MPN patients and 32 737 comparisons without an MPN diagnosis, matched on sex, age, region of residence, and level of education. During the study period, the difference in HRU (rate ratio) between patients and matched comparisons ranged from 1.0 to 1.5 for general practitioner contacts, 0.9 to 2.2 for hospitalizations, 0.9 to 3.8 for inpatient days, 1.0 to 4.0 for outpatient visits, 1.3 to 2.1 for emergency department visits, and 1.0 to 4.1 for treatments/examinations. In conclusion, MPN patients had overall higher HRU than the matched comparisons throughout the follow-up period (maximum 8 years). Further, MPN patients had substantially increased HRU in both the primary and secondary healthcare sector in the 2 years preceding the diagnosis.

Automated analysers underestimate atypical basophil count in myeloid neoplasms

Recent data suggest basophils can adopt an atypical appearance in myeloid disorders including myeloproliferative neoplasms (MPNs) and myeloproliferative/myelodysplastic disease. We hypothesized that automated analysers may not accurately quantitate basophils in myeloid neoplasms based on scatter properties. This study examined basophil counts and properties in myeloid disorders by automated cell analyser, manual differential, and flow cytometry. Patients with myeloid neoplasms and control patients with no myeloid disorder diagnosis at a tertiary care centre were identified. Basophil percentage was compared for automated analyser counts (Sysmex XN9000), manual differential, and flow cytometry. Basophil scatter properties in MPNs were examined using flow cytometry. Thirty-one patients with myeloid neoplasms were included: 58% were male, mean age was 70.2 (±20.7) years, 32% had a diagnosis of chronic myeloid leukaemia with the remaining patients divided among various other forms of myeloid disease (including: essential thrombocythemia, polycythemia vera, unclassifiable MPN, myelodysplastic syndromes). For these patients, mean basophil percentage by automated analyser was significantly lower than manual differential (2.7 ± 2.9 vs. 7.1 ± 4.6, respectively, p< 0.001). No significant difference was found between automated versus manual differential for basophils in control subjects (p= 0.373). For myeloid neoplasm patients, mean basophil percentage was not significantly different between manual differential and flow cytometry (p= 0.116); mean basophil percentage by automated analyser was significantly lower than flow cytometry (2.7 ± 2.9 vs. 5.3 ± 3.7, respectively, p= 0.003). Automated analysers underestimate basophil counts in patients with myeloid neoplasms. Manual differential and flow cytometry are recommended for more accurate quantitation and characterization of aberrant basophils.

Classic myeloproliferative neoplasms in Singapore: A population-based study on incidence, trends, and survival from 1968 to 2017

BackgroundCurrent descriptive epidemiological information on classic myeloproliferative neoplasms (MPNs) is incomplete. Published data among Asian population are particularly sparse. MethodsWe conducted a large population-based study to determine the incidence rates and survival patterns of MPN reported to the Singapore Cancer Registry during the period 1968–2017. Age-standardised incidence rates(ASR), overall survival, 5-/10-year relative survival ratio (RSR) were estimated. Joinpoint regression was used to evaluate quinquennial percent change (QPC) in incidence. ResultsWe identified 2557 individuals diagnosed with MPN including 1031 chronic myeloid leukaemia (CML), 424 polycythaemia vera (PV), 389 essential thrombocythaemia (ET), 134 primary myelofibrosis (PMF) and 579 MPN unclassifiable (MPN-U). The overall respective ASRs per 100,000 for CML, PV, ET, PMF and MPN-U were 1.24, 1.15, 1.07, 0.43, and 0.80 in 2013–2017. Males had higher ASR than females in all MPNs. A gradual rise in incidence trends of CML was observed between 1968 and 2017 (QPC 2.1%, 95% CI −0.9, 5.3). The overall incidence trends of non-CML MPNs including PV (QPC 62.9%, 95% CI 19.3, 122.6), ET (QPC 54.2%, 95% CI 23.5, 92.3) and PMF (QPC 103.5%, 95% CI 19.1, 247.6) increased sharply during 1993–2017. Survival was lower in MPNs compared with expected survival in general population: 5-year RSRs were 0.82 (95% CI 0.78, 0.86), 0.96 (95% CI 0.91, 1.01), 0.96 (95% CI 0.92, 1.01), 0.53 (95% CI 0.43, 0.65), and 0.74 (95% CI 0.68, 0.80) for CML, PV, ET, PMF and MPN-U respectively. ConclusionCML incidence has increased marginally, whereas non-CML MPNs incidences have sharply increased. MPN patients have a lower relative survival compared to the general population, and patients with PV and ET have the most favourable relative survival. Median survival for CML patients has increased dramatically over the last 50 years.

Diagnostic and management strategies for Myeloproliferative Neoplasm-Unclassifiable (MPN-U): An international survey of contemporary practice

Myeloproliferative Neoplasm-Unclassifiable (MPN-U) is defined as an MPN that fails to meet the diagnostic criteria for any of the other defined classical or ‘non-classical’ MPNs. The reported incidence is variable, dependent on appropriate recognition, and the clinical course can be highly variable ranging from an indolent disorder through to an aggressive disease course with a significant risk of thrombosis, bulky splenomegaly, and debilitating symptom burden. Clinicians frequently manage these conditions according to the clinical concerns e.g. splenomegaly akin to the phenotype, but no evidence base exists. Currently, there are no widely accepted guidelines to deal with both the diagnostic work up and subsequent clinical management of this entity. We hence surveyed major International MPN centres to gain an understanding of common challenges in MPN-U management in 2021 and aid identification of considerable practice variations where harmonisation would be desirable. Such information is vital to support future consensus guidelines in addition to informing further collaborative studies.

How I manage myeloproliferative neoplasm-unclassifiable: Practical approaches for 2022 and beyond.

Myeloproliferative neoplasm (MPN)-unclassifiable (MPN-U) or not otherwise specified represents a rare, poorly defined and heterogeneous group of MPNs. Disease incidence is difficult to define but likely represents close to 5% of all MPNs when strict World Health Organisation (WHO) criteria are applied. Dynamic review over time is required to assess if the disease can be re-classified into another MPN entity. A diagnosis of MPN-U leads to many challenges for both the patient and physician alike including lack of agreed monitoring and therapeutic guidelines, validated prognostic markers and licenced therapies coupled with exclusion from clinical trials. MPN-U has an inherent risk of an aggressive clinical course and transformation in some but who, and when to treat in the chronic phase, including identifying who may require more aggressive therapy at an earlier stage, remains elusive. Moreover, despite the significant thrombotic risk, there is no agreement on systematic primary thromboprophylaxis. We hereby provide a contemporary overview of MPN-U in addition to four illustrative cases providing our collective suggested approaches to clinical challenges.

The Classification of Myeloproliferative Neoplasms: Rationale, Historical Background and Future Perspectives with Focus on Unclassifiable Cases.

Simple SummaryMyeloproliferative neoplasms (MPNs) are clonal hematological disorders, characterized by increased proliferation of the myeloid lineages in the bone marrow. Since their original recognition by William Damashek in 1951, MPNs have been extensively investigated at a clinical-pathological and molecular level. This prompted a progressive refinement of their classification and diagnostic criteria. Uncertainties nonetheless remain in a small (yet consistent) subset of cases, characterized by unconventional and/or overlapping clinical-pathological features. Such cases (referred to as MPN, unclassifiable [MPN-U]) encompass a broad spectrum of entities, including early phase MPNs, terminal (i.e., fibrotic) MPNs, MPNs associated with inflammatory or neoplastic disorders, and poorly characterized MPNs with clinical-pathological mismatch or atypical molecular features. In this review, we discuss the rationale behind the classification and diagnostic criteria of MPNs, focusing on the still open issues concerning MPN-U.Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by increased proliferation of one or more myeloid lineages in the bone marrow. The classification and diagnostic criteria of MPNs have undergone relevant changes over the years, reflecting the increased awareness on these conditions and a better understanding of their biological and clinical-pathological features. The current World Health Organization (WHO) Classification acknowledges four main sub-groups of MPNs: (i) Chronic Myeloid Leukemia; (ii) classical Philadelphia-negative MPNs (Polycythemia Vera; Essential Thrombocythemia; Primary Myelofibrosis); (iii) non-classical Philadelphia-negative MPNs (Chronic Neutrophilic Leukemia; Chronic Eosinophilic Leukemia); and (iv) MPNs, unclassifiable (MPN-U). The latter are currently defined as MPNs with clinical-pathological findings not fulfilling the diagnostic criteria for any other entity. The MPN-U spectrum traditionally encompasses early phase MPNs, terminal (i.e., advanced fibrotic) MPNs, and cases associated with inflammatory or neoplastic disorders that obscure the clinical-histological picture. Several lines of evidence and clinical practice suggest the existence of additional myeloid neoplasms that may expand the spectrum of MPN-U. To gain insight into such disorders, this review addresses the history of MPN classification, the evolution of their diagnostic criteria and the complex clinical-pathological and biological features of MPN-U.

Hematopoietic Stem and Progenitor Cell Fitness As a Novel Prognostic and Monitoring Biomarker for JAK2 V617F Myeloproliferative Neoplasms (MPNs)

Hematopoietic Stem and Progenitor Cell Fitness As a Novel Prognostic and Monitoring Biomarker for JAK2 V617F Myeloproliferative Neoplasms (MPNs)

Clinical Discernment, Bone Marrow, and Molecular Diagnostics Are Equally Important to Solve the Phenotypic Mimicry among Subtypes of Myeloproliferative Neoplasms

The 2016 WHO classification integrates clinical, bone marrow (BM)-morphology, and molecular features to define disease entities. This together with the advancements in molecular detection and standardization of BM features enable an accurate diagnosis of myeloproliferative neoplasms (MPN) in the majority of patients. Diagnostic challenges remain due to phenotypic mimicry of MPN, failing specificity of BM-morphology, and the fact that phenotype-driver mutations, such as JAK2V617F, are not exclusive to a particular MPN, and their absence does not preclude any of these. We present a series of cases to illustrate themes to be considered in complex cases of MPN, such as triple-negative (TN)-MPN or MPN-unclassifiable (MPN-U). Eleven patients labelled as TN-MPN or MPN-U were included. Serum tryptase and NGS were part of a systematic/sequential multidisciplinary evaluation. Results were clustered into four categories based on diagnostic entities and/or how these diagnoses were made: (A) With expanding molecular techniques, BCR-ABL1 and karyotyping should not be missed; (B) systemic mastocytosis is underdiagnosed and often missed; (C) benign non-clonal disorders could mimic MPN; and (D) NGS could prove clonality in some “TN”-MPN cases. The prognostic/therapeutic consequences of an accurate diagnosis are immense. In TN-MPN or MPN-U cases, a multidisciplinary re-evaluation integrating molecular results, BM-morphology, and clinical judgment is crucial.

Cigarette Smoking, Complete Blood Count, and Myeloproliferative Neoplasms — a Meta-Analysis

IntroductionThe classic chronic Philadelphia-negative chronic myeloproliferative neoplasms (MPN) are acquired stem cell diseases and include essential thrombocythemia, polycythemia vera, primary myelofibrosis and unclassifiable MPN. Recently, these disorders have been described as “A Human Inflammation Model for Cancer Development”, reflecting chronic inflammation to be a major driving force for clonal evolution and disease progression in MPNs.The objectives of this meta-analysis were to investigate if cigarette smoking is associated with increased blood cell counts (CBC) and incidence of MPN as proof of concept.MethodA systematic review and meta-analysis was undertaken using databases Pubmed, EMBASE, Web of Science and World Health Organization Global Health Library for all relevant papers published until 15th February 2017 on smoking and complete blood counts and on smoking and myeloproliferative neoplasms according to a formal protocol that followed the PRISMA guidelines.Following data extraction, STATA 13.0 was used to calculate raw and standardized mean differences for CBC (in SI units) and pooled odds ratios for MPN with 95% confidence intervals using random and fixed effects models. Main analyses included analyses of current smokers, ex-smokers, and ever smokers vs. non- or never smokers. Heterogeneity was measured using I2 -statistical analysis and Cochrane Q statistic, and reported corresponding p-values and sensitivity analyses included among others gender and smoke dosage(heavy (>=20 cigarettes/day) and light(<20 cigarettes/day)). Funnel plots, Egger's test and Begg's tests were used to assess publication bias.ResultsComplete Blood Count (CBC): A total of 88 references published from 1974-2017 in 10 different languages and including 90 study populations with 533,242 individuals, 222,887 current smokers, 92,001 ex-smokers, and 222,538 non-smokers between the ages of 15 and 89 from 34 countries were analyzed.Meta-analyses showed that the mean of most CBCs were higher in smokers and ex-smokers compared to non-nonsmokers. Differences were larger comparing smokers to non-smokers than comparing ex-smokers to non-smokers, and also larger comparing heavy smokers to non-smokers than comparing light smokers to non-smokers. Furthermore, differences in most CBCs were higher in men compared to women.All analyses presented high heterogeneity in main and sensitivity analyses but no overall publication bias.Myeloproliferative Neoplasms (MPN): We included 5 studies with a total of 1,368,738 individuals and 2017 MPN cases. 2 studies were prospective and 3 studies case-control. Pooled odds ratio for MPN were 1.44 (95%CI: 1.33-1.56; p-value=5*10(-20); I-square=0%, p(heterogeneity)=0.56) comparing smokers to non-smokers, 1.10(0.86-1.41; p-value=0.46; I-square=71%, p(heterogeneity)=0.03) comparing ex-smokers to non-smokers, and 1.30(1.14-1.49; p-value=8*10(-5); I-square=45%, p(heterogeneity)=0.16) comparing ever-smokers to never smokers.ConclusionCurrent smokers, ex-smokers, and ever-smokers have higher mean CBCs and an increased risk of MPN compared to non-smokers. [Display omitted] DisclosuresHasselbalch:AOR Orphan: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Coexisting Oncogenic Signaling Pathway Mutations in Cnl/Acml/CMML/MPN-U

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (unMPN) are rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to poorly defined molecular mechanisms, these diseases have been, until now, diagnosed based on morphology and exclusionary criteria and treated empirically, resulting in dismal outcomes. Recent application of next generation sequencing has shed light on the genetic mechanisms of CNL/aCML/CMML/MPN-U. In particular, Maxson et al. and others have shown that CSF3R membrane proximal and transmembrane mutations are present in 83-89% of CNL and 3.3-44% of aCML patients, and expression of CSF3R membrane proximal mutants is sufficient to recapitulate the clinical features (mature granulocyte accumulation) of CNL in a mouse model. This has led to revision of the WHO diagnostic criteria for CNL, enabling a more accurate diagnosis and facilitating the development of targeted treatment for this disease. Aside from the CSF3R driver mutations, few recurrent signaling pathway mutations have been characterized in this group of diseases that could serve as the basis for such targeted therapeutic strategies. The aim of the current study is to characterize the frequency and distribution of signaling pathway driver mutations in a large cohort of CNL/aCML/CMML/MPN-U patients with the hope of informing diagnosis, prognosis and treatment.Methods and results: We performed whole exome sequencing on a large cohort of patients (n=160) with plausible diagnosis of CNL/aCML/MPN-U/CMML. We screened hotspot variants in genes representing kinases, non-kinase growth factor and cytokine receptors. We observed that 66.9% of patients harbor at least one signaling pathway driver mutations (CSF3R 20.6%, NRAS 17.5%, JAK2 9.4%, CBL 6.5%, PTPTN11 5.6%, ETNK1 5.6, KRAS 4.4%, NF1 3.1%, FLT3 3.1%, SH3B2 3.1%, etc.), largely involving JAK/STAT and RAS signaling pathways. Interestingly, MPL mutations and CALR indels were rare in our cohort. The variants of signaling pathway driver mutations were observed with high allele frequencies: CSF3R (52.1%), JAK2 (48.9%), NRAS (46.3%), PTPN11 (40.7%), CBL (34.4%) and KRAS (29.5%), indicating the majority of these driver mutations are present in the dominant leukemic clones.Interestingly, 21.5% and 3.7% patients harbor two and three different active signaling pathway mutations, respectively. Clonality analysis demonstrated a co-occurrence pattern in more than half of the cases where more than two signaling pathway mutations were identified, underscoring a complex genetic pathogenesis. CSF3R was shown to co-occur with NRAS, SH2B3, PTPN11, NTRK2, CBL, ETNK1 and ABL1 mutations; NRAS mutations were present in combination with CSF3R, KRAS, JAK2, CBL, STAT3, NTRK2, FLT3, and GNB1 mutation. Since CSF3R and RAS mutation co-occur at high frequencies (n=5, 22%) in this cohort. We performed drug sensitivity assays, and we observed reduced drug sensitivity to either JAK or MEK inhibitors in cells transformed by co-existing mutations, whereas drug combinations targeting the co-existing mutations demonstrated synergistic cytotoxicity .Conclusions: In sum, CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with high frequencies of signaling pathway mutations in the major clone. More than 25% of patients demonstrate multiple different pathway mutations in a co-existing pattern, which warrants comprehensive screening of molecular targets and high-efficacy drug combination strategies. DisclosuresTyner:AstraZeneca: Research Funding; Incyte Corporation: Research Funding; Leap Oncology: Consultancy; Syros: Research Funding; Seattle Genetics: Research Funding; Agios Pharmaceuticals: Research Funding; Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceutica: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding.

Clinicopathological characterisation of myeloproliferative neoplasm-unclassifiable (MPN-U): a retrospective analysis from a large UK tertiary referral centre.

Myeloproliferative neoplasm-unclassifiable (MPN-U) presents an MPN-type phenotype that fails to meet diagnostic criteria for other MPN variants. Variability in the clinicopathological phenotypes presents many challenges. Amongst a registry cohort of 1512 patients with MPN, 82 with MPN-U were included, with a median (range) age of 49·7(13-79)years. Albeit heterogeneous, common presentation features included raised lactate dehydrogenase, thrombocytosis and clustered/pleomorphic megakaryocytes on trephine biopsy. Thrombosis was common (21%), necessitating vigilance. The median event-free survival was 11·25years (95% confidence interval 9·3-not reached), significantly shortened in cases with lower platelet counts (<500×109 /l) and a leucocytosis (≥12×109 /l) at presentation. Generation of potential MPN-U prognostic scores is required.