ALBERT

Description: Introduction Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). These are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. MPN are caused by three main recurrent mutations: JAK2V617F, mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL) genes. Interferon alpha (IFNα) treatment induces not only a hematological response in around 70% of ET, PV and early myelofibrosis, but also a significant molecular response on both JAK2V617F- and CALR-mutated cells. However, a complete molecular response is only achieved in around 20% of patients. Our aim is to predict the long-term efficacy of IFNα in JAK2V617F- and CALR-mutated patients by monitoring the fate of the disease-initiating mutated HSC in order to better stratify the molecular responders. Methods A longitudinal observational study (3-5 years) was performed in 46 IFNα-treated patients. The MPN disease distribution was 42% ET, 47% PV and 11% PMF. We detected 33 patients with JAK2V617F mutation, 11 with CALR mutations (7 type 1/type 1-like and 4 type 2/type 2-like), 1 with both JAK2V617F and CALR mutation and 1 with JAK2V617F, CALR mutation and MPLS505N. At 4-month intervals, the JAK2V617For CALR mutation variant allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was determined by studying the presence of the mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38-HSC-enriched, CD90-CD34+CD38- immature and CD34+CD38+committed progenitors). We used a combination of mathematical modeling (Michor et al., Nature, 2005) and Bayesian analysis to infer the long-term behavior of mutated HSC. Results After a median follow-up of 40 months, IFNα targeted more efficiently and more rapidly the HSPC, particularly the HSC-enriched progenitors, than the mature blood cells in JAK2V617Fpatients (p100 µg/week). Moreover, very low proportion of heterozygous mutated HSC compared to high proportion can be targeted more easily in patients. The associated mutations at diagnosis and at the last timepoint were also investigated using an NGS-targeted myeloid panel. Results indicate that IFNα does not induce any further mutations on additional genes and the mathematical approach predicts that associated mutations have no major impact on the ratio of HSC decrease. Conclusion Altogether, using a rigorous method of statistical inference, our results show that IFNα exhaust the human mutated HSC by differentiation in HSPC and mature cells. This is likely due to IFNα inducing a stronger proliferation of mutated compared to wild-type HSC, as previously shown in a mouse model (Mullally et al., Blood, 2013). Our study predicts that IFNα can slowly eradicate the mutated HSC, but this beneficial effect would be more efficient: i) in patients with homozygous JAK2V617F versus those with heterozygous JAK2V617F or CALR-mutated, ii) with high IFNα dose, iii) in patients with very low proportion of heterozygous JAK2V617F and CALR-mutated HSC. Thus, this study will help to stratify patients for IFNα treatment.These results might also explain the different outcomes in current IFNα clinical trials. Disclosures Constantinescu: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AlsaTech: Other: Co-Founde; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics; Wiley & Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine. Kiladjian:AOP Orphan: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Consultancy.

Description: Myeloproliferative neoplasms (MPNs) are clonal malignant disorders characterized by the increased production of mature myeloid cells in blood. The classical MPNs include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Those pathologies are due to the acquisition of gain-of-function mutations leading to the constitutive activation of the cytokine receptor / JAK2 signaling pathway: JAK2V617F in 70% of cases and mutations in the thrombopoietin receptor (MPL) gene in 5% of cases. More recently, around fifty different mutations in the calreticulin (CALR) gene have been described in 30% of ET and PMF with two more frequent mutations called del52 (type 1) and ins5 (type 2). All the CALR mutations induce a frameshiflt to an alternative reading frame in the exon 9 leading to a new C-term tail of the protein with hydrophobic features, and the loss of the KDEL sequence, which is involved in its endoplasmic reticulum retention. The goal of this work was to understand the role of CALR mutants (del52, del46, del34, ins5, del19, del13) in human hematopoiesis. By studying the variant allele frequency (VAF) in 20 patients, we have shown that the CALR mutations are present in all blood mature cells not only in granulocytes and monocytes (CD14+) with a VAF 〉30% but also in B cells (CD19+), NK cells (CD56+) and in some cases in T cells (CD3+). Moreover, we have observed that CALR mutations are present in all hematopoietic progenitors including CD34+CD38-CD90+ (HSC), CD34+CD38-CD90- (immature progenitors) and CD34+CD38+ (committed progenitors) cell fractions after investigating the clonal architecture of the progenitors. CALR mutation was detectable in more than 40% of progenitor cells except in 2 patients (15 patients studied) and with, in some cases, no detectable wild type CALR progenitors. Homozygous CALR mutations were rare except in one case associated with disease progression. Whatever the VAF, there was no significant differences among the different progenitor types and granulocytes. Finally, we observed that all the associated mutations studied (TET2, PHF6, SYNE1, SCARA5, PIK3CD, SETD1B) in 6 patients postdated CALR mutations. We could also show in 15 patients samples that CALR mutants give a specific megakaryocytic progenitor (CFU-MK) spontaneous growth mediated both by MPL and JAK2 activation using specific inhibitors and short hairpin RNAs. The CFU-MK spontaneous growth correlated with a constitutive activation of JAK2/STAT3/5 pathway in megakaryocytes derived from in vitro cultures of CD34+ progenitors. In aggregate, these results show that all CALR mutants studied are present in all human hematopoietic cells including myeloid and lymphoid cells, give an early clonal advantage at the level of the HSC compartment and a specific increased growth of the megakaryocytic lineage via MPL/JAK2 activation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are acquired clonal disorders of hematopoietic stem cells (HSC) leading to the hyperplasia of one or several myeloid lineages. They are due to three main recurrent mutations affecting the JAK/STAT signaling pathway: JAK2V617F and mutations in the calreticulin (CALR) and thrombopoietin receptor (MPL). Interferon alpha (IFNα) is the only drug that not only induces a hematological response in ET, PV and early MF, but also a significant molecular response on both JAK2V617F or CALR-mutated cells. Our broad aim was to understand the mechanism of action of IFNα. Previously, our group and others have shown that IFNα specifically targets JAK2V617F HSC in a chimeric JAK2V617F knock-in mouse model. In this study, we wanted to know how and how fast IFNα impacts the different mutated human hematopoietic compartments. Methods: A prospective study was performed with a cohort of 47 patients treated by IFNα for 3-5 years. The MPN disease distribution was 40% ET, 49% PV and 11% MF. This cohort included 33 JAK2V617F-mutated patients, 11 CALR-mutated patients (7 type 1/type 1-like and 4 type 2/type 2-like), 2 both JAK2V617F- and CALR-mutated patients and 1 MPLW515K-mutated patient. At 4-month intervals, the JAK2V617F or/and CALR mutation allele frequency was measured in mature cells (granulocytes, platelets). Simultaneously, the clonal architecture was also determined by studying the presence of the JAK2V617F or CALR mutations in colonies derived from the different hematopoietic stem and progenitor cell (HSPC) populations (CD90+CD34+CD38- HSC-enriched progenitors, CD90-CD34+CD38- immature progenitors and CD90- CD34+CD38+ committed progenitors). Results: After a median follow-up of 33 months, IFNα targets more efficiently and rapidly the HSPC particularly in HSC-enriched progenitors, than the mature blood cells in JAK2V617F patients (p