SummaryIn the Austrian biodatabase for chronic myelomonocytic leukemia (ABCMML) clinicolaboratory real-life data have been captured from 606 CMML patients from 14 different hospitals over the last 30 years. It is the only large biodatabase worldwide in which functional methods such as semisolid in vitro cultures complement modern molecular methods such as next generation sequencing. This provides the possibility to comprehensively study the biology of CMML. The aim of this study was to compare patient characteristics with published CMML cohorts and to validate established prognostic parameters in order to examine if this real-life database can serve as a representative and useful data source for further research. After exclusion of patients in transformation characteristics of 531 patients were compared with published CMML cohorts. Median values for age, leukocytes, hemoglobin, platelets, lactate dehydrogenase (LDH) and circulating blasts were within the ranges of reported CMML series. Established prognostic parameters including leukocytes, hemoglobin, blasts and adverse cytogenetics were able to discriminate patients with different outcome. Myeloproliferative (MP) as compared to myelodysplastic (MD)-CMML patients had higher values for circulating blasts, LDH, RAS-pathway mutations and for spontaneous myelomonocytic colony growth in vitro as well as more often splenomegaly. This study demonstrates that the patient cohort of the ABCMML shares clinicolaboratory characteristics with reported CMML cohorts from other countries and confirms phenotypic and genotypic differences between MP-CMML and MD-CMML. Therefore, results obtained from molecular and biological analyses using material from the national cohort will also be applicable to other CMML series and thus may have a more general significance.

Mast cell leukemia (MCL) is a rare, life-threatening malignancy defined by a substantial increase in neoplastic mast cells (MCs) in bone marrow (BM) smears, drug-resistance and a poor prognosis. In most patients, the survival-time is less than 1 year. However, exceptional cases may present with a less malignant course. We report on a 49-year-old female patient with MCL Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts diagnosed in 2013. In February 2013, first symptoms, including flushing, headache and diarrhoea, were recorded. In addition, mild anemia was detected. The disease was characterized by a massive increase in well-granulated, mature and often spindle-shaped MCs (80%) in BM smears. The serum tryptase level amounted to 332 ng/mL. Like in most other MCL patients, no skin lesions were detected. However, unlike in other patients, tryptase levels remained stable and no other signs or symptoms of MCL-induced organ damage were found. Sequencing studies revealed an isolated S476I point-mutation in KIT but no mutation in codon 816. The patient received histamine receptor blockers, but refused cytoreductive therapy. After 9 months, still no progression or organ damage was detected. However, progression with transformation to acute MCL occurred after 12 months. We propose that the chronic type of MCL with stable conditions, absence of organ damage and a mature MC morphology, is recognized as a distinct entity that should be distinguished from the acute variant of MCL.

Background: Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy with features of both a myelodysplastic syndrome and a myeloproliferative neoplasm.The pathogenesis of CMML is incompletely understood due to the large heterogeneity of molecular aberrations in genes involved in epigenetic regulation, RNA-splicing and signal transduction including components of RAS and JAK2 signaling. Functional tests may be important to better estimate the contribution of a particular molecular aberration in the pathogenesis of the malignancy. We have originally demonstrated extensive in vitro formation of myeloid colonies (CFU-GM) without addition of exogenous growth factors in a subset of patients with CMML (Geissler et al, Leuk Res 1988). We reported that this spontaneous CFU-GM colony formation in CMML is a GM-CSF dependent in vitro phenomenon (Geissler et al, J Exp Med 1996) and could also show in a small retrospective study that CMML patients with high spontaneous CFU-GM growth (>100/105 PBMNC) have a worse prognosis compared to patients with low myeloid colony formation (Sagaster et al, Ann Hematol 2004) suggesting a clinical significance of our observation. In juvenile myelomonocytic leukemia, in which molecular aberrations are mainly restricted to the RASopathy genes including NRAS, KRAS, NF1, CBL and PTPN11, spontaneous formation of CFU-GM due to GM-CSF-specific hypersensitivity is a hallmark feature of disease, which has been included in the diagnostic criteria. We therefore speculated that high spontaneous myeloid colony formation in CMML might also reflect hyperactivation of the RAS signaling pathway. Aim: Our aim was to study the correlation between spontaneous myeloid colony formation and the presence of mutations in RASopathy genes in patients with CMML. Moreover the relationship of high autonomous CFU-GM formation with phenotypic features of CMML and its clinical outcome was investigated. Patients and Methods: In this study we included 137 CMML patients of the "Austrian Biodatabase for CMML (ABCMML)" in whom CFU-GM data and/or molecular data were available. CFU-GM growth in the absence of exogenous cytokines was assessed in a central laboratory using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Molecular characterization was also performed in a central laboratory using NGS with amplicon-based target enrichment of 39 CMML associated genes. Assuming that clones that are too small are unlikely to significantly impact hematopoiesis only mutations with an allele burden of ≥20% were considered positive in this analysis. Clinical and hematological data were obtained from patients records. Results:High spontaneous CFU-GM growth (≥100/105 PBMNC) was found in 38/135 (28%) CMML patients, of whom 3 were already transformed into secondary AML at the time of in vitro culture testing. There was a significant correlation between high CFU-GM formation in vitro and the presence of mutations in genes involved in the RAS signaling pathway. The incidence of RAS pathway mutations was 72% in CMML patients with high colony growth and 31% in patients with low spontaneous CFU-GM formation (p<0.0001). As shown in Table 1 high spontaneous myeloid colony formation was associated with increased WBC counts, increased blast cells, increased LDH, more pronounced splenomegaly and inferior survival (Fig. 1). There was no significant difference regarding autonomous CFU-GM growth in CMML patients with molecular aberrations in genes of epigenetic regulation and RNA-splicing, respectively. High spontaneous CFU-GM was never observed in CMML patients in whom the JAK2 V617F mutation was the only molecular aberration in signaling pathways (0/8 patients). Furthermore the in vitro conversion from a growth factor dependent to a growth factor independent phenotype by RAS but not by JAK2 could be demonstrated in BaF3 cells (Fig. 2). Conclusion: Our findings indicate that high spontaneous in vitro myeloid colony formation is associated with the presence of RAS pathway mutations, leukocytosis, splenomegaly and reduced survival. These results suggest that CMML with high spontaneous colony growth is a mainly RAS pathway driven malignancy resulting in myeloproliferation and inferior outcome. This may have clinical implications concerning therapeutic strategies aimed at targeting the hyperactive RAS signaling pathway in these patients. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celegene: Honoraria, Research Funding.

Background:Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy of the elderly with a heterogenous molecular pathophysiology. Whereas mutations in components of the RAS pathways are among the most common somatic mutations in CMML the JAK2 V617F mutation which is a typical finding in polycythemia vera and around 50% of patients with essential thrombocythemia and primary myelofibrosis, respectively, is by far less frequently detected in CMML but can be consistently found in a subgroup of patients in larger series. Due to the fact that JAK2 V617F-positive CMML is a rare disease the clinical, hematological and in vitro growth characteristics of this entity are poorly investigated. In the "Austrian Biodatabase for Chronic Myelomonocytic Leukemia (ABCMML)" we retrospectively and prospectively collect clinical, biologic, and molecular information of patients with CMML from different centers in a real life setting. Aims:Our aim was to characterize the clinical, hematological, molecular and biologic features of CMML patients harboring a JAK2 V617F mutation. Methods:The diagnosis of CMML was established according to diagnostic criteria of the World Health Organization (WHO) classification of 2008 (Vardiman et al, Blood 2009). Clinical and hematological data were obtained from patients records. For molecular characterization we used next-generation sequencing with amplicon-based target enrichment of 39 CMML associated genes. Only mutations with an allele burden of >10% were considered positive in this analysis. Autonomous colony-forming units granulocyte/macrophage (CFU-GM) growth in the absence of exogenous cytokines was assessed using semisolid cultures as previously described (Geissler et al, J Exp Med 1996). Results:Up to now targeted NGS data are available in 116 patients and in vitro culture data in 75 patients respectively. We identified 13 CMML patients who had a JAK2 V617F mutation with an allele frequency >10%. Clinical, hematological, and biologic characteristics in these patients were compared with 103 patients who had NGS sequencing and were negative for the JAK2 V617F mutation. As shown in Table 1 JAK2 V617F-positive CMML patients had significantly higher WBC counts, higher hemoglobin values, higher platelet counts and more pronounced splenomegaly as compared to JAK2 V617F-negative patients. On the other hand the percentage on monocytes in peripheral blood and the numbers of CFU-GM growing in vitro without addition of exogenous growth factors were lower in CMML patients with the JAK2 V617F mutation as compared to patients without this mutation. The majority of JAK2 V617F-positive patients had additional mutations that can be also found in JAK2 V617F-negative patients, in particular mutations in genes of epigenetic regulation and RNA-splicing, respectively. As shown in Figure 1 there was a trend towards a better survival of patients with the JAK2 V617F mutation as compared to JAK2 V617F-negative patients (p=0.05). In a JAK2 V617F-positive CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we were able to demonstrate the disappearance of constitutional symptoms and a durable spleen response lasting for over 56 months (Fig. 2). Conclusion:Out data show that CMML patients with the JAK2 V617F mutation have hematological, biologic and clinical characteristics different from JAK2 V617F-negative CMML patients. These findings suggest that JAK2 V617F-positive CMML patients should be regarded as a distinct subgroup which may benefit from specific targeted treatments. Disclosures Geissler: Novartis: Honoraria. Pfeilstöcker:Novartis: Consultancy, Speakers Bureau. Burgstaller:Novartis: Consultancy, Honoraria. Zach:Novartis: Other: Honoraria for Advisory Board. Hörmann:Novartis: Other: Honoraria for Advisory Board. Jäger:Roche: Other: Personal fees, Research Funding. Sperr:Amgen: Honoraria, Research Funding; Novartis: Honoraria. Kusec:Novartis: Other: Honoraria for lectures. Valent:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celegene: Honoraria, Research Funding.