ALBERT

Description: Introduction: Using whole blood transcriptional profiling, we have previously shown deregulation of several interferon-stimulated genes (ISG) and deregulation of immune and inflammation genes in MPNs as well. Most lately, we have shown that deregulated HLA-genes are upregulated by interferon-alpha2 (IFN) treatment. Herein, we for the first time describe the landscape of ISGs during treatment with IFN, the aims being to describe if unique ISG transcriptional signatures might be elicited during IFN treatment with potential differences between subgroups. Methods: Eight patients with ET, 21 patients with PV, and 4 patients with PMF participated in the study. All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Gene expression microarray analysis of whole blood was performed before and after 3 months of treatment. Total RNA was purified from whole blood, amplified to biotin-labeled RNA, and hybridized to Affymetrix HG-U133 2.0 Plus chips recognizing 54,675 probe sets (38,500 genes). Results: We identified 6261, 10,008, and 2828 probe sets to be significantly differentially expressed in ET, PV, and PMF, respectively, in response to treatment with IFN (pvalue 〈 0.05). Twenty-one previously identified ISGs were investigated: Six genes involved the response to virus: ISG15, IFI35, IFI44, IFIH1, MX1, and OAS1, 2 transcriptional regulators: IFI16 and SP110, and 13 IFN-inducible genes: ADAR, CXCL10, IFI6, IFI27, IFI30, IFI44L, IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, PSME1, and PYHIN1. Significant upregulation of 20, 21, and 18 ISGs was found in patients with ET, PV, and PMF, respectively. None of the 21 genes was significantly downregulated in any of the patient groups. The 6 genes involved in response to virus and the 2 transcriptional genes were significantly upregulated in all subgroups. In patients with ET all but one gene (CXCL10) of the 13 IFN-inducible genes were significantly upregulated. In patients with PV all 13 IFN-inducible genes were significantly upregulated and in PMF all but three (CXCL10, IFITM2, PUHIN1) IFN-inducible genes were significantly upregulated. No significant differences between subgroups were recorded (data not shown). Discussion and Conclusions: Eighteen genes were significantly upregulated compared to baseline values in all 3 MPN-subgroups. The CXCL10 gene was not significantly regulated in ET and PMF during IFN treatment but significantly upregulated in PV. CXCL10 is involved in the activation of neutrophils. Accordingly, impaired regulation of CXCL10 on exposure to IFN might compromise recruitment and activation of neutrophils. The excessively high expression levels of IFI27 after IFN are intriguing but might be explained by the reported high circulating levels of regulatory T cells (Tregs) after IFN. Thus, Tregs produce TGF-beta which stimulates IFI27. Indeed, TGF-beta was significantly upregulated in ET and PV but not in PMF, likely due to the low number of PMF patients. Surprisingly, we did not find any differences in ISG responses between MPNs. Indeed, taken into account that the MPNs depict a biological continuum from the early cancer stages (ET/PV) to the advanced "metastatic" myelofibrosis stage, different ISG signatures between the subtypes might be anticipated for several reasons. First, such differentiated signatures might exist merely reflecting the heterogeneity between subgroups in regard to "tumor burden". Second, differences in frequencies and functionality of immune cells between the subgroups as shown in most recent studies might impact the ISG signatures. Third, chronic inflammation, considered the driving force for clonal evolution in MPNs, has been shown to compromise cell responses to IFN, implying that PMF patients with the most pronounced inflammatory state might exhibit blunted ISG responses. However, the low number of PMF patients may account for the non-significant difference between ET/PV and PMF. In conclusion, except for 4 genes (CXCL10, IFITM1, IFITM2, and PYHIN1), our study has shown the ISGs to be significantly upregulated in all MPN- subtypes after 3 months of exposure to IFN. The reasons for the absence of upregulation of the above 4 genes are unknown but chronic inflammation and immune deregulation might be influential. Further transcriptional studies of ISGs during IFN treatment in larger study populations and with longer exposure times are needed to address these issues. Table. Table. Disclosures Hasselbalch: Novartis: Research Funding.

Description: Background α-interferon (IFN) is an effective treatment in polycythemia vera (PV) and essential thrombocythemia (ET). IFN can induce cytogenetic remissions in PV pts. However, 21–25 % of pts discontinue therapy due to side effects. Pegylated IFN has been suggested to have less side effects. Study design and patients: The Nordic MPD Study Group performed an open label phase II feasibility study of PegIntron® treatment in 42 MPD pts, 20 females and 22 males, median age 54 yr (range 29–77). Inclusion criteria were a platelet count of 〉 400x109/L in pts with symptoms or previous thrombosis (n=26) or 〉1000x109/L in asymptomatic pts without previous thrombosis (n=16). 22 pts had PV, 20 ET. 15 pts had previously received cytoreductive therapy; anagrelide (7), hydroxyurea (6), busulfan (1), P32 (1). Previous IFN therapy was not allowed. Median time from diagnosis to study start was 0.74 ys (0.01–30.2). Initial dose was 0.5 μg/kg once weekly. The treatment goal was a platelet count 〈 400x109/L in symptomatic patients and 〈 600 in asymptomatic patients (CR). If CR was not achieved within 12 weeks PegIntron was increased to 1 μg/kg/week. The dose was subsequently decreased to the lowest dose maintaining CR. Pts QoL was investigated using QLQ-C30 and HAD forms. Neutrophil PRV-1 mRNA expression was analyzed by quantitative RT-PCR prior to and after therapy. Results: Response to treatment was evaluated at 1,2,3,6,9 and 12 months. At 6 months, 29/42 pts (69 %) had achieved CR, after a median time of 83 days. The median dose at time of CR was 0.5 μg/kg/week. CR rate was not related to diagnosis or gender. 4/42 pts (10%) discontinued therapy early due to side effects, another 9 pts were taken off study at 6 months due to side effects or insufficient response. Only 4/14 responding PV pts required phlebotomy therapy, compared to 20/22 before PegIntron. 20/29 patients with CR at 6 months maintained CR at 12 months, whereas 9 pts had gone off study due to side effects. The mean platelet count was 875x109/L prior to therapy, and 512, 448, 362x109/L at 3,6 and 12 months in pts on therapy. No thrombotic or bleeding complications were observed. Side effects were common, the majority WHO grades I-II; fatigue (76 %), injection site reaction (64 %), flu-like symptoms (64 %), headache (56 %), muscle pain (51 %), depression (35 %), insomnia (24 %), hair loss (20 %). Mild elevations were noted of serum ALAT, creatinine and TSH in 19, 3 and 3 pts respectively. QoL data will be presented. Changes in PRV-1 expression were determined in 13 PRV-1 positive pts (9 PV, 4 ET) after 6 months and in 2 PV pts after 24 months. PRV-1 expression was normalized in 3/4 ET pts at 6 months and in 1/2 PV pts after 24 months treatment. Conclusion: We found PegIntron to be effective in reducing the platelet count in 29/42 pts (69 %) and 20 pts (48 %) remained in CR at 1 year. 22 pts (52 %) had discontinued therapy at 12 months, mainly due to side effects, a higher rate than in previous trials. Although analyzed in a small number of pts, reversal of PRV-1 positivity nonetheless suggests that PRV-1 quantification may be useful as a molecular marker of therapeutic efficacy.

Description: Introduction: The Philadelphia-negative myeloproliferative neoplasms are associated with deregulation of inflammation and immune genes implying a gene signature of a chronic inflammatory state and immune deregulation. Several studies have demonstrated interferon-alpha2 (IFN) to be highly efficacious in normalizing elevated blood cell counts and in inducing molecular remissions as well. Using whole blood gene expression, we herein for the first time show that IFN profoundly influences the deregulated inflammation- and immune genes giving rise to a gene signature indicative of decreased inflammation and improved regulation of immune genes during IFN treatment. Material and Methods: Gene expression microarray studies have been performed on eight patients with ET, 21 patients with PV, and 4 patients with PMF. All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole-blood and amplified to biotin-labeled aRNA and hybridized to microarray chips. Results: We identified 6261, 10,008, and 2828 probe sets to be significantly differentially expressed in ET, PV, and PMF, respectively, during treatment with IFN (pvalue

Description: Introduction: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPNs but also potentially having an impact upon the development of accelerated (premature) atherosclerosis, which is recorded in several other inflammatory diseases. Using whole blood gene expression profiling we have previously reported massive deregulation of inflammation genes and genes involved in oxidative stress. Herein, we extend our studies to explore the landscape of atherosclerosis genes, which have not been investigated previously in MPNs but may add novel important information in regard to deregulation of these genes of potential importance for the development of premature atherosclerosis and accordingly the heavy cardiovascular disease burden in MPNs. Methods:Global gene expression profiling was performed on 21 control subjects, 19 patients with ET, 41 patients with PV, and 9 patients with PMF. Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole blood, amplified to biotin-labeled aRNA, and hybridized to microarray chips. The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data. Results:We identified 20,439, 25,307, and 17,417 probe sets that were differentially expressed between controls and patients with ET, PV, and PMF, respectively (FDR£0.05). These genes included 84 genes represented on the Qiagen Human Atherosclerosis gene panel. In patients with ET, FABP3, FN1, LIF, LPL, PDGFB, PDGFRB, PPARD, SERPINE1, and TGFB2 were among the 28 upregulated genes, and APOE, BCL2, ITGA5, KLF2, NFKB1, and SELPLG were among the 17 downregulated genes. In patients with PV, 39 atherosclerosis genes were upregulated including VCAM1, FN1, PDGFRB, LPL, TGFB2, SERPINE1, PDGFB, and LIF, and 17 genes were downregulated including APOE, ITGA5, KLF2, NFKB1, SELPLG, and BCL2. In patients with PMF, PDGFB, HBEGF, LIF, PDGFRB, SERPINE1, TGFB2, and VWF were among the 24 upregulated genes, and KLF2, BCL2, IL1R2, NFKB1, ITGB2, ITGA5, IFNAR2, and SELPLG were among the 22 downregulated genes. BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated, whereas BCL2 were progressively significantly downregulated from ET over PV to PMF (all FDR

Description: Patients with chronic myeloproliferative neoplasms, including essential thrombocythemia (ET), polycythemia vera (PV), and chronic myeloid leukemia (CML), are at increased risk of new hematologic malignancies, but their risk of nonhematologic malignancies remains unknown. In the present study, we assessed the risk of both types of malignancies after an ET, PV, or CML diagnosis. We linked 2 population-based nationwide registries, the Danish National Registry of Patients, covering all Danish hospitals and the Danish Cancer Registry, and assessed subsequent cancer risk in a cohort of all 7229 patients diagnosed with a chronic myeloproliferative neoplasm during 1977-2008. We compared the incidence of subsequent cancer in this cohort with that expected on the basis of cancer incidence in the general population (standardized incidence ratio). Overall, ET, PV, and CML patients were at increased risk of developing both new hematologic and nonhematologic cancers. The standardized incidence ratio for developing a nonhematologic cancer was 1.2 (95% confidence interval [95% CI]): 1.0-1.4) for patients with ET, 1.4 (95% CI: 1.3-1.5) for patients with PV, and 1.6 (95% CI: 1.3-2.0) for patients with CML. We conclude that patients with chronic myeloproliferative neoplasms are at increased risk of developing a new malignant disease.

Description: Introduction Blood eosinophilia may arise from clonal intrinsic disorders or reactive extrinsic conditions. The eosinophilic granulocytes (eosinophils) may have diverse physiological functions and cause organ involvement. A dose-dependent relation between eosinophilia and risks of end-organ damage has not been described and it is not known whether numbers below internationally used threshold criteria for eosinophilia in adults (

Description: Background : Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by the presence of the JAK2V617 mutation in virtually all patients. Recently several studies have shown that the JAK2V617F mutational load decreases during treatment with alpha-interferon2 (1–6). Aim: To report on molecular and histomorphological bone marrow responses in seven PV patients with complete molecular remissions during and after long-term treatment with alpha-interferon 2b. Patients: Seven patients treated with alpha-interferon2b for a median of 84 months (range 31–120) are reported. In four of the patients alpha-interferon2b was started at the time of diagnosis and in three patients 9, 36 and 42 months from the time of diagnosis, respectively. Methods: The mutation was determined by allele specific PCR (n=2 only) (7) and quantitative PCR (qPCR) (n=5) (8). In three out of these patients qPCR JAK2V617F was performed on archived bone marrow from diagnosis (2 patients) and on peripheral blood (one patient) prior to treatment with alpha-interferon2b. A complete molecular remission (CMoR) was defined by less than 2 % JAK2 V617F mutated alleles (7). Results: Molecular Responses. All patients obtained a CMoR after a median of 84 months (29–120 months) of treatment with alpha-interferon2b. Subsequently all patients have discontinued alpha-interferon with a follow-up period of median 10 months (range 4–30 months) and sustained complete hematological remission. Furthermore, in three patients molecular responses have recently been updated – April and May 2008 - showing CMoRs in all (1,2 %, 0,9 % 0,1 % mutated alleles, respectively). Bone Marrow Responses. Follow-up bone marrow biopsies were available in five patients. Complete normalization of the bone marrow was seen in three patients after treatment with alpha-interferon2b for 84, 132 and 132 months, respectively. In the bone marrow from the patient being treated with alpha-interferon for 132 months a qPCR JAK2V617 analysis was performed detecting the mutation at a very low level (0,5 % mutated alleles). In two other patients, being treated with alpha-interferon2b for 24 and 120 months, respectively, and having obtained a CmoR in peripheral blood the bone marrow histomorphology showed marked regression of PV-features but in both patients still with focal areas displaying an increased number of morphologically abnormal megakaryocytes. Updated histomorphological and molecular response patterns will be presented. Discussion and Conclusion : Previous studies on the molecular response during alpha-interferon2a treatment have shown that a substantial proportion of patients achieve a significant molecular response after 12 months with a continuous decrease in the JAK2V617F mutation load at 24 and 36 months (1,5,6). This report confirms and extends preliminary data, showing that long-term treatment with alpha-interferon 2b in a subgroup of PV-patients is able to induce complete molecular remissions with normalization of the bone marrow morphology, which may even be sustained after discontinuation of alpha-interferon2b for up to 20 months (5). Prolonged treatment for several years seems necessary to induce such sustained responses, since treatment for only a few months has been reported to be followed by rapid recurrence of clonal hematopoiesis (9). In conclusion, a state of “minimal residual disease” may be achieved in PV by long-term immune therapy using alpha-interferon 2. Our observations call for large prospective clinical studies in which treatment with alpha-interferon is initiated up-front in patients with JAK2-positive PV and allied diseases. These studies should also aim at exploring the minimal dose of alpha-interferon needed to elicit complete molecular responses in order to minimize side effects of the drug and accordingly diminish the high drop-out rates reported in most previous studies.

Description: Abstract 4108 Background: In Philadelphia-negative chronic myeloproliferative neoplasms (MPN) increased microvascular density, bizarre vessel architecture and increased number of pericytes are distinct histopathological features; apart from the characteristic proliferation of myeloid cell lines and the progressive accumulation of connective tissue in the bone marrow. Pericytes express several markers such as CD146, CD271, Smooth Muscle Actin (SMA), Desmin, Platelet-derived growth factor receptor beta and Neuron-glial 2. However, these markers are also expressed in other cell types of which some are related to vascular structures. Immunofluorescence labelling is the golden standard for detection of co-expressed cellular antigens, but due to the crowded cellular environment in bone marrow and excessive autofluorescence, identification of cell types by light microscopy is preferred. Aim: This is a methodological study aiming to identify pericyte marker profiles by light microscopy in bone marrow biopsies, contributing to our understanding of the pathogenesis of MPN. Method and results: Formalin fixed, decalcified and paraffin-embedded blocks of bone marrow trephine specimens from normal donor (n=1) and patients with primary myelofibrosis (PMF) (n=3) were included. Specimens were subjected to an immunohistochemical sequential multi-labelling and erasing technique (SE-technique), inspired by the work of Glass et al. 2009 (J Histochem Cytochem). Briefly, antigens of interest in the first and/or second layer were detected with an immunoperoxidase system and visualised with Amino-Ethyl-Carbazole (AEC). After imaging, erasing of AEC with 96% alcohol and blocking of immunoreagents, the slides were stained with a traditional double immuno-labelling procedure. We successfully applied up to four layers of antibodies using CD146, SMA, CD34, CD271, and Ki67 in different combinations; either displayed as single or single followed by traditional double sequential staining runs (figure 1). In addition to the conventional light microscopy analysis we applied a Photoshop color palette, where the different immunohistochemical reactions in the staining sequence were assigned to the different color channels creating a single composite image. The SE-technique significantly improves morphological studies especially in bone marrow trephines with the cells of interest intermingled with other cells. Additionally, the SE-technique makes it possible to detect more than two antigens regardless of immunoglobulin type or animal host. Conclusion: To our knowledge, the SE-technique described in this study, is the first to multi-label antigens, identifying vessel and pericyte architecture in bone marrow trephines at light microscopic level. This technique may unravel novel aspects of the composition of the microvessel structures in patients with PMF and related neoplasms. The SE-technique displayed as single staining images and Photoshop color palette combined image. Panel A-D shows an identical area in the different steps of the method. A) CD146. Positive pericytes (arrow). B) SMA. Positive pericytes (arrow). C) CD34. Negative pericytes (arrow). D) Combined image with CD146 (green channel), CD34 (red channel), and SMA (blue channel). Coexpression of CD146/CD34 is seen as yellow reaction deposit, and coexpression of CD146/SMA as cyan reaction deposit (arrow). Note, as a negative control, the CD146 positive fat cell in A (arrowhead) - negative in B and C, and SMA positive pericyte in B (arrow) – negative in C. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4118 Introduction: The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2 (enhancer of zeste homolog 2), SUZ12, and EED, in which the SET (suppressor of vaegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. EZH2, a known repressor of gene transcription, has been reported to be overexpressed in many cancers and correlates with poor prognosis. EZH2 may also be involved in disease progression in patients with the classical Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs) encompassing essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Since the potential oncogenic role of EZH2 in CMPNs has never been investigated, we have assessed gene expression of EZH2 in a cohort of patients with CMPNs. Patients and Methods: Using Affymetrix HG-U133 2.0 Plus microarrays, recognizing 54675 probe sets (38500 genes), gene expression profiling has been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). All patients were diagnosed according to the WHO criteria of a CMPN. Total RNA was purified from whole blood and amplified to biotin-labeled RNA and hybridized to microarray chips. Results: We identified 20439, 25307, 17417, and 25421 probe sets which were differentially expressed between controls and patients with ET, PV, PMF, and CPMNs as a whole, respectively (false discovery rate (FDR) adjusted p values 〈 0.05). These genes included EZH2, which was highly significantly upregulated in patients with PMF as compared to controls (2.3 fold upregulated; uncorrected p-value=1.09×10-8 and FDR=1.75×10-6, and between PMF and non-PMF patients (fold change=2.0, FDR 〈 0.0005). No significant differences in EZH2 gene expression were recorded between controls and ET patients, controls and PV patients, or controls and the CMPN group as a whole. Within patients, the EZH2 gene was also differentially expressed with the highest levels being recorded in patients with PMF compared to PV patients (fold change=2.4, FDR 〈 7.5 ×10-6). Discussion and Conclusions: Using global gene expression profiling we have found the EZH2 gene to be significantly upregulated in CMPN patients, with the highest expression levels being found in PMF. We hypothesize that an altered expression of EZH2 may be involved in the transformation of ET and PV into myelofibrosis. It remains to be clarified if deregulation of EZH2 occurs consequent to mutations in the EZH2-gene. Enhanced EZH2 expression may also be associated with silencing of differentiation genes during myelofibrotic and leukemic transformation. An increased expression of EZH2 may provide a proliferative advantage of the malignant clone through interaction with the pathways of key elements controlling cell growth arrest and differentiation, (e.g. nuclear factor kappa beta and - the proteasome pathway). Studies are in progress to elucidate if genomic loss of distinct microRNAs (microRNA 101 leads to overexpression of EZH2 in cancer is associated with overexpression of EZH2 in CMPNs. Highly expressed EZH2 may be a new marker of an aggressive clinical phenotype which might imply EZH2 as a novel biomarker for predicting prognosis. If so, EZH2-blockade might be a novel approach to be incorporated in the strategies for developing epigenetic therapies in patients with CMPNs. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction The discovery of mutations in the calreticulin (CALR) gene in the majority of JAK2 -V617F negative patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) (Klampfl et al., 2013; Nangalia et al., 2013) has improved the diagnostic accuracy considerably, and most recently distinct clinical and hematological characteristics according to mutational status have been described (Park et al., 2015). The perspective is to personalize and optimize treatment according to the molecular and clinical landscape. This may be achieved by obtaining more information on responses in myeloproliferative neoplasms (MPN) to existing treatment strategies as assessed by the allele burden. Mutations in the CALR gene have proven to play a major role in oncogenic and immunologic processes (Lu, Weng, & Lee, 2015). In this context, it is highly relevant to explore the effectiveness of interferon-alpha2 (IFN) in reducing the CALR -mutated clone. Until now, only one paper has reported a decrease in allele burden in two patients during IFN treatment (Cassinat, Verger, & Kiladijan, 2014). The objective of this report is to expand current knowledge on this important topic by describing the mutant CALR allele burden over time in a larger group of IFN-treated patients. Method Clinical data were collected retrospectively from a single institution on all IFN-treated CALR positive MPN patients with sequential determinations of the mutant allele burden. Type 1 and type 2 mutations were initially identified by a previously published fragment analysis (Klampfl et al 2013). We have developed a Taqman qPCR assay for precise determination of the mutant allele burden of type 1 and type 2 mutations. Stored DNA was subsequently analysed to increase follow-up time. Results Twenty-one patients were included. Fifteen patients had a diagnosis of PMF; 7 of these were diagnosed with prefibrotic myelofibrosis. Six patients had ET. The type 1 and 2 mutations were found in 15 and 6 patients, respectively. Median age was 60 years (range 42-79) and the sex ratio (M/F) was 8/13. Fifteen patients (71%) were in ongoing treatment with IFN, whereas treatment was discontinued in 6 (29%) because of side effects. Median time of IFN treatment was 756 days (range 42-3927). The IFN prescribed was either subcutaneous injection of Pegasys® (median: 45 microgram (ug) per week), PegIntron® 25-50 ug per week, or Multiferon® 3 x 3 million IU per week. Median follow up time since the first CALR measurement was 756 days (range 294-2108). Fourteen patients (67%) maintained an unchanged allele burden during follow up; 1 patient (5%) presented a temporary decrease (from 39% to 27% in allele burden) but increased to the initial level within months while still on IFN treatment (presumably due to low compliance); 1 patient (5%) displayed an increase in allele burden during transformation to acute myelogenous leukemia (Figure 1); and 5 patients (24%) exhibited a marked decrease in allele burden (median decrease: 32%, range 18-45) during treatment with IFN (Figure 2). All 5 patients with decreasing allele burden (Table 1) normalized their platelet counts within a median time of 5 weeks (range 4-20) after initiating treatment with IFN. Conclusion Using a novel sensitive assay for the CALR mutant allele burden, we have demonstrated and substantiated the effectiveness of IFN to reduce the allele burden in a larger series of CALR positive patients with PMF and ET. Importantly, we report for the first time on highly heterogeneous response patterns. Our observation of one fourth of the CALR positive patients responding to treatment with IFN strongly suggests that IFN significantly influences the CALR mutational load. Further clinical and molecular studies are urgently needed to explore the mechanisms behind the heterogeneous response patterns and the clinical implications in regard to clonal evolution and disease progression in non-responding patients. We are currently analysing these issues to assess the definite role of IFN in future treatment strategies in CALR positive MPN patients. Table 1. Patients responding to interferon-alpha2 Characteristics Number/median (range) Patients 5 Age, years 53 (42-62) Sex (M/F) 1/4 Diagnosis- Essential thrombocythemia- Primary myelofibrosis- Prefibrotic myelofibrosis 221 Calreticulin mutation type- type 1- type 2 50 Duration of interferon-alpha2 treatment, days 960 (177-2790) Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Cordua: Janssen-Cilag: Other: travel grant. Off Label Use: interferon alpha2 for myeloproliferative neoplasms. Holmström:La Roche Ltd: Other: travel grant. Pallisgaard:Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Novartis: Other: travel grant, Research Funding, Speakers Bureau; Roche: Other: travel grant. Hasselbalch:Novartis: Research Funding.