ALBERT

Description: Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

Description: Genes encoding the RNA splicing factors SF3B1, SRSF2, and U2AF1 are subject to frequent missense mutations in clonal hematopoiesis and diverse neoplastic diseases. Most "spliceosomal" mutations affect specific hotspot residues, resulting in splicing changes that promote disease pathophysiology. However, a subset of patients carry spliceosomal mutations that affect non-hotspot residues, whose potential functional contributions to disease are unstudied. Here, we undertook a systematic characterization of diverse rare and private spliceosomal mutations to infer their likely disease relevance. We utilized isogenic cell lines and primary patient materials to discover that 11 of 14 studied rare and private mutations in SRSF2 and U2AF1 induced distinct splicing alterations, including partially or completely phenocopying the alterations in exon and splice site recognition induced by hotspot mutations or driving "dual" phenocopies that mimicked two co-occurring hotspot mutations. Our data suggest that many rare and private spliceosomal mutations contribute to disease pathogenesis and illustrate the utility of molecular assays to inform precision medicine by inferring the potential disease relevance of newly discovered mutations.

Description: Background: The discovery of JAK2V617F mutation has lead to the proposal of new algorithms to diagnose and classify MPD. Separation of PV from ET could become less clear, especially in JAK2V617F patients, if one considers that mutated PV and ET are similar conditions. However, the short-term risk of thrombosis, and long-term risk of evolution to leukemia have not yet been shown to be similar in JAK2V617F ET and PV. Current WHO criteria for PV require either RCM 〉125% of predicted value, or Hb level 〉18.5 g in men and 16.5 g in women. Some investigators recently proposed a threshold for Ht at 52% in men and 48% in women to diagnose PV in JAK2V617F patients. Diagnosis of ET requires excluding PV, and using Hb or Ht values for this purpose could avoid RCM measurement. Aims: To evaluate the prevalence and prognostic impact of erythrocytosis determined by RCM measurement in MPD patients classified as ET on peripheral blood counts. Methods: We reviewed all RCM measurements performed in patients suspected of MPD in 2 nuclear medicine laboratories during a 10-year period. Results: Among 566 patients referred for RCM measurement, 71 had isolated thrombocytosis (i.e. both Hb and Ht 〈 values used for PV diagnosis as defined above). RCM was normal in 38/71, but was 〉125% of predicted value, revealing inapparent erythrocytosis, in 33 cases. Thus, after RCM measurement, final diagnosis was ET in 53.5% (38/71), and PV in 46.5% (33/71) of the patients, respectively. Pts with elevated RCM had significantly higher Ht (p

Description: Background: PET with fluorodeoxyglucose combined with computed tomography is a major tool for the diagnosis, staging and monitoring of treatment response in clinical oncology. 3′-18Fluoro-3′-deoxy-L-thymidine (18F-FLT) is a nucleoside analog that quickly accumulates in proliferating cells, evaluated in clinical studies in various cancers as a PET radiotracer offering non invasive assessment of cell proliferation in vivo. Preliminary results of a pilot study suggested that this technique could be useful to assess bone marrow (BM) activity and extramedullary hematopoiesis in patients with myelofibrosis (MF). We present the final analysis of the FLT-MF-2009 study (EudraCT Number: 2009-016804-21) confirming that FLT PET could be a new technique useful for MF management. Methods: Main inclusion criteria were: a diagnosis of MF according to WHO criteria; BM biopsy available for centralized review; written informed consent. 18F-FLT PET was performed 1 hour after injection of 18F-FLT (provided by AAA), and consisted in a whole-body acquisition. Two nuclear physicians interpreted images in a blinded fashion independently, qualitatively and according to a visual scale, and patients were classified according to 3 distinct patterns. 18F-FLT uptake was quantified using maximum standardized uptake value (SUVmax) in several sites of the skeleton (axial skeleton, proximal and distal territories of upper and lower limbs), in the spleen and the liver. Analysis of factors affecting the intensity of 18F-FLT uptake in these sites (using PET pattern and SUVmax as the endpoints) was performed by using non-parametric tests. Results: 15 patients (mean age: 62 years) were included between Apr 2011 and Jul 2012. 8 patients had primary (PMF), 3 post-polycythemia vera (PV), and 4 post-essential thrombocythemia (ET) MF. 13 patients had a palpable splenomegaly. 10 patients had the JAK2V617F mutation with a median mutant allele burden of 59% (interquartile range: 29-80%), 1 had a MPL515 mutation. Therapies at time of PET included hydroxyurea (n=1), androgens (n=1), interferon (n=4) and ruxolitinib (n=5); 4 patients had no specific therapy for MF. Three patterns of 18F-FLT PET images were observed. Pattern A (n=3) showed a normal BM activity in the central skeleton, a mild expansion to distal extremities and normal splenic uptake. Pattern B (n= 9) displayed a rather normal pattern of BM activity in the central skeleton associated with marked expansion of BM activity to distal extremities and intense uptake of the tracer in the spleen. Pattern C (n=3) showed a marked reduced hematopoietic activity in the central skeleton but a high uptake in spleen, suggesting the existence of myeloid metaplasia. Quantitative analyses of 18F-FLT uptake using SUVmax permitted the search for correlations with clinical and biological characteristics. Grade 3 fibrosis in BM biopsy was significantly associated with low SUVmax values measured in axial skeleton (p=0.019), proximal upper limbs (p=0.016) and proximal lower limbs (p=0.019). Significantly higher SUVmax values in proximal upper (p=0.014) and lower limbs (p=0.021) were associated with the diagnosis of post-PV- vs. post-ET- or PMF. SUVmax values in proximal upper limbs also correlated with the hemoglobin level (Spearman correlation coefficient: 0.53; p=0.038). No significant correlation was found between SUVmax values in any territory and platelet values, JAK2V617F positivity or allele burden, disease duration. Significantly lower SUVmax values were measured in proximal upper limbs of patients treated with ruxolitinib (3.9 ± 2) compared to patients treated with interferon (11.2 ± 5) or untreated patients (7.7 ± 3.1) (p=0.043). Conclusion: The results of this pilot study suggests that 18F-FLT PET could be a new, objective, non invasive technique useful for the evaluation of malignant hematopoiesis in MF, in terms of diagnosis, staging and for monitoring response to therapy. SUVmax values may discriminate post-PV MF from other forms of MF, and may distinguish patients with grade 3 fibrosis. If this latter finding is confirmed in a larger study, 18F-FLT PET could become a convenient substitute to sequential biopsies for the staging of BM fibrosis during the course of the disease. In addition, distinct SUVmax values were found in patients treated with ruxolitinib, suggesting that 18F-FLT PET could also be useful to monitor the efficacy of therapies in MF. Disclosures Off Label Use: 18F-FLT is an investigational isotope not approved for the imaging of myelofibrosis. Patients treated off-label with interferon were included in the study..

Description: Abstract 1083 Aim: Since combining differentiating agents and chemotherapy, acute promyelocytic leukemia (APL) is associated with a high cure rate. One remaining issue is the significant rates of early death and relapse observed in patients with high count APL (initial white blood cell count [WBC] ≥ 10.109/L). Early death rate might be underestimated in clinical trials, due to an unknown proportion of patients not registered because of initial severity. For this reason, we reviewed individual histories of all patients with APL referred to our institution during the last 10 years (09/2000-06/2010), with a special focus on admission in intensive care unit (ICU) and inclusion or non-inclusion in recruiting APL trials (European group APL-2000 and APL-2006), as well as long-term follow-up. Patients: A total of 100 patients with newly-diagnosed previously untreated APL, including 8 children, were admitted during this time period. Diagnosis was based on morphology and subsequently confirmed by the presence of the t(15;17) translocation and/or PML/RARA fusion transcript. Results: The rate of patients not enrolled within recruiting trials was 29% (n= 29). This rate was higher in children (n= 5/8, 62.5%) than in adults (n= 24/92, 26%) and remained stable during the two protocol periods (n=17/62, 27% for APL-2000; n= 12/38, 32% for APL-2006). Reasons for non-enrollment were inability to give informed consent in 10 patients (mechanical ventilation or neurological deficiency), physician's decision in 5 patients (2 very high leucocytosis, 1 severe infection, 2 severe liver dysfunction), and concomitant disease in 4 patients (2 HIV patients, 2 other cancers), refusal in 5 patients (including 1 Jehovah witness who eventually survived), and various administrative reasons in 5 patients. Non-enrolled patients had similar sex ratio (F/M=15/14 vs 35/36; p=.99), median age (40.5 [range, 4–79] vs 46 years [4-81]; p=.97), and frequency of additional chromosomal abnormalities (24% vs 28%; p=.80) than enrolled patients. Conversely, they had a higher rate of WBC ≥ 10.109/L (n=15/29 vs 22/71; p=.07) or ≥ 50.109/L (n=8/29 vs 5/71; p=.01), a lower rate of platelet count 〈 40.109/L (28/29 vs 46/71; p=.001), and a higher frequency of microgranular M3-variant subtype (11/29 vs 8/71; p=.004) and BCR3 PML-RARA isoform (14/25 vs 24/70; p=.09). Among the 29 non-enrolled patients, 24 nevertheless received the whole planned standard induction therapy, 2 received arsenic trioxide-based induction, and 3 early died before or the day after chemotherapy initiation. Ninety-nine patients were evaluable for response to induction (1 patient ongoing). Due to a higher early death rate (21% vs 3%; p=.007), the complete remission (CR) rate was lower in non-enrolled patients (79% vs 97%; p=.007). At 5 years, event-free survival (EFS) was estimated at 62% (95%CI, 37–79) vs 84% (95%CI, 72–91) (p=.02) and overall survival (OS) at 63% (95%CI, 36–81) vs 85% (95%CI, 72–93) (p=.03) in the non-enrolled and enrolled group, respectively. Once CR had been reached, non-enrolled patients displayed, however, a good post-CR outcome with 5-year remission duration at 78% and OS from CR at 80%. Of note, only one patient from this cohort died in first CR from a second neoplasia. Twenty-six patients (26%) were admitted in ICU for or during induction (13, 8, and 4 of them requiring mechanical ventilation, amine therapy, and dialysis, respectively). Chemotherapy was initiated in ICU in 19 of them. The rate of trial enrollment was 54% (n= 14/26) in ICU patients compared to 77 % (n= 57/74) in non-ICU patients (p=.04). Again, CR rate (p

Description: Introduction: Targeted drugs are needed for HR-MDS/AML, particularly in elderly patients and Venetoclax, approved for some CLL, gives promising results in elderly AML. Assays to predict response to treatment may enable us to deliver personalized treatment. We sought to determine the most informative assay to predict response; viability assays can directly measure the effects of reagents on growth. Progenitor assays can potentially determine if the reagents can target diseased primitive cells. PET scanning can be used to follow response to treatment. Methods: Peripheral blood (PB) or bone marrow (BM) from 7 MDS/AML patients were incubated in a) no treatment, b) ABT-199 (1 µM) (Abbvie), c) GDC-0973 (1 µM) (Genentech) or d) ABT-199+GDC-0973 (1 µM of each) and assessed for viability using the MTT assay (n=2); cell death followed using the Incucyte® Zoom System (Essen Bioscience) (n=2) or methocult progenitor assays (Stem Cell Technologies) (n=4). Having shown that RAS:BCL-2 co-localization correlated with prognosis in MDS/AML patients (Leuk Res 37:312-9, 2013), immunofluorescence was undertaken. A micro PET device dedicated to mice was used to measure BM blast proliferation. After injection of 18F-FLT(a thymidine analogue) in mice untreated (n=7) or ABT-199 (75mg/kg)+GDC-0973(10mg/kg) treated (n=5) normal FVB/N, HR-MDS mice treated with vehicle (n=4), 2-month old HR-MDS before (n=5) and 3-month old before (n=4) and after ABT-199 (75mg/kg)+GDC-0973(10mg/kg) treatment (n=8), PET imaging was performed (Inveon Siemens Medical Systems), analyzed for signal and quantified. Results: Patient details and results are summarized on Table 1. Using the MTT assay 2 PB patient samples were found to be sensitive to ABT-199 treatment (Figure 1A, AS, p=0.00042 and YA, 0.00002) and more sensitive to the combination compared to untreated (AS, p=0.00007 and YA, 0.000003). With the incucyte the BM of one patient (AE) was found to be resistant to both ABT-199 and GDC-0973, but sensitive to the combination (Figure 1B). PB and BM from patient JA were assayed for apoptosis with the incucyte and were found to be sensitive to ABT-199 with increased apoptosis, resistant to GDC-0973 with decreased apoptosis and sensitive to the combination. Four bone marrow samples were tested in the 4 conditions using the progenitor assay (Figure 1C). Three patients were sensitive to GDC-0973, inhibiting any colony formation and the fourth had reduced colony numbers. In this assay patient JA appeared to be sensitive to GDC-0973 treatment whereas the incucyte assay scored this sample to be resistant to apoptosis; thus the cytotoxic effects of GDC-0973 may not be via apoptopsis. As the progenitor assay is likely to score the primitive disease population, this assay may prove more informative than the others without prior selection. One patient (DH) was clearly resistant to ABT-199, whereas the other three (JA, CB and FL) had reduced colony growth. All patients were sensitive to the combination treatment and inhibited colony growth. The RAS:BCL-2 co-localization in the PB revealed no complex in either the Mito or PM upon treatment with ABT-199 alone and some localization in the Mito with GDC-0973. With both ABT-199 and GDC-0973, there were hardly any cells confirming the cytotoxic effects of the combination. As we have previously shown that PM co-localization of the complex is associated with drug resistance (Blood 130:2613, 2017Suppl), we used the combination on our HR-MDS mouse model, where the complex co-localizes in the PM and followed the mice by PET scanning (Figure 1D). Weak signal was visualized in the femurs of untreated and ABT-199+GDC-0973 treated FVB/N mice (FBR 1.17+/-0.34 and 1.02+/-0.08 respectively). Mild PET signal was seen in the femurs of 2 month-old HR-MDS mice, (FBR 1.79+/-0.98). Intense PET signal was seen in the femurs and proximal humerus of HR-MDS mice treated with vehicle (3 month-old, FBR=2.35+/-1.32). Low PET signals were seen in the femurs of 5/8 HR-MDS mice treated with ABT-199+GDC-0973 (FBR=1.93+/-0.84). FBRs of the 3 groups of HR-MDS mice were significantly higher than those of FBV/N groups. Conclusion: Combined Venetoclax (ABT-199) and GDC-0973 targets MDS/AML progenitors and can potentially overcome drug resistance with the disruption of the RAS:BCL-2 complex. Bone marrow disease progression in HR-MDS mice can be monitored with 18F-FLT-PET imaging; PET data shows that the combination slows down disease progression. Disclosures Padua: Abbvie: Research Funding; Genentech: Research Funding. Giraudier:Novartis: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Reata: Equity Ownership; Celgene: Consultancy; Jazz Pharma: Consultancy; Oncolyze: Equity Ownership; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership; Astra Zeneca: Research Funding.

Description: Abstract 436 Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patients. IFNα was shown to induce complete long-term hematologic and molecular remissions in JAK2V617F-positive MPD patients, suggesting a possible curative effect of IFNα. In order to better understand mechanisms of action of this drug, experiments were perfomred on cell lines, patient cells and mice cells harboring a JAK2V617F mutation. We hypothesized that IFNα may target directly MPD cells through binding to its specific receptor, in addition to the potential immunological effect of this molecule and could induce cell cycle entry of the pathological quiescent HSCs. Human cell lines harboring JAK2 mutation or BCR-ABL oncogene were treated with increasing doses of IFNα. A significant anti-proliferative effect at low concentrations (100 IU/ml) on the JAK2V617F-positive HEL cell line was observed. On the contrary, at this low dose IFNα did not influence growth of the BCR-ABL-positive K562 and the non-mutated UT-7 cell lines. This result supported a direct effect of IFNα in JAK2V617F cells. Suppressor of cytokine signaling (SOCS) are potent inhibitors of the JAK-STAT pathway by proceeding to a classic negative regulation loop proteins. Following IFNα stimulation of HEL cells, SOCS1 and SOCS3 mRNAs expression were induced (p=0.00036 and p=0.0012, respectively) and efficient knock-down of either SOCS1 or SOCS3 by shRNAs expression in HEL cells was able to counteract the anti-proliferative effect of IFNα (p=0.028 and p=0.031, respectively). We concluded that SOCS1 and SOCS3 are involved, in IFNα proliferative inhibition activity of HEL cells. To determine whether JAK2V617F confer hypersensitivity to IFNα inhibitory effect, proliferation and genotyping of CD34+ progenitors isolated from the bone marrow of JAK2V617F–positive MPD patients (n=5) and control subjects (n=4) were plated at one cell per well in 96-well plates and counted and genotyped at Day 10-12. A significant decrease of the patients progenitors clonogenicity was observed when exposed to IFNα (10 000 IU/ml, p

Description: Since the description in 2005 of the recurrent V617F JAK2 gene mutation in MPD patients, we have tested more than 1500 patients each year and confirmed the relative frequency of this mutation in distinct MPD subclasses and its usefulness in classyfying diseases such early PV, MIF, ET and thrombosis such Budd Chiari syndrome. In the case of erythrocytosis we notably observed that among patients with true polycythemia (red cell mass measurement increased by 25%), the presence of the mutation significantly distinguished between PV patients (95% V617F positive) and patients with Idiopathic Erythrocytosis (IE, 0% positive); In ET patients about 50% were positive ; In thrombosis, we demonstrated that the V617F mutation is present in about half of the patients with deep vein thrombosis (Budd Chiari syndrome or portal vein thrombosis), reinforcing the guess that occurrence of thrombosis in these patients is caused by an ignored MPD. The description in 2007 of novel recurrent mutations in the exon 12 of the JAK2 gene led us to test whether these new alterations could help in classifying some of the V617F negative patients. We have tested patients with PV (n= 25) of whom 10 had a post PV myelofibrosis, deep vein thrombosis (n= 20) and IE (n= 21). PV patients fulfilled either PVSG or WHO criteria. IE patients were diagnosed on the basis of an elevation of the hematocrit (median hematocrit of 54% (range: 49% to 56%)), a raised red cell mass (median excess of red cell mass was +35% (range: +25% to +104%); no identifiable cause of secondary erythrocytosis (serum Epo level was under or within the normal range in all patients); and absence of PV according to PVSG or WHO criteria (no splenomegaly, median WBC (×109/L) of 6180 (range: 3500–8300) ; median platelet count (x109/L) of 240 (range: 174–358). DNA extracted from peripheral blood has been tested using the allele specific PCR described in Scott et al., NEJM 2007, allowing to detect all of the described mutations. We failed to identify any mutation in patients with IE or deep vein thrombosis. However, 2 female patients diagnosed with PV were identified to harbour the N542-E543del mutation. With the exception of their relative younger age (41 and 45 respectively) and a very elevated hematocrit (58 and 59%) it was not possible to find any common points to these 2 patients that could allow individualizing patients susceptible to harbour exon 12 mutations. One patient had very low Epo level and splenomegaly, while the other patient had increased WBC but no splenomegaly. None of them had myelofibrosis. In conclusion, only 2 out of 25 PV patients harboured JAK2 exon 12 mutations, thus around 5% of PV patients that fulfilled PV criteria are still negative for any mutation. The fact that IE patients are always negative let the question unsolved of whether this entity is a MPD or not. The search for exon 12 JAK2 gene mutation should be restricted to patients which fulfil PV diagnosis criteria and do not exhibit the “classical” V617F mutation. In these cases, the presence of the mutation is very useful in order to well classify these patients as MPD patients.

Description: The Bone Marrow Failure (BMF) syndromes comprise a variety of distinct acquired or inherited clinical entities. Early distinction between syndromes has clear implications in the disease management and outcome. Fanconi anemia (FA), the most frequent cause of inherited BMF, is usually associated with congenital abnormalities, progressive cytopenia, chromosome fragility, and cancer susceptibility. However, due to a high clinical variability and/or the potential emergence of revertant hematopoietic cells (somatic mosaicism), identifying patients with FA or ruling out this diagnosis can be challenging. If undiagnosed, FA patients who initially present with bone marrow failure will die of toxicity after standard-dose conditioning regimens for HSCT. In this study, we evaluated FA diagnosis in patients with BMF but no clear initial evidence of FA, using of a combination of classical and innovative tests in blood and fibroblasts. A cohort of 82 patients with BMF and no strong clinical evidence of FA was analysed (patients with a clear FA diagnosis were not included). Based on the likelihood of an underlying inherited condition associated with the BMF, we classified patients in 3 groups: those likely to have idiopathic aplastic anemia (IAA) [n=38, group 1], those likely to have a constitutional condition other than FA [n=26, group 2], and those likely to have IAA but who had isolated clinical findings which could also be present in FA [n=18, group 3]. Chromosome breakage test and analysis of the FA/BRCA pathway by FANCD2 immunoblot were performed in PBL in all patients [n= 82]. To overcome potential somatic mosaicism, skin primary fibroblasts were analysed [n= 52]. Also, to rule out FA/BRCA downstream groups, we developed a new flow cytometry test based on MMC-sensitivity in fibroblasts. In total, 6 patients with FA were identified: 1/38 in group 1 (aplastic anemia at 10 yo, no positive clinical findings), 2/26 in group 2 (one with MDS at 48 yo with precocious menopause and vocal cord neoplasia at 38 yo; one with hypoplastic MDS at 50 yo), and 3/18 in group 3 (one with short stature and aplastic anemia at 37 yo; one with MDS and borderline physical abnormalities at 26 yo; and one with a single cafe-au-lait spot and aplastic anemia at 10 yo). Chromosomal breakage tests in PBL were sufficient to diagnose 4 of these FA patients (further classified as FA core using FANCD2 immunoblot). Additional fibroblast analyses were necessary to identify 2 more FA patients (both with complete somatic mosaicism) and, importantly, to definitely exclude FA diagnosis in other patients. In conclusion, underdiagnosing FA is rare if careful history and physical exam are done together with chromosome breakage test in PBL. However, in clinical situations where the suspicion of FA persists despite negative breakage tests, then fibroblasts should be tested. Because no cases of FA were found among patients with IAA and negative breakage tests in PBL, we suggest that FA screening can be limited to this technique in this population (group 1). The strategy here presented allowed us to identify a few unexpected FA cases in a cohort of BMF patients, and importantly, to definitely rule out FA in others, with clear clinical impact for patients who undergo HSCT.