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A significant proportion of COVID-19 patients show evidence of hyperinflammation (HI), of which secondary haemophagocytic lymphohistiocytosis (sHLH) is the most severe manifestation and diagnosed with HScore. Using a COVID-relevant modification of the HScore (%HScore), we set out to determine the prevalence of sHLH in 567 COVID-19 inpatient cases. The overall incidence of individuals with an 80% probability of sHLH in our COVID-19 cohort was 1.59% on admission and only rose to 4.05% if calculated at any time during admission. This small cohort as defined by %HScore showed no excess mortality compared with the whole cohort. Overall, %HScores were lower in older patients (p

Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289, Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272

BackgroundThe success of early dexamethasone therapy for hospitalised COVID-19 cases in treatment of Sars-CoV-2 infection may predominantly reflect its anti-inflammatory action against a hyperinflammation (HI) response. It is likely that there is substantial heterogeneity in HI responses in COVID-19.MethodsBlood CRP, ferritin, neutrophil, lymphocyte and platelet counts were scored to assess HI (HI5) and combined with a validated measure of generalised medical deterioration (NEWS2) before day 2. Our primary outcome was 28 day mortality from early treatment with dexamethasone stratified by HI5-NEWS2 status.FindingsOf 1265 patients, high risk of HI (high HI5-NEWS2) (n=367, 29.0%) conferred a strikingly increased mortality (36.0% vs 7.8%; Age adjusted hazard ratio (aHR) 5.9; 95% CI 3.6-9.8, pInterpretationThe HI5-NEWS2 measured at COVID-19 diagnosis, strongly predicts mortality at 28 days. Significant reduction in mortality with early dexamethasone treatment was only observed in the high risk group. Therefore, the HI5-NEWS2 score could be utilised to stratify randomised clinical trials to test whether intensified anti-inflammatory therapy would further benefit high risk patients and whether alternative approaches would benefit low risk groups. Considering its recognised morbidity, we suggest that early dexamethasone should not be routinely prescribed for HI5-NEWS2 low risk individuals with COVID-19 and clinicians should cautiously assess the risk benefit of this intervention.FundingNo external funding.

There is ongoing debate as to the optimum treatment for IgM paraproteinaemic neuropathy, although there is accumulating evidence that rituximab benefits a proportion of patients. We present a case series of 18 patients with IgM paraproteinaemic neuropathy treated with standard R-CP (rituximab, cyclophos- phamide, prednisolone) chemoimmunotherapy. 13 patients were positive for anti-MAG (myelin associ- ated glycoprotein) antibody. The remaining five had a clinical phenotype suggesting that their IgM paraprotein was causative in their neuropathy. Detailed neurological, neurophysiological and serological outcome data were recorded 1 and 2 years post treatment. The treatment was well tolerated. At 2 years, compared to baseline, a statistically significant improvement was seen in paraprotein concentration, anti-MAG titre, MRC sum score and (from the electrophysiology) the distal motor latency. Percentage of patients improving at 2 years was as follows: Overall neuropathy limitation score 56%, MRC sum score 69%, sensory sum score 61%, anti-MAG titre 100%. These responses compare favourably to those seen with rituximab monotherapy, reported in the literature at 30–50%. On long-term follow-up, 3 cases of glio- blastoma were seen, the aetiology of which is unclear. We suggest there is need for further study of the mechanisms both of the clear neurological benefit but also of the potential risk.ncolchester@yahoo.com

Chronic inflammatory demyelinating polyneuropathy (CIDP) has a variable clinical course with 15% remaining refractory to treatment. We report a woman with severe refractory CIDP and coexisting chronic lymphocytic leukaemia (CLL) who improved dramatically after chemoimmunotherapy appropriate for the CLL, including rituximab. A subsequent CIDP relapse after 15 months responded again to similar treatment, and the improvement has been maintained with 3-monthly rituximab infusions as sole ongoing therapy. The case suggests that CIDP refractory to conventional treatment may have associated pathology, in this case haematological malignancy, and that treating the malignancy can effectively treat the CIDP.

ObjectiveIt has been assumed that a significant proportion of COVID-19 patients show evidence of hyperinflammation of which secondary haemophagocytic lymphohistiocytosis (sHLH) is the most severe manifestation. To facilitate diagnosis of sHLH the HScore has been developed and validated. We set out to examine the prevalence of sHLH-like hyperinflammation in COVID-19.MethodsWe retrospectively examined HScore parameters in 626 COVID-19 cases admitted to our institute of which 567 were suitable for analysis and compared these to a cohort of confirmed infection associated sHLH cases. To account for missing data, we calculated the maximum possible HScore of the recorded parameters (%HScore).ResultsEarly measurement of HScore parameters (day -1 to 4 from diagnosis) strongly predicted the %HScore over the course of the admission (p ConclusionSurprisingly, these findings show that sHLH-type hyperinflammation is not prevalent in COVID-19, and %HScores do not predict outcome. Therefore, new algorithms are required to optimise case selection for clinical trials of targeted anti-inflammatory interventions.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0–4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

Despite the identification of acquired genetic mutations associated with Myeloproliferative Neoplasms (MPNs) there is a paucity of information relating to modifiable risk factors that may lead to these mutations. The MOSAICC Study was an exploratory case-control study of polycythemia vera (PV), essential thrombocythemia (ET), and Myelofibrosis (MF). MPN patients and population controls (identified by General Practitioners) and non-blood relative/friend controls were recruited from 2 large UK centers. Participants completed a telephone-based questionnaire analyzed by unconditional logistic regression analysis adjusting for potential confounders. Risk factors for MPNs identified included increasing childhood household density [odds ratio (OR) 2.55, 95% confidence interval (CI) 1.16–5.62], low childhood socioeconomic status (OR 2.30, 95%CI 1.02–5.18) and high pack years smoking (OR 2.19, 95%CI 1.03–4.66) and current smoking restricted to JAK2 positive PV cases (OR 3.73, 95%CI 1.06–13.15). Obesity was linked with ET (OR 2.59, 95%CI 1.02–6.58) confirming results in previous cohort studies. Receipt of multiple CT scans was associated with a strongly increased risk of MPN although with wide confidence intervals (OR 5.38, 95%CI 1.67–17.3). Alcohol intake was inversely associated with risk of PV (OR 0.41, 95%CI 0.19–0.92) and ET (OR 0.48, 95%CI 0.24–0.98). The associations with childhood household density, high pack years smoking and alcohol were also seen in multivariate analysis. This is the largest case control study in MPNs to date and confirms the previously reported associations with obesity and cigarette smoking from cohort studies in addition to novel associations. In particular, the role of smoking and JAK2 mutation cases merits further evaluation.

Significance Bone marrow failure (BMF) is an inherited life-threatening condition characterized by defective hematopoiesis, developmental abnormalities, and predisposition to cancer. BMF caused by ERCC6L2 mutations is considered to be a genome instability syndrome, because DNA repair is compromised in patient cells. In this study, we report BMF cases with biallelic disease-causing variants and provide evidence from patients’ cells that transcription deficiency can explain the genome instability. Specifically, we demonstrate that ERCC6L2 participates in RNA polymerase II-mediated transcription via interaction with DNA-dependent protein kinase (DNA-PK) and resolves DNA–RNA hybrids (R loops). Collectively, our data point to a causal mechanism in BMF in which patients with ERCC6L2 mutations are defective in the repair of transcription-associated DNA damage., Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients’ lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients’ LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients’ LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA–RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

The myeloproliferative neoplasms (MPN) including polycythaemia vera (PV), essential thrombocythaemia and primary myelofibrosis (PMF) are rare diseases contributing to significant morbidity. Symptom management is a prime treatment objective but current symptom assessment tools have not been validated compared to the general population. The MPN-symptom assessment form (MPN-SAF), a reliable and validated clinical tool to assess MPN symptom burden, was administered to MPN patients (n = 106) and, for the first time, population controls (n = 124) as part of a UK case–control study. Mean symptom scores were compared between patients and controls adjusting for potential confounders. Mean patient scores were compared to data collected by the Mayo Clinic, USA on 1,446 international MPN patients to determine patient group representativeness. MPN patients had significantly higher mean scores than controls for 25 of the 26 symptoms measured (P

Biological sample collection is becoming more common in epidemiology research to obtain DNA for genetic analysis. There are many different DNA collection methods but little evidence on their relative effectiveness. Therefore, we took the opportunity of a prospective case-control study in myeloproliferative neoplasms (MPNs) to compare DNA yield from 8.5 mL PAXgene tubes for whole blood collection versus 2 mL Oragene OG-500 saliva collection kits. MPNs include polycythaemia vera, essential thrombocythaemia, and primary myelofibrosis. These are rare diseases and our exploratory case-control study (MOSAICC) sought to improve knowledge regarding their aetiology and to determine optimal methodology for a larger UK-wide study. Overall, 233 participants were recruited to the MOSIACC study, and we collected 187 blood and 214 saliva samples. The mean DNA yield from blood was 659.18 ng/μL, significantly higher than the mean DNA yield from saliva samples (275.79 ng/μL). The higher provision of saliva samples might reflect its non-invasive and more convenient nature, compared to blood sample provision. The differences in mean DNA yields might reflect differences in clinical assistance, adherence to instructions, and health status of individuals. In conclusion, both sample collection techniques are simple, effective, and suitable for DNA collection for genetic analysis in future epidemiological research studies but OG-500 kits offer a less invasive alternative for those who refuse to provide blood.

The guideline group was selected to be representative of UK-based medical experts with an interest in myeloproliferative neoplasms and eosinophilia. PubMed and EMBASE were searched systematically for publications in English until August 2015 using the key words eosinophilia, hypereosinophilia, eosinophilic leukaemia and HES. The writing group produced the draft guideline which was subsequently revised by consensus by members of the General Haematology and Haemato-oncology Task Forces of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of UK haematologists and representatives from the Nordic MPN Study Group, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology (BSH) Committee and comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in Table I. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of eosinophilia.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases including polycythemia vera (PV), essential thrombocythemia (ET), and primary(idiopathic) myelofibrosis (PMF). In this systematic review, we provide a comprehensive report on the incidence and prevalence of MPNs across the globe. Electronic databases (PubMed, EMBASE, MEDLINE, and Web of Science) were searched from their inception to August 2012 for articles reporting MPN incidence or prevalence rates. A random effects meta-analysis was undertaken to produce combined incidence rates for PV, ET, and PMF. Both heterogeneity and small study bias were assessed. Thirty-four studies were included. Reported annual incidence rates ranged from 0.01 to 2.61, 0.21 to 2.27, and 0.22 to 0.99 per 100,000 for PV, ET, and PMF, respectively. The combined annual incidence rates for PV, ET, and PMF were 0.84, 1.03, and 0.47 per 100,000. There was high heterogeneity across disease entities (I(2) 97.1-99.8%) and evidence of publication bias for ET and PMF (Egger test, P = 50.007 and P ≤ 0.001, respectively).The pooled incidence reflects the rarity of MPNs. The calculated pooled incidence rates do not reflect MPN incidence across the globe due to the high unexplained heterogeneity. Improved, widespread registration of MPNs would provide better information for global comparison of the incidence and prevalence of MPNs.

The circulating tumor cells of chronic lymphocytic leukemia (CLL) are characterized by variably low surface IgM (sIgM) levels and signaling capacity, consequent to stimulation by tissue-based antigen. The variability is evident in both CLL subsets with unmutated and mutated immunoglobulin genes and affects tumor cell survival and proliferation. This appears clinically important because CLL with relatively higher sIgM levels/signaling capacity progress more rapidly than those with lower sIgM levels/signaling capacity (D'Avola, Blood 2016), likely due to a larger proliferative component at tissue sites. B-cell receptor (BCR)-signaling inhibitor ibrutinib exerts its therapeutic activity in CLL by irreversibly binding the Bruton's tyrosine kinase (BTK) at Cys481 in the active site. This leads to redistribution of CLL cells from tissue sites into peripheral blood and to a selective recovery of sIgM levels and function, consequent to release from antigen drive at tissue sites (Drennan, Clin Cancer Res 2019). We have now investigated the hypothesis that sIgM levels on the CLL cells affect duration of response to ibrutinib therapy possibly consequent to circumvention of the pharmacological BTK inhibition. Seventy CLL patients participating in the "real-world" observational study at the University of Southampton (NIHR/UKCRN ID: 31076) were investigated for phenotypic, molecular and functional characteristics associated with response to single-agent ibrutinib. Time to progression requiring a new treatment from ibrutinib start (TTNT) was used as primary endpoint to measure duration of response. The levels of sIgM were measured by flow cytometry. Anti-IgM induced signaling was determined by immunoblotting or iCa2+ mobilization. BTK and PLCγ2 mutations were determined using the Illumina NexteraXT kit and a MiSeq at the University of Southampton and validated independently at Karolinska Institutet. Our previous cut-offs of 50 (MFI) or 5 (iCa2+ % mobilization) were used to distinguish patients with high/low sIgM levels or signaling capacity, respectively. Median follow-up from ibrutinib start was 42 months (range 12-70). Pre-ibrutinib sIgM levels (range 7-618, median 65; high sIgM MFI 46/70, 66%) and sIgM signaling capacity (range 1-98; median 45; high sIgM signalers 56/70, 80%) were broadly heterogeneous. However, median values were significantly higher than the general CLL population at diagnosis, as expected in progressive CLL. By univariate analyses of clinical, phenotypic, FISH and immunogenetic characteristics for TTNT, high sIgM was the only parameter predicting shorter duration of response to ibrutinib (p=0.02). Only 2/24 CLL with low sIgM levels (CLLIgM_lo) had progressed (median TTNT not reached) compared to 15/46 CLL with high sIgM (CLLIgM_hi, 14% at 12 months, 18% at 24 months, 34% at 36 months, 44% at 42 months) during therapy. The levels of sIgM correlated significantly with sIgM signaling capacity prior to treatment (r= 0.68; p From in vitro analysis of 13 therapy-naive CLL samples, the degree of inhibition by ibrutinib of anti-IgM induced signals downstream to BTK correlated negatively with sIgM levels (r=-0.68,p=0.01) and signaling (r=-0.71,p=0.009). Anti-IgM induced signals were then measured in 12 patients (7 CLLIgM_hi, 5 CLLIgM_lo) after 1, 4 and 12 weeks of ibrutinib therapy. These patients had not acquired BTK or PLCγ2 mutations and BTK phosphorylation appeared completely inhibited at all time-points. Mean anti-IgM induced iCa2+ mobilization and ERK1/2 phosphorylation were significantly reduced but not abolished during therapy and degree of inhibition was variable between individuals. Remarkably, degree of inhibition of anti-IgM-induced iCa2+ mobilization and pERK1/2 was significantly lower in CLLIgM_hi than in CLLIgM_lo (p These results confirm that sIgM signaling is dependent on sIgM levels and that it can circumvent BTK blockade when sIgM levels are high. They suggest that high sIgM signaling can drive ibrutinib resistance despite ability of ibrutinib to fully occupy the BTK phosphorylation pocket. A possibility is that those CLL cells with high sIgM represent a potentially dangerous subpopulation equipped to migrate to tissue and to receive proliferative stimuli. These cells might be targeted by a combinatorial therapeutic approach with ibrutinib. Disclosures Johnson: Takeda: Honoraria; Kite: Honoraria; Bristol-Myers Squibb: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Epizyme: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria. Duncombe:Abbvie: Other: Advisory Board membership;Educational support; Gilead: Other: Advisory Board membership; Janssen: Other: Advisory Board membership;Educational support; Novartis: Other: Advisory Board membership. Scarfo:Janssen: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Sutton:Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria; Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding. Forconi:Roche: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Gilead Sciences: Research Funding.

Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P

Background Myeloproliferative neoplasms (MPNs) including the classic entities; polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis are rare diseases with unknown aetiology. The MOSAICC study, is an exploratory case–control study in which information was collected through telephone questionnaires and medical records. Methods As part of the study, 106 patients with MPN were asked about their perceived diagnosis and replies correlated with their haematologist’s diagnosis. For the first time, a patient perspective on their MPN diagnosis and classification was obtained. Logistic regression analyses were utilised to evaluate the role of variables in whether or not a patient reported their diagnosis during interview with co-adjustment for these variables. Chi square tests were used to investigate the association between MPN subtype and patient reported categorisation of MPN. Results Overall, 77.4 % of patients reported a diagnosis of MPN. Of those, 39.6 % recognised MPN as a ‘blood condition’, 23.6 % recognised MPN as a ‘cancer’ and 13.2 % acknowledged MPN as an ‘other medical condition’. There was minimal overlap between the categories. Patients with PV were more likely than those with ET to report their disease as a ‘blood condition’. ET patients were significantly more likely than PV patients not to report their condition at all. Patients from a single centre were more likely to report their diagnosis as MPN while age, educational status, and WHO re-classification had no effect. Conclusions The discrepancy between concepts of MPN in patients could result from differing patient interest in their condition, varying information conveyed by treating hematologists, concealment due to denial or financial concerns. Explanations for the differences in patient perception of the nature of their disease, requires further, larger scale investigation.

Myeloproliferative neoplasms (MPNs) are characterized by overproduction of mature functional blood cells and are often associated with an acquired genetic mutation of Janus Kinase 2(V617F). The etiology of MPNs remains unknown. The aim of this article was to review and collate all known published data investigating environmental and lifestyle factors associated with MPNs. Medline, Embase, PubMed, Cochrane, and Web of Science were systematically searched using terms for MPNs and observational study designs to identify studies investigating the risk factors for MPNs published before March 2010. Of 9,156 articles identified, 19 met the selection criteria. Although the studies exhibited heterogeneity, in case definitions, study design, and risk factors investigated, several themes emerged. A strong association was found with Jewish descent, and with a family history of MPNs. Autoimmune conditions, specifically Crohn's disease, were more common in patients with MPNs. Certain occupational groups were significantly associated with MPNs including occupations with potential exposure to benzene and/or petroleum. Blood donation was associated with an increased risk of polycythemia vera specifically. The vast heterogeneity in studies identified as part of this review suggests that large scale systematic assessment of etiological factors associated with MPNs is warranted.

We genotyped 370 subjects with primary myelofibrosis (PMF) and 148 with postpolycythemia vera/postessential thrombocythemia (PPV/PET) MF for mutations of EZH2. Mutational status at diagnosis was correlated with hematologic parameters, clinical manifestations, and outcome. A total of 25 different EZH2 mutations were detected in 5.9% of PMF, 1.2% of PPV-MF, and 9.4% of PET-MF patients; most were exonic heterozygous missense changes. EZH2 mutation coexisted with JAK2V617F or ASXL1 mutation in 12 of 29 (41.4%) and 6 of 27 (22.2%) evaluated patients; TET2 and CBL mutations were found in 2 and 1 patients, respectively. EZH2-mutated PMF patients had significantly higher leukocyte counts, blast-cell counts, and larger spleens at diagnosis, and most of them (52.6%) were in the high-risk International Prognostic Score System (IPSS) category. After a median follow-up of 39 months, 128 patients (25.9%) died, 81 (63.3%) because of leukemia. Leukemia-free survival (LFS) and overall survival (OS) were significantly reduced in EZH2-mutated PMF patients (P = .028 and P < .001, respectively); no such impact was seen for PPV/PET-MF patients, possibly due to the low number of mutated cases. In multivariate analysis, survival of PMF patients was predicted by IPSS high-risk category, a < 25% JAK2V617F allele burden, and EZH2 mutation status. We conclude that EZH2 mutations are independently associated with shorter survival in patients with PMF.

Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

The myeloproliferative neoplasms (MPN) including polycythaemia vera (PV), essential thrombocythaemia and primary myelofibrosis (PMF) are rare diseases contributing to significant morbidity. Symptom management is a prime treatment objective but current symptom assessment tools have not been validated compared to the general population. The MPN-symptom assessment form (MPN-SAF), a reliable and validated clinical tool to assess MPN symptom burden, was administered to MPN patients (n = 106) and, for the first time, population controls (n = 124) as part of a UK case-control study. Mean symptom scores were compared between patients and controls adjusting for potential confounders. Mean patient scores were compared to data collected by the Mayo Clinic, USA on 1,446 international MPN patients to determine patient group representativeness. MPN patients had significantly higher mean scores than controls for 25 of the 26 symptoms measured (P 0.05); fatigue was the most common symptom (92.4% and 78.1%, respectively). Female MPN patients suffered worse symptom burden than male patients (P 0.001) and substantially worse burden than female controls (P 0.001). Compared to the Mayo clinic patients, MPN-UK patients reported similar symptom burden but lower satiety (P = 0.046). Patients with PMF reported the worst symptom burden (88.3%); significantly higher than PV patients (P 0.001). For the first time we report quality of life was worse in MPN-UK patients compared with controls (P 0.001).

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

Current treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti-immunoglobulin M-induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eμ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of diseases including polycythemia vera (PV), essential thrombocythemia (ET), and primary(idiopathic) myelofibrosis (PMF). In this systematic review, we provide a comprehensive report on the incidence and prevalence of MPNs across the globe. Electronic databases (PubMed, EMBASE, MEDLINE, and Web of Science) were searched from their inception to August 2012 for articles reporting MPN incidence or prevalence rates. A random effects meta-analysis was undertaken to produce combined incidence rates for PV, ET, and PMF. Both heterogeneity and small study bias were assessed. Thirty-four studies were included. Reported annual incidence rates ranged from 0.01 to 2.61, 0.21 to 2.27, and 0.22 to 0.99 per 100,000 for PV, ET, and PMF, respectively. The combined annual incidence rates for PV, ET, and PMF were 0.84, 1.03, and 0.47 per 100,000. There was high heterogeneity across disease entities (I(2) 97.1-99.8%) and evidence of publication bias for ET and PMF (Egger test, P = 50.007 and P ≤ 0.001, respectively).The pooled incidence reflects the rarity of MPNs. The calculated pooled incidence rates do not reflect MPN incidence across the globe due to the high unexplained heterogeneity. Improved, widespread registration of MPNs would provide better information for global comparison of the incidence and prevalence of MPNs.

Introduction FCR improves PFS and OS in CLL. Previous non-randomised Phase II trials suggest adding mitoxantrone to FCR (FCM-R) improves outcomes. The rituximab dose in CLL was inferred from lymphoma but in CLL even low doses of rituximab lead to the loss of CD20 expression (“shaving”) suggesting that low dose rituximab with chemotherapy may be effective. Aims The primary objective was to assess whether the CR rate to FCM-miniR was not inferior to FCR. Secondary objectives were MRD eradication, overall response rates and safety. Late endpoints (not reported) were PFS, OS and MRD relapse. Methods ARCTIC was a phase IIB, randomised, controlled, parallel group trial in previously untreated CLL performed in 34 UK centres. 206 patients were to be randomised to FCR or FCM-miniR with an 80% power to show that FCM-miniR did not lead to 10% worse CR rates compared to FCR. A formal interim analysis on the primary endpoint was planned after 103 patients. The schedule for oral FCR was equivalent to iv FCR with iv rituximab given on Day 1 (375mg/m2 Cycle 1; 500mg/m2 Cycles 2-6), oral fludarabine (24mg/m2/day for 5 days; Days 1-5) and oral cyclophosphamide (150mg/m2/day for 5 days; Days 1-5). FCM-miniR included intravenous mitoxantrone (6mg/m2/day) and 100mg rituximab on Day 1 of 6 cycles given 28 days apart. Patients with neutropenia delaying therapy received G-CSF (lenograstim 263mcg/day; Days 7-13) on all remaining cycles. Prophylaxis with co-trimoxazole and acyclovir was used. Results 200 patients were recruited, 100 to each arm. The interim analysis after 103 patients indicated that although the CR rate to FCM-miniR was similar to predicted it was inferior to FCR. The independent DMC advised that the trial would not show non-inferiority for FCM-miniR and recruitment was stopped. A further 97 patients were recruited up to the interim analysis. 21 patients still receiving FCM-miniR were advised to cross-over to FCR. 100 patients received FCR, 79 FCM-miniR and 21 patients randomised to FCM-miniR crossed over to FCR. The treatment arms were well balanced for the following: median age 63 (36-80) and 20% (n=40) over 70; 67.5% male; 17% prog. stage A, 47% stage B and 35.5% stage C. 33.5% had a creatinine clearance under 70ml/min. 60% (87/146) had unmutated Ig genes; 4% (6/164) 17p deletion; and 15% (26/168) 11q deletion. More 11q del patients were randomised to FCM-miniR (15 vs 7). 29.5% (59/200) discontinued therapy early including 30% (n=30) FCR and 35.4% (n=28) FCM-miniR. 141 (70.5%) completed 6 cycles. 93 (49%) of 189 received G-CSF - more with FCM-miniR compared with FCR (54% vs 44%). 117 (58.5%) patients experienced a dose modification; 57% for FCR and 62% for FCM-miniR; the most common being dose delay in 47% of patients. 181 SAE's were reported from 104 patients; 79 events from 49% (49/100) receiving FCR, 80 from 58% (46/79) receiving FCM-miniR and 47% (9/22) cross-over patients. 62.5% of related SAE's were infectious. 3 patients died due to an SAE, 1 FCR and 2 FCM-miniR. We report only the 179 intention-to-treat patients who did not cross-over. To date 146 of 179 patients (82%) have undergone independent central review. 70% (102/146) achieved a CR or CRi; 78% (62/79) FCR and 60% (40/67) FCM-miniR. 13/22 (59%) patients with a baseline creatinine clearance of Conclusion ARCTIC was stopped prematurely as it was clear that the arm containing mitoxantrone and low dose rituximab would not be non-inferior to FCR. However both FCR and FCM-miniR performed well with CR rates of 78% and 60% respectively. This compares favourably to CR rates of 38% for FC in LRF CLL4 Trial and 44% for FCR in the GCLLSG CLL8 Trial. MRD was undetectable in 55% of patients receiving FCR and 46% for FCM-miniR. Oral FCR in front-line CLL results in extremely high CR rates and the eradication of detectable MRD in the majority. Low dose rituximab (100mg per cycle; 600mg total dose compared to 2875mg/m2 for standard FCR) in combination with FCM appears to be effective although FCM-miniR is inferior to full dose FCR. However there is more toxicity associated with the addition of mitoxantrone confounding the comparison of rituximab doses. Disclosures: Hillmen: Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Celgene: Honoraria. Off Label Use: The use of ofatumumab in combination with chlorambucil in previously untreated CLL. The reported trial will support the extension of the ofatumumab licence. Schuh:GSK: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria.

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.

The refusal of Jehovah's Witnesses with leukaemia to accept transfusion provides a major clinical challenge because of the myelosuppressive effects of chemotherapy. Experience in treating five such patients is described. Two patients with acute lymphoblastic leukaemia (ALL) achieved remission following chemotherapy, the first without transfusion support, the second, a minor, receiving transfusion under a court order: the first patient remains in remission 5 years later, whereas the second subsequently relapsed and died. Of three patients with acute myeloid leukaemia (AML), two received chemotherapy: one died of anaemia during induction chemotherapy whereas the second eventually consented to transfusion but died of refractory leukaemia. The third patient died of anaemia despite erythropoietin. We feel Jehovah's Witnesses should not be denied antileukaemic therapy if they fully understand the risks involved. Minimizing phlebotomy, use of antifibrinolytic agents and growth factors may make chemotherapy feasible, especially in ALL where remission may be induced with less myelosuppressive agents. The outlook for those with AML treated with conventional chemotherapy appears poor; alternative approaches to treatment should be considered in these patients.

The guideline group regarding the diagnosis and management of myelofibrosis was selected to be representative of UK-based medical experts, together with a contribution from a single expert from the USA. MEDLINE and EMBASE were searched systematically for publications in English from 1966 until August 2011 using a variety of key words. The writing group produced the draft guideline, which was subsequently revised by consensus of the members of the General Haematology and Haemato-oncology Task Forces of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated where appropriate. The criteria used to state levels and grades of evidence are as outlined in the Procedure for Guidelines commissioned by the BCSH; the 'GRADE' system was used to score strength and quality of evidence. The objective of this guideline is to provide healthcare professionals with clear guidance on the investigation and management of primary myelofibrosis, as well as post-polycythaemic myelofibrosis (post-PV MF) and post-thrombocythemic myelofibrosis (post-ET MF) in both adult and paediatric patients. © 2012 Blackwell Publishing Ltd.

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIMEL and BIML, in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIMEL only. In CLL, anti-IgM–induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIMEL serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIMEL/BIML phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIMEL phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

Introduction: The myeloproliferative neoplasms (MPNs) including polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are rare diseases which contribute to significant morbidity. Symptom management is a prime objective in MPN treatment but current symptom assessment tools have not been validated compared to the general population. Materials and Methods: The MPN Symptom Assessment Form (MPN-SAF) is a reliable and validated clinical tool used to assess MPN symptom burden. The MPN-SAF was administered to MPN patients (n=106) and, for the first time, General Practice and non-blood relative/family controls (n=124) as part of a UK pilot case-control study. Mean scores for individual MPN-SAF items and for the Total Symptom Score were previously compared between cases and controls adjusting for potential confounding variables (Anderson LA, et al. Am J Hematol. 2015). The current analysis employed linear discriminant analysis (a multivariate technique) to compare MPN-10 symptom profiles between controls and cases, and between controls and patient groups by MPN diagnosis (ET n=55, PV n=37, PMF n=14). Results: MPN patients as a single group had significantly different MPN-10 symptom profiles compared to controls (multivariate analysis of variance [MANOVA] p0.30 for all MPN-10 items, and >0.50 for weight loss, fatigue, night sweats, bone pain, abdominal discomfort, inactivity, concentration problems, and itching. This suggests that all MPN-10 items contribute to distinguishing between patients and controls. The linear discriminate function correctly categorized 72% of all subjects. When considering patients by diagnosis, MPN-10 symptom profiles significantly differed across patients with ET, PV, and PMF and controls (MANOVA p0.50 for the same MPN-10 items as the previous analysis; of the second canonical variable for fever; and of the third canonical variable for bone pain. This suggests that MPN-10 items are broadly useful in differentiating between patients and controls, with particular symptoms (e.g., fever and bone pain) contributing to differentiating between patients with different diagnoses. The linear discriminate function correctly categorized 58% of all subjects. Conclusions: MPN patients experience significant morbidity when compared to the general population highlighting the need to manage symptoms effectively. Symptom profiles can not only distinguish MPN patients from general population participants, but also distinguish among patients with ET, PV, and PMF. The results further validate the use of the MPN-SAF as a discriminatory tool to assess MPN symptomatic disease burden. The MOSAICC Study team acknowledges the support of the National Institute for Health Research, through the Northern Ireland Cancer Research Network (NICRN) and for Southampton the Central South Coast Cancer Network (CSCCN). Disclosures Mesa: Novartis. Research- incyte, Gilead, cti, Genentech, promedior, NS Pharma: Consultancy.

Introduction The Classic Myeloproliferative Neoplasms (MPNs), characterised by an over production of one or more cells of the myeloid lineage, are classified into polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). Despite the identification of numerous genetic mutations, a paucity of information relating to medical and lifestyle factors contributing to the aetiology of these diseases remains. Methods The MOSAICC Study was an exploratory case-control study of MPNs. MPN patients were recruited between two sites, Belfast and Southampton, in preparation for a planned UK-wide investigation. Population controls (identified by General Practitioners) and non-blood relative/friend controls were also recruited. Participants completed a telephone-based questionnaire seeking information on a range of medical, environmental, lifestyle and occupational risk factors. Risk factors were assessed using unconditional logistic regression with adjustment for potential confounders. Results Risk factors identified included smoking [≥25 pack years vs. never: odds ratio (OR) 3.14, 95% confidence interval (CI) 1.26-7.87], increasing childhood household density [>2 vs Conclusions This exploratory study has confirmed a reported association between cigarette smoking and MPNs. It has identified some potential novel risk factors including work-based radiation and solvent exposure. The findings of this study need to be replicated in a larger, adequately powered, study in order to identify important risk factors for the classic MPNs. Conflict of Interest The authors declare no conflict of interest. Acknowledgments: The MOSAICC Study team acknowledges the support of the National Institute for Health Research, through the Northern Ireland Cancer Research Network (NICRN) and for Southampton the Central South Coast Cancer Network (CSCCN). The team thank those who have participated in the MOSAICC study and the personnel who assisted in the recruitment of patients. Disclosures Mesa: CTI Biopharma: Research Funding; Promedior: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Pfizer: Research Funding; NS Pharma: Research Funding; Genentech: Research Funding; Gilead: Research Funding.

Purpose: To determine the origin and relationship of the rare IgG+ variant of chronic lymphocytic leukemia (CLL) to the two common IgM+IgD+ subsets that are distinguished by expression of unmutated or mutated VH genes, with the former having a worse prognosis. Experimental Design: IgG+ CLL cells were characterized using phenotypic, functional, and immunogenetic analyses. Results: IgG+ CLL was phenotypically similar to mutated IgM+IgD+ CLL (M-CLL) and variably expressed CD38 (4 of 14). ZAP-70, a tyrosine kinase preferentially expressed in unmutated CLL, was found in only 2 of 14 cases. The ability to signal via surface IgM (sIgM) varies between the main subsets of CLL and is associated with expression of ZAP-70. In IgG+ CLL, 9 of 14 responded to engagement of sIgG with no apparent requirement for expression of CD38 or ZAP-70. However, signal capacity correlated with intensity of sIgG expression. Most switched immunoglobulin variable region genes were somatically mutated without intraclonal variation, and no case expressed activation-induced cytidine deaminase. Derivation from a postgerminal center B cell is, therefore, likely, and a relationship with M-CLL is suggested. This is supported by a shared biased usage of the V4-34 gene. Similar bias in normal B cells developed with age, providing an expanded population for transforming events. However, conserved sequences detected in the CDR3 of V4-34-encoded γ chains were not found M-CLL, indicating no direct path of isotype switch from M-CLL. Conclusion: IgG+ CLL is likely to arise from an age-related expanded pool of B cells, on a path parallel to M-CLL, and perhaps with a similar clinical course.

The tumor B-cell receptor (BCR) is the key to survival and proliferation in chronic lymphocytic leukemia (CLL) and is now a therapeutic target of very effective BCR-associated kinase inhibitors. CLL cells are typically characterized by a variable state of anergy, defined by low surface IgM (sIgM) levels and signaling ability with consequent relatively low proliferation rate. The subset with unmutated IGHV (U-CLL) is generally less anergic with a poorer prognosis than that with mutated IGHV (M-CLL), and appears more sensitive to BCR-inhibitors (Byrd et al., NEJM, 2013). However it remains unclear if the variable anergy reflects the nature of the cell of origin and/or the functional state of the BCR, thereby determining clinical behavior. In this study we investigated if variations of BCR sIgM levels and signaling correlate with clinical behavior of CLL and assessed the phenotypic and genetic associations with the variations. The study included samples at diagnosis or prior to treatment from 222 patients with sIgM/D CLL diagnosed according to the iWCLL2008 criteria (99 months median follow up). sIgM levels on the CD19+/CD5+ CLL cells and signaling [% intracellular Ca2+ mobilization (iCa2+)] were determined using soluble F(ab’)2 polyclonal antibodies (Mockridge at al, Blood, 2007). sIgM levels were analyzed for their association with i) survival, ii) phenotype (CD38, ZAP70 and BCR regulators CD5, CD19, CD20, CD22) and iii) genetic lesions (Del13q, Trisomy12, Del11q/ATM, Del17p/TP53, or NOTCH1 and SF3B1 mutants). Time from diagnosis to progression requiring first treatment (TTFT) was used as primary endpoint, while overall survival (OS) was used as a secondary endpoint to avoid the influences of chemotherapy. Best cut-offs for progression requiring treatment were determined with ROC and Youden’s T-tests (treatment as a state variable). Levels of sIgM (5th-95th percentile 12-378, median 52) varied markedly between patients. Levels were higher at more advanced stage of disease and sIgM expression (MFI>56) and signaling (iCa2+>6%) associated with significantly more rapid TTFT. Although sIgD levels and signaling also associated with shorter TTFT, multivariate Cox regression adjusted for IGHV status, sIgM levels, sIgM signaling, sIgD levels and sIgD signaling revealed that only U-IGHV (p By focusing on sIgM, a multivariate analysis adjusted for IGHV status, sIgM levels, CD38 and ZAP70 confirmed U-IGHV (p=0.002, 95% CI 1.35-3.56) and high sIgM (p=0.003, 95% CI 1.25-3.04) as the only independent predictors of shorter TTFT. Consistently, high sIgM levels associated with a more aggressive CLL even when U-CLL or M-CLL were investigated separately. Phenotypic analyses showed that high sIgM CLL had higher CD20, CD38, ZAP70, and CD22, than low sIgM CLL. Genetic analysis revealed that CLL subsets harboring Trisomy12 or Del17p/TP53 had significantly higher sIgM levels and signaling capacity than Del13q, even when U-CLL or M-CLL were analyzed separately. Trisomy12 U-CLL cases were enriched for NOTCH1 mutations. Consistently, U-CLL with NOTCH1 mutations (with or without Trisomy12) expressed higher sIgM than Del13q U-CLL. This study has several biological and clinical implications. First, it supports the critical role of sIgM in CLL-associated anergy. Our previous studies had shown dynamic variations of sIgM on circulating CLL cells following (auto)antigen engagement in tissue (Mockridge et al., Blood, 2007; Coelho et al. Blood, 2013). The subpopulation with higher sIgM is potentially the most dangerous and the most sensitive to kinase-inhibitors. Second, it documents the dominant role of sIgM levels on cell signaling and on tumor progression in CLL, with relevance even within U-CLL and M-CLL. Third, this study suggests influences of altered genetics on sIgM levels. However BCR-inhibitors overcome the influence of genetic lesions like Del17p/TP53. This points to a dominant role of reversed sIgM anergy in the behavior of CLL in vivo and claims the need of studies to verify sensitivity to kinase-inhibitors in CLL patients with different sIgM levels. Disclosures No relevant conflicts of interest to declare.

Chronic Lymphocytic leukaemia (CLL) is currently incurable using conventional therapies, although new drugs aimed as signalling pathways are having some therapeutic success. Features of CLL cells reveal a balance between anergy, apoptosis or increased proliferation, with outcome determined by the B cell receptor (BCR). Engagement of the BCR triggers formation of the signalosome leading to calcium [Ca+2]i mobilization and ERK phosphorylation. However other microenvironmental factors affect CLL cell growth and survival and may modulate the BCR response. We have focused on IL-4, produced mainly by T cells and known to rescue CLL cells from apoptosis when cultured in vitro. IL-4 strongly induces phosphorylated STAT6 (STAT6-P) within CLL cells in vitro, therefore we analysed STAT6-P expression within lymph nodes of SLL/CLL patients. We detected STAT6-P expression in SLL/CLL lymph nodes predominantly within proliferation centres. Induction is likely to be via IL-4, since IL-13 and CD40L, which are also capable of inducing STAT6-P, did not produce a STAT6-P signal in CLL cells in vitro. To analyze the effect of IL-4 on BCR signalling, CLL samples were exposed to IL-4 for between 1-72h prior to treatment with anti-IgM. Using calcium flux as a marker of BCR signalling we found that IL-4 significantly increased the calcium flux, as compared to the untreated control, in 63.6% (7/11) of the cases. We showed that the increased calcium flux coincided with an upregulation of sIgM expression observed between 24-72h. Furthermore there was no significant up-regulation of sIgD levels even though calcium flux increased in a proportion of cases (6/9) in response to anti-IgD following IL-4 pre-treatment. We further investigated the effect of IL-4 on known positive and negative regulators of BCR signalling. While no significant changes in CD19 expression were found, CD5 expression was significantly downregulated in all cases which showed IL-4-mediated amplification of signal in response to anti-IgM. Next we evaluated phosphorylated ERK (ERK-P) as a marker of BCR signalling. CLL cells were incubated overnight in the presence or absence of IL-4 and subsequently stimulated with soluble anti-IgM for 15 minutes. We found ERK-P expression was augmented following αIgM activation in the presence of IL-4 compared to the control in a proportion of samples (12/15). When CLL cells were treated with the PI3K inhibitor LY294002 prior to the addition of anti-IgM, ERK-P was reduced due to inhibition of the BCR signalosome upstream of ERK. However pre-treating CLL cells with IL-4 enabled the cells to overcome chemical inhibition of PI3K with LY294002 and restored ERK-P expression. Using densitometry analysis a significantly greater signalling capacity quantified by ERK-P expression (P=0.042) was observed in 15/23 patients. Interestingly, 73.3% of those 15 cases were unmutated CLL (U-CLL), the aggressive form of the disease. Taken together, these data shows that IL-4 is able to enhance conventional BCR mediated signalling and may partially overcome chemical inhibition of the classical BCR pathway in CLL cells. The augmentation of BCR signalling is partly mediated by an increase in sIgM receptor density but may also be partly regulated by modulation of the positive and negative regulators of BCR signalling. Together these data indicate an important role of the microenvironment in BCR signalling in CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2906 Chronic Lymphocytic leukemia (CLL) is currently incurable using conventional therapies. CLL cells can evade killing by various therapeutic strategies. However the precise mechanisms are currently unknown. Autophagy is regulated by a complex system of proteins, and is used by both normal and malignant cells as a protective mechanism against cellular stress induced by starvation, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. In malignant cells autophagy was shown to promote tumorigenesis and/or resistance to chemotherapy. Therefore we hypothesized that autophagy may play a role in CLL biology. Autophagy can also promote cell death when stress signals are elevated above a particular threshold for a prolonged period of time. In this study we investigated the basal expression levels of autophagy specific genes and the effect of autophagy specific inhibitors (Bafilomycin, 3-methyladenine and hydroxychloroquine) and inducers (Phenethyl isothiocyanate) on CLL survival. Phenethyl isothiocyanate (PEITC) is about to enter clinical trials for CLL (NCT00968461). We have investigated induction of components of the autophagic pathway following treatment of CLL cells in vitro with a range of chemical inhibitors. Immunoblotting was carried out to investigate components of the autophagy pathway using phosphorylation state-specific and pan-reactive antibodies. Bafilomycin (BAF), 3-methyladenine (3-MA) and hydroxychloroquine (HCQ) toxicity towards CLL samples were evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. PEITC was used at concentrations between 2.5 and 25μM to investigate its effect on signaling. Autophagy was quantitated by immunoblotting of LC3-I and LC3-II. Lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy and is essential for autophagasome formation. Immunoblotting was also performed for ATG3, ATG5 and ATG7, key components of the autophagy pathway. Monodansylcadaverine (MDC) was used with immunofluorescence and FACS analysis to investigate increases in autophagasome formation. Transmission electron microscopy (TEM) was used to confirm double membrane bound autophagosomes. Co-immunoprecipitation was used to evaluate if Beclin-1 was sequestered by Bcl-2 preventing autophagy. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. LC3-I was lipidated to LC3-II (p=0.019) and ATG3 (p=0.021) was upregulated to a greater extent in CLL samples compared with normal B-cell controls at basal levels. This suggested that autophagy was active to a greater extent in CLL samples compared with normal individuals. In addition Beclin was dissociated from Bcl-2 in CLL samples indicating that autophagy was active. Autophagy appears to be a pro-survival mechanism in untreated CLL cells as inhibiting basal levels of autophagy with autophagy inhibitors BAF (50–200nM), 3-MA (5–10mM) and hydroxychlorquine (5–10μM) resulted in CLL apoptosis as shown by MTT, Annexin V/PI analysis and PARP cleavage. Interestingly augmenting autophagy was also capable of inducing apoptosis in CLL samples. Treatment with PEITC caused an increase in punctate staining using MDC which is suggestive of autophagosome formation. We went on to determine that PEITC further induced LC3-II lipidation using immunoblotting and showed a substantial increase in overall LC3 protein expression. PEITC also induced the expression of ATG3, a key protein in the autophagy pathway. We then evaluated autophagosome formation using TEM (Figure 1). Our data showed greater numbers of autophagosomes in the PEITC treated samples compared to the untreated controls. Therefore autophagy in CLL sits on a knife-edge, such that perturbations that either increase pro- death or decrease pro-survival autophagy signals can result in CLL cell death, depending on the duration and intensity of the signal. Figure 1. Transmission electron microscopy of CLL cells CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Figure 1. Transmission electron microscopy of CLL cells . / CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Disclosures: No relevant conflicts of interest to declare.

Abstract 431 Background. Primary myelofibrosis (PMF) has the worst prognosis among myeloproliferative neoplasms with median overall survival (OS) of 4.6y in the International Prognostic Scoring System (IPSS) series (Cervantes F, Blood 2009;113:2895) and 6.5y in patients (pts) seen more recently (Cervantes F, JCO, 2012 in press). OS is predicted by the four risk categories of IPSS, dynamic-IPSS and IPSS-plus system, and these scores are used for therapeutic choices particularly allogeneic stem cell transplantation. Nevertheless, pts heterogeneity still remains within these categories, necessitating improved risk stratification. A number of molecular abnormalities have been reported in PMF pts, but their prognostic relevance is incompletely understood, particularly with regard to transformation to leukemia (AL). The aim of this work was to analyze the prognostic impact of known mutations detected close to diagnosis in an international series of 429 pts. Patients and methods. PMF diagnosis had to satisfy the 2008 WHO criteria. Mutations in JAK2V617F, MPLW515, EZH2, ASXL1, TET2, IDH1/2, DNMT3A, CBL, SRSF2 were genotyped in whole blood or granulocytes using allele specific RTQ-PCR, HRM and direct sequencing; all mutations were confirmed at least twice. Missense, nonsense and frameshift mutations were considered; in case of novel mutations, SNPs were excluded by database searching and when feasible by germline DNA genotyping. The prognostic value of the molecular variables with regard to overall survival (OS) was analyzed by Cox regression and adjusted for the IPSS category. The association of molecular features with the risk of progression to AL was investigated in the framework of competing risks by the Fine & Gray regression method. Replicability of the prognostic models for both OS and progression to AL was assessed by replication in 1000 bootstrap samples randomly taken from the original series. Results. Patient median age was 60y. Median follow-up was 3.7y (95% CI, 0.02–27.9), death occurred in 157 pts (32%). Frequency of pts with constitutional symptoms was 28%, splenomegaly 74%, anemia 27%, leukocytosis 8%, >1% blasts 16%, thrombocytopenia 12%. Abnormal karyotype was found in 24% (of 229 evaluated). IPSS risk category: low-risk 35%, Int-1 30%, Int-2 21%, High-risk 14%. Frequency of mutations was: JAK2V617F 59.8% with 49% of pts having Survival Model. Median survival was 9.7y (CI, 7.9–12.2), 22y in low-risk, 10y Int-1, 6.2y Int-2, 2.5y high-risk (P Leukemia Model. AL occurred in 75 pts (15.2%) after of a median of 3.8y (95%, 0.04–26.5) from diagnosis. Mutations in ASXL1 and IDH1/2 were the only molecular variables associated with higher risk of AL with a SHR of 2.33 (P Conclusions. In this comprehensive series of mutations profiled in PMF pts, mutations in ASXL1 emerged as a powerful prognostic variable for survival refining prognosis in the Int-2/high risk IPSS categories. ASXL1 and IDH1/2 mutations predicted for death due to AL. Therefore, genotyping for ASXL1 and IDH1-2 mutations at diagnosis may help to tailor therapy for pts with IPSS Int-2/high risk PMF. Disclosures: No relevant conflicts of interest to declare.

Abstract 2811 Among chronic myeloproliferative neoplasms (MPN) myelofibrosis (MF) has the worst prognosis. The International Prognostic Score System-IPSS reliably discriminates four categories of patients (pts) with different survival (OS); however, the possibility to identify pts with very high-risk disease, based on intrinsic characteristics of the underlying disease, would be particularly useful for therapeutic choices that include early stem cell transplantation. Several studies have addressed the prognostic value of molecular abnormalities harbored by MF pts, but a part from a negative role of low JAK2V617F allele burden on survival independently reported by the Mayo Clinic and our own group, none of them impacted significantly on survival. Here we have genotyped at diagnosis 370 pts with primary MF (PMF) and 148 with post-polycythemia vera/essential thrombocythemia MF (PPV/PET-MF) for mutations of EZH2 and ASXL1. EZH2 is the catalytic component of polycomb repressive complex 2 involved in trimethylation of H3 lysine 27, while ASXL1 is a member of enhancer of trithorax and polycomb group required for repression of HOX genes through deubiquitination of histone H2A. All 20 EZH2 exons were screened by high-resolution melting followed by direct sequencing whereas direct sequencing was used for ASXL1 exon 12 genotype. Pts median FU was 39 mo (1–340); 128 pts died (25.9%), 81 (18.3%) because of leukemia (AL). Among PMF pts survival differed significantly according to the IPSS category and was also predicted by the lowest V617F quartile. EZH2 mutation. We found 22 PMF (6%), 1 PPV-MF (1.2%) and 6 PET-MF (9.4%) mutated pts, harboring 25 different mutations, 20 exonic (heterozygous in 83%), 5 intronic. Concurrent JAK2 V617F, MPL W515L/K, IDH1/2, TET2, CBL and ASXL1 mutations were found in 41.4%, 0%, 0%, 6.9%, 3.4% and 20.7% of EZH2 -mutated pts. In multivariate analysis EZH2 mutation was associated with leukocytosis (12.3±13.2 vs 18.7±11.5×10e9/L) in PMF pts, but no additional clinical/hematological correlation was discovered. EZH2 mut pts clustered preferentially in the IPSS high-risk category (52.6%; P=.002). More EZH2 mut than wt PMF pts died (51.9% vs 24.4%; P ASXL1 mutation. In a total number of 334 evaluated patients we found 38 different mutations in 49 pts (14.7%), 35 PMF (15%), 5 PPV-MF (8.6 %) and 9 PET-MF (21.4%). Mutations were frameshift, non sense and missense mutations, heterozygous in 46 of 49 cases. Co-expression of JAK2 V617F, MPL W515L/K and EZH2 mutation was found in 55.1%, 4.1% and 12.2%, respectively. Co-espression of ASXL1 and IDH1/2, TET2 or CBL was observed in two, one and one of the evaluable subjects, respectively. ASXL1 mutation was associated with leucocytosis (P=.006), a blast count >1% (43.8% vs 16.6%, P According to EZH2 and ASXL1 mutational status, median OS was significantly shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (34 vs 116 vs 198 vs 272 mo, respectively. P No impact of EZH2 or ASXL1 mutations on OS or LFS was seen in PPV/PET-MF. Overall, these results showed that mutations in genes involved in epigenetic gene regulation such as EZH2 and ASXL1 negatively impact on OS and LFS in pts with PMF, suggesting that screening for these mutations might be useful for risk stratification and treatment decisions. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria.

Background: Macroautophagy (autophagy) has been shown to be both a pro-survival and pro-death signalling pathway. The decision between life or death is dependent on the type and developmental stage of the cell and the duration and intensity of the signal. Autophagy is used by cancer cells as a mechanism to overcome cellular stresses such as starvation, reactive oxygen species (ROS) generation and misfolded protein accumulation and can result in tumorigenesis and resistance to chemotherapy. One of the pathways which regulate responses to ROS/ER stress is the unfolded protein response (UPR). Briefly, upon ER stress, the HSP70 family member BiP is sequestered away from IRE1 , PERK and ATF6, resulting in IRE1 and PERK phosphorylation and activation of downstream signalling pathways. These signals can result in cell survival by upregulating AKT or cell death via induction of CHOP and noxa. However this pathway has not been fully investigated in chronic lymphocytic leukaemia (CLL). Interestingly the UPR is also known to regulate autophagy. The lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy, since LC3-II is essential for formation of the autophagasome and the initiation of autophagy. Beclin-1 is another essential protein involved in canonical autophagy and is sequestered by Bcl-2 preventing autophagy in a number of cell types. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. Methods: Twenty CLL samples were treated with the HSP70 inhibitor 2-phenylacetylenesulphonamide (PAS; 10-20 M), an activator of the UPR, in the presence of the pan-caspase inhibitor ZVAD. In addition 4 control samples (normal B cells) were treated with PAS for the indicated times. Immunoblotting was performed for BiP, HSP70, Phosphorylated eIF2 (eIF2 -P), JNK (JNK-P), Jun (Jun-P), p38MAPK, LC3-I/II, Beclin expression and its co-immunoprecipitation with Bcl-2. Ten CLL cell isolates were treated with 2 autophagy inhibitors bafilomycin (Baf) (50-200 nM) and 3-methyl adenine (3-MA) (2.5-10 mM) and their effects on cell killing evaluated using PARP cleavage, a surrogate marker of caspase 3 activation. Results: CLL cells expressed a greater basal level of LC3-II in a proportion of samples relative to normal B cells. In addition Beclin-1 did not coimmunoprecipitate with Bcl-2 above that shown with the isotype control in CLL cells. Treatment with PAS resulted in an increase in stress which resulted in the upregulation of BiP and HSP70 protein. This subsequently led to phosphorylation of eIF2 indicative of PERK activation, coincidental with an increase in the eIF2 targets LC3-I/II and noxa expression. Treatment with PAS also resulted in an increase in JNK, Jun and p38MAPK phosphorylation, 3 proteins involved in regulating autophagy in mammalian cells. Furthermore, CLL cells treated with the autophagy inhibitors 3-MA and Baf rapidly underwent apoptosis within 24 h. Conclusion: These data suggest that autophagy is active at a basal level in a proportion of CLL cells and is greater than that seen in control B cells. Activation of the UPR with PAS led to an upregulation of proteins known to regulate autophagy and resulted in an increase in LC3-II, a protein routinely used as an indicator of autophagy. Inhibitors of autophagy also rapidly induced apoptosis in CLL cells. These data suggested that autophagy may have a pro-survival role in the biology of CLL. However pro-survival autophagy is balanced on a knife-edge, such that perturbation by inducing or inhibiting the pathway results in CLL cell death.