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Contains fulltext : 290820.pdf (Publisher’s version ) (Open Access) Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34(+) CD19(-) ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS. 01 januari 2023

Background Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. Objectives In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. Methods Nine patients were enrolled and received at least two courses of ara-C (1 g/m(2)/2 h b.i.d. d1-5, i.e., a total of 10 g/m(2) per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m(2) d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. Results The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level 1.0 x 10(9)/L and to platelet recovery >50 x 10(9)/L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. Conclusion Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Iatrogenic immunodeficiency-associated lymphoproliferative disorders (IA-LPD) may arise in patients treated with immunosuppressive drugs for autoimmune disease or other conditions. Polymorphic EBV-positive B-lymphoproliferations often have features mimicking Hodgkin lymphoma and typically a self-limited, indolent course. We present an unusual case with isolated, intracerebral manifestation of polymorphic B-LPD with features of classic Hodgkin-lymphoma in an immunosuppressed patient treated with methotrexate and infliximab, including clinical-radiological features and a detailed description of morphological findings, together with a literature review on reported cases of primary CNS manifestation of cHL and IA-LPD with Hodgkin-like morphology. The patient achieved complete remission following neurosurgery with gross total tumor resection and drug withdrawal without any additional treatment. Post-operative staging revealed no evidence for focal relapse or systemic disease during the 18 months follow-up period. Among the previously reported 24 cases of primary, isolated Hodgkin lymphoma in the central nervous system, three similar cases of iatrogenic, IA-LPDs were identified and are discussed here. Polymorphic B-LPD are destructive lesions with a range of morphologic features and disease manifestations. It is clinically important to recognize the spectrum of proliferations with features of classic Hodgkin lymphoma in immunodeficiency, iatrogenic settings, because they are likely to impact the choice of treatment strategies. Funding Agencies|Karolinska InstituteKarolinska Institutet

Contains fulltext : 290818.pdf (Publisher’s version ) (Open Access) BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice. 01 januari 2023

Outcome in PCNSL patients and its association with PD-L1+ leukocytes in the tumor microenvironment

The percentage of blasts cells in the bone marrow (BM) of MDS patients is one of the key parameters for MDS classification and for the differential diagnosis with acute myeloid leukemia (AML). Currently, the gold standard to determine the blast percentage is conventional cytomorphology. To assess the possible impact of blast cell enumeration in BM biopsies from MDS patients on the final WHO classification using CD34 immunohistochemistry (IHC) a total of 156 BM samples from MDS and MDS-AML patients were studied and compared to blast counts by cytomorphology (CM). Eighty-nine BM aspirates were also studied by flow cytometry (FCM). Percentages of CD34+ blasts by IHC were determined blindly by two hematopathologists. Automated CD34-cell count was performed in 25 cases. Good overall agreement was found for CM and FCM with respect to critical blast thresholds (5%, 10%, 20%) (p p

Current guidelines recommend flow cytometric analysis as part of the diagnostic assessment of patients with cytopenia suspected for myelodysplastic syndrome. Herein we describe the complete work-up of six cases using multimodal integrated diagnostics. Flow cytometry assessments are illustrated by plots from conventional and more recent analysis tools. The cases demonstrate the added value of flow cytometry in case of hypocellular, poor quality, or ambiguous bone marrow cytomorphology. Moreover, they demonstrate how immunophenotyping results support clinical decision-making in inconclusive and clinically 'difficult' cases.

The 13th workshop of the European Bone Marrow Working Group in Utrecht, The Netherlands, was devoted to studying myelodysplastic syndromes (MDS) and their boundaries. The panel received 44 cases submitted to the 3 invited categories, which included: reactive cytopenias with dysplasia, idiopathic cytopenia of undetermined significance, clonal haematopoiesis of indeterminate potential, idiopathic dysplasia of uncertain significance and overt MDS. For this summary, we have selected 17 cases that highlight difficulties in separating true MDS from other causes of cytopenia and the intricate relationship between clonal haematopoiesis and true MDS. In addition, cases of overt MDS with challenging features were also selected. All cases were stained for p53 expression. Using instructive submitted cases we discuss the following: (1) cytopenia with clonal haematopoiesis not fulfilling MDS criteria, (2) cytopenia and/or dysplasia with germline mutations and/or familial history suggesting an underlying gene defect, (3) MDS based on a recurrent chromosomal abnormality and (4) overt MDS with diagnostic difficulties due to concurrent treatment or disease. The lively discussion in the open forum of the workshop illustrated the need for better integrative understanding of the evolution of acquired genetic abnormalities in haematopoiesis, and the challenge of diagnosing true MDS in cytopenic patients with genetic abnormalities, either germline or acquired.

Objectives To report the findings of the 2019 Society for Hematopathology/European Association for Haematopathology Workshop within the categories of reactive eosinophilia, hypereosinophilic syndrome (HES), germline disorders with eosinophilia (GDE), and myeloid and lymphoid neoplasms associated with eosinophilia (excluding entities covered by other studies in this series). Methods The workshop panel reviewed 109 cases, assigned consensus diagnosis, and created diagnosis-specific sessions. Results The most frequent diagnosis was reactive eosinophilia (35), followed by acute leukemia (24). Myeloproliferative neoplasms (MPNs) received 17 submissions, including chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Myelodysplastic syndrome (MDS), MDS/MPN, and therapy-related myeloid neoplasms received 11, while GDE and HES received 12 and 11 submissions, respectively. Conclusions Hypereosinophilia and HES are defined by specific clinical and laboratory criteria. Eosinophilia is commonly reactive. An acute leukemic onset with eosinophilia may suggest core-binding factor acute myeloid leukemia, blast phase of chronic myeloid leukemia, BCR-ABL1–positive leukemia, or t(5;14) B-lymphoblastic leukemia. Eosinophilia is rare in MDS but common in MDS/MPN. CEL, NOS is a clinically aggressive MPN with eosinophilia as the dominant feature. Bone marrow morphology and cytogenetic and/or molecular clonality may distinguish CEL from HES. Molecular testing helps to better subclassify myeloid neoplasms with eosinophilia and to identify patients for targeted treatments.

Intrachromosomal amplification of chromosome 21 (iAMP21) is a cytogenetic subtype associated with relapse and poor prognosis in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). The biology behind the high relapse risk is unknown and the aim of this study was to further characterize the genomic and transcriptional landscape of iAMP21. Using DNA arrays and sequencing, we could identify rearrangements and aberrations characteristic for iAMP21. RNA sequencing revealed that only half of the genes in the minimal region of amplification (20/45) were differentially expressed in iAMP21. Among them were the top overexpressed genes (p < 0.001) in iAMP21 vs. BCP ALL without iAMP21 and three candidate genes could be identified, the tyrosine kinase gene DYRK1A and chromatin remodeling genes CHAF1B and SON. While overexpression of DYRK1A and CHAF1B is associated with poor prognosis in malignant diseases including myeloid leukemia, this is the first study to show significant correlation with iAMP21-positive ALL.

Diagnosis of myelodysplastic syndromes (MDS) is not straightforward when objective data, such as blast excess and abnormal cytogenetics, are lacking. Expert laboratories use flow cytometry (FCM) to help diagnose MDS. However, most of FCM protocols for MDS are complex, requiring a high level of expertise and high cost. We have reported a FCM mini-panel consisting of four FCM parameters (so-called Ogata score), which is simple to conduct and inexpensive. In this paper, to refine this mini-panel, we have introduced a new FCM parameter, which quantifies CD33 expression on CD34+ cells (called Granulocyte/CD34 cell CD33 ratio). Bone marrow cells from MDS without blast excess (low-grade MDS) and controls were stained with CD34, CD45, and CD33 and analyzed for five parameters ("Granulocyte/CD34 cell CD33 ratio" plus four parameters in the Ogata score). By a multivariate logistic regression model, only three parameters, including "Granulocyte/CD34 cell CD33 ratio" had statistically significant power for diagnosing low-grade MDS. Based on the results, we constructed a new scoring system, which showed approximately 50% sensitivity and more than 95% specificity in diagnosing low-grade MDS. Our revised mini-panel is suitable for screening samples suspected for MDS and provides a basis for further improvement in diagnostic FCM protocols for MDS.

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p3% was strongly associated with MDS/CMML (286/293, 98%, p Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS.

OBJECTIVE: The aim of this study was to evaluate the diagnostic and prognostic role of multiparameter flow cytometry (FC) in patients with idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS).METHODS: We performed FC using a standardized panel and two different diagnostic algorithms (Ogata, Wells) in a well-characterized cohort of 79 patients with ICUS/CCUS and compared it with a retrospective blinded morphological evaluation and data from targeted next-generation DNA sequencing of 20 myelodysplastic syndrome (MDS)-related genes.RESULTS: Our data show that FC has low sensitivity in distinguishing CCUS from ICUS patients (40.5% for Ogata score and 59.5% for Wells score). The Wells score was suggestive of dysplasia in ICUS/CCUS patients with concurrent morphological signs of dysplasia in the bone marrow (following re-evaluation by two hematopathologists) and in CCUS patients with a higher mutational burden. Eight patients with ICUS/CCUS from our cohort progressed to another myeloid malignancy (MDS, acute myeloid leukemia, or chronic myelomonocytic leukemia), all showing flow cytometric signs of dysplasia.CONCLUSION: FC performs poorly in diagnosing CCUS versus ICUS. However, it can potentially provide prognostic information in cytopenic patients by identifying a subgroup of patients with a higher grade of dysplasia, higher mutational burden, and higher risk of progression and, together with mutational screening, also identify a group of patients who might require morphological reassessment of dysplastic changes in their bone marrow.

A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53-mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53, RUNX1, and TET2, respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making.

The telomerase reverse transcriptase (TERT) gene rs2736100_C allele has recently been shown to be associated with an increased risk for myeloproliferative neoplasms (MPNs) among Caucasians. However, it is unknown if this association is present in other ethnical populations and whether rs2736100 allele frequencies mirror the incidence of MPNs in a population. Here we genotyped TERT rs2736100 variants in 126 Swedish and 101 Chinese MPN patients and their age-, sex-, and ethnically-matched healthy controls. Healthy Chinese adults had a higher frequency of the A allele and lower frequencies of the C allele compared to Swedish counterparts (57.4 vs 47.0 % for A, 42.6 vs 53.0 % for C, P = 0.006). Both Swedish and Chinese patients harbored significantly higher C allele frequency than their controls (62.7 vs 53.0 % and 57.4 vs 42.6 % for Swedish and Chinese, respectively, P = 0.004). Swedes and Chinese bearing the CC genotype had a significantly increased risk of MPN compared to AA carriers (OR = 2.47; 95 % CI: 1.33–4.57, P = 0.003, for Swedes, and OR = 3.45; 95 % CI: 1.52–7.85, P = 0.005, for Chinese). Further analyses showed that rs2736100_CC was associated with robustly enhanced risk in males only (CC vs AA, OR = 5.11; 95 % CI: 2.19–11.92, P

Purpose: Recurrently mutated genes have been identified in patients with myelodysplastic syndromes (MDS) and, more recently, in patients with unexplained cytopenia. (Kwok et al. Blood 2015, Hansen et al. American Journal of Hematology 2016 and Malcovati et al. Blood 2017). In this study, we investigated the prognostic impact of these mutated genes in patients with idiopathic cytopenia and compared them to a control cohort of patients with low risk MDS. Methods: We included patients with idiopathic cytopenia after routine assessment, without cytogenetic abnormalities. For comparison, a group of low risk MDS patients without cytogenetic abnormalities, excess of blasts or ring sideroblasts were included. All samples were sequenced covering at least the 20 most recurrently mutated genes in MDS, and a subset of cases underwent a blinded morphology review by two hematopathologists. Results: Two hundred and forty nine patients, 171 with idiopathic cytopenia and 78 with low risk MDS, were included in this study. Of these, 80 (47%) and 53 (68%), respectively, had one or more detectable mutations. There was no difference in survival between the groups, however a predefined subset of "adverse mutations" (ASXL1, NRAS, SRSF2, U2AF1, TP53, RUNX1, EZH2, IDH2 and GATA2, adopted from Bejar et al. Current Opinion in Hematology 2017) was associated with inferior survival in the MDS group (p= 0.035), but not in the group with idiopathic cytopenia and at least one mutation (p= 0.43) (Figure 1). However, if an adverse mutation was present in the idiopathic cytopenia group the risk of progression to MDS or AML increased significantly (HR [CI:95%] 12.01 [1.47; 98.23], p= 0.02), after adjusting for age and sex. Thus mutational screening identified the patients with unexplained cytopenia at risk of progressing to an overt myeloid neoplasm (Figure 2). A total of 18 patients (23%) progressed to a myeloid neoplasm during follow up, of those 12 had material available at time of progression. All patients who progressed to AML (n=4) acquired a new driver mutation at time of progression, in contrast to the patients who progressed to MDS or CMML (n=8) without excess of blasts, who showed a clonal expansion or a steady variant allele frequency at the time of progression. TET2 and DNMT3A mutations were more frequent in patients with idiopathic cytopenia, and were associated with less dysplasia of bone marrow cells. A total of 109 cases with idiopathic cytopenia underwent a blinded morphology review by two independent reviewers; ten cases were concordantly reclassified to fulfill the criteria for MDS, and all of these had at least two mutations. None of these have progressed to higher risk MDS and these ten are not included in the 18 patients mentioned above, who progressed to MDS, CMML or AML during follow up. Conclusion: We here show that mutational profiling can identify patients with idiopathic cytopenia who are at risk of progression, but in contrast to low-risk MDS, the presence of adverse mutations in patients with idiopathic cytopenia do not predict inferior survival. Disclosures Hansen: Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees. Grønbæk:Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

Background: Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement ba ...

Objectives To examine contemporary survival patterns in the general population of patients diagnosed with chronic myeloid leukaemia (CML), and to identify patient groups with less than optimal outcomes. Design Prospective population-based cohort. Setting The UK's Haematological Malignancy Research Network (catchment population 3.6 million, with >2000 new haematological malignancies diagnosed annually). Participants All patients newly diagnosed with CML, from September 2004 to August 2011 and followed up to 31 March 2013. Main outcome measure Incidence and survival. Results With a median diagnostic age of 59 years, the CML age standardised (European) incidence was 0.9/100 000 (95% CIs 0.8 to 0.9), 5-year overall survival was 78.9% (72.3 to 84.0) and 5-year relative survival 88.6% (81.0 to 93.3). The efficacy of treatment across all ages was clearly demonstrated; the relative survival curves for those under 60 and over 60 years being closely aligned. Survival findings were similar for men and women, but varied with deprivation; the age and sex adjusted HR being 3.43 (1.89 to 6.22) for deprivation categories 4–5 (less affluent) versus 1–3 (more affluent). None of these differences were attributable to the biological features of the disease. Conclusions When therapy is freely provided, population-based survival for CML is similar to that reported in clinical trials, and age loses its prognostic significance. However, although most of the patients with CML now experience close to normal lifespans, those living in more deprived areas tend to have poorer outcomes, despite receiving the same clinical care. A significant improvement in overall population outcomes could be achieved if these socioeconomic differences, which may reflect the treatment compliance, could be eliminated.

Dendritic cells (DCs) develop in the bone marrow from haematopoietic progenitor cells. Two subsets, plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), have been identified. Little is known regarding DC levels in bone marrow of patients with acute myeloid leukaemia (AML) before and after chemotherapy. We investigated relative pDC and mDC levels in bone marrow from 37 hospital controls and 60 patients with AML [at diagnosis, complete remission (CR) and follow-up] using four-colour flow cytometry. The pDC immunophenotype was characterized as lin-/HLA-DR+/CD123 + and mDC as lin-/HLA-DR+/CD11c+. In 69% of patients with AML, no DCs were detected at diagnosis. At CR, mDC levels were the same in patients with AML and hospital controls while pDC levels were slightly lower. There was no association between minimal residual disease or survival rates and DC levels. Patients with low mDC levels at CR were more likely to suffer from complicated infections, although the difference was not statistically significant. Altogether, there was a profound decrease in DC levels in patients with AML at diagnosis. DC levels increased at CR and were higher than in hospital controls after post-remission therapy, suggesting that DCs recover after repeated chemotherapy. There may be an association between mDC levels and infectious complications.

The cannabinoid receptors are upregulated in many types of cancers, including mantle cell lymphoma (MCL) and have been suggested to constitute novel therapeutic targets. The expression pattern of the key members of the endocannabinoid system was analyzed in a well-characterized MCL patient cohort and correlated to biological features. 107 tumor tissues were analyzed for the mRNA levels of cannabinoid receptors 1 and 2 (CNR1 and CNR2) and the two main enzymes regulating the endocannabinoid anandamide levels in tissue: NAPEPLD and FAAH (participating in synthesis and degradation, respectively). NAPEPLD, CNR1 and CNR2 were overexpressed while FAAH expression was reduced in MCL compared to non-malignant B-cells. Both low CNR1 and high FAAH levels correlated with lymphocytosis (p=0.016 and p=0.022, respectively) and with leukocytosis (p=0.0018 and p=0.047). Weak to moderate CNR1 levels were a feature of SOX11 negative MCL (p=0.006). Both high CNR2 and high FAAH levels correlated to anemia (p=0.0006 and p=0.038, respectively). In conclusion, the relative expression of the anandamide synthesizing and metabolizing enzymes in MCL is heavily perturbed. This finding, together with high expression of cannabinoid receptors, could favor enhanced anandamide signaling and suggest that targeting the endocannabinoid system might be considered as part of lymphoma therapy.

Contains fulltext : 137293.pdf (Publisher’s version ) (Open Access) Del(5q) myelodysplastic syndromes defined by the International Prognostic Scoring System as low- or intermediate-1-risk (lower-risk) are considered to have an indolent course; however, recent data have identified a subgroup of these patients with more aggressive disease and poorer outcomes. Using deep sequencing technology, we previously demonstrated that 18% of patients with lower-risk del(5q) myelodysplastic syndromes carry TP53 mutated subclones rendering them at higher risk of progression. In this study, bone marrow biopsies from 85 patients treated with lenalidomide in the MDS-004 clinical trial were retrospectively assessed for p53 expression by immunohistochemistry in association with outcome. Strong p53 expression in >/= 1% of bone marrow progenitor cells, observed in 35% (30 of 85) of patients, was significantly associated with higher acute myeloid leukemia risk (P=0.0006), shorter overall survival (P=0.0175), and a lower cytogenetic response rate (P=0.009), but not with achievement or duration of 26-week transfusion independence response. In a multivariate analysis, p53-positive immunohistochemistry was the strongest independent predictor of transformation to acute myeloid leukemia (P=0.0035). Pyrosequencing analysis of laser-microdissected cells with strong p53 expression confirmed the TP53 mutation, whereas cells with moderate expression predominantly had wild-type p53. This study validates p53 immunohistochemistry as a strong and clinically useful predictive tool in patients with lower-risk del(5q) myelodysplastic syndromes. This study was based on data from the MDS 004 trial (clinicaltrials.gov identifier: NCT00179621).

OBJECTIVES: Thrombocytopenia is an independent adverse prognostic factor in patients with Myelodysplastic syndromes (MDS). Azacitidine, first-line treatment for the majority of patients with higher-risk MDS, is associated with aggravated thrombocytopenia during the first cycles. Eltrombopag is a novel thrombopoietin receptor agonist, which also has been shown to inhibit proliferation of leukaemia cell lines in vitro. This phase I clinical trial was designed to explore the safety and tolerability of combining eltrombopag with azacitidine in patients with MDS. In addition, we assessed the potential effects of eltrombopag on hematopoietic stem and progenitor cells (HSPCs) from included patients. PATIENTS AND METHODS: Previously untreated patients with MDS eligible for treatment with azacitidine and with a platelet count

Abstract 790 Lenalidomide has emerged as a very effective therapy for del(5q) myelodysplasia. Its mechanism of action, however, has hitherto remained elusive. Interestingly, the more primitive hematopoietic compartment seems to possess a clonal advantage where del(5q) HSC are able to outcompete remaining normal HSC. We have previously demonstrated that lenalidomide is able to abrogate this clonal advantage and found that lenalidomide restored expression of the matricellular protein SPARC, a gene located within the commonly deleted region on chromosome 5q. We hypothesized that the decreased expression of SPARC in del(5q) HSC leads to increased adhesion of HSC to their respective niche cells, translating to increased rates of proliferation, partly explaining the competitive advantage against non-del(5q) HSC. We conducted a prospective study analyzing the del(5q) HSC/progenitor compartment from 23 patients before, during and after (refractory phase) lenalidomide, in order to test whether a hematopoietic stem cell (HSC)-intrinsic decrease of SPARC explains the why and how a clone of cells inherently defective at spawning functioning cellular descendants is not selected against, but rather exhibits a clonal advantage. In addition, we studied whether treatment with lenalidomide induced changes in the microenvironment these cells reside in. We analyzed cell cycle distribution, frequency of apoptosis, and expression of adhesion markers on normal and del(5q) HSPC by multi-parameter flow cytometry. These experiments revealed a slight increase in proliferation of del(5q) versus normal HSC, as well as complex changes in the expression of adhesion markers in HSC of patients treated with lenalidomide. We studied the functional adhesion of normal and del(5q) HSPC to defined matrix components of the microenvironment and observed that HSPC from del(5q) patients exhibited stronger adhesion than normal bone marrow cells to fibronectin and VCAM-1. Recombinant SPARC protein abrogated adhesion to VCAM-1 specifically in a subset of patients, while having no significant effect on normal HSPC. To study whether SPARC plays a role in the clonal dominance, we used lentiviral transduction to overexpress SPARC in HSPC and found that increased expression of SPARC led to severely reduced engraftment in NSG-mice. We also analyzed how lenalidomide impacts the microenvironmental niche. To this end, we compared the gene expression profile of mesenchymal stem cells (MSC) obtained from del(5q) patients by DNA microarray and found only 36 genes to be differentially expressed by more than 2-fold, with 15 and 21 genes up- or downregulated in del(5q) MSC, respectively. Using longitudinal bone marrow biopsies from 5 patients on and off treatment, we analyzed whether lenalidomide induced changes in the frequency of candidate niche cells such as mesenchymal stem cells (MSC), macrophages, endothelium and megakaryocytes, by using immunohistochemical markers for the aforementioned cells. Lenalidomide induced no significant changes in the number of nestin+ MSC but seemed to decrease the number of Factor-VIII+ megakaryocytes. Taken together, these studies suggest that decreased expression of SPARC leads to increased adhesion of del(5q) HSC/progenitor cells to defined components of the microenvironment and may explain why del(5q) HSC are able to outcompete the remaining healthy HSC. Our studies implicate that lenalidomide is able to abrogate this clonal advantage partly via its increase in SPARC expression with a consecutive decrease in adhesion. Disclosures: No relevant conflicts of interest to declare.

BACKGROUND: Patients with chromosome 5 abnormalities and high-risk myelodysplastic syndromes or acute myeloid leukemia have a poor outcome. We hypothesized that increasing doses of lenalidomide may benefit this group of patients by inhibiting the tumor clone, as assessed by fluorescence in situ hybridization for del(5q31).DESIGN AND METHODS: Twenty-eight patients at diagnosis or with relapsed disease and not eligible for standard therapy (16 with acute myeloid leukemia, 12 with intermediate-risk 2 or high-risk myelodysplastic syndrome) were enrolled in this prospective phase II multicenter trial and treated with lenalidomide up to 30 mg daily for 16 weeks. Three patients had isolated del(5q), six had del(5q) plus one additional aberration, 14 had del(5q) and a complex karyotype, four had monosomy 5, and one had del(5q) identified by fluorescence in situ hybridization only.RESULTS: Major and minor cytogenetic responses, assessed by fluorescence in situ hybridization, were achieved in 5/26 (19%) and 2/26 (8%) patients, respectively, who received one or more dose of lenalidomide, while two patients achieved only a bone marrow response. Nine of all 26 patients (35%) and nine of the ten who completed the 16 weeks of trial responded to treatment. Using the International Working Group criteria for acute myeloid leukemia and myelodysplastic syndrome the overall response rate in treated patients with acute myeloid leukemia was 20% (3/15), while that for patients with myelodysplastic syndrome was 36% (4/11). Seven patients stopped therapy due to progressive disease and nine because of complications, most of which were disease-related. Response rates were similar in patients with isolated del(5q) and in those with additional aberrations. Interestingly, patients with TP53 mutations responded less well than those without mutations (2/13 versus 5/9, respectively; P=0.047). No responses were observed among 11 cases with deleterious TP53 mutations.CONCLUSIONS: Our data support a role for higher doses of lenalidomide in poor prognosis patients with myelodysplastic syndrome and acute myeloid leukemia with deletion 5q. (Clinicaltrials.gov identifier NCT00761449).

On average, 30% of patients with myelodysplastic syndrome (MDS) develop overt acute myeloid leukemia (AML) during the course of the disease. There is a continuous search for the best model of individual risk assessment for MDS patients. In this review, we summarize current findings on factors that have been associated with increased risk of AML transformation. These include laboratory values such as high lactate dehydrogenase levels, complex karyotypes, numbers and aberrant immunophenotype of bone marrow blasts, bone marrow-related features such as numbers and distribution of CD34+ cells, and recently established molecular markers. A wide range of described molecular aberrations in MDS, including various gene mutations, chromosomal instability, short telomeres, high levels of gene methylation, and histone modification, partly explains clinical heterogeneity of this disease. Continuous research will bring more insight in the pathogenesis of various MDS categories, making individual risk assessment and tailored therapy for each patient possible.

Purpose To determine the frequency of TP53 mutations and the level of p53 protein expression by immunohistochemistry (IHC) in low-risk myelodysplastic syndromes (MDS) with del(5q) and to assess their impact on disease progression. Patients and Methods Pre- and postprogression bone marrow (BM) samples from 55 consecutive patients with International Prognostic Scoring System low risk (n = 32) or intermediate-1 risk (n = 23) were studied by next-generation sequencing of TP53. IHC for p53 was performed on 148 sequential BM samples. Results TP53 mutations with a median clone size of 11% (range, 1% to 54%) were detected in 10 patients (18%) already at an early phase of the disease. Mutations were equally common in low-risk and intermediate-1–risk patients and were associated with evolution to acute myeloid leukemia (5 of 10 v 7 of 45; P = .045). Nine of 10 patients carrying mutations showed more than 2% BM progenitors with strong p53 staining. The probability of a complete cytogenetic response to lenalidomide was lower in mutated patients (0 of 7 v 12 of 24; P = .024). Conclusion By using sensitive deep-sequencing technology, we demonstrated that TP53 mutated populations may occur at an early disease stage in almost a fifth of low-risk MDS patients with del(5q). Importantly, mutations were present years before disease progression and were associated with an increased risk of leukemic evolution. TP53 mutations could not be predicted by common clinical features but were associated with p53 overexpression. Our findings indicate a previously unrecognized heterogeneity of the disease which may significantly affect clinical decision making.

Introduction and aims The term ICUS is used to describe cases with persistent cytopenia (more than 6 months) without evidence of dysplasia in the bone marrow (BM) smear, and normal cytogenetics. Flow cytometry is currently a standard tool for the diagnosis of myelodysplastic syndromes (1). Our goal was to evaluate the efficacy of flow cytometry in detecting bone marrow dysplasia in the absence of abnormal morphology. Materials and methods A total of 79 patients with ICUS and 12 patients with MDS were analyzed with a standardized flow cytometry panel (2). Data were analyzed using two different algorithms; the Ogata algorithm (3) and Wells algorithm (4). For each of the algorithms, samples were classified as suggestive for dysplasia when scoring equal to or higher than 2. Furthermore, all ICUS patients were screened for mutations using targeted sequencing of 20 genes (DNMT31, TET2, EZH2, SRSF2, CBL1, SF3B1, UAF1, GATA2, IDH1, IDH2, CEBPa1, ZRSR2, TP53, KRAS, NRAS, ETV6, RUNX1, JAK2 and ASXL1) and 57/71 samples were additionally evaluated for signs of dysplasia by two independent hematopathologists. Results The diagnostic value of flow cytometry in MDS was confirmed by our data; the Wells algorithm was slightly superior to Ogata algorithm in predicting MDS (10/12 patients (83.3%) vs. 7/12 patients (58.3%) respectively, fig. 1A) and was therefore the main algorithm used for the further analysis of the ICUS patients. Flow cytometry was suggestive of dysplasia in 34/79 (43%) patients with ICUS. Interestingly, in patients with at least one detected mutation, flow cytometry was positive in 23/42 (54.8%) of the cases, while it was positive in 8/9 (88.9%) patients with more than two mutations (fig. 1C). There was no higher frequency of abnormal flow cytometry in patients with higher risk mutations. Additionally, for the 57 patients evaluated for dysplastic changes, flow cytometric abnormalities were more frequent (p= 0.05) in the 22 patients with moderate/severe dysplasia (15/22 patients or 68.2%, fig. 1D). After a median follow-up of 20 months (range: 3 - 90), a total of eight patients progressed to either MDS, CMML or AML. Interestingly, these eight patients had flow cytometric abnormalities suggestive of dysplasia before developing hematological malignancy. In conclusion, flow cytometry can identify a subgroup of ICUs patients with a higher mutational burden, dysplastic changes and a higher risk for progression to MDS or a more aggressive myeloid disease and has a place in the diagnostic evaluation of patients with idiopathic cytopenia. References: Porwit A, Van De Loosdrecht AA, Bettelheim P, Eidenschink Brodersen L, Burbury K, Cremers E, et al. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes - Proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS. Leukemia. 2014;28(9):1793-8. van Dongen JJM, Lhermitte L, Böttcher S, Almeida J, van der Velden VHJ, Flores-Montero J, et al. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia [Internet]. 2012 Sep [cited 2014 Nov 28];26(9):1908-75. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3437410&tool=pmcentrez&rendertype=abstract Porta MGD, Picone C, Pascutto C, Malcovati L, Tamura H, Handa H, et al. Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: Results of a European LeukemiaNET study. Haematologica. 2012;97(8):1209-17. Wells DA, Benesch M, Loken MR, Vallejo C, Myerson D, Leisenring WM, et al. Myeloid and monocytic dyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood. 2003;102(1):394-403. Figure 1. Figure 1. Disclosures Hansen: Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees. Grønbæk:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen Pharma: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees.

Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement based on cytomorphological examination of the cerebrospinal fluid (CSF) alone is not as sensitive with low cell counts as flow cytometric immunophenotyping (FCI) of the CSF. However, the importance of low CSF blasts counts at diagnosis is uncertain. We sought to determine the significance of FCI in relation to conventional morphological examination.We retrospectively compared FCI of the CSF with cytomorphology at diagnosis or relapse of childhood ALL. All patients were diagnosed 2000-2012 in Stockholm or Umeå, Sweden. Clinical data were collected from medical records and the Nordic leukemia registry. Treatment assignment was based on morphological examination only.The cohort was comprised of 214 patients with ALL. CSF involvement was detected by both methods in 20 patients, in 17 by FCI alone, and in one patient by cytomorphology alone. The relapse rate was higher for patients with negative cytology but positive FCI compared to those without CNS involvement using both methods. The difference was especially marked in the current protocol. However, none of the patients with negative CSF cytology but positive FCI had a CNS relapse.FCI of the CSF increased the detection rate of CNS involvement of ALL approximately two times compared to cytomorphology. Patients with low-level CNS involvement may benefit from additional intensified systemic or CNS-directed therapy, but larger studies are needed.

A high proportion of lower-risk del(5q) MDS patients will respond to treatment with lenalidomide. The estimated duration of transfusion-independence is 2 years including some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after start of treatment. As the molecular mechanisms underlying disease progression in del(5q) MDS remain to be elucidated, we do not know how to predict disease progression or how to monitor patients during lenalidomide treatment. We previously reported that small TP53 mutated subclones predict for an unfavorable outcome in del(5q) patients, and that these subclones expand with disease progression. However, whether or not other somatic mutations or factors related to the bone marrow microenvironment also contribute to disease progression has not been comprehensively assessed. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22), or other treatments including stem cell transplantation (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cells (HSPC) subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. In our cohort, 13 of 35 patients progressed to either higher-risk MDS (n=4) or leukemia (n=9), 12 of whom were treated with lenalidomide. Progression was associated with the detection of a restricted subset of new recurrent mutations, either alone or in combination: TP53 (n=9, p=0.0004), TET2 (n=6, p=0.006), RUNX1 (n=3, p=0.044), and PTPN11 (n=1). Regardless of whether the three mutations (TP53, TET2 and RUNX1) were present in the initial sample or whether they subsequently developed, testing positive for any of them carried a high probability (13/16, 81%) for predicting progression. For 11 out of 13 patients the new mutations were detected prior to the time point of clinical progression and the median time from detection of the mutation to clinical evidence of progression was 42 months (range 0-83.9). Thus, we were able to detect the mutation in the majority of cases well before clinical signs of disease progression. Seven of the nine patients who developed leukemia carried a TP53 mutation. Based on a median sequencing depth of 370 reads, the mutation was considered present pre-treatment in one of these patients and to have developed under treatment in the other six. Using flow cytometry for surveillance of HSPC subsets in lenalidomide-treated patients, we found that neither lenalidomide treatment nor the acquisition of additional mutations led to any uniform profound changes in the hematopoietic hierarchy unless the patient showed clinical signs of progression. Microarray analysis of mesenchymal stromal cells (MSC) exhibited an expression footprint consistent with MSC with high expression of typical MSC markers and absence of hematopoietic gene signatures. However, we observed only minor differences in gene expression between pre- treatment del(5q) and healthy MSC. In conclusion, while flow cytometric analysis of HSPC populations or analysis of the microenvironment had limited predictive value in this cohort of lower-risk del(5q) MDS, all patients who progressed to either higher-risk MDS or leukemia were identified by harboring recurrent mutations in a limited number of genes, i.e., TP53, RUNX1, TET2, and PTPN11. Based on our data, we advocate for conducting a prospective study aimed at investigating in a larger number of del(5q) MDS cases pre- and post-lenalidomide treatment, whether the detection of such mutations can guide clinical decision making, such as suggesting which patients should undergo hematopoietic cell transplantation. Disclosures No relevant conflicts of interest to declare.

Dendritic cells (DC) are antigen-presenting cells that play a pivotal role in coordinating functions of the immune system. Previous studies suggest that bone marrow (BM) failure in myelodysplastic syndromes (MDS) may be in part immune-mediated, and that the high propensity for relapse may reflect decreased immune surveillance. This study aimed to assess the frequency of DC in BM samples from well-annotated untreated MDS patients by using 4-colour flow cytometry. DC levels were markedly reduced in all subtypes of MDS. The clinical impact of this finding on therapy response and relapse after, e.g. allogeneic stem cell transplantation warrants further investigation.

Introduction: Refined risk-classification of patients (pts) with MDS allows for improved treatment selection for individual pts. The Revised International Prognostic Scoring System (IPSS-R) has recently been validated as a prognostic tool in lower-risk MDS pts with deletion 5q [del(5q)], who were treated with LEN in the MDS-004 study (Sekeres et al. Blood Cancer J 2014; in press). P53 nuclear protein expression, as assessed by immunohistochemistry (IHC), predicted overall survival (OS) and risk of progression to acute myeloid leukemia (AML) in lower-risk MDS pts with del(5q) (Saft et al. Haematologica 2014;99:1041-9). This analysis evaluated the prognostic value of adding p53 IHC to IPSS-R to predict OS and AML progression in pts with lower-risk MDS with del(5q). Methods: In a subset of 85 pts from MDS-004 with bone marrow (BM) biopsies available, p53+ staining (≥ 1% IHC+++ BM cells) was visualized by IHC. Twenty-four pts had missing IPSS-R scores; 1 due to lack of baseline cytogenetic data and 23 because of missing exact BM blast percentage. Thus, 61 pts (42 initially treated with LEN and 19 with placebo) had IPSS-R and p53 IHC data available; 89% of pts in the placebo group crossed over to LEN 5 mg at Week 16. The IPSS-R Very Low and Very High risk groups with < 5 pts were combined with the Low and High risk groups, respectively. AML-free survival (AFS), OS, and time to AML progression within p53 IHC status (p53+ vs p53−), and IPSS-R risk groups were characterized by the Kaplan-Meier method with differences evaluated by the log-rank test. Results: Of 61 pts, 38% were p53+. There was a linear increasing trend in the proportion of pts with p53+ across IPSS-R risk groups from Very Low/Low, Intermediate to High/Very High (29%, 47% and 63%, respectively; Cochran-Armitage trend test P = 0.050). The 3 IPSS-R risk groups significantly predicted AFS and OS (log-rank P < 0.001 for both AFS and OS), but not time to AML progression (P = 0.335). Overall, AFS, OS, and time to AML progression differed significantly between p53+ versus p53− pts (23.9 vs 47.9 months for median AFS, P = 0.003; 27.0 vs 50.6 months for median OS, P = 0.005; and 44.3 months vs not reached [NR] for median time to AML progression,P = 0.003). In the IPSS-R Very Low/Low risk group (n = 38), AFS, OS, and time to AML progression were significantly worse in p53+ versus p53− pts (20.1 vs 63.1 months for median AFS, P = 0.011; 28.4 vs 76.8 months for median OS, P = 0.031; and 65.2 months vs NR for median time to AML progression, P = 0.014). Results for all IPSS-R risk groups in pts with p53 and IPSS-R data are presented in the Figure. The lack of significant differences between p53+ versus p53− pts in the Intermediate and High/Very High risk groups is likely due to the small sample size of these groups. Conclusions: In this exploratory subset analysis of lower-risk MDS pts with del(5q), p53 IHC status in the IPSS-R Very Low/Low risk group significantly impacted AFS, OS, and AML progression. These data support the addition of p53 mutational analysis to prognostic risk assessment which should help inform the selection of appropriate treatment for individual MDS pts with del(5q). These results need to be validated in a large sample set, which will be accomplished as part of the ongoing efforts to include prognostic molecular mutations in future updates of IPSS-R Figure 1 AFS (A), OS (B), and time to AML progression (C) in pts with p53 and IPSS-R data (N = 61) Figure 1. AFS (A), OS (B), and time to AML progression (C) in pts with p53 and IPSS-R data (N = 61) Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Shiansong Li: Celgene Corporation: Employment, Equity Ownership. Greenberg:Celgene: Research Funding; Onconova: Research Funding; GSK: Research Funding; Novartis: Research Funding; KaloBios: Research Funding. Sekeres:Amgen Corp.: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Dreyfus:Novartis: Honoraria; Celgene: Honoraria. Fenaux:Novartis: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Swern:Celgene: Employment, Equity Ownership. Sugrue:Celgene: Employment, Equity Ownership. Hellstrom-Lindberg:Celgene: Research Funding.

Abstract 3798 Introduction: Transfusion-dependency (TD) in patients with low- and int-1-risk MDS is associated with increased morbidity and mortality (Malcovati, JCO 2007). Around 30% of such patients respond to Epo +/− G-CSF with a median duration of two years (Jadersten, Blood 2005). Azacitidine (Aza) has been reported to induce transfusion-independence (TI) in patients with lower-risk MDS (Lyons, JCO 2009). However, this treatment has not been systematically evaluated in patients with a well-documented resistance to Epo +/− G-CSF, neither has the benefit of the combination of Aza and Epo been evaluated in a prospective study. Methods: This prospective open-label, non-randomized phase II study will include 30 consecutive patients with IPSS low- or int-1-risk MDS and a RBC transfusion need of ≥4 units q 4 weeks. Patients should be refractory to full-dose Epo + G-CSF for >8 weeks, or considered ineligible for such treatment according to a predictive model based on serum erythropoietin levels and transfusion need (Hellstrom-Lindberg, Br J Hem 2003). Included patients are treated with Aza 75 mg/m2/d for five days q 28 days for six cycles. Patients remaining TD after six cycles are treated with another three Aza cycles, with the addition of Erythropoietin â 60,000 units/week. Primary endpoint is number of patients achieving TI after six cycles and secondary endpoints are number of patients achieving TI after another three cycles of Aza+Epo, effect on bone marrow morphology, peripheral blood parameters, and safety profile. Results: Twenty-eight patients were enrolled from January 2010 until August 2011, another six were screen failures. See table 1 for patient characteristics. Seventeen patients were previously treated with and refractory to Epo (60,000 units/week) + G-CSF (300 μg/week), whereas remaining patients scored “low” in the predictive model and were thus considered refractory upfront. Median number of transfusion units needed was 7 (4–14) during the 8 weeks preceding inclusion. Fifteen patients are so far evaluable for treatment with Aza alone and 6 patients for Aza+Epo. TI was achieved in 2/15 patients (13%) after Aza alone. No additional responses were observed after the addition of EPO. Twenty-three serious adverse events were reported in 12 patients, with neutropenic fever (n=8), infections (n=4), and diarrhea (n=2) being most common. Three patients pre-terminated the study, two due to sustained neutropenia and one due to leukemic transformation. Nadir values after each cycle of Aza were seen at Week 3 for thrombocytes (147±34×109) and at Week 4 for neutrophils (1.4±0.9 x109), respectively, reflecting a cytotoxic effect on hematopoiesis. Discussion: Aza can induce TI in patients with TD lower-risk MDS, but response rate is lower in this group of documented EPO-G-CSF-refractory patients compared to previous reports of less well-controlled cohorts. Since toxicity is substantial, candidate patients for this treatment must be selected carefully. The combination of Aza and Epo remains to be further evaluated. Disclosures: Off Label Use: Azacitidine for lower-risk MDS. Hellström-Lindberg:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clonal heterogeneity has not been described in patients with myelodysplastic syndrome (MDS) of the 5q- syndrome subtype, for which lenalidomide has emerged as a highly potent treatment. Interestingly, transformation to acute myeloid leukemia (AML) occurs more frequently in patients without a cytogenetic response to lenalidomide. We describe two patients with classical 5q- syndrome with complete erythroid and partial cytogenetic response to lenalidomide, who evolved to high-risk MDS and AML, respectively, with complex karyotypes including del(5q). Immunohistochemistry of pre-treatment marrow biopsies revealed small fractions of progenitors with aberrant cytoplasmic nucleophosmin (NPMc+) expression and in one patient also a TP53 mutation. Both lesions may induce a state of genomic instability, and represent novel findings in 5q- syndrome. These subclones remained stable during response, but expanded at transformation, suggesting that pre-existing cells with more malignant genotype may be insensitive to lenalidomide and responsible for disease progression. We are currently investigating a larger patient material for the presence of subclones with molecular lesions in order to assess the potential impact on the risk of leukemic evolution.