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Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet clinical need. Though BTK and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL treated with ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital PCR (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32/49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6/32, BTK mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were also identified in 6/49 responding patients (12%; 5/6 VAF

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 iming to highlight achievements in the diagnostics and treatment of blood disorders and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1 to 2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cellbased Immune Therapies; and Gene Therapy. © 2022 Wolters Kluwer Health. All rights reserved.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

The importance of predisposition to leukaemia in clinical practice is being increasingly recognized. This is emphasized by the establishment of a novel WHO disease category in 2016 called "myeloid neoplasms with germline predisposition". A major syndrome within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic stem cell transplantation is the only curative option; however, chances of survival decrease with progression of immunodeficiency and MDS evolution. Penetrance and expressivity within families carrying GATA2 mutations is often variable, suggesting that co-operating extrinsic events are required to trigger the disease. Predictive tools are lacking, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review we describe the genetic, clinical, and biological aspects of familial GATA2-related MDS, highlighting the importance of developing robust disease preclinical models to improve early detection and clinical decision-making of GATA2 carriers. This work was supported by ERA PerMed GATA2-HuMo Funding Mechanism (Spain: Acció instrumental de SLT011/18/00006 of the Department of Health of the Government of Catalonia, to AG and AB; Hungary: ED-18-1-2019-001 grant from the National Research, Development and Innovation Office to CB; and Germany: German Federal Ministry of Education and Research (BMBF) 2018-123/01KU1904 to MWW), ÚNKP-21-2-I-SE-21 and Hungarian National Academy of Scientist Education grant to KL, TKP2021-NVA-15, TKP2021-EGA-24 and EU's Horizon 2020 research and innovation programme under grant agreement no. 739593 and Elixir Hungary to CB, MSCA No 101029927-scGATA2track (H2020-MSCA-IF-2020) to OM-B, the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO PID2020-15591RB-100), La Marató de TV3 (202001-32), FPS Grant 2018 by Fondazione Pisana per la Scienza ONLUS and CERCA Programme/Generalitat de Catalunya for institutional support to AG, Vera and Joseph Dresner Foundation, Evans MDS Foundation, American Lebanese Syrian Associated Charities, and BMBF MyPred 01GM1911A to MWW. Open access funding enabled and organized by ProjektDEAL.

Germline variants in RUNX1, GATA2 and DDX41 may confer a predisposition to hereditary haematopoietic malignancies (HHMs) such as MDS and AML yet have distinct age ranges of malignancy diagnosis and a highly variable overall risk for leukemogenesis. The increased awareness and identification of carriers of these germline variants, particularly before development of malignancy, has changed the way in which individuals and families need to be managed in the clinic. Individuals need lifelong monitoring and may also need modification to treatments when malignancy does develop, compared to sporadic counterparts. Gaps in understanding pre-malignant states in HHM syndromes have hampered efforts to design effective clinical surveillance regimes, provide personalized pre-emptive treatments, and appropriate counselling to patients.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world. In previous studies, various proportion of patients was found to carry CD11b+ or CD11c+ B cells whose presence was an unfavourable prognostic factor. The exact mechanism however, how these receptors contribute to the pathogenesis of CLL has not been revealed so far. Here we analysed the role of CD11b and CD11c on B cells of CLL patients in the adhesion to fibrinogen and in the migration towards stromal cell derived factor-1 (SDF-1) and studied the role of CR4 in the adherence of the CD11c+ B cell line BJAB. We observed that both CR3 and CR4 mediate adhesion of the malignant B cells. Moreover, we found, that CR4 was strongly involved in the migration of the leukemic cells towards the chemoattractant SDF-1. Our data suggest that CR3 and CR4 are not only passive markers on CLL B cells, but they might contribute to the progression of the disease. Since the role of SDF-1 is prominent in the migration of CLL cells into the bone marrow where their survival is supported, our findings help to understand how the presence of CD11c on leukemic B cells can worsen the prognosis of chronic lymphocytic leukaemia.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

Background Philadelphia negative myeloproliferative neoplasms (MPNs) are characterized by frequent mutations of driver genes including JAK2, CALR and MPL. While the influence of JAK2 V617F mutant allele burden on the clinical phenotype of MPN patients is well-described, the impact of CALR mutant allele burden on clinical features needs further investigation. Patients and methods Quantitative assessment of JAK2 and CALR mutations was performed on diagnostic DNA samples from 425 essential thrombocythemia (ET) and 227 primary myelofibrosis patients using real-time quantitative PCR and fragment length analysis. Characterization of CALR mutations and detection of MPL mutations were performed by Sanger sequencing. Results Twelve novel CALR mutations have been identified. ET patients with CALRmut load exceeding the median value exhibited lower hemoglobin values (12.0 vs. 13.6 g/dL), higher LDH levels (510 vs. 351 IU/L) and higher rate of myelofibrotic transformation (19% vs. 5%). The CALRmut load was higher among ET patients presenting with splenomegaly compared to those without splenomegaly (50.0% vs. 43.5%). Conclusion Our study confirms the clinical significance of driver mutational status and JAK2mut load in MPNs; in addition, unravels a novel clinical association between high CALRmut load and a more proliferative phenotype in ET.

Context Follicular lymphoma (FL) is the most common type of indolent non-Hodgkin lymphoma, with an average overall survival rate of 15 years. The prolonged disease course raises the need for an effective disease monitoring system, which is hampered by the limited specificity and sensitivity of the most widely used, imaging-based PET-CT method. Molecular analysis of tumor-derived circulating DNA (ctDNA), frequently referred to as liquid biopsy, is a promising minimally invasive disease monitoring tool for patients suffering from malignancies. Recent studies interrogating the genomic background of FL have revealed activating mutations in the epigenetic regulator EZH2 gene in 25% of patients. Objective In our study, we tested the potential of liquid biopsy to detect and monitor EZH2 mutations in plasma samples of patients with EZH2-mutant FL treated with immunochemotherapy. Patients and Methods Eighty-five blood plasma samples were collected from 41 EZH2 mutant patients. Cell-free plasma DNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). The EZH2 mutation status was assessed using a multiplex digital droplet PCR (ddPCR, Bio-Rad Laboratories, USA) approach, detecting seven different EZH2 hotspot mutations using only two PCR reactions. The molecular results were correlated with PET-CT images. Results Among the 41 patients, 36 responded to treatment (88%); meanwhile, 5 patients did not (12%). We detected an EZH2 mutation in 89% of the samples collected prior to treatment (17/19). Similarly, 86% of the samples (6/7) collected during therapy from patients not responding to treatment proved to be EZH2 mutated; meanwhile, only 22% of the samples were positive from responding patients (6/33). As expected, all samples collected during remission were EZH2 wild-type. EZH2 variant allele frequencies (VAF) correlated with the total metabolic tumor volume detected on the PET-CT scans. We also demonstrated spatial heterogeneity of EZH2 mutations in two cases, where different EZH2 mutations deriving from distinct anatomical sites could simultaneously be detected in the plasma. The median sensitivity of our ddPCR approach was 0.15%, with EZH2 VAFs ranging between 0.2 and 74%. Conclusions Our results demonstrate that liquid biopsy offers a sensitive, radiation-free, minimally invasive monitoring method for EZH2-mutant FL patients.

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Context: Follicular lymphoma (FL) is the most common type of indolent non-Hodgkin lymphomas, with an average overall survival rate of 15 years. The majority of FL patients experience disease progression or high-grade transformation to a more aggressive lymphoma, raising the need for effective treatment response monitoring, which, however, is hampered by the limited specificity and sensitivity of the most widely used, imaging-based PET-CT method. Molecular analysis of tumor-derived circulating DNA (ctDNA), frequently referred as liquid biopsy, is a promising, minimally invasive, radiation-free disease monitoring tool for patients suffering from a wide range of different tumor types. Recent studies interrogating the genomic background of FL have revealed activating mutations in the epigenetic regulator EZH2 gene in 25% of patients at diagnosis. Objective: In this feasibility study, we tested the power of detection and time-resolved monitoring of EZH2 mutations in ctDNA of FL patients receiving immunochemotherapy (ICT). Methods: Thirty-five blood plasma samples have been collected from 19 EZH2 mutant FL patients treated with ICT. After ctDNA isolation, EZH2 mutations were screened in each sample using digital droplet PCR (Bio-Rad Laboratories, USA). The acquired genetic results were compared with PET-CT-based imaging data where available. Results: EZH2 mutations were detected in plasma samples of four patients suffering from active or transformed FL. All patients with EZH2 wild type plasma samples were in complete or partial remission. In patients with EZH2 mutation-positive ctDNA, allele frequencies of the mutations correlated with the amount of metabolically active tumor sites observed on PET-CT scan images. Variant allele frequencies of EZH2 mutations rapidly declined or dropped below the detection limit upon successful treatment, while in one patient, treatment failure was associated with high EZH2 variant allele frequency. We also demonstrated spatial heterogeneity of EZH2 mutations in another case where different EZH2 mutations deriving from distinct anatomical sites could simultaneously be detected in the plasma. Conclusions: Our results suggest that liquid biopsy-based EZH2 mutation analysis with sensitive digital droplet PCR method offers a real-time, radiation-free, sensitive treatment response monitoring tool for patients with active EZH2 mutant FL.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with >20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Introduction: The irreversible Bruton tyrosine kinase (BTK) inhibitor ibrutinib is changing the paradigm of the treatment of chronic lymphocytic leukemia (CLL) with remarkable outcomes in first line as well as in relapsed CLL, including high risk patients with TP53 aberration. While the majority of patients demonstrate durable responses, with a median follow up of 3 years, approximately 18% of them develop resistance and relapse. Mutations in the BTK and PLCG2 genes emerged as predominant mechanisms conferring secondary ibrutinib resistance with BTK and/or PLCG2 mutations detected in the majority of resistant patients, often even 10 months before the clinical relapse. Given the very poor outcome of patients discontinuing ibrutinib, comprehensive understanding of the mechanisms of ibrutinib resistance and changes in the clonal architecture induced by the selective pressure of the drug are of major clinical importance. To dissect the clonal evolution in the context of all relevant mutation targets in CLL, we performed a temporal mutation profiling of 31 genes in paired pre- and post-treatment samples of patients receiving ibrutinib therapy. Methods: Sequential samples from 18 patients treated with single-agent ibrutinib were included in this study. Pre-treatment peripheral blood samples were available in all patients, with post-treatment samples at 18 months for 6 patients, at 12 months for 7 patients, at 8 months for 3 patients and at 3 months for 2 patients, with a median follow up of 12 months. Targeted deep NGS analysis of 31 recurrently mutated target genes, including ATM, BCOR, BIRC3, BRAF, BTK, CHD2, DDX3X, EGR2, EIF2A, EP300, FBXW7, HIST1H1E, IGLL5, KLHL6, KMT2D, LRP1B, MAPK1, MED12, MGA, MYD88, NFKBIE, NOTCH1, PLCG2, POT1, RIPK1, RPS15, SAMHD1, SF3B1, TP53, XPO1 and ZMYM3 was performed using the TruSeq Custom Amplicon approach (Illumina) on genomic DNA specimens extracted from peripheral blood mononuclear cells. Variant calling was performed using the VariantStudio3.0 application (Illumina) and only variants supported with more than 100 mutant reads were considered. Our cohort represented a heavily pretreated patient group with a median of 2.5 (range: 1-5) lines of prior therapies. Results: The ultra-deep NGS analysis revealed a total of 473 somatic mutations in the 18 paired samples with a median allelic depth of 25.550x across the 31 genes analyzed. The post-treatment samples carried a considerably higher number of mutations compared to the pre-treatment samples (114 vs 359 mutations) with more than half of the variants representing subclonal alterations with variant allele frequencies (VAF) of Conclusions: The ultra-deep NGS analysis performed on paired samples of CLL patients treated with ibrutinib provided a significant insight into the mutational repertoire as well as subclonal dynamics of the leukemic compartment. Furthermore, it has revealed a profound subclonal heterogeneity that seems to be further enhanced by ibrutinib therapy, as demonstrated by a higher frequency of subclonal variants in the post-treatment samples. The clinical significance and the potential role of the novel BTK and PLCG2 variants identified in our study will require a longer follow up time, as currently all patients harboring these subclonal variants are still on ibrutinib therapy. Disclosures No relevant conflicts of interest to declare.

Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.

GATA2 mutations have recently been reported in acute myeloid leukaemia (AML) patients with CEBPA-double mutations. To explore their impact on this favourablerisk disease, we determined GATA2 status in 153 sporadic AML patients and three members of a germ-line CEBPA-mutant family at AML presentation. Overall, 27% (15/55) CEBPA-double, 16% (7/43) CEBPA-single and 0% (0/55) normal karyotype/ CEBPA-wild-type patients were GATA2-mutant. All familial AML patients acquired both a second CEBPA and a GATA2 mutation. CEBPA and GATA2 mutant levels indicated that both mutations were likely to be early events in leukaemogenesis. GATA2 status did not impact on the favourable outcome of CEBPAdouble/ FLT3-inernal tandem duplication-negative patients.

Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis, but early-phase studies of the PI3K p110δ inhibitor GS-1101 have reported inferior responses in MCL compared with other non-Hodgkin lymphomas. Because the relative importance of the class IA PI3K isoforms p110α, p110β, and p110δ in MCL is not clear, we studied expression of these isoforms and assessed their contribution to PI3K signaling in this disease. We found that although p110δ was highly expressed in MCL, p110α showed wide variation and expression increased significantly with relapse. Loss of phosphatase and tensin homolog expression was found in 16% (22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110δ inhibition was sufficient to block B-cell receptor-mediated PI3K activation, combined p110α and p110δ inhibition was necessary to abolish constitutive PI3K activation. In addition, GDC-0941, a predominantly p110α/δ inhibitor, was significantly more active compared with GS-1101 against MCL cell lines and primary samples. We found that a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and this ratio increased significantly with relapse. These findings support the use of dual p110α/p110δ inhibitors in MCL and suggest a role for p110α in disease progression.

In lymph nodes, chronic lymphocytic leukemia (CLL) cells (prolymphocytes and paraimmunoblasts) form proliferation centers (PCs), which are also known as pseudofollicles. To reveal whether PCs play a role in the accumulation of genetic alterations in CLL, we compared deletion at 11q22.3, 13q14.3, and 17p13.1 loci and trisomy 12 by the fluorescence in situ hybridization (FISH) technique in PCs versus surrounding small lymphocytes (SLs) in 12 formalin-fixed paraffin-embedded (FFPE) lymph nodes. The FFPE sections were stained with methylene blue and PCs were marked by laser beam. Subsequent FISH analysis was performed, relocalizing the previously defined regions. Loss of 11q was detected in five cases, loss of 13q in two cases, loss of 17p in two cases, and trisomy 12 in one case. In seven cases PCs contained a significantly higher ratio of cells with genetic alterations compared with the surrounding SLs. Our results show that CLL cells with genetic alterations tend to accumulate in PCs. The clonal expansion of the cell population carrying genetic alterations within PCs may contribute to CLL progression.

Inherited risk determinants for follicular lymphoma (FL) have recently been described in the immune gene-rich human leukocyte antigen region on chromosome 6p. The known importance of host immune response to FL survival led us to evaluate these germline factors in FL outcome. We confirm the association of single nucleotide polymorphisms rs10484561 (P = 3.5 × 10−9) and rs6457327 (P = .008) with risk of FL and demonstrate that rs6457327 predicts both time to (P = .02) and risk of (P < .01) FL transformation independently of clinical variables, including the Follicular Lymphoma International Prognostic Index.

Introduction: Bruton tyrosine kinase inhibitor ibrutinib has altered the therapeutic landscape of chronic lymphocytic leukemia (CLL) with remarkable responses in relapsed or refractory CLL. Despite mostly durable responses, approximately 20% of patients experience disease progression with, in many cases, BTK or phospholipase Cg2 (PLCG2) resistance mutations predating the clinical progression by up to 15 months. Longitudinal studies have shed some light on the temporal aspects of clonal evolution processes leading to ibrutinib resistance, however, the spatial heterogeneity within the various environmental niches has not been described in detail yet. Here, we report the case of a CLL patient in which we are able to document, for the first time, an example of ibrutinib driven spatial convergent evolution leading to disease progression. The patient developed ibrutinib resistance after 21 months of treatment with simultaneous lymphadenomegaly and lymphocytosis. Sanger sequencing revealed a canonical BTK p.C481S mutation in peripherial blood and a PLCG2 p.D993H mutation in the lymph node with the absence of the BTK p.C481S mutation present in the peripherial blood suggesting convergent evolution in terms of the BTK and PLCG2 variants with both commonly associated with ibrutinib resistance. The patient was subsequently treated with venetoclax but due to further progression despite of high-dose salvage chemotherapy and autologous stem cell transplantation the patient died. Methods: Serial samples at ten different timepoints during the disease course were analysed. Genomic DNA was isolated from peripheral blood mononuclear cells and native lymph node tissue. Circulating cell-free DNA (ccfDNA) was isolated from peripheral blood samples. The fraction of tumor cells was determined by flow cytometry. During the Sanger sequencing BTK exons 11, 15, 16, and PLCG2 exon 12, 19, 20, 24, 27 and 30 were examined. Targeted ultra-deep next-generation sequencing (NGS) analysis of the BTK and PLCG2 genes was performed on a MiSeq platform (Illumina). Abundances of the PLCG2 p.P993H and BTK p.C481S variants were quantified using custom assays on a QX200 droplet digital PCR system (ddPCR, BioRad). Results: To dissect this spatial heterogeneity, BTK and PLCG2 mutations were screened by NGS which confirmed the Sanger finding and in addition to the dominant PLCG2 p.D993H variant, identified the BTK p.C481S mutation as a minor clone in the lymph node, with the BTK p.C481S mutation remaining the exclusive for the peripheral blood. Next, we tested the presence of these mutations in the circulating ccfDNA with ddPCR. Notably, both mutations were identified in the ccfDNA with a VAF of 1%. The ddPCR analysis revealed that both variants were absent in the pre-ibrutinib peripheral blood sample, however their emergence predated the clinical progression by 15 months. We observed a gradual expansion of these variants in the sequential samples, demonstrating clonal selection under the selective pressure of ibrutinib. Of note, the ddPCR assay identified the PLCG2 p.D993H mutation as a minor clone (VAF: 0.2%), previously unseen in the peripheral blood by NGS at the time of ibrutinib relapse. Intriguingly, the two subclones displayed differential sensitivity to the venetoclax therapy. In the peripheral blood, we observed a reduction of the BTK p.C481S positive subclone accompanied by expansion of the subclone harbouring PLCG2 p.D993H which was previously dominant in the lymph node. With these dynamic changes in the subclonal architecture, the disease progressed and ultimately led to death of the patient. Conclusions: Clonal evolution is a major driving force of disease progression in CLL. Several studies have traced the effect of treatment on temporal evolutionary trajectories in the context of standard and targeted therapies and identified subclonal heterogeneity and active clonal selection. However, the spatial aspect of subclonal dynamics has not been appreciated until recently. Here, we presented a unique case of CLL, developing ibrutinib resistance via multiple routes simultaneously, with the observed spatial heterogeneity leading to progression and aggressive transformation of the disease. This case highlights the importance of genetic profiling of multiple affected sites as investigations restricted to peripheral blood may underestimate the repertoire of clinically relevant genetic alterations. Disclosures No relevant conflicts of interest to declare.

Background: It has been known for approximately 19 years that germline mutations in RUNX1, lead to familial platelet disorder with predisposition to myeloid malignancy (FPD-MM, OMIM 601399). Since that time researchers have identified a broad range of different RUNX1 mutations, in over 100 families. In large families, the diagnosis of malignancy shows variable penetrance among family members with the same mutation; some carriers of RUNX1 mutations do not develop malignancy. The causes of this heterogeneity are currently not known, complicating counselling and risk analysis for individual carriers. Recent advances in genetic sequencing technology applied by many FPD-MM research groups around the world have highlighted their value in understanding the somatic genetic changes that are associated with development of malignancies in germline RUNX1 mutation carriers. Collectively this information could lead to powerful insights essential for more precise risk assessment, monitoring, and therapeutic intervention. Specifically, a growing catalogue of somatic mutations associated with germline RUNX1 malignancy offers the opportunity for informed monitoring of asymptomatic RUNX1 carriers for additional high-risk somatic mutations, in turn providing the possibility for early therapeutic intervention to arrest the leukemic process. The challenge in advancing these goals for FPD-MM is the relative rarity of the disorder in individual populations. Global data sharing in a highly interactive FPD-MM research community offers a solution to this problem that benefits all patients world-wide. Aims: To create a global RUNX1 network through identifying and contacting researchers and clinicians with known and novel germline RUNX1 families, seeking their collaboration to share genomics data. To create a RUNX1.db portal to collectively house and analyse genomics data from germline RUNX1 carriers, with associated phenotype and clinical information, such that researchers have an ongoing means by which to combine their data with those generated by other groups around the world. Methods: RUNX1 network : Through existing collaborative networks, a systematic review of the literature, and referrals from initial contacts, we have identified a global network of researchers managing FPD-MM cases and families. RUNX1.db: We have adapted a custom-built variant analysis platform, VariantGrid, that is a visual web application and database designed to help scientists manage and analyse DNA variants that can be used to aggregate and analyse multiple datasets. Results: Preliminary analysis of aggregated data from the literature suggests there are features of germline RUNX1 syndrome that can be ascertained. These include frequent somatic mutation of RUNX1 in malignancy development, as well as mutations in genes associated with clonal hematopoiesis that may precede development of overt leukemia. This analysis also suggests that different types of germline RUNX1 mutations may be associated with different combinations of somatic mutations in the tumour. To further this analysis, we have identified over 70 groups internationally that either manage RUNX1 families or have identified potential germline carriers through genomics initiatives. This represents a large and growing resource for both scientific studies and clinical programs to benefit individuals with FPD-MM. Many of these groups have generated NGS data sets from patient samples which will be available for analysis through this portal. Further details and activities of the network, and results from the genomics aggregation database will be presented. Conclusion: We have created a global RUNX1 network, aggregated their data, and generated a RUNX1.db genomics portal for the continuous curation of genomics data from germline RUNX1 carriers. A preliminary analysis has already identified specific features of germline RUNX1 mutated malignancies that have clinical importance. Ongoing scientific and clinical studies through the RUNX1 network will enhance the power of aggregated data analysis, with RUNX1.db providing a central link, driving new insights that benefit patients. Disclosures Natsoulis: Imago BioSciences, Inc.: Consultancy, Equity Ownership. Guzman:Cellectis: Research Funding. DiNardo:Bayer: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Abbvie: Honoraria. Borate:Novartis: Consultancy; Agios: Consultancy. Wei:Amgen: Honoraria, Other: Advisory committee, Research Funding; Pfizer: Honoraria, Other: Advisory committee; Celgene: Honoraria, Other: Advisory committee, Research Funding; Abbvie: Honoraria, Other: Advisory board, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Other: Advisory committee, Research Funding; Novartis: Honoraria, Other: Advisory committee, Research Funding, Speakers Bureau. Dokal:MRC, Bloodwise, Telomerase Activator Sciences: Research Funding; The Gary Woodward Dyskeratosis Congenita Trust: Membership on an entity's Board of Directors or advisory committees; Action Medical Research, European School of Haematology: Membership on an entity's Board of Directors or advisory committees; Barts and The London School of Medicine and Dentistry, Queen Mary University of London,: Employment, Research Funding; Telomerase Activator Sciences: Research Funding; Barts and The London, Queen Mary University of London: Employment; Gary Woodward Dyskeratois Congenita Trust: Membership on an entity's Board of Directors or advisory committees. Hiwase:Novartis: Research Funding; Celgene: Research Funding. Branford:Cepheid: Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Owen:Merck: Honoraria; Teva: Honoraria; AbbVie: Research Funding; Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Research Funding; AstraZeneca: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding. Fitzgibbon:Epizyme: Consultancy, Research Funding; Gilead: Consultancy. Rienhoff:Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kurokawa:Astellas Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Nippon Sinyaku: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Eizai: Research Funding; MSD: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Pfizer: Research Funding; Takeda Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Teijin Pharma: Research Funding; Chugai Pharmaceutical: Research Funding.

In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.

Background : While myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are considered sporadic hematopoietic stem cell clonal disorders, there are rare occurrences of familial cases ( Aims:To investigate the molecular mechanisms underlying the variable penetrance and clinical heterogeneity observed in a GATA2-mutated family. Results:Targeted deep-sequencing of 33 genes frequently mutated in MDS/AML revealed a low overall burden of acquired mutations in the symptomatic carriers with no mutations detected in asymptomatic family members. It was noteworthy that an acquired ASXL1 mutation (p.Gly646TrpfsTer12) was identical in all affected individuals (III.1, III.3 and III.7) (Figure 1) although the variant allele frequency was lower (12%) in III.7 and remained stable (range 12-6%) over a 4 year monitoring period. GATA2 expression was lower in III.7 as assessed by quantitative RT-PCR and strikingly this was associated with monoallelic expression of the mutated GATA2 allele with complete loss of the wild-type (WT) allele expression. Temporal analysis of III.7 at yearly intervals demonstrated reactivation of the WT allele 2 years later, coinciding with a marked improvement in hematological parameters (normal monocyte count, neutrophils >1x109/L). These changes in GATA2 expression were not linked to gross changes in methylation, as assessed by methylation specific PCR and bisulphite sequencing, nor acquisition of additional mutations in the WT promoter. Instead, we believe that allele-specific fluctuations in expression are accompanied by changes in chromatin structure at the promoter. Using a SNP (rs1806462 [C/A]) located in the 5'UTR of GATA2, we assessed allele-specific enrichment of H3K4me3 and H3K27me3 chromatin marks by chromatin immunoprecipitation. Sanger sequencing revealed a significant enhancement in the deposition of H3K4me3 activating chromatin mark on the mutated allele compared to the WT allele at diagnosis and this was reversed at later follow-up, correlating with reactivation of the WT allele expression. There were no discernible allele-specific differences in the H3K27me3 mark across the phenotypes at different time-points. Conclusion: Variable penetrance amongst germline mutation carriers is a feature of many families with inherited forms of MDS/AML and this may be related to the nature of secondary genetic events acquired in at-risk individuals. In this study, however, we show that changes in the WT:mutant allele expression ratio as a result of local and allele-specific changes in chromatin deposition may also influence the penetrance of the inherited mutation. Figure 1 Figure 1. Disclosures Cavenagh: Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Tyrosine kinase inhibitors specific for BCR-ABL, were a major breakthrough in CML therapy. Second generation tyrosine kinase inhibitors (dasatinib, nilotinib) are indicated for imatinib resistant and intolerant patients. Present guidelines recommend continuous drug dosing for maintaining remission. There is no available data concerning the optimal duration of dasatinib therapy. We report the case of an imatinib intolerant patient who succeeded a complete molecular remission with dasatinib. Dasatinib was stopped bacause of intolerance, but complete molecular remission was sustained for one year and minor molecular remission for 27 months after discontinuation of dasatinib.

While most myelodysplastic syndrome/acute myeloid leukemia cases are sporadic, rare familial cases occur and provide some insight into leukemogenesis. The most clearly defined familial cases result from inherited mutations in RUNX1 or CEBPA. Recently, novel germline mutations in GATA2 have been reported. We, therefore, investigated individuals from families with one or more first-degree relatives with myelodysplastic syndrome/acute myeloid leukemia with wild-type RUNX1 and CEBPA, for GATA2 mutations. Screening for other recurrent mutations was also performed. A GATA2 p.Thr354Met mutation was observed in a pedigree in which 2 first-degree cousins developed high-risk myelodys-plastic syndrome with monosomy 7. They were also observed to have acquired identical somatic ASXL1 mutations and both died despite stem cell transplantation. These findings confirm that germline GATA2 mutations predispose to familial myelodysplastic syndrome/acute myeloid leukemia, and that monosomy 7 and ASXL1 mutations may be recurrent secondary genetic abnormalities triggering overt malignancy in these families.

Recent studies have identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non-Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B-cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright © 2010 John Wiley & Sons, Ltd.

Background Follicular lymphoma is characterized by the t(14;18) translocation resulting in constitutive expression of BCL-2 protein; however approximately 10–15% of follicular lymphomas do not express BCL-2 protein, and a small fraction of these cases does not exhibit translocation of the BCL-2 gene either. It is highly debated whether cases of follicular lymphoma without BCL-2 gene rearrangement and expression represent a separate lymphoma entity with distinct biological characteristics, different from the BCL-2-positive cases. Design and Methods To further characterize follicular lymphomas without BCL-2 gene rearrangement and expression, we analyzed and compared the mutational status of IGVH genes as well as other genes ( C-MYC , PAX-5 and RHOH ) frequently involved in the specific type of genomic instability called aberrant somatic hypermutation in 11 cases of BCL-2-negative and 7 cases of BCL-2-positive follicular lymphomas. We also determined the levels of expression of activation-induced cytidine deaminase in these cases. Results The analyzed cases were grade 2 and grade 3A follicular lymphomas. Our findings demonstrate that follicular lymphomas without BCL-2 gene rearrangement and expression are associated with ongoing somatic hypermutation of the IGVH genes, low activity of aberrant somatic hypermutation and elevated activation-induced cytidine deaminase expression. These results were in concordance with the results found in the cases of BCL-2-positive follicular lymphoma. Conclusions Although, BCL-2 protein overexpression is considered to be a critical pathogenic event in the development of follicular lymphoma, our findings suggest that follicular lymphomas with the same morphology, immunophenotype, mutational pattern and activation-induced cytidine deaminase expression may develop without the involvement of BCL-2 gene. The present data support the hypothesis that BCL-2-positive and BCL-2-negative follicular lymphomas (grades 1-3A) represent a homogenous group with different initial but several common additional molecular pathways.

Chronic lymphocytic leukaemia (CLL) with increased prolymphocytes (CLL/PL) has been defined by the World Health Organization (WHO) classification and considered as a progressive and clinically aggressive variant of CLL. To further characterize the biological features of this disease, we performed IgVH gene mutational status, FISH and high-resolution comparative genomic hybridization (HR-CGH) analysis in 17 cases of CLL/PL. All CLL/PL utilized members of VH1, VH3 and VH4 families, with the highest prevalence of the VH1-69 gene. In all but one cases analyzed, the VH genes were unmutated. The FISH and HR-CGH analyses showed frequent occurrence of trisomy 12, del(11)(q23), del(17)(p13) and genetic imbalances, but recurrent genetic lesion characteristic for CLL/PL was not found. The follow-up HR-CGH analysis of two cases showed that increase of prolymphocytes in the course of CLL or CLL/PL are associated with clonal evolution and selection of the tumour clone. In conclusion, this study suggests that CLL/PL is a relatively homogeneous disease regarding VH gene mutation, but heterogeneous regarding genetic lesions. The heterogeneity and high number of genomic imbalances found in CLL/PL suggest that a genome-wide instability of the neoplastic cells may play a role in the development of the disease.

To determine the possible role of aberrant somatic hypermutation (ASHM) and activation-induced cytidine deaminase (AID) expression in the pathogenesis of mediastinal large B-cell lymphoma (MBL), the mutational status of genes affected by ASHM, including c-MYC, PAX-5 and RhoH, was analysed, and the expression level of AID mRNA in tumour specimens from six patients with MBL was determined. Mutations in one or more genes and high expression of AID mRNA were detected in all the six cases of MBL. These results suggest that ASHM and AID expression may have a role in the pathogenesis of MBL.

Background Germline mutations in the N-terminal of CCAAT/enhancer binding protein α (CEBPA) are a feature of autosomal dominant AML. Despite the strong penetrance of these mutations, the age of disease onset varies considerably and is usually precipitated by acquiring a C-terminal mutation. Although biallelic CEBPA-mutations in sporadic AML are associated with favorable clinical outcomes, little is known about long-term survival or the secondary molecular events linked with familial cases. Aims We sought to establish the long term clinical outcomes in familial CEBPA-mutated AML and to examine the patterns of secondary mutations associated with leukemic transformation. Methods and Results Disease specific and follow up information was collected in 16 affected patients, from 7 pedigrees, published between 2004 and 2011. In 94% of patients (n=15), at least 3 years follow up was achieved. All pedigrees had a germline N-terminal CEBPA mutation and 17 of 18 documented disease episodes had an acquired C-terminal mutation. The age at AML diagnosis varied from 2-39 years (median 24.5 yrs) with a single asymptomatic carrier detected (now 23 yrs). With the exception of 1 patient diagnosed in 1963, all cases received combination chemotherapy at diagnosis. Additional consolidation comprised autologous stem cell transplantation (SCT, n=3) and allogeneic SCT in 1 patient failing to achieve CR post induction therapy. Ten patients had at least 1 further disease episode, the first recurrence presenting after a median of 2.1 years (range 0.5-14 yrs), 5 continued in CR and 1 patient was lost to follow up. In 3 out of 4 patients, where CEBPA was screened at recurrence, the acquired C-terminal mutations differed from diagnosis, signifying new episodes of AML. The median overall survival (OS) for the entire cohort was 20 years (1.1-46 yrs, n=16) and 17.3 years for patients with multiple disease episodes, reflecting durable responses to second line therapies. To identify potential co-operating mutations in CEBPA pedigrees, whole exome sequencing (WES) was performed in 7 tumor samples from 5 patients across 3 pedigrees, all with the germline mutation p.P23fs (Figure 1). All tumor DNA samples were sequenced with matched remission or skin DNA as a germline control. The number of acquired mutations in familial tumors was similar to sporadic disease, with 10-22 (median=14) non-synonymous tier 1 mutations per sample. In addition to the acquired C-terminal CEBPA mutation, these included established AML loci such as EZH2, TET2, WT1, GATA2, NRAS, CSF3R and the recently identified cohesin complex gene, SMC3. Of note, FLT3-ITD, NPM1 and DNMT3A mutations were absent in all tumors. A minimum of 2 established mutations were identified in each tumor and, at present, we can only speculate on which additional mutations are ‘driver' or ‘passenger' events. Reflecting findings in sporadic AML, biallelic CEBPA and GATA2 mutations co-occurred in both siblings from Pedigree 1 and were subsequently identified by Sanger sequencing in the child III.2 (Figure 1). All 3 patients continue in long term remission following chemotherapy. We were able to trace the clonal evolution in patient I.2 (Pedigree 3) by WES of 3 consecutive tumor samples which arose over a 17 year period. At diagnosis (Dx) the patient received induction and consolidation chemotherapy and remained disease free for 14 years. The second disease episode (R1) was treated with chemotherapy followed by autologous SCT and the third presentation (R2) was chemo-refractory. Tumor DNA from R2 was clonally related to Dx, sharing 7 identical mutations, including the original C-terminal CEBPA deletion. In contrast, R1 appeared molecularly distinct from Dx and R2, most likely representing a new clone which was subsequently eradicated with treatment. Conclusion This is the first report of long term clinical outcomes in familial CEBPA-mutated AML. Although many patients experienced disease recurrence, our extended follow up showed that OS remained favorable despite multiple episodes of disease. Assessment of C-terminal CEBPA mutations provided a unique insight into the recurrence of AML, with some patients appearing to develop completely new leukemic episodes. Although the penetrance of germline mutations is high, healthy carriers and late onset disease are noted, emphasizing the need for clinical vigilance and screening of all related potential SCT donors. Disclosures: No relevant conflicts of interest to declare.

Abstract 1652 Background: Activation of the phosphoinositide-3 kinase (PI3K) pathway contributes to mantle cell lymphoma (MCL) pathogenesis, but early phase studies of the p110δ selective inhibitor GS-1101 demonstrate inferior responses in MCL compared to CLL and indolent NHL. The relative importance of the class Ia PI3K isoforms - p110α, p110β and p110δ in MCL is not clear. While p110δ is enriched in leucocytes, p110α gene amplification has been reported in 68% of MCL. Loss of PTEN expression occurs in approximately 15% of MCL and evidence in solid tumors suggests an essential role for p110β in PI3K signaling associated with PTEN loss. We previously reported significant up-regulation of p110α expression in relapsed disease as well as significantly greater in vitro cytotoxicity with GDC0941, a predominantly p110α/δ inhibitor, compared to the p110δ selective inhibitor GS-1101 in MCL. To investigate this further, we used isoform selective inhibitors to study the relative contribution of class Ia isoforms to PI3K signaling in the context of B-cell receptor (BCR) activation, p110α over-expression and PTEN deletion in this disease. We also screened tumor cells for activating mutations in PIK3CA and PIK3R1, the genes encoding p110α and p85α respectively. Methods: Peripheral blood mononuclear cells (PBMCs) and lymph nodes from MCL patients were used in addition to two MCL cell lines Jeko1 and Granta519. Goat anti-human IgM f(a'b) fragments were used for BCR stimulation. Immunohistochemistry (IHC) and western blotting were used to assess PTEN expression. In addition to GDC0941 and GS-1101, A66 was used for p110α-selective inhibition and TGX-221 for p110β inhibition. GS-1101 was purchased from Active Biochem and all other inhibitors from Selleck Chem. Changes in downstream targets of PI3K (p-Akt t308, p-Akt s473 and p-S6 s235/236) were evaluated using western blotting. DNA was extracted from 10 primary samples and 2 cell lines. Exons 9, 10, 11, 13, 15 and 16 of PIK3R1 and exons 9 and 20 of PIK3CA, were sequenced and screened for activating mutations. Results: p110δ is expressed at high levels in MCL and p110δ selective inhibition with GS-1101 was sufficient to block BCR-stimulated activation of the PI3K pathway in MCL cells. In order to investigate the relevance of increased p110α in relapsed MCL, that we previously reported, we treated the Granta519 MCL cell line, which exhibits p110α gene amplification and constitutive Akt phosphorylation, with GS-1101 and GDC0941. In GS-1101 treated cells, phosphorylation of Akt was still detectable at low levels after 2 hours of treatment while p-Akt was undetectable in cells treated with equimolar concentrations (1μM) of GDC0941. At 24 hours, GS-1101 treated cells showed an increase in Akt phosphorylation compared to 2 hour levels suggesting ongoing p110α mediated signaling despite p110δ inhibition, whereas p-Akt remained undetectable in GDC-0941 treated cells. These changes were reflected in downstream phosphorylation of ribosomal S6. The addition of the highly selective p110β inhibitor TGX221 to GS-1101 did not have any additional effect on p-Akt whereas adding a p110α selective inhibitor (A66) mimicked the activity of GDC0941, further supporting the role of p110α in p110δ-independent PI3K signaling. We found loss of PTEN expression in 17% (25/147) of MCL biopsies by IHC. There was no change in the pattern of class Ia isoform expression in tumors with loss of PTEN and adding a p110β inhibitor to GS-1101 did not increase cytotoxicity in MCL primaries that had loss of PTEN expression. No activating PIK3CA or PIK3R1 mutations were detected in 10 primary MCL samples and 2 cell lines. Conclusion: In healthy B-cells, p110δ is essential for BCR mediated PI3K signaling but both p110α and p110δ contribute to tonic PI3K signaling. Our studies confirm a similar essential role for p110δ in MCL cells both by its expression and its role in BCR signaling. However, over-expression of p110α in MCL, especially in relapsed disease, increases the contribution of this isoform to tonic PI3K signaling thereby limiting the efficacy of p110δ-selective inhibition. The increased expression of p110α with relapse may reflect reduced dependence of tumor cells on survival signals from their microenvironment. We conclude that whereas PI3K p110β does not appear to play a significant role in MCL, inhibition of both p110α and δ isoforms appears essential for effective PI3K inhibition in this disease. Disclosures: Auer: Pharmacyclics: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Abstract 145 Follicular lymphoma (FL) displays a heterogeneous clinical course with an initial good response followed by multiple relapses and the need for recurrent therapeutic interventions. Histological transformation (tFL) to a more aggressive phenotype remains a critical event in a subset of FL patients (20–30%) and is associated with poor clinical outcomes. Genetic profiling of serial FL biopsies and published cases of donor-derived FL in recipients of bone marrow transplantation suggest that the FL clones detected at diagnosis, relapse and transformation may arise by divergent evolution from a common progenitor B cell (CPC). An understanding of the clonal dynamics during disease progression together with the genetic events present at each time point will prove important for monitoring and effectively targeting these tumors. We performed whole genome sequencing (WGS) using 100bp paired-end reads on tumor DNA of lymph node (LN) biopsies from 6 patients who were selected on the basis of availability of a FL, subsequent tFL biopsy and paired germline sample (8 FL, 6 tFL, 6 germline). All 6 cases had received at least one line of therapy achieving a complete remission (CR) prior to undergoing histological transformation (number of therapies: 2–6; number of relapses: 1–6), with a mean time to transformation of 8.6 years (range: 1.6–15.6 yrs) from diagnosis. Tumor and germline genomes were sequenced at an average coverage of 37× of mapped individual reads. Our analysis identified mutations in the coding and non-coding (5' and 3' UTR, miRNA, lincRNA and CpG islands) regions of the FL and t-FL genome. Variants were validated by Sanger sequencing. Mutations were detected in the coding region of 587 genes. Each tumor had an average of 72 somatic mutations (range: 41–143) with higher frequencies in the tFL genomes (mean: FL 64.8; tFL 83.6), indicating ongoing genetic evolution with disease progression. In addition to identifying somatic mutations in previously described genes involved in histone methylation (MLL2, EZH2) and histone acetylation (CREBBP, MEF2B), we also report mutations in other chromatin regulators including linker and core histones (HIST1H1 B-E, HIST1H2BG) that facilitate folding of chromatin into higher order structure and ZMYM3, a regulator of gene expression at the H3K4me3 mark. Interestingly, all biopsies showed co-occurrence of mutations in MLL2 with at least one additional chromatin regulator, and frequent acquisition of novel epimutations at transformation. Moreover, we observe recurrent mutations in genes involved in the NF-kB pathway, B cell receptor signaling, B cell development, DNA repair and apoptosis regulation. In all cases, we show an overlap of somatic variants (based on comparisons between non-synonymous, synonymous and UTR variants) between the paired FL/tFL biopsies, although this clonal relationship varied across the patients (range: 7.0–86.9%; mean 52.4%). The overall pattern of FL evolution to transformation was consistent with the existence of a shared common origin, the CPC (Figure 1). We did not identify any compelling single mutational event that was restricted to the tFL ‘branch’ that could immediately be linked with transformation. However, the ‘truncal’ CPC mutations present in a patient's biopsies highlighted key biological pathways that are commonly deregulated in both FL and tFL. Although the existence of such complex patterns of heterogeneity and progression has cast doubt on the possibility of developing personalized therapies capable of targeting multiple variants simultaneously in solid tumors, the detection of recurring events common to FL, tFL and the CPC clone in our study, suggests that a stratified medicine approach targeting these specific pathways in FL warrants consideration. Figure 1: Clonal divergence of follicular lymphoma in patient 2 illustrating the presence of shared ‘truncal’ and unique ‘branching’ somatic mutations at various tumour time points. The numbers above each branch indicate the number of non-synonymous mutations. CPC, common progenitor cell; GL, germline; FL-1, 1st relapse; tFL, transformed FL Figure 1:. Clonal divergence of follicular lymphoma in patient 2 illustrating the presence of shared ‘truncal’ and unique ‘branching’ somatic mutations at various tumour time points. The numbers above each branch indicate the number of non-synonymous mutations. CPC, common progenitor cell; GL, germline; FL-1, 1st relapse; tFL, transformed FL Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Merck: Honoraria; Mundipharma: Honoraria; Pharmacyclics: Honoraria.

In lymph nodes the chronic lymphocytic leukaemia (CLL) cells (prolymphocytes and paraimmunoblasts) form pseudofollicles (PFs) which are also known as proliferation centers. To reveal whether PFs play a role in generation of genetic alterations in CLL we compared the cytogenetic prognostic markers by fluorescent in situ hybridization (FISH) technique in PFs versus surrounding small lymphocytes (interfollicular areas - IFs) in 12 formalin-fixed paraffin-embedded (FFPE) lymph nodes. The FFPE sections were stained with methylene blue and PFs were marked by laser beam. Subsequent FISH analysis of deletion at 11q22.3, 13q14.3 and 17p13.1 loci and trisomy 12 was performed relocalizing the previously defined regions. Loss of 11q was detected in 5 cases, loss of 13q in 2 cases, loss of 17p in 2 cases and trisomy 12 was found in 1 case. In 7 cases PFs contained significantly higher ratio of cells with genetic alterations than the surrounding IFs. Our results revealed higher rate of genetic abnormalities in PFs suggesting that these unique compartments play a role in generation or accumulation of genetic abnormalities. The accumulation of genetic lesions in PFs denotes that genetic progression and/or transformation of CLL may begin in these proliferation centers.

The mutational status of the immunoglobuin heavy variable (IGHV) genes is an undisputable strong prognostic factor that subdivides patients with chronic lymphocytic leukemia (CLL) into 2 subgroups, i.e. IGHV-unmutated CLL (U-CLL) and IGHV-mutated CLL (M-CLL). U-CLL and M-CLL have distinct landscapes of genomic aberrations as well as distinct prognosis, since U-CLL is considerably more aggressive than M-CLL. That said, there is considerable clinical heterogeneity among M-CLL patients, ranging from patients without need of treatment to patients requiring early therapeutic intervention, indicating the need to further refine prognosis in this subgroup. In recent years, it has become evident that the prognostic impact of genomic aberrations may differ depending on IGHV gene mutational status. Hence, defining genomic aberrations with prognostic impact in M-CLL patients may help identifying patients with an predicted unfavorable prognosis within this subgroup, with obvious implications for management of follow up and therapy choice. To study the clinical impact of recurrent gene mutations in relation to IGHV gene mutational status, we collected a large, multi-center cohort including 4,674 patients with CLL [median age at diagnosis, 64.5 years; male/female, n=2,962 (63%)/n=1,712 (37%); Binet stage A (n=3,369, 74%), B (n=827, 18%), and C (n=387, 8%); IGHV-mutated (M-CLL, n=2,498, 56%) and IGHV-unmutated (U-CLL, n=1,927, 44%); isolated del(13q) (n=1,868, 41%), trisomy 12 (n=571, 13%), del(11q) (n=503, 11%), and del(17p) (n=249, 5.5%); treated (n=2,745, 59%) and untreated (n=1,929, 41%)] and performed next-generation sequencing (NGS) and/or Sanger sequencing of 9 genes (BIRC3, EGR2, NFKBIE, MYD88, NOTCH1, POT1, SF3B1, TP53, and XPO1) on pre-treatment samples. Overall, pathogenic mutations in any of these genes were detected in 1720/4674 patients (36.8%, using a variant allele frequency cutoff of 5% for NGS), while the remaining patients were wildtype; 2 mutations were observed in 361 patients (7.7%) and 3 or more mutations in 58 patients (1.2%). The mutation frequency for the individual genes was: TP53 (10.4%, including TP53 mutations and/or del(17p)), NOTCH1 (10.1%, 3'UTR mutations not included), SF3B1 (9.3%), XPO1 (3.9%), POT1 (3.8%), NFKBIE (3.7%), BIRC3 (3.0%), EGR2 (2.5%) and MYD88 (2.5%; Figure 1A). Except for MYD88, gene mutations in each of the investigated genes were associated with significantly shorter time-to-first-treatment (TTFT) in univariate analysis. In multivariate analysis of Binet stage A patients (n=3,369; including all genes, IGHV gene mutational status, age at diagnosis and gender), SF3B1 (Hazard Ratio (HR) 1.79; p In summary, different spectra of genetic mutations independently predicted short TTFT in M-CLL and U-CLL, respectively, with SF3B1 mutations as the only aberration found to be significant predictor of shorter time to first treatment in both subgroups. Importantly, mutations within several genes (i.e. SF3B1, NOTCH1, XPO1 and NFKBIE) identified patients in the M-CLL subgroup with a high-risk profile; conversely, TP53 mutations did not affect TTFT in this subgroup. On these grounds, we suggest to include analysis of recurrent gene mutations to identify high-risk patients within the M-CLL subgroup. Figure 1 Figure 1. Disclosures Brieghel: AstraZeneca: Consultancy. Rossi: Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Scarfo: Astra Zeneca: Honoraria; Abbvie: Honoraria; Janssen: Honoraria, Other: Travel grants. Mattsson: Gilead: Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Martinez-Lopez: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Serna: AbbVie, AstraZeneca, Beigene, Gilead, GSK, Janssen, Jazzpharma, Novartis, Roche: Consultancy; ABBVIE, ASTRAZENECA,ROCHE: Research Funding; AbbVie, AstraZeneca, Roche: Speakers Bureau. Hernández Rivas: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Smedby: Jansen-Cilag: Other: part of a research collaboration between Karolinska Institutet and Janssen Pharmaceutica NV for which Karolinska Institutet has received grant support. Bullinger: Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Hexal: Consultancy; Janssen: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Menarini: Consultancy; Novartis: Consultancy, Honoraria; Amgen: Honoraria; Astellas: Honoraria; Sanofi: Honoraria; Seattle Genetics: Honoraria; Bayer: Research Funding. Bosch: TAKEDA: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel. Terol: BMS: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Roche: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hospital Clinico Valencia: Current Employment. Cuneo: AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau. Gaidano: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemann: Novo Nordisk Foundation: Research Funding; CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding. Ghia: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sunesis: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria.