ALBERT

Description: Background: The clinical course in CLL is very heterogeneous ranging from stable disease to a rather rapid progression requiring treatment. The acquisition of genetic abnormalities termed clonal evolution (CE) is likely to correlate with clinical progression and might be used to guide treatment strategies. Aim: The aim of this study was to evaluate the frequency of CE on the cytogenetic (CCE) and molecular genetic (MCE) levels and its association with the IGHV mutation status and clinical outcome. Methods: 179 CLL cases were selected on the basis that chromosome banding analysis (CBA) and mutation analyses in TP53 and SF3B1 all having been performed at least at two time points. The median age at first evaluation was 72 years (range: 46-95). The first time point of analysis was at primary diagnosis (n=131) or during course of disease but prior to any treatment (n=48). In all patients interphase FISH was performed with probes for 17p13 (TP53), 13q14 (D13S25, D13S319, DLEU), 11q22 (ATM), and the centromeric region of chromosome 12 and the IGHV mutation status was evaluated. A total of 465 CBA, 417 TP53 and 424 SF3B1 mutation analyses were evaluated. The median number of samples per patient was 2 (range: 2-9). The time between samples ranged from 1 month to 9.8 years (median 21 months). For all patients clinical follow-up data was available with a median follow-up of 7.4 years and 5-year overall survival (OS) of 88%. Results: At first investigation CBA revealed a normal karyotype in 31 (17%) patients. In cases with an aberrant karyotype the pattern of abnormalities was typical for CLL: del(13q); 51% (homozygous: 15%), +12: 18%, del(11q): 16%, and del(17p): 5%. A complex karyotype (≥3 abnormalities) was present in 18%. The IGHV status was unmutated (IGHV-U) in 56% of cases and TP53 and SF3B1 mutations were detected in 10% and 15%, respectively. CCE was observed in 63/179 patients (35%). The median time to CCE was 46 months (range 3-111). The most frequent abnormalities gained during CCE were loss of 17p (14/63; 22%), 13q (11/63; 18%), and 11q (10/63; 16%). Acquired loss of 17p was more frequent in SF3B1mutated CLL (19% vs 6%, p=0.04). MCE was observed in 29/179 cases (16%). TP53 and SF3B1 mutations were acquired during the course of the disease in 23 (14%) and 7 (5%) cases, respectively. The median time to MCE was 61 months (range 1.5-109). Of note, in 2 cases with TP53 deletion a TP53 mutation was acquired and in 2 cases with TP53 mutation a TP53 deletion was acquired. In 12 CLL both a TP53 deletion and a TP53 mutation were acquired (table). CCE and MCE were significantly associated with IGHV-U (p=0.003; p

Description: Background: The simultaneous detection of a BCR-ABL1 rearrangement and a JAK2V617F mutation in the same patient is a very rare event and has previously been described in case reports or very small series of cases only. Aim: 1) To establish the incidence of cases with concurrent BCR-ABL1 rearrangement and JAK2V617F mutation. 2) Evaluate whether one clone harbours both mutations or whether there are two independent clones. 3) Establish whether these patients have additional concurrent gene mutations and how they influence the evolution of both diseases. Patients and Methods: A total of 27,907 patients with suspected myeloproliferative neoplasms (MPN) where studied in parallel for BCR-ABL1 and JAK2V617F mutation from May 2005 to June 2014 at our institution. BCR-ABL1 analysis was performed by multiplex RT-PCR and JAK2V617F mutation analysis by melting curve based LightCycler assay. A total of 23 patients (0.08%) were positive for both mutations. Eleven patients were male and 12 were female with a median age at diagnosis of 72 years (range 46-80 years). Of fifteen patients 2 or more sample time points were available for follow-up analyses (median follow-up: 4 years, range: 5 months - 9 years). Both BCR-ABL1 and JAK2V617F mutation loads were assessed by quantitative real time PCR. In addition, 22/23 cases were analyzed upon detection of co-occurrence of both clones with a pan-myeloid gene panel consisting of 25 genes: TET2, RUNX1, PHF6, ASXL1, CBL, DNMT3A, SF3B1, TP53, BCOR, BRAF, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, KIT, KRAS, MPL, NPM1, NRAS, SRSF2, U2AF1, and WT1. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sequence NGS platform (Roche 454, Branford, CT). The median coverage per amplicon was 2,215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. Results: At the time point of detection of both mutations morphological assessment was available in 12 patients. The remaining 5 showed features typical for CML. Bone marrow blast count was

Description: Background: Minimal/measurable residual disease (MRD) correlates with outcome in acute myeloid leukemia (AML). Flow cytometry (FC) and quantitative PCR (QPCR) both are recommended by ELN (Schuurhuis et al., Blood 2018) and NCCN guidelines. Comparative MRD data on FC and QPCR are limited. Aim: To assess the relative contribution of FC and QPCR in generating prognostically relevant MRD data in AML. Methods: 936 bone marrow samples of 305 AML patients in cytomorphologic CR were analyzed for MRD by both FC and QPCR (mainly fusion transcripts and NPM1 mutations) in parallel applying ELN guidelines. Patients´ages at diagnosis ranged from 20 to 89 years (median 57), sex ratio (f/m) was 155/150. As anticipated due to selection for MRD assessment risk according to ELN was favorable in 191 (63%), intermediate in 92 (30%) and poor in 18 (6%). Accordingly, 5 year event-free survival (EFS) was 68% and 5 year overall survival (OS) 68% at 2.4 years median follow-up. Samples for MRD assessment were drawn between 7 days and 8.5 years after diagnosis (median 5.8 months). MRD samples were categorized into 5 intervals: I1, 2 years, 148 (16%). Results: First, we focused on the percentages of MRD as determined by FC (%FC). %FC ranged from 0% to 5% (median, 0.003%). Overall, %FC strongly correlated with EFS (p

Description: Introduction: CEBPA mutations (mut) occur in 5-14% of patients with acute myeloid leukemia (AML). A difference in prognosis between CEBPA single- (sm) and CEBPA double-mutated (dm) cases has been reported with longer overall and event-free survival in dm cases. This may be due to differences in the amount and composition of concomitant mutations. Aims: 1) Elucidate the cause of the clinical difference between CEBPAsm and dm cases. 2) Evaluate the clonal architecture of CEBPAmut AML in a well characterized cohort (Fasan et al., Leukemia 2014). Patients: We investigated concomitant mutations in a cohort of 229 CEBPAmut AML cases (129 sm and 100 dm). The cohort was composed of 113 females and 116 males, median age was 67.1 y (range: 15.7-87.6 y). 218 cases showed intermediate risk (n=162 normal and n=56 aberrant karyotype), 11 cases adverse risk cytogenetics. Results: CEBPAdm (median age 56.8 y, range 15.7–87.6 y) were significantly younger compared to sm (median age 64.7 y, range 20.4–87.0 y, p=0.001). Furthermore, CEBPAdm occurred significantly more often in females than in males (61.0% vs 39.0%; p=0.002). With regard to cytomorphology and cytogenetics, no significant differences between CEBPAsm and dm cases were seen. Concomitant mutations were present in significantly more CEBPAsm cases compared to CEBPAdm cases (93.0% vs 79.0%; p=0.003). Furthermore, the amount of additional mutations was higher in CEBPAsm (mean: 2.1 mutations, range: 1-6) compared to CEBPAdm (1.3 mutations, range: 1-4). ASXL1 (p=0.009), DNMT3A (p

Description: Abstract 1595 Background: Immunohistochemistry for Cyclin D1 (CCND1) expression is routine in cases suggestive of mantle cell lymphoma (MCL). Most MCL are t(11;14)/IGH-CCND1-positive by FISH. PCR based detection of the fusion transcript is hampered by widespread breakpoints. Only few data is available on quantitative real-time PCR (RQ-PCR) for CCND1 expression measurement. Aims: To assess CCND1 mRNA expression and correlate it with t(11;14) in mature B-cell neoplasms. Methods: We established a RQ-PCR assay for CCND1 mRNA measurement and investigated 451 cases: 142 MCL (in all cases IGH-CCND1 confirmed by FISH), 76 chronic lymphocytic leukemia (43 typical CLL, 33 CLL/PL), 20 hairy cell leukemia (HCL), 13 hairy cell leukemia-variant (HCL-v), 20 splenic marginal zone lymphoma (SMZL), 91 other mature B-cell neoplasms. CCND1 background expression was assessed in 29 pts with other hematological neoplasms and 60 healthy individuals. FISH and/or chromosome banding analysis for the t(11;14) was available in 364 pts. Bone marrow (BM, n=267) or peripheral blood (PB; n=184) samples were analyzed by cytomorphology, multiparameter flow cytometry (MFC), FISH, and RQ-PCR. CCND1 mRNA expression was given by RQ-PCR in comparison to ABL1 mRNA expression (%CCND1/ABL1). Limited dilution of high expressers into cDNA of healthy controls revealed a sensitivity of the assay of up to 0.1 %. Results: IGH-CCND1 translocation carriers had higher %CCND1/ABL1 than those without which hold true in the total cohort (mean±SD, 420.4±740.3 vs 17.8±128.3; p

Description: Guiding antileukemic treatment in patients with acute myeloid leukemia (AML) is increasingly based on levels of minimal residual disease (MRD) which can be quantified with high sensitivity by multiparameter flow cytometry (MFC). The optimum checkpoint for determination of MRD during the course of therapy, however, has not yet been determined. We applied MFC using a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIPs) at diagnosis and to quantify MRD by individually selected antibody combinations. The prognostic impact of MRD levels was assessed in comparison to cytogenetics and age. Patients received double induction, consolidation, and maintenance therapies and underwent allogeneic stem cell transplantation if they were younger than 60 years and had a matched related donor. In 286 patients with newly diagnosed and untreated AML MFC-based assessment for the presence of LAIP has been performed. The median percentage of LAIP-positive bone marrow cells at diagnosis was 16.04% (range, 2.54%–76.14%). All individual LAIPs were applied to 26 normal bone marrow samples to estimate sensitivity based on the median percentages of LAIP-positive normal bone marrow cells which ranged from 0.00% to 1.01% (median, 0.02%). A total of 550 follow-up samples has been analyzed in these patients at different checkpoints (CP1, up to day 21 after start of therapy, n=85; CP2, day 22–60, n=122; CP3, day 61–120, n=158; CP4, day 121–365, n=137; CP5, after day 365, n=48). In order to adjust for differences in the percentages of LAIP-positive bone marrow cells at diagnosis the logarithmic difference (LD) between diagnosis and follow-up was calculated for each follow-up sample. The median LDs at the respective checkpoints were: CP1, 2.02; CP2, 2.29; CP3, 2.39; CP4, 2.53; and CP5, 2.81. Separation of patients according to the respective median LDs resulted in differences in event-free survival (EFS; CP1: 21.1 vs. 9.1 months, p=0.0711; CP2: 14.2 vs. 9.3 months, p=0.0095; CP3: 30.9 vs. 13.5 months, p=0.0055; CP4: median not reached vs. 14.1 months, p

Description: Discriminating between cytopenia(s) due to myelodysplastic syndromes (MDS) and due to other (non-clonal) causes can be challenging, especially when dysplasia as assessed by cytomorphology is minimal, and when other MDS-specific features (such as ring sideroblasts or cytogenetic aberrations) are absent. Current recommendations for diagnosing MDS endorse flow cytometry (FC) as a valuable and informative diagnostic tool. Most FC protocols focus on analyzing the progenitor cells and the maturing myelomonocytic lineage. However, one of the most frequently observed symptoms in MDS is anemia, which is often associated with erythrodysplasia. Therefore, flow cytometric features of nucleated erythroid cells may complement current validated FC tools. The international, multicenter study within the European LeukemiaNet MDS-FC working group (ELNet-IMDS-Flow) reported herein focused on defining those erythroid parameters that enable discrimination of dyserythropoiesis associated with MDS from erythropoiesis in non-clonal cytopenias. This analysis was based on ELNet iMDS-flow guidelines for studying nucleated erythroid cells and their expression of CD117, CD71, CD36, CD235a and CD105. [Westers et al., Leukemia 2012] Nineteen centers (members of the ELNet-iMDS-flow) collected FC data on the erythroid lineage in mainly low grade MDS cases and pathological and normal controls. Bone marrow aspirates were taken after informed consent in accordance with the Declaration of Helsinki and local ethics committee approval. Data from a learning cohort were compared among MDS patients and controls; the results were validated in a separate cohort. The learning cohort comprised 685 cases and the validation cohort 352 cases; in total 191 normal controls, 443 pathological controls, and 403 MDS cases were included. The data revealed that the analysis of the expression pattern of CD71 and that of CD36 on erythroid cells in combination with the percentage of CD117+ erythroid progenitors provides the best discrimination between MDS and non-clonal cytopenia. The selected markers were used to build an FC erythroid dysplasia score which displayed a sensitivity of 59% (95% CI: 49-68%) and a specificity of 84% (95% CI: 77-89%). Of note, not every MDS case shows signs of erythrodysplasia by cytomorphology whereas some non-clonal conditions do. Evaluation of the results in the validation cohort displayed a specificity of 77% (95% CI: 29-50%) and a sensitivity of 39% (95% CI: 66-85%) for separating pathologic controls and MDS cases based on FC erythroid dysplasia. Most “FC-dysplastic” cases in the pathological control group involved reactive conditions and cytopenia associated with infections. The majority of the “FC-dysplastic” controls demonstrated abnormal CD71 expression, which argues against the application of single aberrancies to indicate dysplasia. Considering only the presence of multiple erythroid aberrancies as erythroid dysplasia by FC increased the specificity to 96% and 95% in the learning and validation cohorts, respectively; however, at the cost of a markedly reduced sensitivity (37% and 21%, respectively). Ultimately, analysis of the erythroid and myeloid lineages should be combined to increase both sensitivity and specificity. In summary, the defined erythroid marker combination may aid the diagnostic work-up of cytopenic cases with suspected MDS, particularly in combination with flow cytometric evaluation of the progenitor cells and maturing myelomonocytic lineage. This will be implemented in an upcoming multicenter data collection exercise within ELNet iMDS-flow. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Loosdrecht:Celgene: Consultancy.