Anne Tierens

Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment.

Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10−3 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10−3, which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10−4 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.

Residual disease detected by flow cytometry is an independent predictor of survival in childhood acute myeloid leukaemia; results of the NOPHO-AML 2004 study.

Early response after induction is a prognostic factor for disease outcome in childhood acute myeloid leukaemia (AML). Residual disease (RD) detection by multiparameter flow cytometry (MFC) was performed at day 15 and before consolidation therapy in 101 patients enrolled in the Nordic Society of Paediatric Haemato‐Oncology AML 2004 study. A multicentre laboratory approach to RD analysis was used. Event‐free survival (EFS) and overall survival (OS) was significantly different in patients with and without RD at both time points, using a 0·1% RD cut‐off level. RD‐negative and ‐positive patients after first induction showed a 5‐year EFS of 65 ± 7% and 22 ± 7%, respectively (P < 0·001) and an OS of 77 ± 6% (P = 0·025) and 51 ± 8%. RD‐negative and ‐positive patients at start of consolidation therapy had a 5‐year EFS of 57 ± 7% and 11 ± 7%, respectively (P < 0·001) and an OS of 78 ± 6% and 28 ± 11%) (P < 0·001). In multivariate analysis only RD was significantly correlated with survival. RD before consolidation therapy was the strongest independent prognostic factor for EFS [hazard ratio (HR):5·0; 95% confidence interval (CI):1·9–13·3] and OS (HR:7·0; 95%CI:2·0–24·5). In conclusion, RD before consolidation therapy identifies patients at high risk of relapse in need of intensified treatment. In addition, RD detection can be performed in a multicentre setting and can be implemented in future trials.

Blastic plasmacytoid dendritic cell neoplasm with leukemic presentation: 10‐Color flow cytometry diagnosis and HyperCVAD therapy

Few studies describe the comprehensive immunophenotypic pattern of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in the bone marrow and its treatment. This retrospective analysis evaluates the diagnostic flow cytometry (FCM) pattern and outcome of nine patients diagnosed with BPDCN. A four‐tube 10‐color FCM panel used for diagnosis of acute leukemia (AL), showed cells in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA‐DR, CD71. Seven patients received front‐line induction therapy with HyperCVAD with an overall response rate of 86%. Five of six responders underwent planned allogeneic hematopoietic cell transplantation (allo‐HCT). For a median follow up of 13.3 months, the 1‐year disease free survival and overall survival were 56 and 67%, respectively. An accurate diagnosis of BPDCN can be made by 10‐color FCM using a four‐tube AL panel demonstrating a characteristic pattern of antigen expression. Front‐line induction chemotherapy with HyperCVAD can yield high remission rates, but allo‐HCT is required for long‐term durable remissions. Am. J. Hematol. 91:283–286, 2016.

Immunoglobulin heavy and light chain gene features are correlated with primary cold agglutinin disease onset and activity

Immunoglobulin heavy chain ( IGH ) and light chain gene sequences of 27 patients with primary cold agglutinin disease (CAD) were studied to find features explaining the heterogeneity of clinical presentation and disease activity.nnCAD is a hemolytic anemia mediated by monoclonal IgM anti-I

Lymphoplasmacytic lymphoma and marginal zone lymphoma in the bone marrow: paratrabecular involvement as an important distinguishing feature.

OBJECTIVESnThe differential diagnosis between bone marrow involvement by lymphoplasmacytic lymphoma (LPL) and marginal zone lymphoma (MZL) is challenging because histology and immunophenotype of both diseases overlap. We revisited the diagnostic pathology features of both diseases in the bone marrow.nnnMETHODSnWe studied a series of bone marrow trephine biopsy specimens from 59 patients with Waldenström macroglobulinemia without extramedullary involvement and bone marrow biopsy specimens from 23 patients with well-characterized MZL who also had bone marrow involvement. H&E- and immunoperoxidase-stained sections of bone marrow trephine biopsy specimens as well as flow cytometry and classic cytogenetics performed on aspirations were reviewed. The study was complemented with MYD88 L265P mutation analysis of all samples.nnnRESULTSnThe most distinguishing features of LPL with respect to MZL were focal paratrabecular involvement (P < .001), the presence of lymphoplasmacytoid cells (P < .001) and Dutcher bodies (P < .001), increased numbers of mast cells (P < .001), and the MYD88 L265P mutation (P < .001).nnnCONCLUSIONSnLPL can be reliably distinguished from MZL in the bone marrow by using a combination of pathology characteristics. Our findings stress the diagnostic importance of using the combination of the following parameters for a correct LPL diagnosis: paratrabecular infiltration, the presence of lymphoplasmacytoid cells and cells with Dutcher bodies, and an increased number of mast cells in addition to the presence of MYD88 mutation.

Frequent somatic mutations of KMT2D (MLL2) and CARD11 genes in primary cold agglutinin disease

Additional Supporting Information may be found in the online version of this article: Fig S1. Survival data in 446* patients with primary myelofibrosis, stratified by absolute monocyte count and the Dynamic International Prognostic Scoring System (DIPSS)plus. Table SI. Multivariable analysis of overall survival in 291 patients with primary myelofibrosis with available information on absolute monocyte count, Dynamic International Prognostic Scoring System-plus risk status, driver mutational status and ASXL1/SRSF2 mutational status.

RUNX1 truncation resulting from a cryptic and novel t(6;21)(q25;q22) chromosome translocation in acute myeloid leukemia: A case report

Fluorescence in situ hybridization examination of a pediatric AML patient whose bone marrow cells carried trisomy 4 and FLT3-ITD mutation, demonstrated that part of the RUNX1 probe had unexpectedly moved to chromosome band 6q25 indicating a cryptic t(6;21)(q25;q22) translocation. RNA sequencing showed fusion of exon 7 of RUNX1 with an intergenic sequence of 6q25 close to the MIR1202 locus, something that was verified by RT-PCR together with Sanger sequencing. The RUNX1 fusion transcript encodes a truncated protein containing the Runt homology domain responsible for both heterodimerization with CBFB and DNA binding, but lacking the proline-, serine-, and threonine-rich (PST) region which is the transcription activation domain at the C terminal end. Which genetic event (+4, FLT3-ITD, t(6;21)-RUNX1 truncation or other, undetected acquired changes) was more pathogenetically important in the present case of AML, remains unknown. The case illustrates that submicroscopic chromosomal rearrangements may accompany visible numerical changes and perhaps should be actively looked for whenever a single trisomy is found. An active search for them may provide both pathogenetic and prognostic novel information.

Outcome after intensive reinduction therapy and allogeneic stem cell transplant in paediatric relapsed acute myeloid leukaemia

Given that 30–40% of children with acute myeloid leukaemia (AML) relapse after primary therapy it is important to define prognostic factors and identify optimal therapy. From 1993 to 2012, 543 children from the Nordic countries were treated according to two consecutive protocols: 208 children relapsed. The influence of disease characteristics, first line treatment, relapse therapy and duration of first remission on outcome was analysed. Second complete remission (CR2) was achieved in 146 (70%) patients. Estimated 5‐year overall survival (OS5y) was 39 ± 4% for the whole group and 43 ± 4% for the 190 patients given re‐induction therapy, of whom 76% received regimens that included fludarabine, cytarabine (FLA) ± anthracyclines, 18% received Nordic Society for Paediatric Haematology and Oncology (NOPHO) upfront blocks and 5% received other regimens. Late relapse ≥1 year from diagnosis, no allogeneic stem cell transplantation (SCT) in first remission and core binding factor AML were independent favourable prognostic factors for survival. For the 128 children (124 in CR2) that received SCT as consolidation therapy after relapse, OS5y was 61 ± 5%. Four of 19 children (21%) survived without receiving SCT as part of relapse therapy. Our data show that intensive re‐induction followed by SCT can give cure rates of 40% in children with relapsed AML.

Rare MLL-ELL fusion transcripts in childhood acute myeloid leukemia—association with young age and myeloid sarcomas?

BackgroundThe chromosomal translocation t(11;19)(q23;p13) with a breakpoint within subband 19p13.1 is found mainly in acute myeloid leukemia (AML) and results in the MLL-ELL fusion gene. Variations in the structure of MLL-ELL seem to influence the leukemogenic potency of the fusion in vivo and may lie behind differences in clinical features. The number of cases reported so far is very limited and the addition of more information about MLL-ELL variants is essential if the possible clinical significance of rare fusions is to be determined.Case presentationCytogenetic and molecular genetic analyses were done on the bone marrow cells of a 20-month-old boy with an unusual form of myelomonocytic AML with multiple myeloid sarcomas infiltrating bone and soft tissues. The G-banding analysis together with FISH yielded the karyotype 47,XY, +6,t(8;19;11)(q24;p13;q23). FISH analysis also demonstrated that MLL was split. RNA-sequencing showed that the translocation had generated an MLL-ELL chimera in which exon 9 of MLL (nt 4241 in sequence with accession number NM_005933.3) was fused to exon 6 of ELL (nt 817 in sequence with accession number NM_006532.3). RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript.ConclusionsBased on our findings and information on a few previously reported patients, we speculate that young age, myelomonoblastic AML, and the presence of extramedullary disease may be typical of children with rare MLL-ELL fusion transcripts.

Accurate and sensitive analysis of minimal residual disease in acute myeloid leukemia using deep sequencing of single nucleotide variations

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0.02% was achieved. The assay was linear in MRD range (0.03% to 1%) with good precision [CV, 4.1% (2.2% to 5.7%) at VAF 1% and 13.3% (8.8% to 19.4%) at VAF 0.1%], and low relative bias [7.9% (2.5% to 15.3%) at VAF 1%]. When applied to six childhood AML cases and compared with multiparameter flow cytometry for MRD analysis, deep sequencing showed concordance and superior sensitivity. Further high concordance was found with expression of fusion transcripts RUNX1-RUNX1T1 and KMT2A-MLLT10. The deep sequencing assay also detected mutations in blood when VAF in bone marrow exceeded 0.1% (nxa0=xa019). In conclusion, deep sequencing enables reliable detection of low levels of residual leukemic cells. Introduction of this method in patient care will allow for highly sensitive MRD surveillance in virtually every patient with AML.