Introduction: Since the publication of the International Guidelines (Borowitz, 2010; Illingworth, 2018), no study has assessed the long-term evolution of paroxysmal nocturnal hemoglobinuria (PNH) ...clones using high-resolution flow cytometry. The sole evaluation, performed by Sugimori et al, using a 2-color flow cytometry test, showed the disappearance of PNH clones in 24% of patients with bone marrow (BM) failure over 5 years (Sugimori, 2009). A diagnostic practice harmonization using high-resolution flow cytometry has spread in France since 2013 through an on-going inter-laboratory comparison program (Debliquis, 2015). Thus, our HPNAFC group has been able to initiate a French nation-wide multicenter prospective observational study.
Objective: We aimed to assess the evolution of PNH clones over a long term period using mostly high sensitivity test, which is required for minor clone assessment, with validated flow cytometry data.
Methods: All patients of any age with a PNH clone or GPI-deficient cells ≥0.01%, newly- or previously-diagnosed, detected in France from February 29th 2016, could be included in this Observatory, provided that the center had validated a PNH flow cytometry quality control. For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. Referent cytometrist of each center included patients in the e-CRF available on the HPNAFC website providing clinical and biological information as well as flow cytometry raw data files. This study was approved by the national research ethics board.
Results: As of July 15th 2019, 48 participating flow cytometry laboratories across France have enrolled 356 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. All cases have been carefully reviewed by the 2 principal investigators, who both thoroughly re-examined flow cytometry data and the e-CRF filling that led to the update of roughly one third of the submitted files. This enabled the validation of 200 patients at diagnosis, the remaining 156 being ongoing. One hundred and three of the 200 validated patients displayed at least one follow-up point (more than 3 months apart from the diagnosis) with a clone size determined at diagnosis (see flow chart figure 1A). For 8/103 patients, exchanges with centers are still ongoing. Thus, we were able to assess the evolution of PNH clones of 95 patients with 2 range: 1-8 follow-up points over a period of 4.1 0.3-14.2 years, corresponding to 200 validated follow-up points. The patient median age at diagnosis was 40 years old 10-85 with 3 pediatric cases (<18y) and a M/F sex ratio of 0.86. Diagnoses were made between 2003 and 2018 with clinical information available in 97% of cases: 19 patients (20%) had hemolytic anemia and most patients (n=73, 77%) displayed BM failure including aplastic anemia (n=62), myelodysplastic syndrome (n=7) and unexplained cytopenia(s) (n=4). No case of thrombosis was included. All patients with hemolytic anemia showed an increasing clone size over time including the two who were not treated with eculizumab (Figure 1B; median size at diagnosis on neutrophils: 81.3% vs median size over 5 years: 96.3%). The median clone size at diagnosis for patients with BM failure was 1.5% on neutrophils with a very wide range 0.01-97.87, almost half of them being less than 1%. When comparing the diagnosis point with the latest follow-up point, PNH clone size increased in 37 patients, decreased in 16 of them and remained stable in 20 cases (Figure 1C, D). Nine of the 37 patients reached a PNH clone size above 50% and 4 of them received a treatment by eculizumab in a median delay of 5 years 1.5-6.0. Interestingly, no patient showed spontaneous disappearance of PNH clones, pending the use of a high-resolution flow cytometry test. The only five patients with undetectable PNH clones (Figure 1D, red lines) were those who underwent BM transplantation.
Conclusion: This multicenter study based on robust flow cytometry analysis showed no disappearance of PNH clones, including minor ones, over a long period of time, regardless of the clinical manifestations, except for patients who underwent BM transplantation. Moreover, PNH clone size increased in half of patients with BM failure, justifying a long term PNH clone size monitoring, even in these patients.
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Le Garff-Tavernier:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Honoraria; Takeda: Honoraria; Pfizer: Honoraria; Gilead: Honoraria; Genzyme: Honoraria. Socie:Alexion: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Drenou:Alexion: Consultancy, Honoraria. Wagner Ballon:Alexion: Consultancy, Honoraria.
Oncogenesis and ontogeny of blastic plasmacytoid dendritic cell neoplasm (BPDCN) remain uncertain, between canonical plasmacytoid dendritic cells (pDCs) and AXL+ SIGLEC6+ DCs (AS-DCs). We compared 12 ...BPDCN to 164 acute leukemia by Affymetrix HG-U133 Plus 2.0 arrays: BPDCN were closer to B-cell acute lymphoblastic leukemia (ALL), with enrichment in pDC, B-cell signatures, vesicular transport, deubiquitination pathways, and AS-DC signatures, but only in some cases. Importantly, 1 T-cell ALL clustered with BPDCN, with compatible morphology, immunophenotype (cCD3+ sCD3− CD123+ cTCL1+ CD304+), and genetics. Many oncogenetic pathways are deregulated in BPDCN compared with normal pDC, such as cell-cycle kinases, and importantly, the transcription factor SOX4, involved in B ontogeny, pDC ontogeny, and cancer cell invasion. High-throughput sequencing (HaloPlex) showed myeloid mutations (TET2, 62%; ASXL1, 46%; ZRSR2, 31%) associated with lymphoid mutations (IKZF1), whereas single-nucleotide polymorphism (SNP) array (Affymetrix SNP array 6.0) revealed frequent losses (mean: 9 per patient) involving key hematological oncogenes (RB1, IKZF1/2/3, ETV6, NR3C1, CDKN2A/B, TP53) and immune response genes (IFNGR, TGFB, CLEC4C, IFNA cluster). Various markers suggest an AS-DC origin, but not in all patients, and some of these abnormalities are related to the leukemogenesis process, such as the 9p deletion, leading to decreased expression of genes encoding type I interferons. In addition, the AS-DC profile is only found in a subgroup of patients. Overall, the cellular ontogenic origin of BPDCN remains to be characterized, and these results highlight the heterogeneity of BPDCN, with a risk of a diagnostic trap.
•BPDCNs have varying profiles evoking either pDC or AS-DC signatures, and phenotype can sometimes be confusing, notably with T-cell ALL.•Genetics is heterogeneous: a lymphoid-like profile (IKZF1) is highly prevalent, whereas immune defects can mimic AS-DC characteristics.
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Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate ...into plasma cells
and
. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers particularly solid tumors (3-5) can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare for a review of the few reported cases, see Ref. (6). The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (V
and D
gene replacement), BCR editing, and somatic mutations-events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements clonality assessment, V(D)J sequencing, and somatic hypermutation analysis in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same V
and J
genes but different D
genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.
Schistocyte counts are a cornerstone of the diagnosis of thrombotic microangiopathy syndrome (TMA). Their manual quantification is complex and alternative automated methods suffer from pitfalls that ...limit their use. We report a method combining imaging flow cytometry (IFC) and artificial intelligence for the direct label-free and operator-independent quantification of schistocytes in whole blood.
We used 135,045 IFC images from blood acquisition among 14 patients to extract 188 features with IDEAS® software and 128 features from a convolutional neural network (CNN) with Keras framework in order to train a support vector machine (SVM) blood elements’ classifier used for schistocytes quantification.
Keras features showed better accuracy (94.03%, CI: 93.75-94.31%) than ideas features (91.54%, CI: 91.21-91.87%) in recognising whole-blood elements, and together they showed the best accuracy (95.64%, CI: 95.39-95.88%). We obtained an excellent correlation (0.93, CI: 0.90-0.96) between three haematologists and our method on a cohort of 102 patient samples. All patients with schistocytosis (>1% schistocytes) were detected with excellent specificity (91.3%, CI: 82.0-96.7%) and sensitivity (100%, CI: 89.4-100.0%). We confirmed these results with a similar specificity (91.1%, CI: 78.8-97.5%) and sensitivity (100%, CI: 88.1-100.0%) on a validation cohort (n=74) analysed in an independent healthcare centre. Simultaneous analysis of 16 samples in both study centres showed a very good correlation between the 2 imaging flow cytometers (Y=1.001x).
We demonstrate that IFC can represent a reliable tool for operator-independent schistocyte quantification with no pre-analytical processing which is of most importance in emergency situations such as TMA.
None.
Summary
Monoclonal gammopathy of unknown significance (MGUS), smouldering multiple myeloma (SMM), and multiple myeloma (MM) are very common neoplasms. However, it is often difficult to distinguish ...between these entities. In the present study, we aimed to classify the most powerful markers that could improve diagnosis by multiparametric flow cytometry (MFC). The present study included 348 patients based on two independent cohorts. We first assessed how representative the data were in the discovery cohort (123 MM, 97 MGUS) and then analysed their respective plasma cell (PC) phenotype in order to obtain a set of correlations with a hypersphere visualisation. Cluster of differentiation (CD)27 and CD38 were differentially expressed in MGUS and MM (P < 0·001). We found by a gradient boosting machine method that the percentage of abnormal PCs and the ratio PC/CD117 positive precursors were the most influential parameters at diagnosis to distinguish MGUS and MM. Finally, we designed a decisional algorithm allowing a predictive classification ≥95% when PC dyscrasias were suspected, without any misclassification between MGUS and SMM. We validated this algorithm in an independent cohort of PC dyscrasias (n = 87 MM, n = 41 MGUS). This artificial intelligence model is freely available online as a diagnostic tool application website for all MFC centers worldwide (https://aihematology.shinyapps.io/PCdyscrasiasToolDg/).
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a ...retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)–like, acute lymphoid leukemia (ALL)–like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone CHOP)–like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.
•Criteria for a cytological and phenotypical diagnosis of BPDCN are defined.•First-line treatment with Aspa-MTX followed by allo-transplant is the best way to achieve prolonged survival even in elderly.
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Introduction
Although the median age at diagnosis of acute myeloid leukemia (AML) is 67 years, with approximately one third of patients aged 75 years or older, limited treatment options are available ...for the elderly. There is no standard of care for older patients with AML unfit for intensive chemotherapy. In this case, despite specific treatment such as low-dose cytarabine (LDA) and 5 azacytidine (5AZA), outcome remains extremely poor, without cure (Thomas X, Current Treat Options Oncol 2017, 18(1):2). The goal of our study is to establish a prognostic model of survival in order to identify whether the absence of specific treatment may not be harmful in a subset of patients.
Patients and Methods
The French Hauts-de-France AML observatory is a population-based database reporting AML cases diagnosed and supported in 9 Hospital Centers from the French region Haut-de-France. From January 2008 to December 2016, 572 patients older than 75 years were included in the observatory. Among them 324 patients received best supportive care (BSC) alone, 142 hypomethylating agents, 82 low doses aracytine, and 24 other treatments. As a general consensus accepted in all participating centers, BSC was proposed in unfit patients, after performance status and comprehensive geriatric assessment according to results of a preliminary fast geriatric assessment oncodage G8 (Bellera CA, Ann Oncol 2012, 23(8):2166-72). Clinical data were collected in each center. The study was conducted according to the Declaration of Helsinki and was approved by the Human Research Committee of Lille and the internal review board of the Lille University Hospital Tumor Bank (certification NF 96900-2014/65453-1).
Results
In the BSC group, median age at diagnosis was 82 years (interquartile range IQR 78-86). Median WBC count was 7.3 x10^3/mL (IQR 2.18-42.5) with a median peripheral blood blasts percentage of 21% (IQR 6-59.5). Median bone marrow blasts was 51% (IQR 30-75). Karyotype was available for 181 patients. Two patients were in the favorable, 111 patients (61%) were in the intermediate, 68 patients (38%) were in the unfavorable cytogenetics group and 58 patients (32%) had a complex karyotype.
For the BSC group, median survival was 3.2 months (IC 2.3- 4) with a 18% 12-months survival estimate.
In univariate Cox models, WBC count (p<0.0001), circulating blasts percentage (P=0.0001), bone marrow blasts percentage (P=0.0001) and complex karyotype (p= 0.05) were predictive of a shorter survival. Neither age nor unfavorable karyotype retained prognostic value.
Multivariate analysis selected two independent covariates namely circulating blasts percentage without identifiable cutoff (p=0.0002), and complex karyotype (p=0.05). Prediction of risk according to this proportional hazard model showed that patients at lowest risk could be defined by the absence of both complex karyotype and circulating blasts. The survival of this subgroup of unfit patients who received BSC (7% of patients, median 16 months) was not significantly different from the survival of fit patients with the same characteristics (absence of both complex karyotype and circulating blast) who received 5AZA or LDA (median survival of 26.6 months, p=0.07) (Figure 1.).
Remaining patients (with complex karyotype or circulating blast), of the BSC cohort had a shorter survival than patients treated with 5AZA or LDA (3.7 months vs 13 months, p<0.0001).
Conclusion
In a population of elderly AML patients (> 75 years), 18% are alive at 12 months without any chemotherapy. Our results indicate that the absence of chemotherapy may not be detrimental in a small subset of unfit patients identified by the absence of both complex karyotype and circulating blasts. However further studies are mandatory for characterizing most of patients alive at 12 months with BSC.
Figure 1. Survival of patients with both complex karyotype and circulating blasts
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No relevant conflicts of interest to declare.
1 Laboratoire dHématologie, CHU, Bordeaux, France
2 Laboratoire dHématologie, CHU, Dijon, France
3 Murdoch University, Perth, Australia
4 University of Texas MD Anderson Cancer Center, Houston, ...Texas, USA
5 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
6 Uniklinik Eppendorf, Hamburg, Germany
7 Molecular Genetics, Royal Devon and Exeter Hospital, Exeter, UK
8 Molecular Diagnostics, Jeroen Bosch Hospital, s-Hertogenbosch, Netherlands
9 Laboratoire dHématologie, CHU, Clermont-Ferrand, France
10 Hematology Division, University of Utah School of Medicine, Salt Lake City, Utah, USA
11 Laboratoire dHématologie, CHU, Brest, France
12 Laboratoire de Cytologie Clinique et Cytogénétique, CHU, Nîmes, France
13 Laboratoire d'Hématologie, Hôtel-Dieu, Paris, France
14 Department of Hematology, University of Pavia and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
15 Experimental Hematology, Department of Biomedicine, Basel University Hospital, Basel, Switzerland
16 Laboratoire dHématologie, CHU, Nantes, France
Correspondence: Sylvie Hermouet, Laboratoire dHématologie, Centre Hospitalier Universitaire, Institut de Biologie, 9 quai Moncousu, 44093 Nantes, France., E-mail: sylvie.hermouet{at}chu-nantes.fr
Background: Many different techniques have been designed for the quantification of JAK2 V617F allelic burden, sometimes producing discrepant results.
Design and Methods: JAK2 V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing.
Results: A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2 V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2 V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2 V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques – one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean±2SD – with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2 V617F.
Conclusions: Techniques expressing the allelic burden as JAK2 V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.
Key words: JAK2V617F, quantification, standardization, allele-specific PCR, myeloproliferative diseases, multicenter study.
Related Article
Clinical relevance of JAK2 (V617F) mutant allele burden
Francesco Passamonti, Elisa Rumi
Haematologica 2009 94: 7-10.
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Peripheral blood monocytes include three subsets defined by CD14 and CD16 surface markers. An increase in the CD14
CD16
classical monocyte fraction ≥ 94% of the total monocytes was proposed to ...rapidly and efficiently distinguish chronic myelomonocytic leukemia from reactive monocytosis. The robustness of this assay required a multicenter validation. The flow cytometry assay designed to quantify peripheral blood monocyte subsets was implemented by multiple diagnosis laboratories in France. A nationwide survey was performed to evaluate its performance. All the 48 French laboratories answered the questionnaire, revealing that 63% use this assay routinely. Central blind reanalysis of 329 cytometry files collected from five laboratories demonstrated an excellent correlation in classical monocyte fraction measurement (r = 0.93; p < 0.0001). The cutoff value of 94% classical monocytes being the critical readout for diagnosis, we then compared 115 patients with classical monocytes ≥ 94% and 214 patients with a fraction < 94% between initial analysis and reanalysis. An agreement was obtained in 311 files. Finally, an overt diagnosis, available for 86 files, confirmed a good sensitivity (93.6%) and specificity (89.7%). This survey demonstrates the robustness of the flow assay with limited variability of classical monocyte percentage between centers, validates the 94% cutoff value, and confirms its sensitivity and specificity.
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