Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry.

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group "CytHem-LMMC" are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.

ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

BACKGROUND: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB. METHODS: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available. RESULTS: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB. CONCLUSIONS: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples.

[Myeloid neoplasms associated with rearrangement of PDGFRB: A rare and tricky disease]. / La néoplasie myéloïde associée à un réarrangement de PDGFRB : une pathologie rare de diagnostic difficile.

In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×109/L. Bone marrow aspiration was rich and showed a heterogeneous distribution of myeloid cells with clusters of promyelocytes and proerythroblasts associated with numerous eosinophils and spindle-shaped mast cells but without excess of blasts, dysplasia nor maturation skewing. These aspects suggested an atypical myeloproliferative neoplasm. Bone marrow biopsy was performed showing also a very high cellularity with area of myeloid and erythroid precursors associated with numerous spindle-shaped mast cells. Diagnoses of unclassified myeloid neoplasm and/or systemic mastocytosis were then proposed. Further chromosome analysis showed a t(5;8) translocation with PDGFRB rearrangement revealed in fluorescent in situ hybridization. Patient was treated with imatinib and intravenous immunoglobulin therapy allowing a significant improvement in neurological symptoms and biological results. Patient condition is currently stable after six lines of treatment. This rare hematopoietic neoplasm displays unusual histological and cytological features and can mimic other myeloproliferative neoplasm. Specific cytogenetics analysis should be considered for such cases with hypereosinophilia to select patients that may benefit from targeted therapy.

Evaluation of two new highly multiplexed PCR assays as an alternative to next-generation sequencing for IDH1/2 mutation detection.

IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development of a fast and sensitive assay to detect IDH1R132 , IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays - an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next-generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin-fixed paraffin-embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132 , IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost-effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti-IDH1/2-targeted therapies.

Fusion Gene Detection and Quantification by Asymmetric Capture Sequencing (aCAP-Seq).

Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, µBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.

Optimization of CSF biological investigations for CNS lymphoma diagnosis.

Diagnosis of lymphoma leptomeningeal dissemination is challenging and relies on a wide array of methods. So far, no consensus biological guidelines are available. This increases the chance of intra- and interpractice variations, despite the shared concern to perform the minimum amount of tests while preserving clinically relevant results.We evaluated a training cohort of 371 cerebrospinal fluid (CSF) samples from patients with putative lymphomatous central nervous system (CNS) localization using conventional cytology (CC), flow cytometry (FCM), molecular clonality assesment by PCR and cytokine quantification (CQ). This led us to propose a biological algorithm, which was then verified on a validation cohort of 197 samples. The samples were classified according to the clinical context and the results of each technique were compared. Using all four techniques was not useful for exclusion diagnosis of CNS lymphoma (CNSL), but they proved complementary for cases with suspected CNSL. This was particularly true for CQ in primary CNSL. Overall, diagnosis can be obtained with a two-step approach. The first step comprises CC and FCM, as results are available quickly and FCM is a sensitive method. Both PCR and CQ can be postponed and performed in a second step, depending on the results from the first step and the clinical context.The proposed algorithm missed none of the CNSL samples of the validation cohort. Moreover, applying this algorithm would have spared 30% of PCR tests and 20% of CQ over a one-year period, without compromising clinical management.

Multicenter validation of the flow measurement of classical monocyte fraction for chronic myelomonocytic leukemia diagnosis.

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.

High sensitivity of the Hematoflow™ solution for chronic myelomonocytic leukemia screening.

BACKGROUND: Accumulation of classical monocytes CD14++ CD16- (also called MO1) ≥ 94% can accurately distinguish chronic myelomonocytic leukemia (CMML) from reactive monocytosis. The HematoFlow™ solution, able to quantify CD16 negative monocytes, could be a useful tool to manage monocytosis which remains a common issue in routine laboratories. METHODS: Classical monocytes were quantified from 153 whole blood samples collected on EDTA using both flow cytometry methods, either MO1 percentage determination by the multiparameter assay previously published and regarded here as the reference method, or CD16 negative monocyte percentage determination by the means of HematoFlow™. RESULTS: Both methods of classical monocyte percentage determination were highly and significantly correlated (r = 0.87, P < 0.0001). The HematoFlow™ solution leant toward an overestimation of the genuine classical monocyte percentages obtained by the reference method. Percentages of CD16 negative monocytes provided by HematoFlow were higher than 94% for all the 73 patients displaying classical monocytes MO1 found ≥94% by the reference method, indicating a sensitivity of 100%. Furthermore, the calculation of CD16 negative monocyte percentage can be easily computerized and integrated to the middleware. CONCLUSIONS: We propose a new application of the Hematoflow™ solution that can be used as a flag system for monocytosis management and CMML detection. © 2017 International Clinical Cytometry Society.