Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis.
ABSTRACT This article describes a procedure for performing routine three-color flow cytometric analysis for acute leukemia on lysed whole bone marrow preparations. This technique uses the combination of CD45 intensity and right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleated red blood cells. On this display, leukemic cells occupy a unique blast region characterized by intermediate CD45 density and low RALS, which, in normal marrows, contains less than 5% of the total cells. This approach was applied to 39 cases of acute leukemia and 8 cases of myelodysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukemic cell counts over a wide range. Moreover, the pattern seen on the CD45-RALS display was different for different French-American-British subtypes of leukemia, suggesting that this pattern might be useful for categorization. When CD45-peridin chlorophyll alpha protein was combined with other pairs of fluorescein isothiocyanate- and phycoerythrin-conjugated reagents, it was possible to set an analysis window on the leukemic blasts and display dual-parameter (ie, green vs. red fluorescence) data regarding expression of two additional markers on the leukemic population. This gating strategy was superior to traditional forward-angle versus RALS displays in that it did a better job of isolating the leukemic cells analytically.
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ABSTRACT: Real-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10(-4) in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.British Journal of Haematology 10/2013; 164(2). DOI:10.1111/bjh.12614 · 4.96 Impact Factor
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ABSTRACT: The concept of minimal residual disease in acute myeloid leukaemia has been steadily developed pre-clinically, with quantitative polymerase chain reaction (qPCR) leading the way with highly validated assays for patient-based risk stratification at post-treatment time points, which are being integrated in clinical trials both at evaluation of first complete remission (CR1) and after attaining CR1. Moreover, multicolour flow cytometry (MFC) has been increasingly employed in identifying leukaemia-associated immunophenotypes (LAIPs) with significant progress being made in standardization. In translating these widely varying methodologies to parameters useful for individualized patient decision-making, one of the obstacles has been that the assays entail varying sensitivities dependent on a number of variables. For qPCR, sensitivity depends on target type (i.e. fusion transcript, mutated gene or even overexpressed gene) and - in the case of overexpressed genes - on expression in healthy haematopoiesis. For MFC, sensitivity is likewise largely a function on whether the same phenotype is seen in normal immature cells and, in addition, antigen drift/shift with LAIPs changing at relapse is a well-known problem. In considering which sensitivity to opt for, a further variable is the situation of patient, most importantly the level of cytoreduction intended. Here we will attempt to give an overview of these pertinent questions intended for the practicing haematologist, focusing on where the field is heading at the clinical level.British Journal of Haematology 06/2012; 158(5):569-80. DOI:10.1111/j.1365-2141.2012.09203.x · 4.96 Impact Factor
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ABSTRACT: The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20mg/m(2) on days 1-5 and temsirolimus 25mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3·5months, and median overall survival was 4months (9·1months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; P=0·0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen.British Journal of Haematology 11/2011; 156(2):205-12. DOI:10.1111/j.1365-2141.2011.08940.x · 4.96 Impact Factor