Multiparametric Flow Cytometry versus Conventional Cytology in the Study of Leptomeningeal Involvement in Malignant Hematological Diseases.

BACKGROUND: CNS involvement is a complication in hematologic malignant neoplasms. The advantage of multiparametric flow cytometry (MFC) over conventional cytology (CC) in detecting occult leptomeningeal disease in CSF has been proven previously, as reported in the literature. In this study, we reviewed the experience of our laboratory in evaluating CSF specimens by MFC and CC after refinement of technical procedures. METHODS: MFC analysis was performed in 159 specimens. In 91 specimens, simultaneous CC and MFC analysis was requested and results compared. RESULTS: Neoplastic cells were identified in 27 (17.0%) of the total samples and in 17 (18.7%) of the paired specimens group by MFC, compared with 2 (2.2%) specimens with positive results as determined by CC. MFC enabled identification of malignant cells in low-cellularity specimens (<5 cells/µL) and all neoplasm categories. CONCLUSION: MFC allowed the detection of minimal numbers of tumor cells in CSF specimens from individuals with leukemia and lymphoma in whom CC had not been able to identify those tumor cells.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

A drug potency signature links progression of chronic lymphocytic leukemia to mitochondria-related stress responses and metabolic reprogramming under hypoxia.

Chronic lymphocytic leukemia (CLL) cells change their metabolic program between normoxia and hypoxia, possibly affecting cytotoxic drug potency by altering mitochondria-related cell stress responses (MRCSR) including mitophagy, mitochondrial biogenesis, and mitochondrial proteostasis. We evaluated in CLL cells from nine patients, the single and multiple-combined drug potency of arsenic trioxide (ATO), valproic acid (VPA), vincristine (VCR) and MG132 as four pharmacological sensors influencing mitochondrial apoptosis, mitochondrial biogenesis, mitophagy, and mitochondrial proteostasis respectively, under normoxia and hypoxia to force hypoxia-induced metabolic reprogramming (HMR). Untreated cells from all patients remained viable under O2 levels below 0.5% for 72 h. We obtained 21 measures of drug potency and interaction at 50% effect level that we denoted drug potency signature (DPS). Using the comparative DPS between normoxia and hypoxia, two non-supervised classification algorithms discriminated CLL patients with active disease (ADT) and stable disease (NAD) and showed complete consistency with their clinical characteristics. In ADT group under hypoxia, the potency of MG132 was increased, the interaction of ATO + VPA and ATO + VPA + VCR shifted towards antagonism, and ATO + VPA + VCR + MG132 shifted towards synergism, indicating a prominent role of mitochondrial proteostasis. Classification of patients based on DPS, depended on the contrasting response of drugs under hypoxia and normoxia, owing to HMR. Using these drugs as pharmacological sensors, we linked the metabolic arrangement of CLL cells under hypoxia, to potency of drugs targeting MRCSR, and to the clinical features of individual patients, therefore providing new sources of data on disease progression, drug response and risk prognosis.