Barbara Denys

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5–20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

A Real-Time Polymerase Chain Reaction Assay for Rapid, Sensitive, and Specific Quantification of the JAK2V617F Mutation Using a Locked Nucleic Acid-Modified Oligonucleotide

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

Clinical and biological characterization of patients with low (0.1–2%) JAK2V617F allele burden at diagnosis

Detection of the JAK2 V617F mutation is of major help in the diagnosis of myeloproliferative neoplasms (MPNs).[1][1]–[3][2] Techniques using allele-specific quantitative PCR (AS-qPCR) can reliably and consistently detect down to 0.001% mutated alleles.[4][3] Moreover, a study of healthy blood

Leukaemic stem cell load at diagnosis predicts the development of relapse in young acute myeloid leukaemia patients

Giosu e Costa Apollinaire Ngankeu Rami I. Aqeilan Carlo M. Croce Francesco Bertoni Stefano Alcaro Francesco Trapasso Lymphoma and Genomics Research Programme, The Institute of Oncology Research, Bellinzona, Switzerland, Biomedical Section, Tecnologica Research Institute, Crotone, Italy, Laboratory for Biomedical Neurosciences Ente Cantonale Ospedaliero, Bellinzona, Switzerland, Department of Experimental and Clinical Medicine, University ‘Magna Græcia’ of Catanzaro, Catanzaro, Italy, Departimento Scienze della Vita, University ‘Magna Græcia’ of Catanzaro, Catanzaro, Italy, Department of Molecular Immunology, Virology and Medical Genetics, The Ohio State University, Columbus, OH, USA, and The Lautenberg Centre for Immunology and Cancer Research, Institute for Medical Research, The Hebrew University, Jerusalem, Israel. E-mail: eugenio.gaudio@ior.iosi.ch; Trapasso@unicz.it

A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

Standardization of BCR‐ABL1 messenger RNA quantification by real‐time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR‐ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory‐specific conversion factor (CF), co‐ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR‐ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results.

The YPEL5-PPP1CB fusion transcript is detected in different hematological malignancies and in normal samples.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in Western adults. It was suggested that transcripts from a reciprocal trans-splicing event between YPEL5 and PPP1CB were present exclusively in CLL patients (more than 90%). Here we show that the YPEL5–PPP1CB fusion is not specific for CLL but is also detected in other hematological malignancies such as chronic myeloid leukemia, monoclonal B cell lymphocytosis or acute leukemia and also in normal samples. As such, it is unlikely that the YPEL5–PPP1CB fusion is a good drug target in CLL or a suitable target to monitor disease.

The NF1 hotspot in acute myeloid leukemia: what’s in a name?

Eisfeld and colleagues [1] recently described recurrent mutations in NF1 in patients with acute myeloid leukemia (AML), with a higher frequency than seen in previous studies. They show that mutations in hotspot Thr676 are associated with lower complete remission rates and shorter overall survival in patients <60 years. One of the factors that may have contributed to the previous oversight of this hotspot in previous studies, is its location in a mononucleotide cytosine repeat, which can easily be missed due to a faulty interpretation of sequencing data. Changes in mononucleotide repeats are cumbersome in their technical analysis but also in their nomenclature. Initiatives by the Human Genome Variation Society (HGVS) are trying to limit the nomenclature variability by providing clear guidelines [2] how to describe such variants. The authors refer to the NF1 hotspot as “p.Thr676fs*24 [c.2026dupC], Thr676”. This annotation is in conflict with the HGVS guidelines in several ways: as the DNA sequence is determined, this should be prominent, and the derived amino acid sequence should be between brackets (as it is extrapolated from the change at the DNA level). Applying this rule, the annotation would change to “c.2026dupC; p. (Thr676fs*24)”. However, no reference sequence is provided, and nucleotide 2026 is not a C in the two reference sequences (NM_000267.3 and NM_001042492.2) used to determine the Locus Reference Genomic transcripts [3]. In both transcripts, the C-stretch runs from nucleotides 2027 to 2033. As stated in the HGVS guidelines [2], for duplications in stretches of repeated sequences, the most 3′ residue is arbitrarily assigned to have been changed, resulting in a reassignment of the NF1 hotspot to c.2033 in the DNA sequence and from Thr676 to Ile679 in the protein. In conclusion, the HGVS compliant nomenclature of an additional cytosine in this C-stretch is NM_001042492.2: c.2033dup; p.(Ile679Aspfs*21). The Ile679 amino acid annotation is also used in the AACR GENIE dataset, to which the authors refer to confirm their results. The recognition of Ile679 as a hotspot for NF1 mutations, if confirmed in other studies, would benefit significantly from the use of a uniform nomenclature, following the HGVS guidelines.

Cancer-related mRNA expression analysis using a novel flow cytometry-based assay

Cancer‐related gene expression data mostly originate from unfractionated bulk samples, leading to “expression averaging” of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single‐cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM‐based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels.

RNA-based FLT3-ITD allelic ratio is associated with outcome and ex vivo response to FLT3 inhibitors in pediatric AML

Controversy exists whether internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-internal tandem duplication [ITD]) allelic ratio (AR) and/or length of the ITD should be taken into account for risk stratification of pediatric acute myeloid leukemia (AML) and whether it should be measured on RNA or DNA. Moreover, the ITD status may be of relevance for selecting patients eligible for FLT3 inhibitors. Here, we included 172 pediatric AML patients, of whom 36 (21%) harbored FLT3-ITD as determined on both RNA and DNA. Although there was a good correlation between both parameters ARspearman = 0.62 (95% confidence interval, 0.22-0.87) and ITDlengthspearman = 0.98 (95% confidence interval, 0.90-1.00), only AR ≥ 0.5 and length ≥48 base pairs (bps) based on RNA measurements were significantly associated with overall survival (AR: Plogrank = .008; ITDlength: Plogrank = .011). In large ITDs (>156 bp on DNA) a remarkable 90-bp difference exists between DNA and RNA, including intron 14, which is spliced out in RNA. Ex vivo exposure (n = 30) to FLT3 inhibitors, in particular to the FLT3-specific inhibitor gilteritinib, showed that colony-forming capacity was significantly more reduced in FLT3-ITD-AR ≥ 0.5 compared with ITD-AR-low and ITD- patient samples (P < .001). RNA-based FLT3-ITD measurements are recommended for risk stratification, and the relevance of AR regarding eligibility for FLT3-targeted therapy warrants further study.