What do we mean by sensitivity when we talk about detecting minimal residual disease?

Eing

Kuçi

et al. 2009

Bone Marrow Transplant

Enrichment of cell subpopulations is a prerequisite for lineage-specific chimerism analysis (LCA), a frequent approach in follow-up after allo-SCT. An efficient enrichment technique is Magnetic Cell Sorting (MACS) using the AutoMACS separator. However, evaluation of purity, recovery and applicability for PCR-based chimerism analysis of MACS-enriched subpopulations from post-transplant peripheral blood, providing reduced cell numbers and/or unbalanced proportions of subpopulations, is currently unavailable. We performed enrichment of CD3-, CD14-, CD15-, CD19-and CD56-positive subpopulations using 'Whole Blood MicroBeads' and AutoMACS separator in 137 prospectively collected peripheral blood samples from 15 paediatric patients after allo-CD3-/CD19-depleted SCT. Purity was assessed by immune phenotyping. Recovery and applicability for chimerism analysis was evaluated. Excellent purity 490% was achieved in CD14-, CD15-positive cells in 81%, 95% of the isolates and in 86% of CD3 and CD19 isolates, if ACC was 4400 cells per ll. Median purity of CD56-positive isolates was 78.9%. Recovery 490% was between 93 (CD56) and 37% (CD15). Conventional and real-time PCR-based chimerism analysis was feasible in virtually all samples. Isolation of cell subpopulations by automated cell enrichment in post-transplant peripheral blood is feasible and fast providing excellent purity and recovery for routine lineage-specific chimerism analysis.

Section: Real-time Pcrmentioning

confidence: 99%

Enrichment of cell subpopulations applying automated MACS technique: purity, recovery and applicability for PCR-based chimerism analysis

Eing

Kuçi

et al. 2009

Bone Marrow Transplant

“…The quantifiable limit, denoted as the lowest possible threshold 35 was 25 copies for both the plasmids.…”

Section: Reproducibility and Quantifiable Limit Of Standard Dilutionsmentioning

confidence: 99%

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study

Gruhn²,

Coliva

et al. 2009

Leukemia

Self Cite

A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within o0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E þ 04 Abelson (ABL) expression was 3.5E þ 03 compared with that of 2.3E þ 01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.

“…Flow cytometry can be used to detect a combination of antigens present on leukemic cells. Monitoring residual leukemic cells with this method can provide strong prognostic information, but it is difficult to standardize and results strongly depend on the laboratory that is performing the analysis (11)(12)(13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

Prospective Validation of a New Method of Monitoring Minimal Residual Disease in Childhood Acute Myelogenous Leukemia

Steinbach

Bader

Clinical Cancer Research

et al. 2015

Purpose: This study evaluated the prognostic impact of a novel, simple, and standardized assay for monitoring minimal residual disease (MRD) in pediatric acute myelogenous leukemia (AML).Experimental Design: The expression of seven leukemia-associated genes (WT1, PRAME, CCL23, GAGED2, MSLN, SPAG6, and ST18) was measured by TaqMan Low Density Arrays in 112 patients and 52 healthy controls. Patients were treated according to the multicenter study AML-BFM 2004. Samples were collected prospectively at standard time points. The laboratory that measured MRD was blinded to patient outcome.Results: Relapse-free survival (RFS) was 95% (N ¼ 19; SE ¼ 5%) if expression of all genes was down to normal on day 15, 63% (N ¼ 41; SE ¼ 8%) if expression was normalized on day 28, and 38% (N ¼ 21; SE ¼ 11%) in patients who still showed elevated expression on day 28. The prognostic impact of MRD remained significant (P ¼ 0.002) when patients were stratified for the AML-BFM 2004 risk group. Multivariate analysis identified the MRD risk group and day 28 cytology as the only independent prognostic factors. Patients with a cytologic nonremission on day 28, which was confirmed by MRD, had a dismal prognosis. Only 1 out of 8 patients survived without relapse.Conclusions: This novel method of monitoring MRD has a strong prognostic impact that is independent from established risk factors in childhood AML.