Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL

Abstract

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Improved outcome of children transplanted for high-risk leukemia by using a new strategy of cyclosporine-based GVHD prophylaxis

“…22 Multiparameter flow cytometry was performed on fresh cells after erythrocyte lysis using staining with Ab panels, as described previously in ALL. 23 At diagnosis, leukemia-associated immunophenotypes were determined on 10 000 to 15 000 nucleated cells. For follow-up samples, as described above, 2.5 × 10 5 non-gated events or more stained nucleated cells were analyzed using FACSCalibur flow cytometers (BD Bioscience, San Jose CA, USA).…”

“…The MRD positivity was defined as ⩾ 10 − 3 . [22][23][24][25] Statistical analysis All statistical analyses were performed using SPSS for Windows (version 11.0; SPSS, Chicago, IL, USA). Pearson's χ 2 tests were used to compare possible differences in qualitative variables between groups of patients.…”

Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study

“…Multi-color flow cytometry assays with at least 6 colors have been applied to MRD monitoring in acute lymphoblastic leukemia, 14,15 multiple myeloma, [16][17][18] chronic lymphocytic leukemia, 19 and hairy cell leukemia. 20 To date, there are no established criteria for MRD quantification by MFC in MCL.…”

Determinación de la clonalidad en tejidos humanos

“…En general, la EMR es un factor pronóstico importante en leucemias y linfomas (43,44). La finalidad de las pruebas para determinación de EMR es detectar hasta el último blasto.…”

“…Para determinar la EMR por medio de las pruebas de reordenamientos de Ig y TCR, se aplican los protocolos sobre una muestra diagnóstica rica en células tumorales, y se secuencian los reordenamientos más representativos; a partir de los resultados se diseñan primers específicos y se ponen en marcha ensayos cuantitativos por PCR (44). Un 30% de los casos presentan evolución clonal y requieren seguimiento por varios rearreglos (45); sin embargo, puede decirse que el rastreo por medio de esta técnica no solo es muy preciso, sino personalizado, además de considerarse como el estándar de oro (46).…”