Minimal residual disease detection in cryopreserved ovarian tissue by multicolor flow cytometry in acute myeloid leukemia

Tristan Zver, Magalie Alvergnas-Vieille, Francine Garnache-Ottou, Christophe Ferrand, Christophe Roux, Clotilde Amiot, Tristan Zver, Magalie Alvergnas-Vieille, Francine Garnache-Ottou, Christophe Ferrand, Christophe Roux, Clotilde Amiot

No abstract available

Keywords: MFC; acute myeloid leukemia; ovarian MRD detection.

Figures

Figure 1.

Validation of an experimental model of AML cell dilutions within reference ovarian cells. (A and B) The dilution experiments consisted in adding a decreasing number of AML leukemic cells to non-contaminated ovarian single-cell population. n: number of experiments per dilution point performed using AML blood or BM cells from independent patients. The X-axes represent theoretical values of AML cell dilution in reference ovarian cells and the Y-axes represent experimental values quantified by MFC (axes in the log scale). The mean value for each dilution level is represented by a horizontal bar. (A) Modeling of AML cell detection in reference ovarian cell suspension by MFC using CD13-CD33, CD14 or HLA DR and expression of these AML markers used in hematology on reference ovarian cells. (B) Modeling of AML cell detection in reference ovarian cell suspension by MFC using CD361, CD33 alone or CD43 and expression of these more appropriate AML markers for MRD assessment on reference ovarian cells. (C) Comparison of MRD results obtained by MFC and RT-qPCR in dilution experiments. MRD results obtained with MFC were compared with RT-qPCR for the 10-fold serial dilutions of leukemic cells from 6 AML patients with NPM1 mutation A in reference ovarian cells. The number of samples is indicated for each part of the graph. (a) Concordance of 19 positive MRD values (> 1×10−4) obtained by MFC and RT-qPCR. The correlation coefficient was calculated with the Spearman’s rank correlation test. (b) No samples were MFC positive (>1×10−4) and RT-qPCR negative (<1×10−4). (c) For one sample, RT-qPCR was positive below 1×10−4 whereas MFC was negative (< 1×10−4) and for one other sample, RT-qPCR was below the maximum sensitivity (Smax) and MFC was negative. (d) Samples for which RT-qPCR was positive (> 1×10−4) and MFC was negative (< 1×10−4). MRD: minimal residual disease; MFC: multicolor flow cytometry; RT-qPCR: quantitative polymerase chain reaction; AML: acute myeloid leukemia; BM: bone marrow; MRD: minimal residual disease; Smax : maximum sensitivity.

Figure 2.

Positive MRD detection by MFC in the cortical ovarian tissue and medulla of Patient #1. Only viable cells (SYTO13+/7AAD−/CD45low) are displayed in all dot plots (pink). (A) AML patient cells at diagnosis express the following markers: CD11b+/CD43+/CD15+ (P1∩P2∩P3, black). (B) Cortical ovarian cells from the same patient at hematologica remission: among 300,000 events acquired, we identified 56 LAIP+ events with the same phenotype as AML cells at diagnosis: MRD level is quantified at 2×10−4. (C) Medulla ovarian cells from the same ovarian sample: among 460,000 events acquired, we identified 213 LAIP+ events, corresponding to an MRD level of 5×10−4. MRD: minimal residual disease; MFC: multicolor flow cytometry.