Children with acute lymphoblastic leukemia (ALL) with у0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigenreceptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10 4 or more normal cells), accurate (r 2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r 2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels у1 in 10 4 . The percentages of leukemic cells measured by the two methods correlated well (r 2 = 0.978). Of the remaining 50 samples, 48 had MRD levels Ͻ1 in 10 4 . In only two samples results were discordant: 2 in 10 4 and 5 in 10 4 leukemic cells by PCR but Ͻ1 in 10 4 by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.
Hematologic relapse remains the greatest obstacle to the cure of children with acute lymphoblastic leukemia (ALL). Recent studies have shown that patients with increased risk of relapse can be identified by measuring residual leukemic cells, called minimal residual disease (MRD), during clinical remission. Current PCR methods, however, for measuring MRD are cumbersome and time-consuming. To improve and simplify MRD assessment, we developed a real-time quantitative PCR (RQ-PCR) assay for detection of leukemic cells that harbor the TAL-1 deletion. We studied serial dilutions of leukemic DNA and found the assay had a sensitivity of detection of one leukemic cell among 100 000 normal cells. We then investigated 23 samples from eight children with ALL in clinical remission. We quantified residual leukemic cells by using the TAL-1 RQ-PCR assay and by using limiting dilution analysis. In 17 samples, both methods detected MRD levels у0.001%. The percentages of leukemic cells measured by the two methods correlated well (r 2 ؍ 0.926). In the remaining six samples, both methods detected fewer than 0.001% leukemic cells. We conclude the TAL-1 RQ-PCR assay can be used for rapid, sensitive and accurate assessment of MRD in T-lineage ALL with the TAL-1 deletion. Leukemia (2001) 15, 166-170.
Hallmark features of adaptive CD4+ T-cells include differentiation into multiple effector T helper (Th) fates and subsequent emergence of memory states. Due to the heterogeneous nature of CD4+ T-cells, determining possible developmental relationships between effector and memory states in vivo has remained a challenge. Previously we employed single-cell RNA-sequencing (scRNA-seq) to delineate the molecular trajectories of Plasmodium-specific TCR transgenic CD4+ T cells during the first week of experimental malaria. We observed a fate bifurcation as cells progressed from an intermediate, activated state to either effector Th1 or T follicular helper (Tfh) cells. My current project tests the hypothesis that CD4+ Th cells directly convert into memory CD4+ T cells during malaria, and aims to define the underlying transcriptomic processes. Tracking endogenous TCR sequences via TraCeR analysis firstly confirmed that sibling clones populated both Th1 and Tfh fates. Next, by comparing transcriptomes, and performing computational lineage tracing of the scRNA-seq data, we observed a direct transition from Th1 and Tfh effector states to memory states. This was characterised by gradual transcriptomic quiescence during the first month of infection, as well as transcriptomic coalescence of Th1 and Tfh fates, but with retention of Th1/Tfh bifurcated structure in memory cells. Finally, premature cure of infection with anti-malarial drugs altered transcriptomic pathways taken by cells towards those with better Th1 recall and germinal centre Tfh phenotypes, respectively. Thus, by examining transcriptional dynamics using scRNA-seq, I am tracing the molecular pathways taken by effector CD4+ T cells towards functional memory states.
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