Flow Cytometric Minimal Residual Disease Research Articles

Predictive Value of Flow Cytometric MRD Analysis in Childhood Acute Lymphoblastic Leukaemia at the End of Remission Induction Therapy.

Patients with acute lymphoblastic leukaemia (ALL) in morphological remission may still have up to 1010 residual malignant cells. Detection of minimal residual disease (MRD) at the end of induction therapy allows better estimation of the leukaemic burden and can help selection of appropriate therapeutic strategies. Flow cytometric (FC) detection of MRD is based on the identification of immunophenotypic combinations expressed on leukaemic cells but not on normal hematopoietic cells - leukaemia associated immunophenotypes (LAIPs). We prospectively analysed bone marrow samples from 77 patients who presented with ALL to our unit between 1999–2003 and attained morphological remission. These patients were treated on a standard protocol. Multiparameter FC identification of LAIPs was performed at various time points, as dictated by the treatment protocol. Our results show that flow cytometric MRD at the end of induction therapy is an independent and the most significant predictor of relapse, both on univariate and multivariate analysis. The relapse risk was 4% if day 28 MRD was <0.01% and 50% if day 28 MRD was >0.01% (p<0.05). We conclude that flow cytometric based MRD assays can be used to assess early response to treatment and predict relapse in a similar way to molecular MRD analysis at the end of induction therapy. Flow cytometric analysis of MRD offers the advantages of being cheaper, more widely available and has quicker turnaround times.

Standardized 4 Color Flow Cytometric Minimal Residual Disease Detection Failed To Overcome Regeneration Problems but Identifies Early Blast Clearence Predictive of Molecular Remission after Induction in Childhood B Lineage Leukemia.

Several studies showed a good correlation between minimal residual disease (MRD) obtained by flow cytometry (FC) and by PCR (Ig/TCR rearrangements). However, before FC is widely applied for therapeutic decisions we need exact and standardized criteria. Therefore, we pre-defined B-lineage cell subsets using 4 or 3-color FC and we established background levels for each subset in pre-defined time-points on therapy.Methods. Children with ALL were treated by ALL IC BFM2002 protocol. This international flow cytometric trial MiniMini contained 4 laboratories (Croatia 36 patients, Israel 48, Hong Kong 33 and Czechia 136). 442 samples were measured simultaneously by PCR and 4color FC. Pre-defined subsets (28 values) were measured at 5 time points (d0, d8, d15, d33 and week 12). Cut off for positivity and negativity of individual subpopulations were assessed as follows: 1) negative samples by PCR for respective time point were randomly divided into training and testing cohort 2) 98.5 percentile of individual subpopulation in training cohort was selected as a cut off for positivity and negativity 3) cut off values were tested on testing cohort and PCR positive samples (total 337 samples), sample was considered positive when having at least one value above cut off (when more values were above cut off, highest value was considered as MRD).Results. When all time points were analyzed together, sensitivity of FC MRD was 82% and specificity 88% There are no negative patients at day 8 in bone marrow by FC or by PCR. 5 samples at day 15 were positive by PCR (<=10^−4) and negative by FC, no sample was false positive by FC, sensitivity was 98%, no sample was negative by both methods. At day 33 and at week 12, sensitivity dramatically dropped to 34 and 13%, specificity was 80% and 100%. We next asked whether the early blast clearance assessed by FC MRD predicts positivity by PCR after induction. Univariate analysis showed that the blast clearance by FC both at days 8 and 15 predicts the PCR-based molecular response at day 33 and/or week 12. Patients with PCR-positive MRD at day 33 and at week 12 had more often over 50% blasts at day 8 BM (Fischer test p=0.0013 and p=0.04) and/or more than 5% blasts at day 15 by FC (p=0.0015 and p=0.0036).Conclusion. With a minimized subjective bias and exact background values set for each cellular subset and timepoint, we did not reach satisfactory sensitivity at day 33 and at week 12. The FC-assessed leukemia clearance at days 8 and 15 predicts MRD by PCR at day 33 and at week 12. Since a non leukemic background influences sensitivity of FC MRD at day 33 and at week 12, complex approach using 6 and more color combinations is needed for precise distinction between leukemic and non leukemic B cells.

Flow cytometric protein expression profiling as a systematic approach for developing disease-specific assays: identification of a chronic lymphocytic leukaemia-specific assay for use in rituximab-containing regimens

Depletion of disease below the levels detected by sensitive minimal residual disease (MRD) assays is associated with prolonged survival in chronic lymphocytic leukaemia (CLL). Flow cytometric MRD assays are now sufficiently sensitive and rapid to guide the duration of therapy in CLL, but generally rely on assessment of CD20 expression, which cannot be accurately measured during and after therapeutic approaches containing rituximab. The aim of this study was to use analytical software developed for microarray analysis to provide a systematic approach for MRD flow assay development. Samples from CLL patients (n=49), normal controls (n=21) and other B-lymphoproliferative disorders (n=12) were assessed with a panel of 66 antibodies. The DNA-Chip analysis program was used to identify discriminating antibodies, with hierarchical cluster analysis to identify complementary combinations. An iterative process was used: increasing numbers of patients were assessed with smaller, more targeted antibody panels until a highly specific combination (CD81/CD22/CD19/CD5) was identified. This combination was as sensitive and specific as previously reported assays and potentially applicable to blood and marrow samples from patients treated with current therapeutic approaches including rituximab. This approach to the identification of disease-specific antibody combinations for MRD analysis is readily applicable to a variety of haematological disorders.

The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia

6533 Background: Multiparametric flow-cytometry is frequently used to assess the levels of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients achieving complete remission after intensive chemotherapy. In our previous experience, MRD negativity after consolidation cycle, as defined by a level of bone marrow residual leukemic cells ≤3.5x10−4, was associated with a significantly longer relapse free survival (RFS) and overall survival (OS). The aims of the present study were to confirm, in a larger series of patients, the prognostic relevance of the post consolidation MRD assessment and to validate the threshold of 3.5x10−4. Methods: 100 patients affected with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age &lt;61 yrs) or AML13/AML15 (age &gt;61 yrs), consisting in intensive induction and consolidation cycles. Median age was 52 years (range 18–78), all FAB subtypes were represented with the exception of APL cases. A Maximally Selected Rank Statistic analysis was used to select the best threshold. Results: The statistical test confirmed that the level of 3.5×10−4 residual leukemic cells was the most significant in discriminating categories of risk. In fact, levels of MRD measured at the post-consolidation time-point identified 2 distinct subgroups: 36 patients MRD negative and 56 MRD positive. MRD negative patients had a better outcome in terms of relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008), regardless of MRD status after induction. In fact, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. The multivariate analysis, including karyotype, age, MDR1 phenotype and post induction and post consolidation MRD levels, indicated that the post-consolidation MRD status was an independent factor affecting relapse rate (P&lt;0.001), OS (P=0.039) and RFS (P=0.008). Conclusions: We conclude that 1) the threshold of 3.5x10−4 is valid in discriminating risk categories in AML; 2) MRD assessment at post-consolidation check-point is critical to predict disease outcome. The incorporation in clinical trials of flow-cytometric MRD determination may allow a more accurate risk stratification of AML patients. No significant financial relationships to disclose.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

Prognostic significance and modalities of flow cytometric minimal residual disease detection in childhood acute lymphoblastic leukemia

AbstractDetection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) predicts outcome. Previous studies were invariably based on relative quantification and did not investigate sample-inherent parameters that influence test accuracy, which makes comparisons and clinical conclusions cumbersome. Hence, we conducted a prospective, population-based MRD study in 108 sequentially recruited children with ALL uniformly treated with the ALL-Berlin-Frankfurt-Münster (ALL-BFM) 95 protocol in Austria (median follow-up of 40 months). Using sensitive, limited antibody panel flow cytometry applicable to 97% of patients, we investigated 329 bone marrow samples from 4 treatment time points. MRD was quantified by blast percentages among nucleated cells (NCs) and by absolute counts (per microliter). Covariables such as NC count, normal B cells, and an estimate of the test sensitivity were also recorded. Presence and distinct levels of MRD correlated with a high probability of early relapse at each of the time points studied. Sequential monitoring at day 33 and week 12 was most useful for predicting outcome independently from clinical risk groups: patients with persistent disease (≥ 1 blast/µL) had a 100% probability of relapse, compared to 6% in all others. Absolute MRD quantification was more appropriate than relative, due to considerable variations in total NC counts between samples. Regeneration of normal immature B cells after periods of rest from treatment limited the test sensitivity. In conclusion, MRD detection by flow cytometry is a strong and independent outcome indicator in childhood ALL. Standardization regarding absolute quantification on the basis of NCs and assessment during periods of continuous treatment promise to increase the accuracy, simplicity, and cost efficiency of the approach.