Flow Cytometry-based Minimal Residual Disease Research Articles

Clinical Outcomes of B Cell Acute Lymphoblastic Leukemia Patients Treated with Haploidentical Stem Cells Combined with Umbilical Cord Blood Transplantation

Clinical outcomes of hematopoietic stem cell transplantation (HSCT) using a haploidentical stem cell graft and an unrelated umbilical cord blood unit (haplo-cord HSCT) for the treatment of B cell acute lymphoblastic leukemia (B-ALL) remain unclear. This study was conducted to explore the clinical outcomes of haplo-cord HSCT in patients with B-ALL. A total of 112 B-ALL patients who underwent haplo-cord HSCT and 64 B-ALL patients who underwent haploidentical HSCT (haplo-HSCT) at our center between 2010 and 2020 were retrospectively included in this study, and clinical outcomes and prognostic factors were further analyzed. Of the 112 haplo-cord HSCT recipients, 106 (94.6%) achieved complete haploidentical chimerism and 6 (5.4%) had mixed cord blood chimerism. No differences in neutrophil and platelet recovery or in the incidences of graft-versus-host disease, cytomegalovirus/Epstein-Barr virus viremia, bloodstream infection, or hemorrhagic cystitis were observed between the haplo-cord HSCT and haplo-HSCT groups. Compared with the haplo-HSCT group, the haplo-cord HSCT group had a higher absolute number of CD3+ cells (P=.029) and a lower ratio of CD3+CD4+ /CD3+CD8+ cells (P=.049) at 1 month post-transplantation. Moreover, the haplo-cord HSCT group had lower minimal residual disease (MRD) levels at 1 month (P=.020) and 100 days (P=.038) post-transplantation and a better 3-year prognosis (overall survival, P=.016; disease-free survival, P=.041; cumulative incidence of relapse [CIR], P=.016). The CIRs in patients with adverse genomic features (P=.040) or flow cytometry-based MRD (FCM-MRD) ≥1×10-4 (P=.033) were improved by haplo-cord HSCT. Multivariate analysis revealed that haplo-cord HSCT could independently improve the 3-year OS, DFS, and CIR of B-ALL patients (OS, P=.029; DFS, P=.024; CIR, P=.024). In addition, allo-HSCT at first complete remission was an independent parameter associated with 3-year OS for B-ALL patients (P=.014). An FCM-MRD ≥1×10-4 pre-HSCT could independently predict unfavorable 3-year DFS and CIR (DFS, P=.020; CIR, P=.036) in B-ALL patients. Our data suggest that the use of haplo-cord HSCT can independently improve survival in patients with B-ALL.

A Phase II Study of 5-Azacytidine (AZA) and Venetoclax As Maintenance Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission

A Phase II Study of 5-Azacytidine (AZA) and Venetoclax As Maintenance Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission

Clinicoepidemiologic Profile and Outcome Predicted by Minimal Residual Disease in Children With Mixed-phenotype Acute Leukemia Treated on a Modified MCP-841 Protocol at a Tertiary Cancer Institute in India.

Mixed-phenotype acute leukemia (MPAL) accounts for 1.2% to 5% of acute leukemia across age groups with intermediate prognosis. We evaluated clinicoepidemiologic profiles and outcomes of MPAL. Records of children younger than 15 years of age with acute leukemia from January 2010 to December 2016 were reviewed on the basis of the MPAL WHO 2008 criteria. Treatment was uniform with a modified MCP-841 protocol. Descriptive analysis tools were used. Outcomes were measured by the Kaplan-Meier method on MedCalc, version 14.8.1. Among 3830 children with acute leukemia in the study period, 2892 received treatment from our center, of whom 24 (0.83%) had MPAL, median age 9 years, with a male:female ratio of 3:1, and median white blood cell of 13.4×10/L. Common immunophenotypes were B/myeloid-12 (50%), T/myeloid-9 (37.5%), and B/T-lymphoid-3 (12.5%). Some B/myeloid cases had abnormal cytogenetics. Seventeen patients were evaluable for outcome. Sixteen patients underwent postinduction bone marrow and 13 (81%) achieved morphologic remission. Thirteen patients underwent flow cytometry-based minimal residual disease evaluation; 9 (69%) were <0.01% (4 postinduction, 5 postconsolidation), and 67% of these had sustained remission till the last follow-up. None underwent bone marrow transplant. The projected 3-year event-free and overall survival rates were 40% and 48%, respectively (median follow-up: 22 mo). MPAL represented <1% of childhood acute leukemia. acute lymphoblastic leukemia-type chemotherapy that incorporated high-dose cytarabine was effective in achieving an minimal residual disease-negativity rate of 69% in evaluated patients, which was also predictive of better outcome.

Validation of a modified pre-lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B-non Hodgkin lymphoma.

Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.

Flow-Cytometry Based Detection of Any Minimal Residual Disease (FC-MRD) in Children with T-Acute Lymphoblastic Leukemias (T-ALL) Is a Powerful Indicator of Outcome

Background: Minimal Residual Disease (MRD) is a powerful predictor of event-free survival in acute lymphoblastic leukemia (ALL). However, as T-ALL is less common, MRD studies are limited, often with small cohorts, and even fewer have been done by flowcytometry-based MRD (FC-MRD). There have also been different time-points such as Post-Induction (PI-MRD), and late or Post-Consolidation (PC-MRD) that have shown significant correlation with overall, event, and relapse-free survival (OS, EFS &amp; RFS). As the available literature is still not conclusive, we investigated the impact of FC-MRD on survival in childhood T-ALL at a large tertiary cancer care centre in India. Methods: Children less than 15-years age diagnosed with T-ALL from Jan-2014 to May 2018 were treated uniformly on a modified MCP-841 protocol that included a 4-drug induction (vincristine, prednisolone, l-asparaginase, daunomycin), and consolidation with high-dose cytarabine (24gm/m2 in 3 equal legs for children below 3-years, and 16gm/m2 in two equal legs, for 3 or more years age). Post-consolidation therapy followed the pattern of Interim Maintenance, two Delayed Intensification cycles, and 18 months of Maintenance. Central Nervous System (CNS)-directed therapy consisted of intrathecal methotrexate in all cycles, and those with CNS involvement received additional Cranial Radiation of 18Gy. In early-thymic-precursor (ETP-ALL) immunophenotype patients, dexamethasone replaced prednisolone in Induction. The protocol did not include High-Dose Methotrexate. FC-MRD was estimated by 10-color FC-MRD assay on Navios flow-cytometer (Beckman Coulter, Inc.) at Post-Induction(day-35), and Post-Consolidation(day-78) for those PI-MRD positive(+ve). FC-MRD was analyzed on Kaluza® software v-1.3. Any detectable value was taken as positive. Statistical analysis was performed using MedCalc Statistical Software®. Results: Of 368 eligible patients diagnosed at our centre in the study period, 81 did not undergo PI-MRD (14- Induction deaths, 13- treatment abandonment, 54- referral to other institute/ time-point missed/ did not consent/ other reasons). Another 18 abandoned treatment within 100 days of diagnosis and were also excluded. In the remaining 269, median age was 10-years (range:1-15), M:F-3.8:1. Median presenting WBC was 96.7 x 103/cmm (range:1.14-849), and thirteen patients had ETP. PI-MRD was positive in 125(46%) patients (median MRD -0.3%, range:0.0007-43.6%) of which 58(46.4%) developed medullary relapse, compared to 17 of 144(11.6%) for PI-MRD negative(-ve) patients, with Hazard Ratio (HR) for risk of medullary-relapse for PI-MRD+ve being 5.02(95% CI:3.16-7.96; p&lt;0.0001). Median RFS was 20.5 months (95% CI:16.2-34.7) for PI-MRD+ve patients, while median was not-reached for PI-MRD-ve. PI-MRD+ve patients were at high risk for all events (medullary relapse, extramedullary relapse and death) with an incidence of 53.6% versus 27.8%; p&lt;0.0001, and HR of 2.36 (95% CI:1.6-3.47; p&lt;0.0001). Probability of EFS at 30 months was 68.9% for PI-MRD+ve and 41.1% for PI-MRD-ve patients(Fig-1), while there was no significant difference in 30-month OS, which were 78.7% and 77.9% respectively. Most relapsed/ refractory patients were unable to undergo intensive salvage regimens and/ or hematopoietic stem cell transplants due to socio-economic constraints. When their disease progressed, they were mostly sent home on palliative care on an oral metronomic chemotherapy protocol, on which they survived for varying periods of times. PC-MRD was available in 90 PI-MRD+ patients, and was positive in 28(31%), with median MRD level of 0.055% (range:0.0008-27.6%). PC-MRD+ve patients had significantly shorter RFS (median-14.3 months, 95% CI:7.1-49.7 months), with risk of relapse HR of 2.36 (95% CI:1.6-3.47; p&lt;0.0001). Univariate and multivariate analysis using Cox-hazard model for age, hyperleukocytosis and ETP-immunophenotype showed that PI-MRD was the most significant and an independent high-risk factor for relapse with HR for multivariate analysis being 5.5(95% CI:2.85-11.45; p&lt;0.0001). Conclusion: We conclude that 10-color FC-MRD done Post-Induction and Post-Consolidation detecting any residual disease reliably identifies those at highest risk of relapse and any other event. PI-MRD+ is an independent, and also the most important risk-factor for any event, and if PC-MRD is also positive, relapse occurs early. Figure 1 Disclosures No relevant conflicts of interest to declare.

Flow Cytometry Based MRD and Its Impact on Survival Outcome in Children and Young Adults with ALL: A Prospective Study from a Tertiary Cancer Centre in Southern India.

Presence of minimal residual disease (MRD) following induction chemotherapy is a well-recognized risk factor to predict relapse in acute lymphoblastic leukemia (ALL). There is paucity of data on MRD and outcome in ALL from India. We share our experience in establishing a flow cytometry-based MRD assay for ALL with emphasis on determination of the number of patients who had MRD on day 35 of induction therapy and its correlation with outcome and other prognostic factors. We prospectively studied MRD in patients with ALL less than 25years who achieved morphological complete remission with induction therapy. The initial series consisted of 104 patients with ALL. Ninety-two patients had bone marrow samples collected on day 35 of remission induction chemotherapy that was adequate for MRD. Strategy of monitoring MRD was based on flow cytometry using six color staining according the leukemia associated immunophenotype found at diagnosis. Data analysis was done using Fisher exact test. The median age was 8.5years (range 0.9-22years). Thirty-seven out of ninety-two patients (40.2%) had MRD at end of induction. MRD on day 35 was between 0.01 and 0.1% in 18.9% of patients, between 0.1 and 1% in 59.5% and more than 1% in 21.6% patients. Among the patients who had MRD, 16.7% had favourable cytogenetics, 60% had intermediate and 13.3% had high-risk cytogenetics. The presence or absence of residual leukemia by flow cytometry at day 35 was not significantly related to age (p = 1.0), male gender (p = 0.08) hyperleukocytosis (p = 0.25) or day 8 blast clearance (p = 0.21). However, T cell phenotype (p < 0.001) was significantly associated with MRD. The 5-year event free survival (EFS) for patients who had MRD versus those who did not was 69% and 61.1% respectively (p = 0.41). The 5-year overall survival (OS) for patients who had MRD versus those who did not was 72.5% and 61.1% respectively (p = 0.33). Flow cytometric techniques can be applied to monitor MRD in patients of ALL undergoing induction therapy. Our results suggest MRD correlates with certain known prognostic factors. Though the EFS and OS was lower in MRD positive patients, the results were not statistically significant probably because of the small sample size.

CD7 is expressed on a subset of normal CD34-positive myeloid precursors.

To improve monitoring of myeloid neoplasms by flow cytometry-based minimal residual disease (MRD) analysis, we analyzed the significance of leukemia-associated immunophenotype (LAIP) markers in 44 patients. In a pilot study cohort, peripheral blood or bone marrow samples from 13 patients with myeloid neoplasms and one case of B lymphoblastic leukemia in complete hematologic remission after allogeneic bone marrow or stem cell transplantation were subjected to selection for leukemia-specific phenotypes by fluorescence-activated cell sorting using individual marker combinations, followed by PCR-based chimerism analysis. The feasibility of this method could be demonstrated, with selection being successful in 12 cases, including two cases where mixed chimerism was found exclusively in sorted cells. Interestingly, four specimens displayed full donor chimerism in cells expressing the presumably aberrant combination CD34+ /CD7+ . Further analyses, including assessment of an independent cohort of 25 patients not affected by neoplastic bone marrow infiltration, revealed that normal myeloid precursors usually include a population coexpressing CD34, CD13, CD33, and CD7. We conclude that the combination CD34+ /CD7+ might not be suitable as an LAIP for MRD diagnostics and that a subset of normal myeloid precursors in the bone marrow expresses CD7.

Clearance of Somatic Mutations at Remission and the Risk of Relapse in Acute Myeloid Leukemia.

Purpose The aim of the current study was to determine whether the degree of mutation clearance at remission predicts the risk of relapse in patients with acute myeloid leukemia (AML). Patients and Methods One hundred thirty-one previously untreated patients with AML who received intensive induction chemotherapy and attained morphologic complete remission (CR) at day 30 were studied. Pretreatment and CR bone marrow were analyzed using targeted capture DNA sequencing. We analyzed the association between mutation clearance (MC) on the basis of variant allele frequency (VAF) at CR (MC2.5: if the VAF of residual mutations was < 2.5%; MC1.0: if the VAF was < 1%; and complete MC [CMC]: if no detectable residual mutations) and event-free survival, overall survival (OS), and cumulative incidence of relapse (CIR). Results MC1.0 and CMC were associated with significantly better OS (2-year OS: 75% v 61% in MC1.0 v non-MC1.0; P = .0465; 2-year OS: 77% v 60% in CMC v non-CMC; P = .0303) and lower CIR (2-year CIR: 26% v 46% in MC1.0 v non-MC 1.0; P = .0349; 2 year-CIR: 24% v 46% in CMC v non-CMC; P = .03), whereas there was no significant difference in any of the above outcomes by MC2.5. Multivariable analysis adjusting for age, cytogenetic risk, allogeneic stem-cell transplantation, and flow cytometry-based minimal residual disease revealed that patients with CMC had significantly better event-free survival (hazard ratio [HR], 0.43; P = .0083), OS (HR, 0.47; P = .04), and CIR (HR, 0.27; P < .001) than did patients without CMC. These prognostic associations were stronger when preleukemic mutations, such as DNMT3A, TET2, and ASXL1, were removed from the analysis. Conclusion Clearance of somatic mutation at CR, particularly in nonpreleukemic genes, was associated with significantly better survival and less risk of relapse. Somatic mutations in nonpreleukemic genes may function as a molecular minimal residual disease marker in AML.

Intra-Tumoral Blast Heterogeneity and Implications for Minimal Residual Disease Detection in T-Cell Acute Lymphoblastic Leukemia

Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity.Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients).Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts.Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations.Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (>20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments.Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells.The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Issue highlights—March 2013

Issue highlights—March 2013

Standardizing minimal residual disease by flow cytometry for precursor B lineage acute lymphoblastic leukemia in a developing country

In addition to standard risk criteria at diagnosis, minimal residual disease (MRD) following initiation of therapy is a well-recognized risk factor to predict relapse. Literature from developing countries addressing therapeutic or laboratory practices related to MRD, is largely lacking. In a first paper from India, we describe our experience in establishing a flow cytometry-based MRD assay for precursor B lineage ALL (BCP-ALL) with emphasis on the assay standardization and cost. Normal templates for B cell development were established in 10 control patients using CD45, CD11a, CD38, CD20, CD10, CD19, CD58, CD34, CD123, and CD22. BCP-ALL samples (n = 42) were characterized at diagnosis to identify a suitable marker for follow-up during mid (D+21) and end of induction (D+33). Both, multiparametric immunophenotyping and single marker detection of LAIP were used for data analysis. In 95.2% of BCP-ALL at least two informative markers could be obtained when a minimum of four cocktail combinations were used. The combination CD20, CD10, CD45, and CD19 was the most useful (71.4%) followed by combinations containing CD38 (66.7%), CD22 (57.1%), CD11a (52.4%), and CD58 (33.3%). Using our approach, 60 and 47% of patients had detectable MRD at mid and end induction time points, respectively. We have described a relatively cost effective MRD panel which is applicable to over 90% of patients. We hope that this data would encourage more centers in India and other resource constrained health delivery systems to develop MRD assays.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.