15-color highly sensitive flow cytometry assay for post anti-CD19 targeted therapy (anti-CD19-CAR-T and blinatumomab) measurable residual disease assessment in B-lymphoblastic leukemia/lymphoma: Real-world applicability and challenges.

OBJECTIVES: Measurable residual disease (MRD) is the most relevant predictor of disease-free survival in B-cell acute lymphoblastic leukemia (B-ALL). We aimed to establish a highly sensitive flow cytometry (MFC)-based B-ALL-MRD (BMRD) assay for patients receiving anti-CD19 immunotherapy with an alternate gating approach and to document the prevalence and immunophenotype of recurrently occurring low-level mimics and confounding populations. METHODS: We standardized a 15-color highly-sensitive BMRD assay with an alternate CD19-free gating approach. The study included 137 MRD samples from 43 relapsed/refractory B-ALL patients considered for anti-CD19 immunotherapy. RESULTS: The 15-color BMRD assay with CD22/CD24/CD81/CD33-based gating approach was routinely applicable in 137 BM samples and could achieve a sensitivity of 0.0005%. MRD was detected in 29.9% (41/137) samples with 31.7% (13/41) of them showing <.01% MRD. Recurrently occurring low-level cells that showed immunophenotypic overlap with leukemic B-blasts included: (a) CD19+CD10+CD34+CD22+CD24+CD81+CD123+CD304+ plasmacytoid dendritic cells, (b) CD73bright/CD304bright/CD81bright mesenchymal stromal/stem cells (CD10+) and endothelial cells (CD34+CD24+), (c) CD22dim/CD34+/CD38dim/CD81dim/CD19-/CD10-/CD24- early lymphoid progenitor/precursor type-1 cells (ELP-1) and (d) CD22+/CD34+/CD10heterogeneous/CD38moderate/CD81moderate/CD19-/CD24- stage-0 B-cell precursors or ELP-2 cells. CONCLUSIONS: We standardized a highly sensitive 15-color BMRD assay with a non-CD19-based gating strategy for patients receiving anti-CD19 immunotherapy. We also described the immunophenotypes of recurrently occurring low-level populations that can be misinterpreted as MRD in real-world practice.

Assessment of proxy-reported responses as predictors of motor and sensory peripheral neuropathy in children with B-lymphoblastic leukemia.

Chemotherapy-induced peripheral neuropathy (CIPN), a common condition in children with acute lymphoblastic leukemia, can be challenging to diagnose. Using data from Children's Oncology Group AALL0932 physical function study, we sought to determine if parent/guardian proxy-reported responses from the Pediatric Outcomes Data Collection Instrument could identify children with motor or sensory CIPN diagnosed by physical/occupational therapists (PT/OT). Four variables moderately discriminated between children with and without motor CIPN (c-index 0.76, 95% confidence interval [CI]: 0.64-0.84), but sensory and optimism-corrected models had weak discrimination (c-index sensory models 0.65, 95% CI: 0.54-0.74). New proxy-report measures are needed to identify children with PT/OT diagnosed CIPN.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Molecular classification improves risk assessment in adult BCR-ABL1-negative B-ALL.

Genomic classification has improved risk assignment of pediatric, but not adult B-lineage acute lymphoblastic leukemia (B-ALL). The international UKALLXII/ECOG-ACRIN E2993 (#NCT00002514) trial accrued 1229 adolescent/adult patients with BCR-ABL1- B-ALL (aged 14 to 65 years). Although 93% of patients achieved remission, 41% relapsed at a median of 13 months (range, 28 days to 12 years). Five-year overall survival (OS) was 42% (95% confidence interval, 39, 44). Transcriptome sequencing, gene expression profiling, cytogenetics, and fusion polymerase chain reaction enabled genomic subtyping of 282 patient samples, of which 264 were eligible for trial, accounting for 64.5% of E2993 patients. Among patients with outcome data, 29.5% with favorable outcomes (5-year OS 65% to 80%) were deemed standard risk (DUX4-rearranged [9.2%], ETV6-RUNX1/-like [2.3%], TCF3-PBX1 [6.9%], PAX5 P80R [4.1%], high-hyperdiploid [6.9%]); 50.2% had high-risk genotypes with 5-year OS of 0% to 27% (Ph-like [21.2%], KMT2A-AFF1 [12%], low-hypodiploid/near-haploid [14.3%], BCL2/MYC-rearranged [2.8%]); 20.3% had intermediate-risk genotypes with 5-year OS of 33% to 45% (PAX5alt [12.4%], ZNF384/-like [5.1%], MEF2D-rearranged [2.8%]). IKZF1 alterations occurred in 86% of Ph-like, and TP53 mutations in patients who were low-hypodiploid (54%) and BCL2/MYC-rearranged (33%) but were not independently associated with outcome. Of patients considered high risk based on presenting age and white blood cell count, 40% harbored subtype-defining genetic alterations associated with standard- or intermediate-risk outcomes. We identified distinct immunophenotypic features for DUX4-rearranged, PAX5 P80R, ZNF384-R/-like, and Ph-like genotypes. These data in a large adult B-ALL cohort treated with a non-risk-adapted approach on a single trial show the prognostic importance of genomic analyses, which may translate into future therapeutic benefits.

Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

Blast size-specific flowcytometric ploidy assessment using FxCycleTM Violet dye and its correlation with conventional cytogenetic ploidy in pediatric precursor B-lineage acute lymphoblastic leukemia patients.

INTRODUCTION: Numerical chromosomal abnormalities (aneuploidies), present in approximately 30%-50% of pediatric precursor B-lineage acute lymphoblastic leukemia (B-ALL) patients, are commonly identified through a laborious conventional cytogenetic (CG) technique. Flow cytometry (FCM) can identify both physical and fluorescent properties of cells together, and by using fluorescent nucleic-acid-binding dyes, FCM can identify variations in total nucleic-acid content of cells. FxCycleTM Violet dye (FxCV) is a selective DNA-binding dye which permits simultaneous multiparametric immunophenotyping and cell-cycle/ploidy assessment in a single assay. To date, only two studies have demonstrated the feasibility of FxCV-aided FCM-ploidy analysis in B-ALL patients and only one of these studies have compared their results with CG-ploidy. METHODOLOGY: Blast size-specific FCM-ploidy was prospectively analyzed using FxCV-dye in 109 pediatric B-ALL patients, and the results were compared with concurrent CG-ploidy status. RESULTS: FCM-ploidy categorization was feasible in 98% of samples tested and the results were 82% concordant with CG-ploidy status. We observed significant correlation between DNA content and blast size (r = .823, P < .001) and could demonstrate size differences between diploid vs low-hyperdiploid (P = .025), diploid vs high-hyperdiploid (P < .001) and low- vs high-hyperdiploid blasts (P = .007). CONCLUSION: FCM-ploidy assessment using FxCV dye is a reliable assay and the results closely concur with CG-based ploidy stratification and risk assessment. Using blast size-assisted DNA content analysis, the results of FCM-ploidy analysis can be further fine-tuned.

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

CD304/neuropilin-1 is a very useful and dependable marker for the measurable residual disease assessment of B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Measurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC-based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin-1, in the assessment of MFC-based MRD. METHODS: Expression patterns of CD304 were studied in leukemic blasts from BCP-ALL patients and in normal precursor B cells (NPBC) from uninvolved non-BCP-ALL bone marrow samples using 10-color MFC. MRD was monitored at end-of-induction (EOI; Days 35-40) and end-of-consolidation (Day 78-80) time points. RESULTS: We studied CD304 expression in 300 pediatric BCP-ALL patients and found it positive in BCP-ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6-RUNX1 (p < .001) as well as BCR-ABL1 (p = .019) and inversely associated with TCF3-PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI-MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low-level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI-MRD samples. CONCLUSIONS: CD304 is commonly expressed in leukemic blasts of BCP-ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC-based MRD assay in BCP-ALL.

Multiplexed Digital Detection of B-Cell Acute Lymphoblastic Leukemia Fusion Transcripts Using the NanoString nCounter System.

Chromosomal rearrangements resulting in fusion transcripts have been reported in precursor B-cell acute lymphoblastic leukemia (B-ALL). The identification of fusion events is crucial in the diagnosis of B-ALL. In this study, we used NanoString nCounter technology to design, validate, and evaluate a multiplex panel for the detection of B-ALL fusion transcripts. Fifty-one B-ALL fusion transcripts reported in children in the literature were included in the design of the NanoString panel. Twenty-six fusion transcripts were validated using 64 positive-control samples and 74 negative-control samples with 100% sensitivity and 99% specificity in comparison to RT-PCR. Our results support a potential role of NanoString's technology as a robust and cost-effective technique that could be used in the detection of fusion transcripts and implemented into the diagnostic algorithm of B-ALL.

Early (Day 15 Post Diagnosis) Peripheral Blood Assessment of Measurable Residual Disease in Flow Cytometry is a Strong Predictor of Outcome in Childhood B-Lineage Lymphoblastic Leukemia.

BACKGROUND: In children with acute lymphoblastic leukemia (ALL) low levels of minimal residual disease (MRD) after induction, essentially assessed in the bone marrow, have been shown to be of good prognosis. However, only few studies have tested the peripheral blood for MRD. METHODS: Here, we report the impact on survival of peripheral blood (PB) MRD assessment by multiparameter flow cytometry (MFC) at early time points of treatment in 125 B-ALL children, compared to Day 35 molecular bone marrow (BM) MRD. Patients were sampled for MFC one week postdiagnosis after a pre-phase of corticotherapy (Day 8), then after one week of chemotherapy (Day 15). The study enrolled 67 boys and 58 girls with a median follow-up of 52 months. Over the duration of the study, 20 patients relapsed and eight died. MFC was performed based on the leukemia-associated immunophenotype at diagnosis, using panels of 10 antibodies. RESULTS: Although, PB MFC-MRD had no prognostic impact at Day 8, Day 15 MRD negativity was associated with a significantly better 4 years DFS (91.6 ± 3% vs. 67.6 ± 9% P = 0.0013). Furthermore, while MFC and molecular data were concordant in most cases, patients with detectable PB MRD on Day 15, yet negative in BM on Day 35 had a significantly lower DFS (P < 0.0001). CONCLUSION: This study demonstrates that the less invasive procedure of MFC-MRD assessment in PB can be informative for childhood ALL patients at the early point of Day 15 of the treatment schedule. © 2019 International Clinical Cytometry Society.

Flow cytometric minimal residual disease assessment of peripheral blood in acute lymphoblastic leukaemia patients has potential for early detection of relapsed extramedullary disease.

OBJECTIVES: To evaluate peripheral blood (PB) for minimal residual disease (MRD) assessment in adults with acute lymphoblastic leukaemia (ALL). METHODS: We analysed 76 matched bone marrow (BM) aspirate and PB specimens independently for the presence of ALL MRD by six-colour flow cytometry (FC). RESULTS: The overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. We identified two cases with evidence of leukaemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM. CONCLUSIONS: The use of PB MRD as a non-invasive method for monitoring of systemic relapse may have added clinical and diagnostic value in patients with high risk of extramedullary disease.

B Lymphoblastic Leukemia Minimal Residual Disease Assessment by Flow Cytometric Analysis.

Among the most thoroughly evaluated modalities for assessment of minimal residual disease (MRD) in B acute lymphoblastic leukemia is multiparameter flow cytometry. Flow cytometric evaluation of MRD for B-ALL requires complete understanding of the immunophenotype of hematogones, the normal counterpart of leukemic B lymphoblasts. Assessment of multiple flow cytometry markers, in concert with each other in multidimensional histograms, is necessary to distinguish hematogones from malignant blasts. Emerging therapies targeting CD19 and other B-cell markers can disrupt the most frequently MRD assessment, requiring a revised approach as use of targeted therapies becomes widespread.

Combined IKZF1 and IG markers as new tools for diagnosis and minimal residual disease assessment in Tunisian B-ALL.

INTRODUCTION: The monitoring of minimal residual disease (MRD) approach in patients diagnosed with B-acute lymphoblastic leukemia (B-ALL) allows an early detection of residual clones inducing relapses and therefore appropriate therapy strategy. The molecular markers may identify and quantify the residual blasts in B-ALL with normal cytology. In this study, we aimed to use combined IKZF1, IGH and IGK immunoglobulin genes for diagnosis and MRD monitoring in B-ALL sample using MLPA, multiplex PCR and real-time quantitative PCR. MATERIAL: We showed that multiplex PCR and MLPA are necessary and complementary to detect IKZF1 deletions. RESULTS: We have identified at the diagnosis clonal IGH rearrangement (VH3-JH5) and IKZF1 deletion (Δ4-7), which we have used it for MRD evaluation after induction chemotherapy. Despite the absence of chromosome abnormality, the patient may be classified in high-risk group with a relapse rate of residual blasts>10-4 and sensitivity up to 10-5. This molecular approach enabled the patient's stratification, which was overlooked by classical methods. CONCLUSION: The combined IKZF1 and immunoglobulin genes will be used as appropriate molecular tools for diagnosis and MRD assessment of B-lineage leukemias and introduced as a routine tests in Tunisian clinical laboratories. They will be useful to stratify patients into risk groups leading to better treatment strategy.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Minimal residual disease assessment in childhood acute lymphoblastic leukaemia: a Swedish multi-centre study comparing real-time polymerase chain reaction and multicolour flow cytometry.

Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.

PCR-based clonality assessment in patients with lymphocytic leukaemias: a single-institution experience.

PCR-based clonality testing can be performed in all lymphoproliferations by analysing gene rearrangements of antigen receptors, rearrangements that are unique for each kind of lymphocyte. Reactive lymphoproliferations have polyclonally rearranged Ig/TCR genes, whereas malignant proliferations (leukaemias and lymphomas) show clonal rearrangements. The aim of this study was to assess the clinical benefits of clonality testing with previously evaluated consensus primers in leukaemia patients. The study included peripheral blood and bone marrow samples of 67 leukaemia patients (32 B-CLL, 24 B-ALL and 11 T-ALL). Clonality testing was based on PCR amplification of rearranged IgH and TCR genes. During diagnosis, monoclonal pattern was found in all analysed B-CLL and T-ALL samples. Testing in B-ALL patients showed positive results in all bone marrow and one peripheral blood samples. Results of clonality testing in B-CLL patients during follow-up were concordant between peripheral blood and bone marrow. Obtained results corresponded to clinical course in all but one patient. In B-ALL group, results of molecular testing in peripheral blood and bone marrow confirmed remission estimated according to clinical criteria in all except one patient. Before any clinical sign of relapse, monoclonal pattern was found in six/seven patients by bone marrow and in three/seven patients by peripheral blood analysis, respectively. Results of molecular monitoring in T-ALL patients did not confirme clinical evaluation in two patients. Obtained results indicate high accuracy of re-evaluated primers for clonality assessment in ALL and CLL patients at the time of diagnosis. Results of clonality testing in B-ALL patients indicate that bone marrow analysis has higher sensitivity compared to analysis of peripheral blood.