Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Minimal residual disease quantification by flow cytometry provides reliable risk stratification in T-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10-3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4-9.0, p = 0.008) for day 29 FCM-MRD ≥ 10-3 and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10-3 compared with MRD < 10-3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10-4-<10-3 had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10-4 or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10-4 had a cumulative incidence of relapse of 2.3% (95% CI 0-6.8, n = 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.

Infused CD34 cell dose, but not tumour cell content of peripheral blood progenitor cell grafts, predicts clinical outcome in patients with diffuse large B-cell lymphoma and follicular lymphoma grade 3 treated with high-dose therapy.

Previously, we have shown that patients with diffuse large B-cell lymphoma (DLBCL) transplanted with contaminated bone marrow (BM) generally have a poor outcome. Whether this is also the case when peripheral blood progenitor cell (PBPC) grafts are used is not known. Forty-three patients with chemosensitive DLBCL or follicular lymphoma grade 3 (FLgr3) were treated with high-dose therapy (HDT) and autologous stem cell support. Nine patients received purged grafts. Quantitative real-time polymerase chain reaction (QRT-PCR) for either the BCL2/IgH translocation or allele specific oligonucleotide (ASO) QRT-PCR for the immunoglobulin heavy chain (IgH) complementarity-determining region 3 were used. Nine of 25 (36%) PBPC grafts contained tumour cells as tested by QRT-PCR, including two grafts purged by CD34(+) cell enrichment combined with B-cell depletion. The level of contamination of the PBPC/CD34(+) cells ranged from 0 to 8.28%. No relationship could be shown between the total number of tumour cells infused and relapse. Patients receiving PCR-positive or PCR-negative PBPC grafts had similar progression-free survival (PFS) (P = 0.49). However, a significant difference was seen in PFS and overall survival (OS) for the patients given >/=6.1 x 10(6) CD34(+) cells/kg compared with those given <6.1 x 10(6) CD34(+) cells/kg (P = 0.01 and P < 0.05 respectively).

High-dose therapy supported with immunomagnetic purged autologous bone marrow in high-grade B cell non-Hodgkin's lymphoma.

From August 1987 to March 1995, 25 patients with high-grade B cell non-Hodgkin's lymphoma (NHL) were treated with high-dose therapy (HDT) followed by bone marrow purged with immunomagnetic beads. At the time of transplantation, 20 patients were in sensitive relapse and five in first complete or partial remission. Ten patients had secondary high-grade NHL transformed from low-grade NHL. The HDT consisted of TBI followed by high-dose cyclophosphamide. All patients engrafted, except for two patients with early treatment-related death. Eleven patients relapsed, of whom nine died of lymphoma, and two are alive in new CR. The estimated event-free and overall survivals at 5 years were 40% and 48%, respectively, with a median follow-up of 48 months (range 1-123). Eight of the tumours contained the translocation t(14;18) at the major breakpoint region (MBR) of BCL-2. In these patients the presence of tumour cells in the bone marrow graft before and after purging were assessed by PCR. Four of five patients infused with non-detectable minimal residual disease in their autografts are in complete remission, while two of three patients reinfused with t(14;18) positive cells after purging, experienced a fast and aggressive relapse. As found by others, our data suggest that reinfusion of tumour-free autografts obtained by efficient in vivo purging using chemotherapy before harvesting, and/or by in vitropurging of the stem cell products, influence the patients remission status after HDT.