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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 CIR5yadjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10−2versus 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 > 10−4associated 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.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1–45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69–0.81), being 0.82 (0.74–0.88) for patients 1.0–9.9 years, 0.76 (0.66–0.86) for those 10.0–17.9 years, and 0.65 (0.55–0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1–9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1–45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

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 ≥ 10−3and 5.6 (95% CI 2.0–16, p= 0.001) for PCR-MRD ≥ 10−3compared with MRD < 10−3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10−4–<10−3had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10−4or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10−4had 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.

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The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situhybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C3TA2and T2AG3repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T2AG3strand seemed to occur somewhat later than that of the C3TA2strand in some samples. (GTG)nand other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.