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.

T-cell acute lymphoblastic leukemia in patients 1-45 years treated with the pediatric NOPHO ALL2008 protocol.

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.

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.

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.

Bone marrow hyaluronan and reticulin in patients with malignant disorders.

Myelofibrosis is commonly seen in patients with chronic myeloproliferative diseases and sometimes in myelodysplastic syndrome, acute leukaemia and lymphoproliferative diseases. The fibrotic process is evaluated by grading the amount of collagen deposited in the bone marrow interstitium. The established method to evaluate bone marrow fibrosis is staining for reticulin to visualise the collagen fibres. However, the extra cellular matrix does not only contain collagens but also other components, e.g. glycosaminoglycans of which hyaluronan is the most abundant. Hyaluronan is important for structural and cellular functions. Earlier studies have shown that there is a positive correlation between hyaluronan and reticulin staining in healthy volunteers and in patients with de novo acute myeloid leukaemia. In this study bone marrow biopsies from 43 patients with a malignant disease involving the bone marrow were compared with 18 patients with a malignant disease not involving the bone marrow. The intensity of hyaluronan grading was significantly higher in the patients with disease involving the bone marrow compared to the healthy controls but not compared to the patients without disease involving the bone marrow. The staining intensity of reticulin in the bone marrow was significantly higher in the patients with disease involving the bone marrow, compared to those without disease involving the bone marrow and to the controls. In all patients and the controls there was a correlation between hyaluronan and reticulin.

Association between telomere length and V(H) gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications.

The immunoglobulin V(H) gene mutation status can divide B-cell chronic lymphocytic leukaemia (CLL) into two entities with a different clinical course. Cases with unmutated V(H) genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated V(H) genes, that is, post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Interestingly, telomerase becomes activated during the GC reaction and an elongation of the telomeres occurs in GC B cells. We performed telomere length and V(H) gene analysis in a series of 61 CLL cases, in order to investigate if the unique telomere lengthening shown in GC B cells could reflect the telomere status in the two subsets of mutated and unmutated CLL. A novel association was found between V(H) gene mutation status and telomere length, since significantly shorter telomeres were demonstrated in the unmutated group compared to the mutated group (mean length 4.3 vs 6.3 kbp). Shorter telomeres also constituted a subgroup with a worse prognosis than cases with longer telomeres (median survival 59 vs 159 months). Furthermore, the Ig gene sequence data revealed that samples with high mutations frequency (>6%) had long telomeres ( approximately 8 kbp). Thus, both the telomere and V(H) gene mutation status in CLL appear linked, which may reflect the proliferative history of the clonal cells with regard to the GC reaction.

Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.

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 situ hybridization 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 C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and 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.

Telomerase regulation and telomere dynamics in germinal centers.

Telomere length maintenance, usually executed by telomerase, is a prerequisite for an extended or infinite division potential. Nevertheless most telomerase positive normal cells exhibit telomere shortening. This study details the telomerase expression and telomere dynamics in purified tonsil B cell subsets during the germinal center (GC) reaction. Significant telomere lengthening was observed as naive B cells matured to centroblasts and when centroblasts matured further to centrocytes, resulting in an increase in telomere length of about 4 kbp determined by Southern blotting. Immunopurified cell populations were also studied by fluorescence in situ hybridization and flow cytometry (flow-FISH) confirming that the GC B cells exhibited lengthened telomeres. These data were further verified in unpurified tonsil cells by combining flow-FISH and immunophenotyping using selected surface markers. Centroblasts expressed high levels of telomerase activity, which was increased in centrocytes, whereas resting naive, activated naive and memory B cells were telomerase activity negative. Expression levels of the catalytic subunit (hTERT) RNA paralleled the telomerase activity levels. The unique telomere elongation in GC B cells permits extensive proliferation during the GC reaction and provides the memory cells with a substantial increase in division potential. Understanding the telomere biology of GC cells is important in defining requirements for telomere elongation in vivo, with implications for the normal immune system as well as for lymphomas, and could provide insights into how the division potential of cells can be manipulated in vitro.

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

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.