Recent studies on the immunoglobulin variable heavy chain (IgVH) genes have revealed that B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 clinical entities with either somatically ...mutated or unmutated VH genes. We have analyzed the VH gene mutation status and VH gene usage in 119 B-CLL cases and correlated them to overall survival. A novel finding was the preferential use of the VH3-21 gene in mutated cases, whereas biased VH1-69 gene usage was found in unmutated cases as previously reported. Interestingly, the subset of mutated cases using the VH3-21 gene displayed distinctive genotypic/phenotypic characteristics with shorter average length of the complementarity determining region 3 and clonal expression of λ light chains. In addition, this mutated subset showed significantly shorter survival than other mutated cases and a similar clinical course to unmutated cases. We therefore suggest that B-CLL cases with mutated VH3-21 genes may constitute an additional entity of B-CLL.
Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. ...Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.
Summary
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.
Summary
Cytotoxic CD4+ T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4+ T regulatory cells (Tregs) ...can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4+ T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4+ T‐cell subgroups were investigated in patients with B‐cell malignancies. Peripheral blood was collected from patients with CLL, various B‐cell lymphomas, healthy adult donors, children with precursor B‐cell acute lymphoblastic leukaemia (pre‐B ALL) and from healthy children. CD4+ T cells (CD3+ CD4+ FoxP3−), Tregs (CD3+ CD4+ CD127low FoxP3+) and CD127high FoxP3+ T cells (CD3+ CD4+ CD127high FoxP3+) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T‐cell subgroups compared with healthy donors. Similar results were found in patients with B‐cell lymphomas whereas the CD107a expression in children with pre‐B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4+ T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4+ T cells, including Tregs, are present in patients with B‐cell malignancy and may be an important factor in immune‐related disease control.
Abstract In this study, we followed minimal residual disease (MRD) in eight children with Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) using (i) flow cytometry (FCM), (ii) real-time ...quantitative PCR of IG/TCR gene rearrangements and (iii) RT-PCR detecting fusion gene transcripts. In six of the eight cases the kinetics of MRD clearance was comparable. One of the two discordant cases could be explained by presence of an alternative fusion transcript. The other discordant case showed high BCR-ABL1 RNA level while the other methods did not detect any MRD. In our limited material quantitative RT-PCR of fusion gene transcripts seemed particularly useful to measure MRD in Ph+ ALL. However, BCR-ABL1 expression may not reflect the percentage of leukemic cells as FCM and IG/TCR rearrangement quantification do, and these methods are thus complementary.
Fibrosis may complicate bone marrow (BM) disease and in its advanced stage negatively affect the outcome of the patients due to BM failure, particularly in myeloproliferative disorders. Studies of ...the potential importance of BM fibrosis are rare in childhood acute lymphoblastic leukemia (ALL). We have previously shown a prognostic impact of BM fibrosis measured as reticulin fibre density (RFD) at diagnosis in childhood ALL. To further investigate the consequence of BM fibrosis in childhood ALL in relation to immunophenotype, cytogenetic findings and minimal residual disease (MRD) we retrospectively evaluated the RFD in 139 diagnostic BM biopsies from patients with a total mean follow up time of five years and eight months from two childhood oncology centers in Sweden. Patients with pre-B-ALL showed a higher mean RFD (19.1%) compared to patients with T-ALL (11.4%, p < 0.001). This was true also when comparing high-risk (HR) pre-B-ALL patients (18.1%) with the T-ALL patients (p = 0.002). RFD correlated inversely with the white blood cell count in the HR group (r = −0.41, p = 0.009), probably reflecting our finding of low degree of fibrosis in T-ALL. The cytogenetic analysis revealed that for patients with a hyperdiploid ALL (modal chromosome number: 51–61, n: 41) in the low-risk (LR) group, mean RFD was higher for relapsed patients (22.6%) compared to patients in continuous complete remission (17.2%, p = 0.019). The probability of disease free survival for the same group using RFD cutoff of 21.1%, representing the upper third of the material, was 85%±8% for patients with hyperdiploid ALL and low RFD compared to 51%±14% for patients with hyperdiploid ALL and high RFD (p = 0.01, Figure 1). Data for 32 of the patients demonstrated an association between high RFD at diagnosis and high minimal residual disease (MRD) on day 29 after start of treatment. Patients with FACS-MRD > 10−3 day 29 displayed higher mean RFD at diagnosis (21.2%) compared to patients with FACS-MRD < 10−3 (16.7%, p = 0.027, Figure 2). When analyzing the PCR-MRD data day 29 the findings were similar. We conclude that RFD is higher in pre-B-ALL compared to T-ALL, that RFD has prognostic impact in LR patients with hyperdiploid ALL and that high RFD at diagnosis is associated to high MRD on treatment day 29. All these findings are to our knowledge novel, suggesting that evaluation of BM fibrosis at diagnosis is a potentially new therapy stratifying factor and support the need of further research on BM fibrosis in childhood ALL and expanded use of BM biopsy at diagnosis.
Display omitted Display omitted
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
) 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
and 5.6 (95% CI 2.0-16, p = 0.001) for PCR-MRD ≥ 10
compared with MRD < 10
. Patients stratified to intermediate-risk therapy on day 29 with MRD 10
-<10
had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10
or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10
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.
Abstract Leukemic cells from 230 children with newly diagnosed B-cell precursor ALL were tested for in vitro drug resistance to a panel of anti-cancer drugs. Minimal residual disease (MRD) was ...measured by RQ-PCR. During follow-up, 24 relapses occurred in the 159 children with MRD <0.1% day 29. The risk of any relapse was correlated to vincristine and doxorubicin resistance, with a relative risk of 3.7 (95% CI 1.3–10.5; p = 0.016) for patients resistant to both drugs. There was a significant correlation also for the subgroup with extra-medullary relapses. Our findings indicate that analysis of drug resistance can add prognostic information to other known risk-factors including MRD.
PDF
