Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10⁺CD19⁺ cells and CD20^?CD22⁺ cells in the CD34⁺ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34⁺CD10⁺CD19⁺ and CD34⁺CD20^?CD22⁺ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.     

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10⁺CD19⁺ cells and CD20⁻CD22⁺ cells in the CD34⁺ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34⁺CD10⁺CD19⁺ and CD34⁺CD20⁻CD22⁺ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.     

Prognostic impact of karyotypic findings in childhood acute lymphoblastic leukaemia: a Nordic series comparing two treatment periods. For the Nordic Society of Paediatric Haematology and Oncology (NOPHO) Leukaemia Cytogenetic Study Group

The prognostic impact of acquired chromosome abnormalities was evaluated in a population-based consecutive series of 768 children (< 15 years of age) with acute lymphoblastic leukaemia (ALL). The study cohort included all cases of cytogenetically abnormal childhood ALL diagnosed between 1986 and 1997 in the five Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The probability of event-free survival (pEFS) for the total cohort was 0. 72 +/- 0.02. When comparing the two treatment periods of July 1986 to December 1991 and January 1992 to December 1997, a better survival was seen for the latter time period (pEFS of 0.69 +/- 0.02 vs. 0.76 +/- 0.02, P = 0.05). Hypodiploidy with less than 45 chromosomes, t(9;22)(q34;q11) and 11q23 translocations were associated with a dismal outcome during the whole study period (pEFS of 0.57 +/- 0.12, 0.41 +/- 0.14 and 0.37 +/- 0.10 respectively). The poor prognostic influence of 11q23 rearrangements seemed to be restricted to infants and older children (> 10 years), who differed significantly from children aged 1-10 years in this regard (P < 0. 01). Patients with t(9;22)-positive ALL seemed to benefit from allogeneic bone marrow transplantation in first remission (P = 0.05). The pEFS for children with t(1;19)(q23;p13)-positive ALL was intermediate (0.63 +/- 0.17), with a tendency to a better outcome for patients with the unbalanced variant der(19)t(1;19). Hyperdiploid ALL patients, subdivided into moderate hyperdiploidy (47-51 chromosomes), massive hyperdiploidy (52-60 chromosomes) and cases in the tri-/tetraploid range (> 60 chromosomes) had the best outcome in the last treatment period (pEFS of 0.81 +/- 0.06, 0.80 +/- 0.04 and 0.88 +/- 0.07 respectively), unless t(1;19), t(8;14), t(9;22) or 11q23 translocations were present. In a multivariate analysis including white blood cell (WBC) count, immunophenotype, age, mediastinal mass, central nervous system involvement and leukaemia karyotype, only WBC and modal chromosome number were shown to be significant independent risk factors (P < 0.01).     

Philadelphia positive acute lymphoblastic leukaemia of childhood

On current chemotherapeutic regimens, children with Philadelphia positive acute lymphoblastic leukaemia show a heterogeneous response to treatment. A few respond quickly to treatment and achieve long-term remission. Some fail to achieve remission after induction and the majority respond slowly to treatment. Relapse on treatment is common and remission is sustained in a proportion of cases only after allogeneic stem cell transplantation (allo-SCT). The use of imatinib along with conventional cytoreductive therapy, prior to allo-SCT appears to be the most promising strategy. The future lies in the molecular evaluation of response to treatment and combination targeted chemotherapy.     

Predicting relapse risk in childhood acute lymphoblastic leukaemia

Intensive multi‐agent chemotherapy regimens and the introduction of risk‐stratified therapy have substantially improved cure rates for children with acute lymphoblastic leukaemia (ALL). Current risk allocation schemas are imperfect, as some children are classified as lower‐risk and treated with less intensive therapy relapse, while others deemed higher‐risk are probably over‐treated. Most cooperative groups previously used morphological clearance of blasts in blood and marrow during the initial phases of chemotherapy as a primary factor for risk group allocation; however, this has largely been replaced by the detection of minimal residual disease (MRD). Other than age and white blood cell count (WBC) at presentation, many clinical variables previously used for risk group allocation are no longer prognostic, as MRD and the presence of sentinel genetic lesions are more reliable at predicting outcome. Currently, a number of sentinel genetic lesions are used by most cooperative groups for risk stratification; however, in the near future patients will probably be risk‐stratified using genomic signatures and clustering algorithms, rather than individual genetic alterations. This review will describe the clinical, biological, and response‐based features known to predict relapse risk in childhood ALL, including those currently used and those likely to be used in the near future to risk‐stratify therapy.     

Intraocular relapse of childhood acute lymphoblastic leukaemia

Relapse of childhood acute lymphoblastic leukaemia (ALL) involving the eye is a rare but challenging problem. Twenty cases occurred in patients treated on the Medical Research Council United Kingdom Acute Lymphoblastic Leukaemia XI and ALL97 trials between 1991 and 2001, representing 2.2% of ALL relapses. Seventeen occurred as a first relapse, either in isolation or combined with relapse at another site, and three occurred as a second relapse. All patients with intraocular disease at first relapse were treated with both chemotherapy and radiotherapy, but the doses and protocols used varied. Eleven of these 17 patients are alive and in complete remission with a median follow up of 4 years 2 months from relapse. All 11 children that were treated with a full chemotherapy relapse protocol, together with local radiotherapy have survived. Patients treated with chemotherapy of shorter duration and intensity, despite radiotherapy and/or bone marrow transplantation, did poorly with only one survivor, currently in chronic relapse. Consequently, we suggest that children with eye relapse of ALL be treated with an intensive relapse chemotherapy protocol with local ocular radiotherapy, whether the relapse occurs in isolation or in combination with relapse at another site.     

Prognostic significance of cell surface markers in childhood acute lymphoblastic leukaemia

Examination of surface markers on leukaemic blasts from 51 children with ALL revealed that ALL is a heterogeneous disease. The majority (68 X) of patients with ALL lack surface markers (null leukaemia); 28% could be classed as T cell as they form rosettes with sheep RBC and 4% have been shown to possess surface immunoglobulins and hence are classed as B cells. The children with null cell leukaemia have a better prognosis than T and B cell types.     

Flow cytometric analysis of BCL-2 can distinguish small numbers of acute lymphoblastic leukaemia cells from B-cell precursors

Flow cytometric identification of small numbers of precursor B-cell acute lymphoblastic leukaemia (B-ALL) cells in post-treatment marrow specimens could benefit from the identification of additional, easily detectable markers that could be used in most cases. In this study, we evaluate whether bcl-2 expression quantified by four-colour flow cytometry can be effectively used to discriminate precursor B-ALL blasts from normal B-cell precursors (haematogones) and function as a leukaemia-specific marker. Levels of bcl-2 in the 22 precursor B-ALL cases studied were found to be significantly higher (over sixfold higher on average) than those present in haematogone populations from 22 control marrow specimens. Higher relative levels of bcl-2 expression in the B-ALL cases were maintained with respect to both immature CD34+ and more mature CD34- haematogone subsets, and appeared stable. Dilutional studies indicated that multiparameter flow cytometry analysis using bcl-2 could identify precursor B-ALL blasts representing as few as 1% of CD19+ cells or 0.01% of total leucocytes in bone marrow specimens containing substantial numbers of haematogones. This study suggests that bcl-2 may be an important marker for flow cytometric detection and quantification of small numbers of residual precursor B-ALL cells in bone marrow specimens.     

Can threshold for MPO by flow cytometry be reduced in classifying acute leukaemia? A comparison of flow cytometric and cytochemical myeloperoxidase using different flow cytometric cut-offs

Abstract

Objectives

Myeloperoxidase (MPO) detection either by enzyme cytochemistry (cMPO) or flow cytometry (fMPO) plays a major role in acute leukaemia (AL) diagnosis as per World Health Organization (WHO) 2008 classification. Although 3% cMPO was recommended as positivity, no specific cut-off had been mentioned by WHO for fMPO. Various authors recommend different cut-offs ranging from 3 to 28% for fMPO. The aim of this study was to analyse fMPO cut-offs ranging from 3 to 10% in classifying AL and to assess whether a new cut-off could be suggested.

Methods

Totally, 216 cases of AL were retrospectively analysed for fMPO ranging from 3 to 10% and compared with gold standard. Presence of cMPO (≥3%) and/or expression of two or more pan-myeloid markers (CD13, CD33, and CD117) in the absence of CD19 and CD3 were kept as gold standard for diagnosis of acute myeloid leukaemia (AML).

Results

Sensitivities for classifying AL as AML/mixed phenotypic acute leukaemia (MPAL) at 3, 5.4, and 10% were 98.3, 98.3, and 96.6%, respectively, whereas specificities at this cut-off were 22.2, 91, and 71%, respectively.

Discussion

Only few studies have been done in this aspect to define a consistent cut-off for fMPO for proper classification of acute leukaemias. This was one of the largest and few studies available till date in this regard.

Conclusion

The newer cut-off for fMPO (5.4%) emerged out from our study with best sensitivity and specificity for accurately classifying AL cases into acute lymphoblastic leukaemia, AML, and MPAL.     

Concurrent detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR

Minimal (i.e. submicroscopic) residual disease (MRD) predicts outcome in childhood acute lymphoblastic leukaemia (ALL). To be used clinically, MRD assays must be reliable and accurate. Two well-established techniques, flow cytometry (FC) and polymerase chain reaction (PCR), can detect leukaemic cells with a sensitivity of 0.01% (10(-4)). We analysed diagnostic samples of 45 ALL-patients (37 B-lineage ALL, eight T-lineage ALL) by four-colour FC and real-time PCR. Leukaemia-associated immunophenotypes, at a sensitivity of MRD detection by FC at the 0.01% level, were identified in 41 cases (91%); antigen-receptor gene rearrangements suitable for MRD detection with a sensitivity of 0.01% or better by PCR were identified in 38 cases (84%). The combined use of FC and PCR allowed MRD monitoring in all 45 patients. In 105 follow-up samples, MRD estimates by both methods were highly concordant, with a deviation factor of <5 by Bland-Altman analysis. Importantly, the concordance between FC and PCR was also observed in regenerating bone marrow samples containing high proportions of CD19(+) cells, and in samples studied 24 h after collection. We conclude that both MRD assays yield generally concordant results. Their combined use should enable MRD monitoring in virtually all patients and prevent false-negative results due to clonal evolution or phenotypic shifts.     

Secondary acute lymphoblastic leukaemia is constitutional and probably not related to prior therapy

Very little is known about secondary acute lymphoblastic leukaemia (s‐ALL). This retrospective analysis studied a cohort of s‐ALL patients treated at a single centre between 1994 and 2013, while comparing therapy‐associated ALL (t‐ALL) and antecedent malignancy ALL (am‐ALL) patients. Thirty‐two patients with s‐ALL were identified. The overall incidence was 9·4% among ALL adults while T‐cell s‐ALL was rare (12% of s‐ALLs). The median time interval between two malignant diagnoses was 5·3 years (range: 0·1–28). In contrast to previous reports, most of the s‐ALLs were CD10 +  and without KMT2A (MLL) abnormalities. The overall survival (OS) rates of the entire cohort at 12 and 24 months from ALL diagnosis was 49% and 25%, respectively. Most patients (n = 23, 72%) received prior chemo‐/radio‐therapy for their first malignancy (t‐ALL) and only 9 (28%) did not (am‐ALL). No significant difference was found in the incidence of B‐/T‐ lineage ALL, extramedullary disease, blood count, and the rate of Philadelphia‐positive ALL, nor in the rates of complete remission (P = 0·55) and OS (P = 0·97). This similarity, together with high incidence of family malignancy in both groups, raise the possibility that s‐ALL patients may have an inherent predisposition to malignancies and a history of previous therapy may be of lesser importance in the pathogenesis of s‐ALL.     

Glycophorin A mutations and risk of secondary leukaemia in patients treated for childhood acute lymphoblastic leukaemia

Survivors of childhood acute lymphoblastic leukaemia (ALL) have a higher than expected risk of developing secondary acute myeloid leukaemia (AML). The glycophorin A (GPA) mutation assay measures the frequency of variant NO and NN erythrocytes in MN heterozygotes. A raised variant frequency (Vf) has been shown in patients treated with chemotherapy known to be at risk of secondary leukaemia. ALL patients were investigated for increased Vf using the GPA assay. Vf's at diagnosis were not significantly different from controls (NO Vf P  = 0.193; NN Vf P  = 0.790). During treatment Vf's increased significantly (NO Vf P  = 0.001; NN Vf P  = 0.001). NO Vf returned to control values ( P  = 0.169) within 5 years from diagnosis but NN Vf remained significantly raised ( P  = 0.014). Three study patients developed secondary AML. At diagnosis of AML all three had significantly increased Vf. The first had a significantly raised Vf at routine follow-up 19 years following diagnosis of ALL then developed AML 3.5 years later. The second had a significantly raised NN Vf at diagnosis of ALL indicating possibly prior exposure to a mutagen or defective DNA repair involving erythroid stem cells. We conclude that a raised Vf detected by the GPA assay can act as a marker for the development of secondary induced leukaemia and can be used to screen individuals at a known high risk of this complication.     

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.     

Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis‐relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated β‐catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β‐catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre‐treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt‐inhibition may be a promising therapeutic approach.     

The UK experience in treating relapsed childhood acute lymphoblastic leukaemia: a report on the medical research council UKALLR1 study

We have examined the toxicity and overall outcome of the Medical Research Council UKALL R1 protocol for 256 patients with relapsed childhood acute lymphoblastic leukaemia (ALL). Second remission was achieved in over 95% of patients. Two patients died during induction and seven patients died of resistant disease. The overall actuarial event-free survival (EFS) at 5 years for all patients experiencing a first relapse was 46% (95% CI 40-52). Duration of first remission, site of relapse, age at diagnosis and sex emerged as factors of prognostic significance. Five-year EFS was only 7% for children relapsing in the bone marrow within 2 years of diagnosis, but was 77% for those relapsing without bone marrow involvement > 2.5 years from diagnosis. All analyses in this report are by treatment received. For those receiving chemotherapy alone, the 5-year EFS was 48%; for autologous bone marrow transplantation (BMT), the 5-year EFS was 47%; for unrelated donor BMT, it was 52%; and for related donor BMT, the 5-year EFS was 45%. The groups, however, were not comparable with respect to risk factor profile, and therefore direct comparison of EFS is misleading. Adjustment for time to transplant and prognostic factors was used to reduce the effects of biases between treatment groups, but did not suggest benefit for any particular treatment. There was failure of our planned randomization scheme in this trial with only 9% of those eligible being randomized, which highlights the difficulties in running randomized trials especially in patients who have relapsed from a previous trial. The optimal treatment for relapsed ALL therefore remains uncertain. Alternative approaches are clearly needed for those with early bone marrow relapse if outcome is to improve.     

Personalization of dexamethasone therapy in childhood acute lymphoblastic leukaemia

Dexamethasone is a key component in the treatment of childhood acute lymphoblastic leukaemia (ALL). Despite playing a key role in the improved survival of ALL over several decades, intensification of dexamethasone therapy has also contributed to the increased toxicity associated with treatment, which is now seen to be at unacceptable levels given the favourable disease prognosis. Therefore the focus for treatment is now shifting towards reducing toxicity whilst maintaining current survival rates. As approximately 50% of patients were successfully treated on less intensive protocols of the 1980s, it has been questioned whether therapy intensification is necessary in all patients. Furthermore, there remains a subset of children who are still not cured of their disease. New strategies are therefore needed to identify patients who could benefit from dose reduction or intensification. However, adjusting a potentially life threatening therapy is a challenging task, particularly given the heterogeneous nature of ALL. This review focuses on the potential for patient stratification based on our current knowledge of dexamethasone pharmacokinetics, pharmacogenetics and the action of dexamethasone at the cellular level. A carefully designed, combined approach is needed if we are to achieve the aim of improved personalization of dexamethasone therapy for future patients.     

The prognostic significance of morphological features in childhood acute lymphoblastic leukaemia

A consecutive series of 209 children with acute lymphoblastic leukaemia (ALL) presenting to a regional referral unit between 1970 and 1977 was studied. The following morphological features in the initial bone marrow were recorded: blast size, percentage periodic acid-Schiff (PAS) and oil-red-O (ORO) positivity, percentage of blasts with vacuoles, and acid phosphatase positivity. The blasts were also coded according to the FAB (LI-3) classification. When analysed separately, increasing blast size was significantly related to the length of first remission (P= 0.01). However, this was almost entirely due to its association with the FAB L2 type of disease which also had a highly significant influence on length of first remission (P> 0.0001) independent of all other factors. Patients with L3 disease had blasts with heavy vacuolation, ORO positivity and displayed monoclonal surface immunoglobulin and their prognosis was very poor. The percentage of vacuolated blasts and ORO positivity otherwise showed no influence on prognosis. Fine granularity of PAS staining in lymphoblasts showed no prognostic value but the trend of longer remission duration with increase in percentage of PAS coarse granularity and blocks, was statistically significant (P= 0.006). This relationship was partly due to a correlation with the presence of mediastinal mass and L2 disease, but was still independent of all other prognostic factors. In a smaller number (39) of the series of patients on whom cell surface markers were available, we were unable to demonstrate a correlation between T-derived blasts and the L1 and L2 classification; but there was a strong correlation with polar acid phosphatase positivity in T-derived blasts.