The Effect of L-asparaginase Level Monitoring on Minimal Residual Disease of Childhood Acute Lymphoblastic Leukaemia: A Study from Eastern India

Objective:Research in Eastern India especially among children and adolescents for acute lymphoblastic leukemia (ALL) have not been well documented until recently when it was conducted at a cancer institute of tertiary care with primary objectives of examining and correlating different cell surface markers involved with respect to disease surveillance thereby highlighting it as a strong prognostic marker for future diagnosis and treatment.Materials and Methods:A total of 500 consecutively selected ALL patients were diagnosed and treated according to National Cancer Institute protocol (MCP 841) for a period of 24-88 months during this hospital-based study.Results:Of the total, 50.4% had a higher incidence of T-ALL and 47.6% had pro-B, B-cell precursor ALL. Disease free survival and event free survival were remarkably higher in B-ALL adolescent patients as compared to T-ALL, who had significantly lower overall survival ratio. Prevalence of T-ALL was also observed in relapse cases for adolescent patients.Conclusions:We conclude that there is an increased prevalence of T-ALL among adolescents in Eastern India. Immunophenotypic analysis might help in proper evaluation and prediction of treatment outcomes with an increased thrust on studying age-specific incident rates enabling well planned future treatments for improved and better outcome.

1106 - Study of Acute Leukemia Pattern by Flow Cytometric Analysis: An Experience from Eastern India

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.

Introduction The improved prognosis of pediatric B-cell acute lymphoblastic leukemia (pBALL) is considered as a good progress of medical science in the field of oncology and hematology. Minimal residual disease (MRD) refers to presence of disease in molecular level is a newer practice with respect to the detection of complete remission by conventional pathologic analysis. Prognostic value of MRD in pediatric ALL (p-ALL) is well known. Objectives This study was aimed to describe clinical outcomes and prognosis, that is, overall survival and relapse in the patients with pBALL with respect to minimal residual disease detection on day 15, day 29, and postconsolidations in a tertiary care center in eastern India. Materials and Methods Eight color flow cytometry was used to detect MRD in this study. This contained markers such as CD 19, CD 34, CD 10, CD58, CD 45, CD13, anti-TDT, CD33. Eight panels included were (1) CMPO-FITC/cCD79a-PE/cCd3ECD, (2) CD20-FITC/cCD10-PE/cCd-19ECD, (3) CD34-FITC/cCD117-PE/cCd45 ECD/CD2 PC 5, (4) CD15 FITC/CD33PE/CD45ECD, (5) CD14 FITC/CD13 PE/CD45ECD, (6) HLADR FITC/CD7 PE/CD45 ECD, (7) TdT FITC/CD45 ECD (IF CD34 NEG), and (8) CD58 FITC/CD 45 ECD (IF BOTH CD34 AND TdT NEG; were used to prepare the marker. Results The study included 52 patients. In the 52 patients, 59.6% patients are alive with a p-value of 0.031. MRD was checked on every 15th and the 29th day and postconsolidation of the treatment where in day 15 (p = 0.023), it was 53.4% positive and 46.5% negative. On day 29 (p = 0.031), MRD was 22.5% positive and 77.5% negative, in post consolidation, it was positive in 20% and negative is 80%. MRD value below 0.01 is taken as negative and above is taken as positive. The overall survival (OS) is of 32.88 + 8.59 with a 6 to 36 months of duration. In In relapsed cases, no hemorrhagic relapse was found and two CNS relapse were found. Conclusion It was a study of 52 patients of pBALL with a detection of MRD by FCM. MRD-negative patients had a good prognosis and comparatively lower rate of relapse than the one with positive MRD. Effort should be made to adhere to recommendation of MRD testing in clinical guidelines.

The management of patients with leukaemia: the role of cytogenetics in this molecular era.

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Impact of polymorphisms in apoptosis-related genes on the outcome of childhood acute lymphoblastic leukaemia.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Molecular and MRD-Based Characterization of Acute Lymphoblastic Leukemia in Mexico: Experience from the Mexico in Alliance with St. Jude “Bridge Project”

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Immunophenotype (IPT) and cytogenetics are essential for diagnosis, risk stratification, and management for ALL. Evaluating the burden of immunophenotypic and cytogenetic profile of pediatric ALL patients. A descriptive cross-sectional study was conducted on 100 patients of ALL (1-18 completed years) attending a tertiary-care center in Kolkata, Eastern India. Ninety-six percent of patients had B-cell ALL (94.00% pre-B ALL and 2.00% Pro-B ALL) and 4.0% had T-ALL. 60% B-cell ALL were CD19/CD10 positive, 10% were CD79a positive, 9% were only CD19 positive, and 7% were only CD10 positive. Thirty-three percent of T-ALL were CD3+, whereas 22% were positive each for CD4 and CD7. 51.0% of patients had diploid, 46.0% hyperdiploid, and 3.0% hypodiploid karyotype. Among hyperdiploids, 98% had good prednisolone response and 89% had measurable residual disease (MRD) <0.01. The most commonly diagnosed ALL by IPT was pre-B ALL. Among the detectable cytogenetic abnormalities, t(12; 21) ETV6-RUNX1 was the most common. ZNF-384 gene arrangement was also detected in our study. t(12;21) ETV6-RUNX1 had a good treatment response, while t(9;22) BCR-ABL, t(1;19) TCF3-PBX1, iAMP-21, MLL gene rearrangement, and ZNF-384 gene arrangement had poor treatment response in terms of MRD.

Impact of Minimal Residual Disease at Unrelated Cord Blood Transplantation In Children with Acute Lymphoblastic Leukemia In Remission: a Study on Behalf of Eurocord-EBMT and EBMT-PDWP

Minimal Residual Disease Monitoring In Acute Lymphoblastic Leukemia Patients Using Immune Receptor Sequencing

Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

Introduction: The overall survival in pediatric acute lymphoblastic leukemia (ALL) ranges from 45 to 81% in India. Aggressive chemotherapy protocols like MCP841 have improved the outcome and it can be delivered with minimal supportive care. This study retrospectively analyses the clinical profile and overall survival of patients treated by this protocol. Objective: This single-center study aims to estimate the event-free survival of patients treated accordingly to the MCP841 protocol with high-dose cytarabine (HDAC) at 2 g/m2 as the backbone, along with the risk-stratified incidence and cause of mortality in childhood ALL. Material and Methods: Records of 156 patients aged 1 to 19 years, newly diagnosed with ALL from June 2009 to August 2013 who were treated according to the forementioned protocol were analyzed. Risk stratification for both precursor B-cell ALL (B-ALL) and T-cell ALL (T-ALL) was done, followed by an analysis of the correlation of risk-stratified groups with mortality and survival outcomes. Result: Precursor B-ALL was found in 70% patients (including 69.7% [n = 76] standard risk, 20.1% [n = 22] intermediate risk, and 10% [n = 11] high risk), while 30% had T-ALL (including 74.4% [n = 35] standard risk and 25.5% [n = 12] high risk). Death during induction occurred in 0.04% (n = 5) precursor B-ALL and 23% (n = 11) T-ALL patients. The causes were infection in 62.5%, hemorrhage in 25%, and cortical venous thrombosis in 12.5%. Among those who attained remission (89.7%, n =140), relapse occurred in 26% (n = 27) precursor B-ALL and 28% (n = 10) T-ALL patients. Approximately 31% patients died in the postinduction phase, with progressive disease due to relapse being the most common cause and bone marrow the most common site. Event-free survival at 168 months for overall population, precursor B-ALL, and T-ALL was 59, 62.4, and 51.1%, respectively. Conclusion: A comparable survival outcome in par with similar centers in developing countries with the MCP841 protocol was found. Infections are a major cause of mortality during treatment, especially when associated with malnourishment. Relapsed disease and poor salvage rates remain a major hurdle to achieving better survival in developing countries; however, better supportive care and infection control measures along with implementing risk-stratified high-dose chemotherapy protocols might improve outcome in the future.

The monitoring of minimal residual disease (MRD) during treatment of acute lymphoblastic leukaemia (ALL) provides significant prognostic information allowing early treatment stratification. In children, studies have demonstrated that the prognostic value of MRD measurement, using flow cytometric or molecular techniques, is superior to other traditional markers of disease, such as age, white cell count and genotype (Borowitz et al, 2008; Conter et al, 2010), and MRD monitoring has now been incorporated into most paediatric regimens. Initial studies measured MRD post-induction treatment or at later time-points, allowing intensification of therapy in patients deemed to be at high risk of treatment failure or relapse (Cavé et al, 1998; Coustan-Smith et al, 2000). However, more recently there has been interest in the use of MRD measurement at earlier time-points in an attempt to provide more timely prognostic information and several studies have shown that MRD levels at day 8 or day 15 of treatment predict outcome (Coustan-Smith et al, 2002; Borowitz et al, 2008; Volejnikova et al, 2011). Furthermore, these studies have investigated the use of peripheral blood (PB), in place of bone marrow (BM), as a less invasive means of MRD assessment. Within the United Kingdom, the current and preceding paediatric ALL protocols (UKALL 2011 and UKALL 2003 respectively) use post-induction molecular BM MRD analysis to guide treatment. We have previously shown that post-induction flow cytometric BM MRD analysis is highly predictive of outcome (Motwani et al, 2009) but as yet, the use of MRD measurement at an earlier time-point has not been investigated in the context of the UK treatment regimen. To address this we assessed MRD by flow cytometry using Day 8 (D8) and/or Day 15 (D15) paired PB and BM samples taken from children being treated for ALL at our unit on the UK Medical Research Council UKALL 2003 trial or ALL R3 relapse trial. Samples were analysed as previously described (Irving et al, 2009). Briefly, analysis was performed using 4-colour flow cytometry with the Beckman Coulter (High Wycombe, UK) FC500 analyser using a large panel of monoclonal antibodies to identify all leukaemia-associated immunophenotypes (LAIP). For follow up samples a minimum of two markers were identified for each patient and sequential gating was used for analysis. About 500 000 to 106 cells were acquired for each antibody combination for analysis to be sufficiently sensitive (0·01%). The number of residual leukaemic cells was calculated as the percentage of blasts present within total nucleated cells counted. Sensitivity experiments were performed using dilutions of leukaemic blasts in normal BM or PB and demonstrated a sensitivity of 0·01%. Fifty paired BM and PB samples from 46 patients (41 newly diagnosed, five relapse) were included in the study (age 2–22 years, mean 7·8 years). Of these, 22 were taken at D8 and 28 at D15. Forty-two samples were from patients with B-cell ALL, whilst eight were from patients with T-cell ALL. The most frequent LAIPs used to identify blasts in B-ALL were CD58 (90% of cases), CD45 (75%), CD38 (86%) and CD123 (77%). In T-ALL the most common LAIP combinations were CD34 (75%), CD5/CD56 (62·5%), CD5/CD2 (50%) and CD5/CD99 (50%). As seen in previous studies, B-ALL MRD levels in PB blood were approximately 1·3-log lower than BM MRD levels (D8 mean 2·27% vs. 27·8%; D15 levels 0·63% vs. 7·99%) (Volejnikova et al, 2011). T-ALL levels were approximately 0·5-log lower. Importantly, though, the level of MRD in BM and PB in B-ALL showed a strong correlation (r = 0·77) (Fig 1). As patient numbers were small and follow-up short, there were insufficient relapses to allow correlation with long-term outcome. To address this we examined correlation between the early MRD level and the post-induction molecular MRD level, the current time-point used for risk stratification in the UK trial. Both BM and PB MRD levels were found to be higher at D8 and D15 in those patients later shown to be high risk. Interestingly, PB showed a greater difference than BM at D8 (P = 0·02), whereas BM was more significant at D15 (P = 0·022; Fig 2). Further analysis was carried out to establish a cut-off value that would allow accurate prediction of the post-induction risk group. At D8, a PB MRD >1% identified 100% (5/5) of those that were in the high-risk group post-induction, significantly more than those with PB MRD <1% at D8, of which only 36·4% (4/11) were allocated to the high risk group (P = 0·023). Similarly, at D15, in those patients with a BM MRD >1%, 62·5% (5/8) went on to be classified as high risk, whilst only 21·4% (3/14) of those with a BM MRD <1% were classified as high-risk (P = 0·03). These results clearly suggest that high-risk patients could be identified earlier in their treatment using PB MRD. Importantly, this early prediction of risk could offer an opportunity to intensify treatment in this high-risk group, potentially enhancing disease clearance during the induction phase. Interestingly, D8/15 MRD levels showed no correlation with the peripheral blast count or white cell count at diagnosis, suggesting that assessing MRD at this early time point provides a true measure of treatment sensitivity. Additionally, if numbers are increased to allow correlation with outcome, early MRD results may identify particularly low or high-risk patients, in whom beneficial alterations to treatment may be possible. Although small, this preliminary study further supports that early assessment of MRD using flow cytometric techniques enhances treatment stratification in paediatric ALL. Furthermore, it shows that PB sampling may provide a markedly less invasive means of MRD assessment in these patients. Validation in a larger cohort of patients is warranted. DOC collected and analysed data and wrote the paper. JJ performed flow cytometry, collected and analysed data and wrote the paper. MB and LE performed flow cytometry and collected data. SEL designed the study and wrote the paper. This work was supported by grants from Leukaemia and Lymphoma Research and the Birmingham Children's Hospital Research Foundation. The authors have no conflict of interests.