Evaluation of Minimal Residual Disease (MRD) by Quantification of TEL-AML1 Transcripts Is a Powerful Prognostic Tool in Children with T(12;21) Positive Acute Lymphoblastic Leukemia (ALL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 322-322
Author(s):  
Jean-Michel Cayuela ◽  
Paola Ballerini ◽  
Marina Romeo ◽  
Vahid Asnafi ◽  
Marie-Francoise Auclerc ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Decision Making ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Induction Treatment ◽  
Prognostic Tool ◽  
Time Points ◽  
Increased Risk ◽  
Level 2 ◽  
Risk Of Relapse

Abstract TEL-AML1 fusion transcripts are found in 25% of children with B-cell precursor ALL (BCP-ALL). From June 1993 to December 1999, 1195 children with BCP-ALL were included in the FRALLE 93 protocol. Out of these, 792 were evaluated for TEL-AML1 transcript expression. There is no difference in terms of initial features, DFS, EFS, survival between evaluated (792) and non evaluated (403) patients. Out of the 792 pts, 191 (24%) expressed TEL-AML1 transcripts at diagnosis. To assess the potential prognostic value of TEL-AML1 transcripts quantification, we have retrospectively analysed follow up marrow samples using Europe Against Cancer procedures for real time quantitative RT-PCR assay, on ABI PRISM 7700 (2 reference labs) and Light Cycler apparatus (1 reference lab). Out of the 191 TEL-AML1+ve pts, 83 were evaluated for MRD at different time points after induction therapy (median = D41 (34–55) (53 evaluable pts), at D111 (62–158) (62 pts), at D216 (159–325) (33 pts) and at D838 (365–1287) (49 pts). According to normalized Ct values, samples were attributed to 4 MRD level ranging from 0 to 3 and defined as follows: 0: Ct>40 ; 1 : 36<Ct≤40 ; 2 : 33<Ct≤36 ; 3 : Ct≤33, corresponding respectively to undetectable MRD ; MRD<10-4 ; 10-4≤MRD<10-3 ; MRD≥ 10-3, with respect to dilution of REH cDNA. Distribution of pts according to MRD level at different time points after induction treatment are summarized in the following table. Seventeen relapses have occurred at a median time of 41 months (17–73)(bone marrow: 7, BM + other: 5, testis: 3, CNS: 2). A level 2 positivity at the end of induction was associated with an increased risk of relapse of 3.31(95%CI:1.02 – 10.76, p =.047) while level 3 positivity was associated with a relative risk of 9.52 (95%CI: 2.91 – 31.08, p =.0002). Positivity at D111 was associated with an increased risk of relapse of 8.6 (2.0 – 38.5, p = 0.0042), whatever the level. Combination of data obtained at D41 and D111 allows to distinguish 3 subsets of pts with decreasing relapse-free survival: from 97.5% (95%CI: 85–100%) in pts with no positivity at D111 whatever the D41 result, to 75% (95%CI: 58–92%) in pts with MRD +ve at D111 with low level at D41 and 42% (95%CI: 14–69%) in pts with MRD +ve at D111 with level 2 or 3 at D41 (p<.0001). No other prognostic factor was found (age, sex, WBC, D8 steroid response, D21 bone marrow response) which renders the MRD profile unique in this matter. Conclusion: RQ-PCR-based MRD detection is a powerful prognostic tool in TEL-AML1+ve leukemia. Combination of two time points allows a relevant stratification of pts according to the risk of relapse, compatible with clinical decision making towards intensification or deescalation in the setting of controlled trials FU time point Number of pts in MRD classes (number of relapses) 0 1 2 3 Not evaluated D41 27 (3) 11 (2) 10 (4) 5 (4) 30 (4) D111 40 (2) 14 (6) 7 (2) 1 (1) 21 (6) D216 29 (2) 2 (1) 1 (0) 1 (1) 50 (13) D838 47 (8) 1 (0) 1 (1) 0 34 (8)

scholarly journals Validation of MRD Quantification By Flow Cytometry for Pediatric BCP ALL Relapsed Patients Treated on the Intreall Protocol

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1414-1414 ◽  
Author(s):  
Ester Mejstrikova ◽  
Julie Irving ◽  
Leonid Karawajew ◽  
Marian Case ◽  
Jenny Jesson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Clinical Decision Making ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Induction Treatment ◽  
High Dose ◽  
Risk Patients ◽  
To Receive

Abstract At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols. Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p<0.0001) for patients treated on the BFM protocol, compared to 0.82 (p<0.0001) for those on UK ALL R3. Risk category concordance was 88% (ALL-REZ BFM) and 88% (UKALLR3). For the 21 discordant samples, 5 were MRD positive by flow but negative by PCR and 17 were negative by flow and positive by PCR. When analysing the accuracy, with which flow MRD classified specimens identically as PCR, the sensitivity of flow MRD in the ALL-REZ BFM protocol was 81% (cut off 0.1%) and in UK ALL R3 was 79% (cut off 0.01%). Specificity values were 93% versus 100%, respectively. Although sample processing and quantification of MRD differ between PCR and FC MRD, in both re-induction protocols, there was good correlation of MRD levels assessed by flow cytometry and PCR, validating the use of Flow MRD as a method of choice in patients without PCR targets in the IntReALL trial. Flow MRD also has the advantage of enabling levels of CD22 to be assayed on MRD cells, prior to treatment with Epratuzumab. This research has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278514 - IntReALL", Deutsche Kinderkrebsstiftung for its funding support of the ALL-REZ BFM 2002 clinical trial and the minimal residual disease studies by PCR and the Deutsche Jose Carreras Leukämiestiftung for support of the international principal investigator, Leukaemia and Lymphoma Research and North East Children's Cancer Research Fund, NT 13462-4, NV15-28525A, NV15-26588A, UNCE 204012. Disclosures No relevant conflicts of interest to declare.

Flow Cytometric Minimal Residual Disease Levels After First Inducton Can Define T-Acute Lymphoblastic Leukemia Patients with High Risk of Relapse

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4817-4817 ◽  
Author(s):  
Veselka Nikolova ◽  
Velizar Shivarov ◽  
Ricardo Morilla
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Treatment Options ◽  
Phenotypic Expression ◽  
Time Points ◽  
Group 2 ◽  
Time Point ◽  
Group 1

Abstract Abstract 4817 T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding treatment options. This requires techniques more sensitive than the level of sensitivity of light microscopic technique such as multicolour flow cytometry (FCM). Immunophenotypic alterations called leukemia associated immunophenotypic patterns (LAIP) (i.e.aberrant myeloid markers) and ectopic phenotypic expression (i.e. appearance of immature phenotypes such as TdT, CD1a and CD3 outside their normal site in the thymus) are of benefit to track the residual leukemic cells in T-ALL. A retrospective data analysis of MRD was done comprising T-ALL patients diagnosed and followed-up at the Institute of Cancer Research/Royal Marsden Hospital by means of 3-colour flow cytometry (3C FCM).The aim was to answer a question whether the 3C FCM can reliably split patients into two groups (positive, MRD+ and negative, MRD-) and predict a subsequent relapse and to define a right time point for performing MRD tests. Eight patients were enrolled in the study following the inclusion criteria: (i) complete remission after 1st induction phase of chemotherapy, (ii) presence of LAIP or an ectopic phenotypic expression, and (iii) monitored at defined time points after initial treatment. MRD was measured during the first year of treatment as follows: at the end of phase 1 induction (day 29–35, MRD1), before the start of consolidation (3 months, MRD2), after consolidation (MRD3), during the maintenance therapy (12 months, MRD4). Immunophenotyping was performed on lysed-washed bone marrow samples using CD45 gating strategy and originally defined blast gates at diagnosis. The phenotypes to be followed-up included: TdT+/CytCD3+, CD34+/CYTCD3+, TdT+/CD2+, CD8+/CD10+, CD2+/CD10+, CD7+/CD10+, CD7+/CD33+, CD7+CD34+. Patients were divided into 2 groups in relation to subsequent relapse. Group 1 included 6 patients without relapse. Patient characteristics of the group were: male:female 5:1, mean age 17.7 years, overall survival (OS) 59 months, relapse free survival (RFS) 85 months. Group 2, relapsed patients, included 2 men, mean age 56 years, OS 13 months, RFS 8.5 months. According to the EGIL classification system the 2 men in Group 2 were with an early T-precursor phenotype, whilst Group 1 was heterogenous but cortical-T-ALL predominated. Cytogenetics/FISH and RQ-PCR studies were performed at diagnosis and showed normal karyotype in only one of the Group 2 patients. MRD results showed a difference between the two groups as regards MRD1 and MRD2 time points. Group 1 patients had negative or low MRD levels (below 0.18%) in their MRD1 bone marrow - MRD-, n=4 and MRD+,n=2 (0.18% and 0.12% respectively, sensitivity 0.04%). Those of them who were tested at MRD2 and MRD3 were negative. Both patients in Group 2 showed higher levels of MRD positivity at MRD1 (1% of total bone marrow cells), the first one of them also being positive at MRD2 and the second one becoming MRD+ at MRD4 time point. Although turning to MRD- at MRD3 time point both Group 2 patients relapsed 2.5 and 4.5 months, respectively, after the end of consolidation treatment. Additionally, Group 1 patients had a significantly longer RFS than Group 2 (median 58 months RFS vs. 8.5 months; P <0.001). Conclusions: Reliable detection of MRD in T-ALL is possible by 3C FCM using a combination of TdT and a T cell marker (cytCD3 or mCD3) as such a combination is normally found exclusively in the thymus. The higher MRD-positive levels in Group 2 reflect the more resistant disease in this group and higher probability of early relapse and shortened overall survival. Early T-cell precursor phenotype in these patients appeared to be a subtype at very high risk for treatment failure irrespective of the lack or the presence of genetic lesions. Based on MRD positivity above 0.18% at time points MRD1 or both MRD1 and MRD2 these patients need reassessment of treatment options and more intensive therapy has to be considered for relapse prevention. Finally, the results of our retrospective study suggest the usefulness of implementation of MRD testing by FCM for taking clinical decisions in the prospective clinical trials for novel therapies for T-ALL. Acknowledgments: The study was supported by the Union for International Cancer Control, Geneva, Switzerland (Grant ICRETT-080–2011) Disclosures: No relevant conflicts of interest to declare.

Role Of Day-15 Peripheral Blood MRD Assessment In Pediatric B-ALL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1384-1384
Author(s):  
Karthik B.K Bommannan ◽  
Man Updesh Singh Sachdeva ◽  
Parveen Bose ◽  
Deepak Bansal ◽  
Ram Kumar Marwaha ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Minimal Residual ◽  
Risk Of Relapse

Abstract Introduction Minimal residual disease (MRD) has emerged as an independent prognostic factor for patients of acute lymphoblastic leukemia (ALL). There is a strong correlation between MRD levels in bone marrow and the risk of relapse in childhood & adult leukemias 1, 2. Bone marrow MRD (BM-MRD) level of ≥ 0.01% is considered as positive and a mid-induction MRD of ≥ 1% is associated with high risk of relapse 3. Recently, the concept of peripheral blood MRD (PB-MRD), as a replacement for BM-MRD, has hit the lime light. In pediatric B-ALL, presence of PB-MRD is associated with a high relapse rate in comparison to cases which are PB-MRD negative 4, 5. This study was aimed to compare the levels of mid-induction (day 15) MRD levels in bone marrow and peripheral blood of pediatric B-ALL patients with a hypothesis that PB-MRD levels correlate with BM-MRD levels, and thus can predict BM-MRD levels for further management of the patient. Methods Forty newly diagnosed CD19+CD10+CD34+/- pediatric B-ALL patients under Vincristine, L-Asparaginase and Dexamethasone, were assessed for MRD levels on their paired day 15 PB & BM samples using six colour flow cytometry. With informed consent, both the samples were collected in EDTA vacutainers and lyse-stain-wash technique was used to prepare a single six colour tube comprising of SYTO 13/ CD34PE/ CD20PerCP/ CD19 PECy7/ CD10APC/ CD45APCH7 for each sample. The processed samples were run on BD FACS Canto II with acquisition of 1 million events or till the tubes were empty. Analysis was done using BD FACS Diva software and MRD of ≥ 0.01% was considered positive. Results Among 40 pairs of day 15 PB and BM samples, 25 (62.5%) were BM-MRD positive. Sixteen pairs (40%) had PB-MRD and BM-MRD co-positivity, 9 pairs (22.5%) had isolated BM-MRD positivity and 15 pairs (37.5%) were MRD negative in both PB and BM samples. In other words, among the 25 BM-MRD positive cases, simultaneous PB-MRD was positive in 16 patients (64%) and none of the samples had isolated PB-MRD positivity. Overall analysis of MRD positive cases showed a direct correlation between PB-MRD and BM-MRD (ρ = +0.684, p < 0.000) and BM-MRD levels were 7 times higher than the PB-MRD. In addition, ROC analysis with PB-MRD of ≥ 0.01% as a cut-off, revealed that, the most likelihood of PB-MRD being positive was when BM-MRD was ≥ 0.31%. Conclusions In contrast to the sparsely available literature, our study shows a significant correlation between PB & BM-MRD levels in day 15 paired samples of B-ALL cases. The MRD levels were 7 times higher in BM as compared to PB and PB-MRD was mostly positive with BM-MRD of ≥0.31%. In other words, day 15 PB-MRD positivity indirectly indicates that there is a minimum BM-MRD of 0.31%. Since literature reports prognostic significance of mid-induction BM-MRD at levels ≥1%, on day 15, an assessment of peripheral blood MRD alone, might yield clinically relevant prognostic information. A paired analysis at different time points might also establish a similar correlation as seen in the present study, eliminating the need of BM-MRD during further follow ups of the patient. This will help in avoiding an invasive procedure and improve patient compliance. References 1. Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L, et al. Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. haematologica. 2009;94(6):870-4. 2. Coustan-Smith E, Sancho J, Behm FG, Hancock ML, Razzouk BI, Ribeiro RC, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood. 2002;100(1):52-8. 3. Basso G, Veltroni M, Valsecchi MG, Dworzak MN, Ratei R, Silvestri D, et al. Risk of relapse of childhood acute lymphoblastic leukemia is predicted by flow cytometric measurement of residual disease on day 15 bone marrow. Journal of Clinical Oncology. 2009;27(31):5168-74. 4. Elain CS, Sancho J, Michael LH, Bassem. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood. 2002;100 (7):2399-402. 5. Brisco MJ, Sykes PJ, Hughes E, Dolman G, Neoh SH, Peng LM, et al. Monitoring minimal residual disease in peripheral blood in B lineage acute lymphoblastic leukaemia. British journal of haematology. 1997;99(2):314-9. Disclosures: No relevant conflicts of interest to declare.

Patterns of Individual Tumor Response Testing (ITRT) and Bone Marrow Minimal Residual Disease (MRD) On Day 15 of Therapy in Childhood Precursor-B-Lineage Acute Lymphoblastic Leukemia (ALL): MRD and Ex Vivo Drug Resistance Have Similar Predictive Value of Relapse.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2448-2448
Author(s):  
Jan Styczynski ◽  
Anna Jaworska-Posadzy ◽  
Malgorzata Kubicka ◽  
Robert Debski ◽  
Beata Kurylo-Rafinska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Multivariate Analysis ◽  
Residual Disease ◽  
Ex Vivo ◽  
Cox Model ◽  
Lymphoblastic Leukemia ◽  
Minimal Residual ◽  
Risk Of Relapse

Abstract Abstract 2448 Introduction: The speed of blast clearance during therapy is a major prognostic factor of outcome in childhood acute lymphoblastic leukemia (ALL). Blast count in the peripheral blood on day 8, or in the bone marrow on day 15 and day 33, have been widely used to deliver risk-directed therapy. Another approach to measure the speed of leukemia clearance is the detection of minimal residual disease during induction therapy, as well as at days 33 and 78 of therapy. In vitro measurements of drug resistance (called recently as ITRT, individual tumor resistance testing) in leukemic cells obtained at diagnosis have been of prognostic significance in the prediction of clinical outcome in selected groups of patients. Objective: The analysis of the prognostic impact of (A) residual disease (MRD) at day 15 of induction therapy; (B) in vitro drug resistance at diagnosis (ITRT), (C) correlation of MRD and ITRT, and (D) multivariate analysis of prognostic role of MRD, ITRT, initial factors and initial therapy response to the risk of relapse. Patients and Methods: A total number of 87 children (aged 1–18 years) diagnosed for pre-B-ALL, treated either with ALL-BMF-90 or ALL-IC-2002 protocol were included into the study. ITRT was tested at diagnosis by the MTT assay. Residual disease at day 15 was measured by flow cytometry and determined for cut-off value BML15<0.5%. The median follow-up was 8.9 yrs (range, 0–11.5). Following drugs were tested: prednisolone, dexamethasone, vincristine, L-asparaginase, daunorubicin, doxorubicin, etoposide and cytarabine. PVA score was determined as combined ITRT profile to prednisolone, vincristine and L-asparaginase. Results: (A) The overall pDFS was 0.721±0.052 and the mean survival 9.1 yrs (95%CI=8.2–9.9). Patients with BML15<0.5% had pDFS=0.816±0.055, while those with BML15>0.5% had pDFS=0.542±0.098 (p=0.009, log-rank). The risk of relapse in BML15-positive patients was 3.0-fold higher (1.3–7.1, p=0.013). (B) pDFS was significantly better for patients with sensitive ITRT profile to: PVA (1.00±0.00 vs 0.61±0.06, p=0.002), prednisolone (0.89±0.05 vs 0.54±0.08, p=00002), vincristine (0.84±0.06 vs 0.61±0.08, p=0.035), daunorubicin (0.094±0.04 vs 0.51±0.08, p=0.00002), and L-asparaginase (0.84±0.06 vs 0.59±0.08, p=0.009). In multivariate analysis in Cox model, the prognostic value was retained only for ITRT for prednisolone (p=0.013, HR=0.08, 95%CI=0.01–0.6) and daunorubicin (p=0.004, HR=0.05, 95%CI=0.01–0.4), while ITRT for PVA score was below of significance (p=0.068, HR=0.03, 95%CI=0.01–1.3). (C) Patients with MRD-positive ALL at day 15 (BML15>0.5%) had higher ITRT for following drugs: doxorubicin (p=0.005, RR=1.8, Mann-Whitney U test), L-asparaginase (p=0.029, RR=3.2), and etoposide (p=0.055, RR=4.1), while no differences were found for other drugs. In multivariate logistic regression, the significance impact to development of BML15>0.5% was found for doxorubicin (p=0.035, OR=0.33) and etoposide (p=0.048, OR=0.14). (D) In multivariate analysis in Cox model for relapse risk, three factors had predictive value: BML15>0.5% (p=0.010, OR=3.3, 95%CI=1.3–8.2), ITRT for prednisolone (p=0.012, OR=4.4, 95%CI=1.4–13) and ITRT for daunorubicin (p=0.018, OR=5.9, 95%CI=1.4–26), while age, prednisolone-poor-response at day 8, BM response at day 15, BM response at day 33, and BCR-ABL rearrangement had no significant value. Conclusions: Patients with residual disease at day 15 had 3-fold higher risk of relapse. Patients with resistant ITRT profile to prednisolone and daunorubicin had respectively 12- and 20-fold higher risk of relapse. Presence of residual blasts at day 15 correlates with ITRT to etoposide and doxorubicin. Finally, persistence of blast in marrow at day 15 (BML15>0.5%) and ex vivo drug resistance (ITRT) to prednisolone and to daunorubicin were the strongest prognostic factors predicting relapse in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Outcome After First Relapse Or Refractory Acute Lymphoblastic Leukemia In Children From 1996 To 2011 – Comparison Of Two Different Treatment Strategies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3381-3381
Author(s):  
Francesco Ceppi ◽  
Michel Duval ◽  
Caroline Laverdiere ◽  
Jean-Marie Leclerc ◽  
Yveline Delvas ◽  
...  
Keyword(s):  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Treatment Strategies ◽  
Late Relapse ◽  
Induction Treatment ◽  
Unrelated Donor ◽  
Refractory Disease ◽  
Early Relapse ◽  
First Relapse ◽  
Risk Of Relapse

Abstract Introduction Newly diagnosed acute lymphoblastic leukemia (ALL) has achieved a cure rate of 80-85% in developed countries. Nevertheless, up to 20% of patients still relapse or have a refractory disease. Approximately 50% of these patients are long term survivors. In our institution, the strategy for relapsed patients had changed from hematopoietic stem cell transplantation (HSCT) after reinduction to all patients to a more tailored approach, offering HSCT to patients at high risk of relapse (e.g. those relapsing early after or during treatment) or for those with late marrow relapse with a related donor after a re-induction treatment. The aims of this study was to analyse the results of treatment for children with ALL in first relapse or refractory disease and to compare the outcome based on the strategy of treatment: re-induction followed by HSCT for all patients versus re-induction followed by HSCT or chemotherapy according the risk of relapse. Patients and Methods This retrospective study analysed treatment and outcome of children (up to 18 years-old) with a first relapse or refractory ALL treated in a single institution from 1996 until 2011. All patients were previously treated according to DFCI protocol 95, 00 or 05. Two treatment strategies have been used for patients refractory or with marrow relapse. Until 2007, they were all treated with a re-induction treatment (usually the same protocol used as first-line induction) followed by an allogeneic HSCT with a related or unrelated donor once complete remission was attained. Starting from 2007, patients with a late marrow relapse were treated with chemotherapy alone (or with HSCT if an HLA sibling donor was available), and patients refractory or with an early relapse received at least 3 cycles of chemotherapy before HSCT (with a related or unrelated donor), to reduce pre-transplant residual disease. Results 64 patients had been diagnosed with a first relapse (n=55) or refractory (n=11) ALL whereas 453 patients were diagnosed with ALL during this fifteen years period. At first diagnosis, median WBC was 20 x109/L, 54 (84.5%) patients were B-ALL, 21 (33%) had a positive CNS and 26 (40.5%) had a favourable or a normal karyotype. According to risk stratification at first diagnosis, 26 (40.5%), 30 (47%) and 8 (12.5%) patients were classified as standard, high- and very high risk, respectively. Median ages at relapse diagnosis were 5.9 (range 1.3-17.1) for relapsed and 8.4 (range 1.4-18.4) years for refractory patients. At relapse, median WBC was 6 x109/L and 39 (61%), 14 (22%) and 11 (17%) presented with an isolated marrow, combined and extramedullary (EM) relapse, respectively. Median interval between diagnosis and relapse was 32.5 (range 0.6-135.1) months and 11 (17 %) were classified as refractory, 25 (39%) were classified as early relapse (<36 months from diagnosis to relapse) and 28 (44%) as late relapse (≥ 36 months from diagnosis to relapse). Until 2007, 29 (85 %) patients with marrow relapse received an allogeneic HSCT. After 2007, 14 (70%) received an allo-HSCT and 6 (30%) were treated with chemotherapy after induction. Estimated 3-year event-free survival (EFS) for the whole population was 53% (28% for early and 84% for late relapse [p>0.001]). Before and after 2007, estimated 3-year EFS was respectively 46% and 65% (p=0.3). Conclusion Introduction of a more intensive re-induction therapy and the treatment of the late relapse with chemotherapy alone had permitted to improve the survival of children with relapse/refractory leukemia, probably due to a better control of minimal residual disease (MRD). MRD-guided reinduction therapy is currently the standard of care for these patients and new therapeutics approaches are still needed for patients with early relapse. Disclosures: No relevant conflicts of interest to declare.

Clinical Utility of Next-Generation Sequencing-Based Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2611-2611
Author(s):  
Yuko Sekiya ◽  
Yusuke Okuno ◽  
Hideki Muramatsu ◽  
Atsushi Narita ◽  
Kyogo Suzuki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Risk Factor ◽  
Clinical Utility ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Significant Risk ◽  
Significant Risk Factor ◽  
Time Points ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Purpose Next-generation sequencing (NGS)-based monitoring of minimal residual disease (MRD) was developed to increase the sensitivity and specificity of standard MRD detection methods. However, few published studies have tested the clinical utility of this novel technique. We assessed the clinical utility of NGS-MRD in a uniformly treated cohort of patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). PATIENTS AND METHODS We enrolled 79 unselected patients with pediatric BCP-ALL. Bone marrow samples were collected at the time of diagnosis, on days 33 and 80, at pre- and post-maintenance therapy time points (4-5 and 24 months, respectively), and upon relapse. Genomic DNA was extracted from frozen bone marrow mononuclear cells at each time point. We used diagnostic samples to define the immunoglobulin heavy chain (IGH), complementarity-determining region 3 (CDR3), and T-cell receptor gamma chain (TCRG) loci. From these samples, we detected leukemia-specific CDR3 sequences in >5.0% of all sequence reads. In addition, we performed a multiplex polymerase chain reaction (PCR) to determine the IGH, CDR3, and TCRG loci and subsequently assessed MRD using NGS. The result was considered positive for NGS-MRD if the leukemia-specific CDR3 sequence was detected. The resulting positive MRD values were categorized as "low positive" (<10−4) or "high positive" (≥10−4). RESULTS We detected leukemia-specific CDR3 sequences in 72 of 79 patients (91%). MRD was measured in 232 samples and we obtained positive results in 59 samples. MRD was detected in 51% (28/55) samples on day 33, and the frequencies of positive MRD decreased to 25% (16/65), 19% (11/58), and 7.4% (4/54) samples at day 80, 4-5 months, and 24 months, respectively. Each of the four patients with a positive MRD at 24 months relapsed shortly after detection. In a univariate analysis, the MRD values at day 80 {risk ratio [RR; 95% confidence interval (CI)] = 7.438 (2.561-21.6), p < 0.001}, 4-5 months [RR (95% CI) = 10.24 (3.374-31.06), p < 0.001], and 24 months [RR (95% CI) = 19.26 (4.974-74.59), p < 0.001] showed a statistically significant association with inferior leukemia-free survival (LFS). The classification of patients as either low or high positive for NGS-MRD at day 80 was a significant risk factor for poor LFS [low positive, RR (95% CI) = 6.63 (2.01-21.82), p = 0.002; high positive, RR (95% CI) = 9.40 (2.32-38.17), p = 0.002]. Furthermore, both low and high positivity for MRD at 4-5 months was also a significant risk factor for poor LFS [low positive, RR (95% CI) = 10.32 (3.07-34.70), p < 0.001; high positive, RR (95% CI) = 10.04 (2.00-50.34), p = 0.005]. In an assessment of three multivariate Cox proportional hazard models, we found that both low and high positive NGS-MRD values at day 80 [low positive, RR (95% CI) = 6.05 (1.80-20.39), p = 0.0037; high positive, RR (95% CI) = 8.20 (1.92-35.07), p = 0.002] and at 4-5 months [low positive, RR (95% CI) = 12.98 (3.49-48.28), p < 0.001; high positive, RR (95% CI) = 23.16 (3.28-163.7), p < 0.001] were independent covariates predictive of poor LFS. CONCLUSION We detected leukemia-specific CDR3 rearrangements in 91% of our cohorts, which was comparable with the frequencies detected using sensitive real-time quantitative (RQ)-PCR methods. In both univariate and multivariate analyses, low and high positive NGS-MRD results were significantly associated with poor LFS. In addition, we found that MRD positivity at later time points (4-5 and 24 months) was predictive of a high incidence of relapse and poor LFS. Therefore, NGS-MRD can identify a greater number of patients who are at a high risk of relapse and candidates for intensified chemotherapy or allogeneic HSCT. Our study demonstrates the potential superiority of NGS over the current standard method of MRD monitoring. However, standardization, quality control, and validation of this new technology are warranted prior to its use in routine practice. Disclosures No relevant conflicts of interest to declare.

Impact of Minimal Residual Disease, As Detected by Multiparametric Flow Cytometry Pre and Post Transplantation, On Outcome of Myeloablative Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 592-592 ◽  
Author(s):  
Merav Bar ◽  
Brent L. Wood ◽  
Jerald P. Radich ◽  
Kristine C. Doney ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Overall Survival ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Flow Data ◽  
Multiparametric Flow Cytometry ◽  
Increased Risk ◽  
Blast Count ◽  
One Year ◽  
Minimal Residual ◽  
Risk Of Relapse

Abstract Abstract 592 Relapse occurs in approximately 30% of patients with acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic transplantation (allo-HCT), with relapse rate of more than 60% in high-risk patients. The outcome of relapsed patients is usually poor despite salvage therapies. To improve the outcome of ALL patients after HCT identification of markers to predict impending relapse is necessary. In this study we retrospectively evaluated the prognostic value of minimal residual disease (MRD) monitoring by seven-color multiparametric flow cytometry (MFC) in 159 patients with ALL, who underwent myeloablative allo-HCT in our center between April 2006 and March 2011. Sixty two patients were younger than 18 years at time of transplant, 90 patients were in CR1, 57 patients were in CR2, and 12 patients were in ≥CR3 at time of transplant. 133 patients had B-ALL, 24 patients had T-ALL, and 2 patients had biphenotypic leukemia. Among the B-ALL patients 34 patients had Ph+ disease. Seven-color MFC was performed on bone marrow aspirates before and after HCT. MRD was defined as a cell population showing deviation from normal antigen expression patterns compared with normal or regenerating marrow, with total blast count lower than 5%. Overt hematologic relapse was identified as total blast count ≥5%. 153 patients had pre-HCT flow data within 50 days before transplant. Among these patients MRD pre-HCT increased the risk of relapse (HR=3.17; 95% CI, 1.60–6.28; p=.002) and mortality (HR=2.39; 95% CI, 1.46–3.90, p=.0005) relative to the risk without MRD before HCT. These results were qualitatively unchanged after adjusting for the presence of abnormal cytogenetics or positive BCR-ABL by molecular testing. One year estimates of relapse for patients without and with MRD pre-HCT were 12% and 27%, respectively, and one year estimates of overall survival (OS) were 75% and 44% respectively. Three years estimated relapse were 16% and 33% and 3 years estimated OS were 68% and 40% for patients without and with MRD pre-HCT respectively (Figures 1, 2). 137 patients had “day-28” flow data, where MFC was done between day 17 and day 40 post-transplant. Among patients who had not relapsed by the time of their day-28 MFC, the risk of relapse was higher among patients with positive day-28 MFC compared to those with a negative day-28 MFC (HR=3.55; 95 CI,1.58–8.02; p=.002). Patients with MRD at day 28 were also at increased risk of subsequent mortality compared to those without MRD at day 28 (HR=2.47; 95% CI, 1.23–4.96; p=.01). These results were qualitatively unchanged after adjusting for the presence of abnormal cytogenetics. One year estimates of relapse for patients without and with MRD post-HCT were 15% and 39%, respectively, and 1-year estimates of OS were 76% and 41%, respectively. Three years estimated relapse were 19% and 45% and 3 years estimated OS were 70% and 33% for patients without and with MRD post-HCT, respectively. Modeling post-HCT flow values as a time-dependent covariate also showed an increased risk of relapse (HR=5.27; 95% CI, 2.16–12.87, p=.0003) and death (HR=3.21; 95% CI, 1.58–6.53, p=.001) among patients who had MRD compared to those who did not. These associations remained qualitatively the same after adjusting for presence of pre-HCT MRD (HR=6.04 for relapse; HR=3.53 for overall mortality). These data suggest that pre- or post-HCT MRD, as detected by seven-color MFC, is associated with increased risk of relapse and death after myeloablative HCT for ALL. Figure 1. Cumulative incidence of relapse after HCT based on MRD status before HCT Figure 1. Cumulative incidence of relapse after HCT based on MRD status before HCT Figure 2. Overall survival after HCT based on MRD status before HCT Figure 2. Overall survival after HCT based on MRD status before HCT Disclosures: No relevant conflicts of interest to declare.

CRLF2 /Tslpr Overexpressing Acute Lymphoblastic Leukemia Relapse Is Driven By Chemotherapy-Induced TSLP from Bone Marrow Stromal Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1432-1432
Author(s):  
Christopher Daniel Chien ◽  
Sang Minh Nguyen ◽  
Haiying Qin ◽  
Elad Jacoby ◽  
Terry J. Fry
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Inflammatory Cytokines ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Cells ◽  
Cytotoxic Agents ◽  
Increased Risk ◽  
Early Progression ◽  
Risk Of Relapse

Abstract Despite 5-year survival rates nearing 90%, leukemia is the most frequent cause of death from cancer in children with treatment failure primarily caused by relapse after remission. Therefore, it is critical that we identify new therapies that specifically address leukemic persistence during therapy. A cohort of acute lymphoblastic leukemia (ALL) patients that have an increased risk of relapse and consequently poorer overall survival have leukemia that overexpresses cytokine receptor-like factor 2/thymic stromal lymphopoietin receptor (CRLF2/TSLPR). CRLF2/TSLPR overexpressing patients have rates of relapse nearly double the rate of non-overexpressing patients despite comparable remission rates to low-risk patients and account for half of the cases of high risk Philadelphia-like ALL. We hypothesize that TSLP/TSLPR axis may promote relapse of ALL through overexpressed TSLPR, which sensitizes leukemic blasts to low levels of TSLP in bone marrow (BM) niches promoting survival. To test this theory we generated a TSLPR overexpressing syngeneic murine leukemia (TSLPRhigh) through viral transduction of a transplantable pre-B cell ALL line (TSLPRlow). This TSLPRhigh leukemia has comparable TSLPR expression levels to what is found on human ALL that overexpress TSLPR and the transduced TSLPR is functional with increased phosphorylation of STAT5 protein in response to TSLP stimulation. When ALL lines were injected into immunocompetent mice, we observed an 8 fold difference in the percentage of TSLPRhigh vs. TSLPRlow ALL in the BM 5 days after injection, when leukemia accounted for less than 5% of the BM corresponding to an early stage of ALL progression. Interestingly, in vitro and in vivo cell growth and late-stage lethality were no different between the TSLPRhigh and TLSPRlow ALL indicating that TSLP does not alter ALL proliferation. From this data we can infer that TSLP/TSLPR signaling is likely most critical at early stages of leukemia development when BM stromal niches are intact. Hypothesizing that these niches are the source of TSLP ligand we sought to identify the cells responsible for secreting TSLP in the BM. Indeed, basal levels of TSLP mRNA are present in the BM, but the expression is quite low. Interestingly, TSLP mRNA and protein were markedly induced in murine BM stromal cell lines and primary BMSCs by the inflammatory cytokines IL-1a and TNF-a as has previously been reported for other types of TSLP-producing human cells. Since it is well established that cytotoxic chemotherapeutics can mediate an inflammatory response in patients, we investigated whether cytotoxic agents can cause release of inflammatory cytokines from BM cell populations. We treated primary murine BMSCs, BM hematopoietic cells, and ALL lines with low doses of chemotherapy and observed that the pyrimidine analog cytarabine (Ara-C) was particularly potent in upregulating IL-1a expression from BM hematopoietic cells and ALL and not in BMSCs suggesting that inflammatory cytokine release by hematopoietic cells may induce TSLP production by BMSCs. We set out to determine if IL-1a administration to mice could enhance the early progression of ALL in vivo. Indeed, we found that TSLP mRNA and protein were elevated in the BM and serum of mice injected with IL-1a respectively and that there was significant increase in the percentage of early ALL infiltration in the BM of mice bearing TSLPRhigh ALL but not TSLPRlow ALL suggesting a dependence on high levels of expression of TSLPR to respond to IL-1a -induced TSLP production. In addition, we confirmed the necessity of TSLPR-driven early ALL progression on the presence of the TSLP ligand as we observed no significant increase in TSLPRhigh ALL in TSLP deficient mice after stimulation with IL-1a. Furthermore, the early increase in TSLPRhigh leukemia burden can be reversed by using a TSLP blocking antibody demonstrating that targeting the TSLP/TSLPR axis may be therapeutically relevant. These data demonstrate that TSLP secreted in the BM induced by inflammatory cytokines such as IL-1a can drive accelerated early progression of ALL. These inflammatory cytokines can be induced by cytotoxic agents suggesting that chemotherapy can indirectly provide an unintended advantage to TSLPR overexpressing ALL. Finally, we postulate that therapies targeting the TSLP signaling axis would decrease the risk of relapse in TSLPRhigh ALL particularly in the context of standard therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Low Expression of p53 Protein in Bone Marrow after Hematopoietic Stem Cell Transplantation Indicates Risk of Relapse in Pediatric Patients with Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4996-4996
Author(s):  
Kristin Mattsson ◽  
Emma Honkaniemi ◽  
Birgitta Sander ◽  
Britt Gustafsson
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
P53 Protein ◽  
Lymphoblastic Leukemia ◽  
Median Percentage ◽  
Hematopoietic Stem ◽  
P53 Protein Expression ◽  
Increased Risk ◽  
Pediatric All ◽  
Risk Of Relapse

Abstract Advances in treatment over the last decades have resulted in almost 90% 5-year survival of children with acute lymphoblastic leukemia (ALL). Nonetheless, a minor group with unfavorable prognosis becomes candidates for hematopoietic stem cell transplantation (HSCT). After HSCT, blast count, minimal residual disease (MRD) and chimerism analysis are used as markers for successful engraftment and risk of relapse. However, additional markers are needed to complement the existing ones, as 30 % of ALL-patients still experience relapse after HSCT. Mutations in the tumor suppressor gene TP53 are detected in 2% at diagnosis of pediatric ALLs, but in 11-28 % after relapse. The aim of this study was to evaluate if alterations in the expressions of the TP53 encoded protein p53, analyzed by immunohistochemistry, could act as a prognostic marker in pediatric ALL patients post HSCT. Paraffin-embedded bone marrow samples were collected retrospectively from 49 children diagnosed with pre-B ALL and, treated with HSCT at Karolinska University Hospital between 1997-2010. Patients samples were requested from six different times during the course of the disease; time of diagnosis, before HSCT and at routine controls after HSCT at 0-3, >3-6, >6-12 and >12-24 months. For no patient data was available from all time periods. In total 176 samples were collected. A control group consisted of fifty-five children, where a bone marrow biopsy had been performed due to suspected bone marrow disorder, but a malignant blood disease had been excluded. Tissue micro array (TMA) prepared microscope slides were stained for the p53 protein using a monoclonal mouse anti-human antibody (clone DO-7, Leica), which detects both the wild-type and mutant forms of p53. Protein expression was examined by two independent examiners in a light microscope at high magnification (40X). Cells positive for the p53-protein identified by brown nuclear staining, were counted and calculated as a percentage of total cells per sample for each antibody respectively. Mean number of cells counted per sample was 695. Some samples were lost prior to final analysis due to either; sample core lost during TMA (n=12) or, sample had <100 cells which was minimum number of cells for inclusion in analysis (n=10). The final analysis included 154 bone marrow samples from 49 patients and 55 control samples. Data was analyzed in Statsoft Statistica 12.7. Nonlinear logistic regression was used to evaluate predictive value of the protein. Data was analyzed independently at each of the following times; diagnosis, time of HSCT and at 0-3, >3-6, >6-12 and >12-24 months post HSCT. A p value < 0.05 was considered significant. Interestingly, we found that a low percentage of cells positive for p53 protein expression in the bone marrow at >3-6 months after HSCT was associated with increased risk of relapse in pediatric ALL patients, odds ratio 0.82 (confidence interval 0.68-0.99) p= 0.041. Figure I shows the range of p53 expression in the two groups individually at each time. At >3-6 months after HSCT, the cut off value was calculated to 3.3% p53 positive cells, and was correct for 16 of 20 non-relapse cases and for 9 of 14 relapse cases (overall 73.5% correct). All relapses occurred between 6-50 months after HSCT (mean=20, median=14). The lower amount of p53 positive cells in the relapsed patients compared to the non-relapsed patients could possibly be due to an underlying mutation in TP53 that has altered the ability of producing a protein. This would be in similarity with the literature where it is suggested that TP53 mutations are more common at relapse in pediatric ALL patients. In summary, a lower expression of p53 protein in the bone marrow at >3-6 months after HSCT was significantly associated with increased risk of relapse. Evaluation of p53 protein expression by immunohistochemistry, in bone marrow from pediatric ALL-patients that undergo HSCT may be a potential additional marker for predicting relapse. Figure 1. Range and median percentage of p53 positive cells at different times in bone marrow from children with pre-B ALL. At >3-6 months after HSCT a higher quantity of p53 positive cells indicated a relapse free prognosis. Figure 1. Range and median percentage of p53 positive cells at different times in bone marrow from children with pre-B ALL. At >3-6 months after HSCT a higher quantity of p53 positive cells indicated a relapse free prognosis. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Improving Stratification for Children With Late Bone Marrow B-Cell Acute Lymphoblastic Leukemia Relapses With Refined Response Classification and Integration of Genetics

2019 ◽  
Vol 37 (36) ◽  
pp. 3493-3506 ◽  
Author(s):  
Cornelia Eckert ◽  
Stefanie Groeneveld-Krentz ◽  
Renate Kirschner-Schwabe ◽  
Nikola Hagedorn ◽  
Christiane Chen-Santel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Induction Treatment ◽  
Poor Responders ◽  
Hematopoietic Stem ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Response Classification

PURPOSE Minimal residual disease (MRD) helps to accurately assess when children with late bone marrow relapses of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) will benefit from allogeneic hematopoietic stem-cell transplantation (allo-HSCT). More detailed dissection of MRD response heterogeneity and the specific genetic aberrations could improve current practice. PATIENTS AND METHODS MRD was assessed after induction treatment and at different times during relapse treatment until allo-HSCT (indicated in poor responders to induction; MRD ≥ 10−3) for patients being treated for late BCP-ALL bone marrow relapses (n = 413; median follow-up, 9.4 years) in the ALL-REZ BFM 2002 trial/registry (ClinicalTrials.gov identifier: NCT00114348 ). RESULTS Patients with both good (MRD < 10−3) and poor responses to induction treatment reached excellent event-free survival (EFS; 72% v 65%) and overall survival (OS; 82% v 74%). Patients with MRD of 10−2 or greater after induction had reduced EFS (56%), and their MRD persisted until allo-HSCT more frequently than it did in patients with MRD of 10−3 or greater to less than 10−2 ( P = .037). Patients with 25% or more leukemic blasts after induction (early nonresponders) had the poorest prognosis (EFS, 22%). Interestingly, patients with MRD of 10−3 or greater before allo-HSCT (late nonresponders) still had an EFS of 50% and OS of 63%, which in principle justifies allo-HSCT in these patients. From a panel of selected candidate genes, TP53 alterations (frequency, 8%) were the only genetic alteration with independent prognostic value in any MRD-based response subgroup. CONCLUSION After induction treatment, MRD-based treatment stratification resulted in excellent survival in patients with late relapsed BCP-ALL. Prognosis could be further improved in very poor responders by intensifying treatment directly after induction. TP53 alterations can be defined as a novel genetic high-risk marker in all MRD response groups in late relapsed BCP-ALL. Here we identified early and late nonresponders to be considered as events in future trials.

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