ALBERT

Description: Introduction MRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background. Aims To develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints. Methods Two round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files. For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution. Results We sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed. Discussion We present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks. The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis. Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. Disclosures No relevant conflicts of interest to declare.

Description: The expression of CD27, in the absence of CD44 is found in TEL/AML1+ acute lymphoblastic leukemia (ALL) but not in other B precursor subtypes of ALL (Vaskova et al., Leukemia 19(5), 2005). Since CD27 had not been shown in human B precursors, we searched for such cells among B precursors in nonmalignant bone marrow. In all 14 specimens of children without any evidence of malignant disease, we found CD27+CD44−CD10+CD19+ B precursors (6.3±3.9%). The subpopulation of CD27−CD44+ cells that corresponds to most other ALL subtypes was more frequent (26±14.4%). Two other subpopulations which rarely develop into B precursor leukemia were also present: double-positive (DP) (5.5±1.8%) and double-negative (DN) (61.8±14.9%). We asked, whether the 4 subpopulations represent consequent differentiation stages, therefore the expression of CD27 and CD44 combined with CD10 and CD19 was studied by polychromatic flow cytometry together with the differentiation markers CD34, terminal deoxyribonucleotidyl transferase (TdT), CD20, cytoplasmic IgM and cytoplasmic VpreB (CD179a). The percentage of CD34+ cells is the highest in CD27+CD44− and decreases gradually in DP, CD27−CD44+ and DN subpopulations (73.1±20.7%; 43±16.6%; 13.1±8.2%; 4.4±2.9%). A similar trend is found in the percentage of CD10bright cells, which become virtually missing in CD27− B precursor stages (41.3±15.4%; 7.9±8.1%; 1.4±1.1%; 2.7±1.8%). This sequence of developmental stages was further supported by a gradual loss of intracellular TdT and VpreB and by the increase of cytoplasmic IgM+ cells. We sorted these subpopulations to compare their recombination potential by measuring TdT and RAG-1 mRNA expression by RQ-RT-PCR. Similarly with the protein level, TdT mRNA expression decreases in concordance with the suggested developmental stages. Interestingly, the DP cells cease to transcript RAG-1, suggesting that these cells are in the stage of suppressed RAG-1 expression after completed immunoglobulin (Ig) heavy chain rearrangement. RAG-1 is re-expressed during Ig light chain rearrangement, seen as the reappearance in the CD27−CD44+ subpopulation. Since the cells with a downregulated RAG-1 are known to be frequent among the large proliferating cells we analyzed the percentage of large cells. The DP subpopulation contains the highest percentage of these cells (63.5±9.9%). In all four subpopulations, heavy chain gene (both segments VH1-3-JH and VH4-7-JH) rearrangements were detected, suggesting that heavy chain genes start to rearrange before or at the CD27+CD44− stage. We investigated the light chain rearrangements using the system detecting the intron RSS-Kde rearrangements, which appear in the late phase of Ig light chain rearrangement. Rearranged light chain genes appear at the CD27−CD44+ stage, whereas they are virtually missing at earlier stages. These results show that CD27 molecule, which is so far regarded a marker of memory B cells is expressed also in the early stage of B cell development during Ig heavy chain rearrangement completion. The expression of CD27 and CD44 define differentiation stages that very tightly correlate with Ig recombination maturity as well as with specific subtypes of B precursor leukemia.

Description: Abstract 756FN2 Bone marrow (BM) aspiration at the end of induction therapy plays a crucial role for the evaluation of remission and the minimal residual disease (MRD), both critical for treatment stratification in modern treatment protocols for paediatric acute lymphoblastic leukaemia (ALL). However, the aspiration is repeated in 15–20% of patients, either due to non-representative morphology or to insufficient material needed for MRD analysis. We prospectively analysed 320 paediatric ALL patients treated according to ALL-BFM 2000 (n=301) or ALL IC-BFM 2002 (n=19) protocols with repeated BM aspiration at the end of induction therapy, on treatment day 33. Fourteen patients had more than one re-puncture. The median follow-up was 69 months, 45 (14%) patients had an event (relapse/death). The cause for the repeated BM aspiration was non-representative morphology (32%), insufficient material for MRD analysis (33%) or both (35% cases). In order to evaluate prognostic significance of the re-punctures and to determine which of the repeated samples should be used for the final treatment stratification we analysed MRD levels and MRD stratification, morphology, leukocyte count (WBC) and the length of treatment delay caused by waiting for the repeated aspiration. MRD data were collected and interpreted according to the EuroMRD guidelines in one central reference laboratory per each participating country. Morphology was evaluated centrally using an own scoring system (with a max value of 26 points). Treatment delay between the original and the last aspiration was one-third longer in patients with subsequent event compared to patients remaining in complete remission (CR) (median 8 (range 2 – 21) vs. 6 (1 - 28) days, respectively; p=0.020). Patients with a subsequent event had significantly higher WBC at the time of the last repeated BM aspiration, compared to patients without event (p=0.019), while there was no difference relative to the original aspiration (p=0.9). Analysis of the BM morphology at the original aspiration showed no significant difference between patients with an event vs. those in CR. However, the repeated aspiration of patients with a subsequent event had significantly better morphology (median 18.5/26 vs. 15/26 points, p=0.0012) mainly due to higher cellularity (p=0.003) and number of megakaryocytes (p=0.048). MRD levels were identical or decreased in 88% and increased in 12% of cases comparing the original aspiration to the repeated aspiration. In 63 patients (20%) the different MRD levels would lead to different treatment stratification. Higher MRD was associated with treatment failure; the best predictive values for subsequent event were obtained using the MRD results of the original aspiration (p=3.1e-07) or the highest of the detected MRD levels (p=6.0e-07). The last aspiration before proceeding with treatment had the lowest, though still a highly significant predictive value (p=8.6e-06). Corresponding results are obtained when MRD levels are substituted by final MRD risk stratification into standard, medium or high risk (p

Description: FC is still not employed in MRD based treatment protocols. One problem is lack of standardization suitable for prospective trials involving multiple clinical centers and FC laboratories. Therefore, we established a MiniMini Project, an international collateral study within the ALL IC BFM 2002 treatment protocol for childhood acute lymphoblastic leukemia (ALL). The MiniMini Project provides a mainframe of minimal panel of monoclonal antibody combinations to evaluate MRD by FC. Patients (pts) are stratified according non-MRD criteria (prednisone response at day 8 in peripheral blood (PB), percentage of blasts at day 15 and day 33 in bone marrow (BM), leukocytosis, and age at diagnosis and presence of BCR/ABL or MLL/AF4 fusions). Identical immunophenotypic populations are reported in all pts regardless presenting phenotype. Each laboratory investigates at least 2 pts with B lineage ALL by the T ALL combinations and vice versa. These “cross-lineage controls” together with data on subpopulations that are negative at diagnosis were used to set the specificity cutoff values at each time point (diagnosis, day 8 BM and PB, day 15, day 33 day 52 BM). MRD levels obtained by Ig/TCR rearrangements RQ-PCR in 32 pts (24 pts BCP ALL, 8 pts T ALL) were used to define specificity thresholds. 185 pts were investigated in the participating laboratories. We used data from first Czech cohort of pts (92 pts in total, 16 pts T lineage, 74 pts B lineage, in standard risk group (SRG), n=36, IRG, n=40 and HRG, n=16) in whom clinical data as well as standard FC analysis results were available. We compared morphological percentage of blasts (used for stratification) to a level of residual disease by FC. There was high concordance in SRG of both methods, except 1 patient redirected into IRG group (M3 BM vs. only 14% of blasts by FC). In IRG, concordance was in 92.5% of pts, 3 pts should be placed in HRG group according FC. 98.9% of pts morphologically in complete remission at day 33 were confirmed by FC. Although FC data confirm a significant difference between PGR and PPR in PB specimens at day 8 (p=0.0014), there is an overlap in percentage of leukemic cells between these categories. In total, MRD level above 0.1% was observed in BM of 100, 99, 84, 32 and 3.5 % pts in days 0, 8, 15, 33 and 52, respectively and in PB of 95% pts at day 8. Our first results show feasibility of FC standardization. The choice of subpopulations and the cutoff points will be validated in an independent cohort within the same Project.

Description: Minimal residual disease (MRD) testing based on a unique Ig/TCR gene rearrangement pattern of each patient’s leukaemia turned out to be an independent tool to determine treatment response and the risk of relapse in paediatric acute lymphoblastic leukaemia (ALL). Since 07/2000, MRD information at week 5 and 12 of therapy has been used for stratification in ALL-BFM 2000 trial. In parallel, ALL IC-BFM 2002 has been designed by the International-BFM Group to test the morphological assessment of the early treatment response. Patients are stratified according to the blast proportion in peripheral blood (PB) at day 8 and in bone marrow (BM) at day 15 and 33 of therapy, age, initial WBC and the presence of BCR/ABL and MLL/AF4 fusion. One of the goals of the study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. In the Czech Republic, 73 patients were treated according to ALL IC-BFM 2002 protocol from 11/2002 to 12/2003, 29 in the standard-risk (SR), 35 in the intermediate-risk (IR) and 9 in the high-risk (HR) group. The SR, HR and all T-cell ALL patients were examined for clonal Ig/TCR rearrangements. RQ-PCR patient-specific systems were designed for each of these patients according to the ESG-MRD-ALL criteria. For 39 of the 40 patients tested (97.5%) at least one target with minimal sensitivity of 10(−4) was identified. MRD was evaluated in BM samples from 34 patients at several time points inclusive of the mandatory 5 and 12 week ones. Simultaneously the PB specimens of the T-ALL patients were tested. In total, 205 BM and 64 PB specimens were included. In 7 patients of 24 in the SR group, MRD positivity at week 5 and/or at week 12 was observed (ranging between 9.7x10(−4) and 1.5x10(−2)), thus identifying patients who would not qualify to the MRD-based SR group in ALL-BFM 2000 despite the identical induction regimen. In T-ALL patients, PB-MRD levels paralleled those in BM. MRD results showed no separation of MRD levels between IR- and HR-stratified T-ALL patients. These preliminary findings reveal a significant difference between the stratification results of ALL IC-BFM 2002 and ALL-BFM 2000. A fast response as measured by the morphology criterion (M1 or M2 bone marrow at day 15) together with other low-risk features does not necessarily correspond with rapid MRD clearance. The complete analysis of MRD is planned for the international consortium participating in the ALL IC-BFM 2002 protocol.

Description: Introduction Germline mutations in GATA2 were recently identified as causative for several overlapping syndromes: MonoMAC (monocytopenia, mycobacterial infections), DCML (dendritic cells, monocytes, B and NK cells deficiency), Emberger syndrome (lymphedema, sensorineural deafness, multiple warts) and familiar myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Of note, GATA2 mutations were also found in children and young adults with “primary” MDS. Aplastic anemia (AA) constitutes an important differential diagnosis to pediatric MDS, particularly in patients with normal cytogenetics. Because of heterogeneous phenotype of GATA2 mutated patients, defining a set of typical findings would help in their earlier identification and understanding the natural course of the disease. Therefore we aimed to analyze monocytes and lymphocyte subpopulations with the emphasis on B cell lineage by flow cytometry (FC) and polymerase chain reaction (PCR) in all pediatric patients with GATA2 mutation diagnosed in the Czech Republic. Patients and methods Eleven pediatric patients were found to harbor GATA2 mutations in the Czech Republic so far. Three mutations were intronic. There was a clear male predominance (9/11). In 7 patients the disease manifested with MDS in childhood, 2 female patients were followed for immunodeficiency and developed MDS in adulthood. One another patient was diagnosed with interstitial lung disease and chronic EBV infection. His brother, carrying the same mutation, has mild neutropenia. Bone marrow (BM) and peripheral blood (PB) samples were analyzed by FC. The level of intronRSS-Kde recombination excision circles (KREC) and T-cell receptor excision circles (TREC) for assessment of proliferation history of B and T cells was examined by PCR. The control group comprised 26 GATA2 wild-type MDS (“other MDS”) patients and 36 AA patients. Results Disturbance of B cell compartment was the most frequently observed anomaly in the patients with GATA2 mutation. We observed a decrease of absolute and relative B cell numbers in PB and BM (n=9/11). In BM there was a decrease of immature CD10pos B cells (n=10) with proportional increase of plasma cells. Peripheral blood B cell immunophenotype was shifted towards memory B cells (n=5/7). Presence of normal B cell precursors CD19pos10pos34pos in BM was observed only in 1 patient in part of follow-up samples. Atypical malignant B lymphoblasts were present in another patient, whose MDS quickly progressed to AML with a clear switch to B lymphoid phenotype. Despite significantly reduced number of B cells the levels of IgG were normal in majority of patients. Only 2 patients had IgG hypogammaglobuliemia, in one patient with chronic active EBV infection IgG hypergammaglobulinemia was present. Slightly decreased IgA level was present in 6 patients. Although B cell numbers in other MDS control patients were significantly lower compared to AA, still the decrease was less prominent in comparison with GATA2. The decrease of immature and naive B cells in patients with GATA2 mutation was reflected in very low level of KREC in PB and BM. Stored newborn dry blood spots from 4 patients were evaluated for TREC and KREC numbers. Strikingly, only one patient had negative KREC levels (the youngest patient from our cohort with MDS diagnosed at age 4). The remaining 3 patients had normal TREC and KREC levels at birth. Thus, the deterioration of de novo production of B cells occurred supposedly postnatally in most patients. Low KREC levels were also present in some patients with other MDS (n=5). Relative monocytopenia was found in 2 patients, low NK cells were present in 6 patients. T cells were mostly of naive non-activated phenotype. Conclusions Changes in B cell compartment are the most characteristic feature in patients with GATA2 mutation. Decreased number of B cells together with a shift towards mature phenotype and decreased level of KREC reflect history of substantial B cell proliferation in an environment of impaired production. This process appears to happen postnatally and resemble normal ageing process, which is accelerated due to progenitor cell impairment. Immunophenotyping is a useful tool in identifying patients for GATA2 sequencing. Supported by GAUK 802214, IGA NT/14534-3, NT/13462-4, UNCE 204012, GAČR P301/10/1877 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1282 Poster Board I-304 Ikaros, encoded by the IKZF1 gene, is a zinc-finger transcription factor crucial for normal differentiation of the lymphoid lineage. Multiple isoforms of Ikaros are generated by alternative splicing in lymphoid progenitors. Recent studies based primarily on high-risk (HR) patients with ALL, including Philadelphia-positive cases, have shown an inferior prognosis of patients with Ikaros alterations, leading in most cases to the expression of short, non-DNA binding isoforms of IKZF1. There is only a limited information about the overall frequency and prognostic impact of IKZF1 alterations in non-selected, BCR/ABL-negative ALL cohorts. Using a simplified yet efficient approach based on expression assay described by Iaccobucci et al. together with Agilent-on-chip semi-quantitative electrophoresis, we examined the expression of IKZF1 transcript variants in diagnostic bone marow (BM) samples from 94 children with Ph- ALL. The patients were diagnosed between November 2002 and December 2004 and treated by ALL IC-BFM 2002 protocol. Based on the analysis of peripheral blood from healthy donors and remission BM samples, we determined physiological range for relative expression of IKZF1 isoforms. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK8) and functional IK1 and IK2 isoforms was significantly elevated in 26 of 94 patients (28%). There were no associations between elevated short/long isoforms ratio and age, WBC, ALL IC risk group, TEL/AML1 or hyperdiploidy. Considering the key role of Ikaros in lymphoid lineage specification, we tested whether its altered expression was related to the expression of myeloid markers on leukemic blasts. Neither short/long isoforms ratio, nor single transcript variant expression had any relation to the level of myeloperoxidase (MPO), CD13, CD33, CD65, CD117, CD14 or CD15 expression estimated by flow cytometry. Patients having the short/long isoforms ratio more than 80% had a 5-year RFS 66.7±13.6% compared to 87.5±4.1% in other patients (p=0.04). The main difference between leukemic and normal samples was observed in the relative expression of IK6 dominant-negative isoform. Using a cut-off of 10%, 14 of 94 (15%) ALL samples had increased IK6 expression in relation to other isoforms. Only 2 of 94 patients (2%) expressed IK6 alone. Elevated relative IK6 expression in the range 10-20% (6 patients) had no prognostic impact. Using a cut-off of 20%, 5-year RFS survival was 90.2± 3.3% in the group with low IK6 expression compared to 37.5±17.1% in patients with the high expression (p

Description: Sensitive quantification of minimal residual disease (MRD) is an established method to assess treatment response in patients with bone marrow (BM) relapse of acute lymphoblastic leukemia (ALL). We have investigated, whether MRD can also determine the tumor load in the BM at cytologically isolated extramedullary (IEM) relapse and can consequently be associated with prognosis in this subgroup. The extent of sub-cytological BM involvement at relapse diagnosis has been quantified in 53 patients with first relapse in the central nervous system (n=36), and in the testes (n=17) treated in Germany (n=33; 09′94 - 12′03) and Czech Republic (n=9; 10′01 - 04′04), or in France (n=11; 12′90 - 11′03) according to protocols ALL-REZ BFM, or to COOPRALL, respectively. MRD has been sensitively assessed by real-time-quantitative PCR using T-cell-receptor and immunoglobulin gene-rearrangements detected in the extramedullary or initial BM material. Stability of initial clonal markers was confirmed. A MRD-level of 10−2: n=10. MRD-status was not associated with the relevant prognostic parameters sex, time-point of relapse, immunophenotype, and site, whereas it was associated with age at initial diagnosis (p=.057), and BCR/ABL translocation (p=.011). The probability of event-free survival (EFS) at 5 years was .55+/−.15 and the rate of subsequent relapses was 18% in patients without MRD detection at a level of 10−4 compared to .16+/−.10 and 52% in those with =/〉10−4 (p(log-rank)=.16 and p=.028 respectively). Our study reveals a large range of subcytological BM involvement in patients with IEM relapses of ALL. A relation to single prognostic factors and to the relapse rate can be found, but not to EFS. The study was kindly supported by ‘Deutsche Kinderkrebsstiftung’, Germany, by ‘Kompetenznetz der Paediatrischen Onkologie’, BMBF, Germany; by ‘programme hospitalier de recherche clinique’ (PHRC 2002), France and by GAUK 62/2004, Czech Republic.

Description: Minimal residual disease (MRD) assessment via next generation sequencing (NGS) of immunoglobulin (Ig) and T-cell receptor (TR) gene rearrangements for lymphoid malignancies is currently under extensive development. NGS MRD has a potential to overcome the limitations of current techniques; laboriousness and difficult interpretation of qPCR for Ig/TR and low sensitivity of flow cytometry. However, amplicon-based NGS MRD has potential pitfalls that have to be addressed before it can be safely introduced for clinical decision making. Multi-center concordance in the experimental setting, quality control and interpretation of the results need to be achieved in order to surpass the advantages of qPCR, which is currently rigorously standardized within the EuroMRD consortium. Our aim was to test the stability and reproducibility of an optimized Ig heavy chain (IGH) based NGS approach for MRD assessment in a multi-center setting within the EuroClonality NGS Consortium on two different sequencing platforms. A one-step PCR library preparation approach was tested in seven institutions (Kiel, Salamanca, Milano, Bristol, London, Prague, Torino). Serial dilutions (10-1 to 10-5) of diagnostic DNA into polyclonal DNA as well as follow-up samples of 30 B-cell precursor ALLs with known complete IGH rearrangements were sequenced on the MiSeq. Serial dilutions of five different diagnostic ALL samples and libraries from polyclonal control were sequenced in parallel on both the MiSeq and Ion Torrent platforms. All samples were spiked with pre-defined copy numbers of five reference IGH sequences as a calibrator. FR2 primers, harboring platform-specific sequencing adapters, were used during the one-step PCR with 500ng of DNA per sample (75,000 copies). Negative and positive controls (27 pooled B-cell lines) were used for testing assay stability and reproducibility among the labs. Purpose-built bioinformatics methods were applied to analyze data. MRD results were compared to results of EuroMRD-based qPCR results. A total of 333 libraries were sequenced in 29 deep sequencing runs producing 194 million reads. The IGH gene rearrangements of all 27 pooled positive B-cell line controls were identified in all centers. NGS MRD analysis in 116 ALL follow-up samples revealed MRD positivity in 69/116 samples vs. 66/116 samples in qPCR, with discrepancies concerning samples with low MRD (R2=0.81). The dilution experiments gave similar results for both platforms, with a minimum sensitivity of 10-4 (as currently required by most treatment protocols using qPCR) for all tested assays. The correlation between MRD levels obtained by the two NGS platforms was good (R2=0.84). Ratios of reads containing reference IGH sequences were highly consistent in intra- and inter-laboratory analyses, independent of the total number of reads in the sample. When comparing platforms, in 10-1 dilution samples sequenced on MiSeq the ratio of reads harboring reference sequences was 2.1 to 2.7 times lower than in remaining dilutions, while on the Ion Torrent it was only 0.9 to 1.3 times, reflecting the competition with the leukemic clone. The correlation of the amounts of spiked-in sequences with the representation of reads harboring these sequences was slightly better for the Ion Torrent (R2=0.88) than for the MiSeq (R2=0.79). Amplification efficiency of each primer was checked by analyzing libraries from healthy polyclonal control. All primer sequences were present in all samples on both platforms, however, the differences between four libraries prepared from the same sample sequenced on the MiSeq were 2.6 times higher than in one library from this sample sequenced in five replicates on the Ion Torrent. The newly developed IGH assay shows robust intra and inter-laboratory reproducibility, which is the first step towards the safe use of this new MRD technique in a multi-center setting. The distribution of reference sequences and sequences of primers confirmed that the main source of differences between platform strategies is the library preparation and not the platform itself. Using the same amount of DNA, the sensitivity of the method is similar to qPCR. The performance and costs of the assay are similar for both the MiSeq and Ion Torrent. MRD analysis via NGS has therefore a great potential to replace qPCR as the gold standard for MRD-guided therapy in ALL, provided that thorough standardization can be achieved. Support: NV15-30626A, GBP302/12/G101. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics..

Description: More than 800 children with acute lymphoblastic leukaemia (ALL) are treated every year according to ALL IC-BFM 2002 protocol, which was designed by the International-BFM Group as a parallel to MRD-based ALL/AIEOP BFM 2000 study. The ALL-IC BFM 2002 risk group stratification comprises blast proportion in peripheral blood (PB) after 7 days of prednisone and one IT-MTX (prednisone response) and bone marrow (BM) morphology evaluation at days 15 and 33 of therapy together with age, initial WBC and presence of BCR/ABL and MLL/AF4 fusion. One of the aims of the ALL IC-BFM 2002 study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. We analyzed a total of 203 patients treated according to the ALL IC-BFM 2002 in the Czech Republic, Israel, Hong Kong and Uruguay for the presence of clonal antigen receptor rearrangements. MRD was evaluated in 175 patients at several time-points of therapy including mandatory points at week 5 and 12, which are used in the ALL/AIEOP BFM 2000 stratification. In total, 654 follow-up BM specimens and 80 PB samples were tested. In the univariate analysis, a good molecular response defined as MRD negativity at both week 5 and 12 was associated with the age of 1–6 years (p=0.0001), WBC