Kurzfassung: Detection of minimal residual disease (MRD) in adult ALL is an independent risk parameter and early indicator of an impending relapse. MRD diagnostics have been incorporated in many ALL treatment protocols as a tool for pre-emptive treatment or risk group stratification. Currently, real-time quantitative (RQ)-PCR of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements is regarded as the most sensitive and standardized method to quantify MRD in BCR-ABL negative ALL. However, IG/TR oligoclonality and clonal evolution may lead to false negative results, while sensitivity varies depending on background amplification and is generally limited to 1E-05. Next-Generation Sequencing (NGS) of IG/TR gene rearrangements might overcome these limitations and potentially provide further increases in sensitivity, specificity, accuracy and reproducibility. We have performed a comparison of the two approaches on 130 diagnostic (DG), relapse (REL) and remission (FU) samples of 14 adult patients (pts) with B-cell precursor ALL. To test the NGS sensitivity, we partly selected follow-up samples for (low-level) RQ-PCR MRD positivity or a high pre-test probability of residual disease (either due to a later relapse or MRD detection in temporal relation to the analyzed sample). Patients and Methods 130 samples (97 bone marrow, 31 peripheral blood, 2 stem cell apheresis; 15 DG, 107 FU, 8 REL samples) were analyzed from 14 pts with B-cell precursor ALL. IG/TR-based RQ-PCR was carried out within routine diagnostics in the context of prospective clinical trials [Brüggemann et al., Blood 2006], according to the EuroMRD criteria [van der Velden et al., Leukemia 2007] at the MRD reference laboratory in Kiel. NGS was performed at the Sequenta facilities in South San Francisco. Using universal primer sets, IGH, IGK, TRB, TRG, and TRD variable, (diversity), and joining gene segments were amplified from DG and REL DNA. PCR products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in DG samples. The presence of the tumor-specific clonotype was then quantitated in FU samples. A quantitative and standardized measure of clone level among all leukocytes was determined using internal reference DNA. Comparability of MRD results by RQ-PCR and NGS was assessed using bivariate correlations between methods analysis (software R 3.0.1 with cor.test). Results Sanger sequencing of multiplex PCR detected 60 clonal IGH, IGK, TRB, TRG and/or TRD gene rearrangements at diagnosis (1-6/patient), NGS identified 57 index sequences (1-6/patient). Using NGS, relapse samples were analyzed for clonal evolution: 16/19 index rearrangements of relapsing pts were stable at relapse while 3/19 rearrangements (2 IGH VH-JH and 1 IGK) were lost at relapse. Based on Sanger sequence information of clonal rearrangements 1-3 RQ-PCR assays/patient were established while NGS focused on 1-4 clonal markers/patient. MRD was quantified in 107 FU in remission using both tools. A mean of 223,086±48,030 cells were analyzed per target using RQ-PCR, whereas NGS used 575,776±304,653 cells. An excellent concordance between the two methods was observed (p 〈 0.0001, r2 = 0.974) with 46/107 samples being MRD positive and 45/107 MRD negative with both tools. Sequencing detected MRD in 13 samples that were negative by RQ-PCR whereas RQ-PCR revealed MRD in 3 NGS negative samples. All discrepancies affected samples with low MRD levels (below quantitative range (RQ-PCR) and levels below 8E-06 (NGS)) being consistent with Poisson sampling. In addition, both methods showed quantitative and qualitative inter-target differences with 11/49 RQ-PCR and 32/59 NGS positive samples being negative for at least one target illustrating the importance of focusing on more than one index sequence for RQ-PCR and NGS. Conclusions Significant concordance was observed between NGS and RQ-PCR, with NGS having similar or slightly better sensitivity compared to standardized RQ-PCR. This can be partly attributed to the higher number of cells analyzed by NGS and points out that sensitivity advantages afforded by the NGS platform may be achieved by higher input cell amounts. Prospective analyses of unselected cases must be performed to verify the clinical impact of low level NGS-based MRD detection. Disclosures: Pepin: Sequenta Inc.: Employment, Stockholder Other. Carlton:Sequenta Inc.: Employment, Stockholder Other. Faham:Sequenta Inc.: Employment, Stockholder Other.

Kurzfassung: Introduction: Treatment with Rituximab has improved the outcome for patients with B-cell precursor ALL (B-ALL) and is conventionally restricted to patients with CD20 expression on leukemic blasts above a level of 20%. However, it is not known whether this arbitrary cutoff is biologically meaningful, or if also patients with lower CD20 expression levels profit from Rituximab treatment. In addition, CD20 expression levels might differ between different compartments and can change during early treatment. Therefore, in the current German Multicentric Acute Lymphoblastic Leukemia (GMALL) 08/2013 trial Rituximab is applied to all BCR-ABL1-negative B-ALL patients during induction and early consolidation irrespective of the initial blast CD20 expression. In this study, CD20 expression on blasts at diagnosis and after a 5-day dexamethasone prephase is thoroughly characterized and correlated to MRD response after induction I including one dose of Rituximab to evaluate its impact on response to Rituximab. Methods: Comparative quantification of CD20 expression levels was performed at diagnosis (blood and bone marrow (BM)) and at the end of the prephase (blood) and correlated to minimal residual disease (MRD) response after early Rituximab treatment. Samples were subjected to the EuroFlow standardized stain, lyse and wash and instrument setting procedures (van Dongen et al., 2012) and stained with a 3-tube, 8-color panel, containing the markers CD45, CD20, CD10, CD66c, CD3, CD19, CD71, CD9, CD13+33, CD34, CD22, CD11a, and CD38. CD20 median fluorescence intensities (CD20-MFI) as well as percentages of CD20+ B-ALL blasts among all blasts (%CD20+) were measured. MRD was quantified at day 22 after induction I using real-time quantitative PCR of clonal immune gene rearrangements. MRD response after induction was defined as MRD decrease to a level below 10-4, MRD persistence as detection of quantifiable MRD ≥10-4. Results: FCM results of a total of 166 samples of 84 B-ALL patients were evaluated. CD20 expression of circulating blasts significantly increased after a 5-day prephase in common/pre-B-ALL (c/pre-B) but not in pro-B-ALL in a cohort of 36 paired peripheral blood samples (Fig. 1 A-B, n=8 pro-B-ALL, paired t-test of CD20-MFI and %CD20: p=.1016 and p=.1660, Fig. 2 A-B, n=28 c/pre-B-ALL, p=.0029 and p=0.0060). Interestingly, the increase of CD20 expression on B-ALL blasts contrasted that on mature normal B-cells, where a decrease occurred in pro-B-ALL and c/pre-B-ALL samples at day 6 (paired t-test: p=.0485 and p 〈 .0001, respectively) (Fig.1A, 2A). In 24 paired blood/BM pre-treatment samples the percentage of CD20+ blasts was significantly higher in blood in c/pre-B-ALL (n=19: paired t-test: p=.0009), but not in pro-B-ALL (n=5: paired t-test: p=.095) (Fig. 1C, 2C). The significance levels were not consistently reached for the comparison of CD20-MFI. Nevertheless, we show that in 3/24 (12.5%) patients the percentage of CD20+ B-ALL blasts in BM did not reach the arbitrary cutoff of 20% whereas the matching diagnostic blood did. Finally, CD20 expression levels from 25 BM and 30 pre-treatment blood samples and 38 blood samples after dexamethasone prephase from 54 patients were assessed (in total 93 samples). In further calculations we correlated MRD response values after one dose of Rituximab with the CD20 expression levels on blasts. In detail, the highest measured percentage value of %CD20 per patient in the group of MRD responders was compared to the highest measured %CD20 per patient in the group of MRD persisters. CD20 expression levels were significantly lower in 33 MRD persisters compared to 21 MRD responders (Fig 3, p=.0336). The difference was mainly attributable to a high frequency of MRD persisters in pro-B-ALL compared to c/pre-B-ALL (MRD persistence in pro-B-ALL 8/9 (89%) compared to 25/45 (56%) in c/pre-B-ALL). Conclusion: We measured significant differences in CD20 expression levels on leukemic blasts (i) between pre-treatment blood and BM and (ii) between pre-treatment blood and blood after prephase in patients with c/pre-B-ALL challenging the conventional eligibility criteria for the CD20 targeted treatment. MRD responders tended to have higher CD20 expression levels compared to MRD persisters. Extension of the patient cohort is ongoing to confirm this observation. Furthermore, we will analyze the impact of 3 rituximab applications after induction phase II. Disclosures Ritgen: abbvie: Research Funding; Roche: Honoraria, Research Funding. Viardot:Gilead Kite: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy. Kneba:Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Goekbuget:Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:Incyte: Consultancy; Amgen: Consultancy, Research Funding, Speakers Bureau; PRMA: Consultancy; Affimed: Research Funding; Regeneron: Research Funding; Pfizer: Speakers Bureau; Roche: Speakers Bureau.

Kurzfassung: Introduction Rituximab (R) administration results in significant outcome improvement in B cell precursor acute lymphoblastic leukemia (B-ALL) patients (pts), but is usually restricted to pts with ≥20% CD20+ leukemic blasts. Yet, this arbitrary cut-off is not proven biologically sensible. Moreover, CD20 expression might differ between blood (pb) and bone marrow (bm) and varies under prednisone during early treatment. In the present GMALL08/2013 trial R is administered to all BCR-ABL1-negative B-ALL pts irrespective of the initial leukemic CD20 expression. We assessed the initial and post-prephase CD20 expression in GMALL08/2013 pts and correlated the values with MRD response after Induction I (Ind I) and Consolidation I (Cons I). A historical B-ALL GMALL07/2003 cohort without R treatment and with available CD20 expression and MRD data was used to evaluate for R-unrelated effects. Methods Comparative immunophenotypic quantification of CD20 expression in 207 B-ALL pts was performed at diagnosis (pb d0 and/or bm d0) and/or (a/o) after a 5-day dexamethasone- and cyclophosphamide-containing prephase (pb d6) under EuroFlow standardized procedures. CD20 median fluorescence intensities (CD20-MFI) and percentages of CD20+ B-ALL blasts/all blasts (%CD20+ BL) were assessed. Minimal residual disease (MRD) was determined after Ind I (after 1x R) and Cons I (after 4x R) by quantitative PCR for clone-specific immune gene rearrangements to stratify pts as molecular complete response (MolCR, MRD negativity, assay sensitivity at least 1x10 -4), molecular intermediate response (MolIR, MRD positive non-quantifiable or positive & lt;1x10 -4) and molecular failure (MolFAIL, MRD ≥1x10 -4). Results bm d0/pb d0. In 91 paired bm d0/pb d0 samples %CD20+ BL as well as the CD20-MFI were significantly higher in pb in common/pre-B ALL (c/pre-B ALL) (n=76: paired t-test: p & lt;.0001 and p & lt;.0001) and in normal mature B cells (CD20-MFI paired t-test; pro-B/ c/pre-B p=0.026/p & lt;.0001), but not in pro-B ALL (n=15: paired t-test: p=.81 and p=.76) (Fig. 1A, 2A). Notably, in 6/76 (7.9%) c/pre-B ALL pts the %CD20+ BL in bm d0 was lower and in corresponding pb d0 higher than the arbitrary cut-off of 20%. pb d0/pb d6. CD20 expression of circulating blasts significantly increased after a 5-day prephase in c/pre-B ALL but not in pro-B ALL in 106 paired pb d0/pb d6 samples (paired t-test of CD20-MFI and %CD20+ BL; n=20 pro-B ALL, p=.09 and p=.25; n=86 c/pre-B ALL, p & lt;.0001 and p & lt;.0001). Normal mature B cells presented with the opposite effect (CD20-MFI paired t-test; pro-B/ c/pre-B: p=0.005/p & lt;.0001) (Fig. 1B, 2B). Notably, the %CD20+ BL in pb d0 was lower and in corresponding pb d6 higher than the arbitrary cut-off of 20% in 2/20 (10.0%) pro-B ALL and 12/86 (13.9%) c/pre-B ALL pts. Molecular response under R. The values of %CD20+ BL were correlated with MRD response after Ind I and Cons I (Fig. 3). Since the CD20 expression in the present cohort was shown to be significantly modulated in a drug- and compartment-dependent manner, we used the highest measured value of %CD20+ BL out of the three available values per patient (bm d0, pb d0 a/o pb d6). In the historical cohort (n=145) one value per patient for %CD20+ BL was available. Due to low CD20 expression pro-B ALL did not show any differences in the %CD20+ BL among the risk groups in the present (Ind I n=23, Cons I n=20) and the historical (Ind I n=11; Cons I n=11) cohort. The differences in %CD20+ BL in relation to molecular response were significant in c/pre-B ALL between MolCR and MolFAIL after Ind I (n=127) and Cons I (n=120) (Mann-Whitney test: p=.0002 and p=.0028) and of lower significance between MolIR and MolFAIL after Ind I (p=.013) and between MolCR and MolIR after Cons I (p=.029) in the present cohort. Within the historical cohort (Ind I n=145, Cons I n=143) no significant differences were observed. Conclusions Leukemic CD20 expression was modulated between compartments (bm d0/pb d0) and showed a significant increase in a drug-dependent manner in c/pre-B ALL (pb d0/pb d6) probably in response to dexamethasone. The results might challenge the conventional eligibility criteria for CD20 targeted treatment in c/pre-B ALL. MRD persisters showed lower initial CD20 expression compared to MRD responders in the present cohort consistently receiving R, but not in the historical cohort without R treatment. Accordingly, R seems to improve the early MRD response predominantly in pts with higher CD20 expression. Supported by DJCLS Figure 1 Figure 1. Disclosures Szczepanowski: Amgen: Speakers Bureau. Trautmann: Amgen: Speakers Bureau. Ritgen: Roche: Consultancy, Other: Travel support, Research Funding; Abbvie: Consultancy, Other: Travel support, Research Funding; Chugai: Consultancy; MSD: Consultancy, Other: Travel support; Celgene: Other: Travel support. Nachtkamp: Celgene: Other: Travel Support; bsh medical: Speakers Bureau; Jazz: Speakers Bureau. Viardot: Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; University Hospital of Ulm: Current Employment. Baldus: Jazz: Honoraria; Celgene/BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Schwartz: Morphosys: Research Funding; Gilead: Other: Travel grants, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Travel grants, Speakers Bureau; BTG International Inc: Membership on an entity's Board of Directors or advisory committees; MSD Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Basilea: Other: Travel grants. Goekbuget: Pfizer: Consultancy, Other: Research funding for institution; Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Gilead/Kite: Consultancy; Novartis: Consultancy, Other: Research funding for Institution; Jazz Pharmaceuticals: Other: Research funding for institution; Incyte: Other: Research funding for Institution; Cellestia: Consultancy; Erytech: Consultancy; Morphosys: Consultancy; Servier: Consultancy, Other; Abbvie: Other. Brüggemann: Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Incyte: Other: Advisory Board; Janssen: Speakers Bureau. OffLabel Disclosure: Rituximab administration to patients with CD20-negative ( & lt;20% CD20+ blasts/all blasts) BCR-ABL-negative B-ALL.