Littérature scientifique sur le sujet « Surrogate endpoint, Validation, Leukemia »

Abstract Introduction The need to optimize clinical evaluation of new drugs stimulates researchers and regulatory bodies to consider novel endpoints that facilitate assessment of the efficacy of a new drug earlier in time than do traditional endpoints. To be a useful marker of efficacy, an early endpoint must be an accurate surrogate for the true clinical endpoint. Minimal residual disease (MRD) is a strong prognostic factor for Event Free Survival (EFS) in children with newly diagnosed acute lymphoblastic leukemia (ALL) and is used routinely to assess treatment response and stratify treatment intensity. However, it is not known whether or not early MRD response is an accurate surrogate endpoint for EFS in evaluating the efficacy of treatment interventions. This study addresses for the first time in childhood ALL the formal validation of surrogacy of MRD levels at the end of induction treatment by a meta-analytic approach on individual data from two large phase III trials with a randomized question on type of steroids in induction (dexamethasone 10mg/m2/day vs prednisone 60mg/m2/day). Material and Methods We performed a meta-analysis of individual data of 2955 B-ALL patients from AIEOP-BFM-ALL2000 (NCT00613457, NCT00430118), and 945 and 930 high risk B-ALL patients randomized for steroids after being allocated either to Capizzi or High-dose Methotrexate in COG AALL0232 (NCT00075725; separately considered due to the significant quantitative interaction between Methotrexate regimens and type of steroid). The trials included evaluation of MRD at day +33 (PCR-MRD) and +29 (flow-cytometry MRD), respectively, with a sensitivity of at least 10-4. The three categories MRD level (negative, low positive i.e. <5x10-4 and positive ≥5x10-4), was assessed as surrogate for the EFS endpoint (time to event defined as resistance at the end of induction, relapse, death in remission, second malignancy). A two-level modelling approach was used to estimate the association between MRD and EFS and between the treatment effect on MRD (proportional odds model) and on EFS (proportional hazard model). The quality of the surrogate at the individual level was assessed on the basis of the bivariate Plackett copula model, with a parameter representing the global odds ratio. The quality of the surrogate at the trial level was assessed on the basis of the coefficient of determination R2trial from a linear regression through the estimated treatment effects. Results The main results on MRD and EFS by trial and treatment are in table 1. The meta-analytic approach shows that MRD at the end of induction is a poor surrogate for treatment effect on EFS (Figure 1) at the trial level, with R2trial=0.09 (95% CI: 0-0.29), while, at the individual level, it shows a considerable prognostic association with EFS, after adjusting for treatment, with a 3.9 odds ratio of failure for patients with higher compared to lower MRD levels (95% CI: 3.4-4.4). Additional sensitivity analyses on relevant subgroups generally confirmed the previous findings both at the trial and patient level association. Conclusions Using a meta-analytic approach, we found that MRD, in 3 categories defined according to standard cut-points, is a poor surrogate for EFS at the trial level, thus indicating that the effect of the randomized steroids (dexamethasone vs. prednisone in induction) on the MRD level at the end of induction does not reliably predict the effect of the intervention on EFS. In contrast, the analysis shows a strong and highly significant association between end induction MRD level and EFS time for individual patients, regardless of treatment, confirming the prognostic effect of early MRD response on clinical outcome. This study shows, for the first time, the limitation of a strong prognostic factor in being a surrogate in the context of front line ALL treatments. The impact of type of steroid on MRD distribution at the end of induction is relatively limited and subsequent treatment complexity and intensity, partly tailored on MRD itself as a key criterion used to modulate the intensity of post-induction therapy, may dilute a potential surrogacy. These data suggest that clinicians and regulatory bodies should be cautious in using early MRD response in the context of complex multiagent therapy as an early surrogate endpoint to evaluate the effect of a randomized treatment intervention on long-term EFS. Disclosures Moricke: JazzPharma: Honoraria, Other: financial support of travel costs. Biondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Cellgene: Other: Advisory Board.

Background: In order to facilitate sound economic evaluations of novel treatments, health-economic models of polycythemia vera (PV) must combine effects on surrogate endpoints in trials with disease progression (DP) and mortality in long-term cohort data. Objective: We validate an economic model for PV that uses Janus Kinase 2 (JAK2) burden as a surrogate endpoint to predict DP (thrombosis, myelofibrosis, and acute leukemia) and overall survival (OS) based on progression-specific mortality. Methods: Long-term observational studies that include information about baseline JAK2 burden were identified via PubMed searches and used to validate the model. Kaplan-Meier (KM) OS curves were extracted using a digitizing software. External validity of the model was analyzed by visually comparing OS curves of the model with the KM curves of the included studies, as well as calculating differences in mean OS estimated as area under the curve (AUC). Results: The model’s predictions of cumulative DP were somewhat lower than the published studies. Over 20 years’ time, our base case model predicted a mean OS for a PV patient (15.0–16.5 years), which was in line with the published studies (15.8–17.5 years). Modeled mean OS was almost two years longer (1.6–1.9 years) for patients with JAK2 <50% than patients with JAK2 ≥50%. Only three long-term observational studies that satisfied the predefined criteria were found and could be used in the validation, but these studies did not capture JAK2 evolution over time. Improved model predictions of DP and mortality based on the longitudinal evolution of JAK2 could be derived from real-world data sources. Such data are currently scarce and future observational studies should be designed to capture the long-term impact of JAK2 on DP and mortality in PV.

Abstract Background: Overall survival (OS) remains the definitive primary efficacy endpoint to evaluate previously untreated acute myeloid leukemia (AML) therapies, but it requires prolonged follow-up. An earlier endpoint assessed post-treatment would expedite clinical trial conduct and accelerate patient access to effective new therapies. Our objective was to formally evaluate event-free survival (EFS) as a surrogate endpoint for OS in untreated AML. Methods: Individual patient data were analyzed from 2,475 patients (pts) from 4 multicenter, randomized controlled phase III trials of active treatment in previously untreated AML using anthracycline and cytarabine induction chemotherapy as the concurrent control (CALGB 10201, n=506, enrollment period 2003-2006, age 60-88 years (y); CALGB 10603, n=717, enrollment period 2008-2015, age 18-60 y, FLT3-mutated pts only; SWOG 0106, n=595, enrollment period 2004-2009, age 18-60 y; ECOG-ACRIN 1900, n=657, enrollment period 2002-2008, age 17-60 y). Individual patient-level surrogacy examines the association between the individual patients' EFS and OS time after adjusting for treatment effect, and was assessed using the copula bivariate survival model (Kendall's tau). Trial-level surrogacy measures how precisely the treatment effect on OS can be predicted on the basis of observed treatment effect on EFS, and was evaluated using both linear regression (R2WLS) weighted by trial size and Copula bivariate (R2Copula) models. Pre-specified criteria for surrogacy required either R2WLS or R2Copula ≥0.80, neither below 0.7, with either lower bound 95% Confidence Interval (CI) >0.60. Sensitivity analyses were conducted using different EFS definitions (Table 1). Results: With a median follow-up of 50.2 months for the 896 patients still alive, the median OS and EFS across all four trials were 20.9 months (95% CI: 19.0-22.7) and 5.6 months (95% CI: 4.5-6.4), respectively. Trial-level surrogacy for EFS was strong (R2WLS=0.79; R2Copula=0.89), indicating a high correlation of treatment effect between EFS and OS. At the individual patient-level, however, EFS showed weak association with OS (tau= 0.52), compared to the strength of trial-level surrogacy. The discrepancy between patient-level EFS and OS was greatest among patients who did not achieve a CR, followed by those who achieved a CR but relapsed (Figure 1). Sensitivity analysis on alternative EFS definitions showed that the trial-level surrogacy was similar, but individual patient-level surrogacy varied across different EFS definitions (Table 1). This is consistent with what we previously reported (ASH 2016): EFS estimates differed considerably based on the definition of induction failure (IF) in a single arm setting, but this had minimal impact on the estimation of the treatment effect using EFS in randomized trials. In addition, when considering only relapse and death as events (definition 4), both individual patient- and trial-level correlations were high. Conclusions: Correlation between EFS and OS was impacted by patients not achieving CR during induction. Despite the lack of patient-level correlation, a strong correlation between hazard ratios for treatment effects was observed between EFS and OS on the trial level. Hence, it remains debatable whether EFS represents a clinical benefit in itself for patient with untreated AML considering the strong correlation in treatment effects. Further validation is needed due to the small number of trials included and the heterogeneity across trials. Acknowledgment: We gratefully acknowledge the important contributions of the late Dr. Stephen H. Petersdorf, SWOG S0106 Study Chair. Support: U10CA180821, U10CA180882, U10CA180794, U10CA180820, U10CA180888; Clinicaltrials.gov Identifiers: NCT00085124 (10201), NCT00651261 (10603), NCT01253070 (11001), NCT00085709 (SWOG S0106), and NCT00049517 (ECOG-ACRIN E1900) Disclosures Uy: Curis: Consultancy; GlycoMimetics: Consultancy. Larson:Pfizer: Consultancy, Research Funding; BristolMyers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Ariad/Takeda: Consultancy, Research Funding.

Abstract Introduction. Minimal residual disease (MRD) is an objective measure of disease status defined by the number of leukemic cells in the blood or bone marrow of leukemic patients. In recent clinical studies of chronic lymphocytic leukemia (CLL), undetectable MRD levels (< 1 tumor cell/10,000 leukocytes) have been shown to correlate with prolonged progression free survival (PFS) and overall survival, independent of treatment or known risk factors. MRD assessment has been proposed as an alternative to PFS as a primary endpoint in frontline CLL pivotal studies to evaluate the efficacy of novel therapies at an earlier time-point. Thorough standardization and validation are needed to use MRD as a primary surrogate endpoint. Allele specific oligonucleotide (ASO)-PCR of immunoglobulin (IG) gene rearrangements is a method for quantifying MRD using patient-specific PCR primers and has been standardized by the EuroMRD Consortium (www.EuroMRD.org). Given that each patient has individualized PCR primers designed for their leukemic clone, this posed a unique challenge for the analytical validation studies to demonstrate that the assays are uniform in their reproducibility and analytical sensitivity to measure MRD across patients with CLL. Here we report a comprehensive, IVD-guided analytical validation of the ASO-PCR technique according to the guidance of regulatory authorities. We provide evidence that the ASO-PCR methodology can reproducibly measure MRD to the required threshold of 10-4, across patients with CLL. Results. Performance of ASO-PCR was assessed using a combination of retrospective data from the CLL11 clinical trial and prospectively performed experiments. Patient assays from 60 CLL patients were tested in two EuroMRD laboratories to demonstrate linearity across the measurement range of 10-1 to 10-5, and a limit of detection of 6.3x10-5, which is below the cut-off of 10-4 used for defining MRD negativity. Concordance of the method to an orthogonal method was determined from the previously published comparison of flow cytometry with ASO-PCR (Boettcher et al., Leukemia 2009; 23: 2007) with 93.8 % overall agreement between both methods (n=452). Agreement of MRD status was >97% when comparing individually designed ASO primers for the same patient within the lab. The overall agreement between the two different laboratories using independently designed ASO-PCR assays was 93.5%. Precision was assessed above and below the threshold of 10-4 using ASO-PCR assays of 3 individual patient samples diluted to appropriate MRD levels listed in Table 1. Theexperiment was designed to mimic sources of variation by evaluating MRD samples over the course of the clinical study (3 days x 2 operators x 3 patients x 2 laboratories x 3 replicates). Overall variability was estimated using a mixed effects model including fixed patient effects and random effects for operator and day. Based on the known MRD distribution of frontline CLL patients, we estimate acceptable overall variability on the order of 80% CV at lower concentrations (≤ 3.2x10-4) and 40% CV at higher concentrations (> 3.2x10-4). This precision estimate provides reasonable misclassification rates (< 5%) due to the fact that the majority of patients had MRD levels either well above or below 10-4 level. Experiments also addressed stability of patient specimens and critical assay components. Table 1. Precision of ASO-PCR results obtained at MRD levels 10-2 to 10-5Table 1.Estimated Total CV (%) Averaged Across 3 patientsMRD levelKielErasmus1.00E-028.7034.791.00E-0310.0235.743.20E-0415.8233.271.00E-0431.7036.383.20E-0589.8978.801.00E-05258.06277.27 Conclusion. The analytical validation studies described here provide evidence that the ASO-PCR methodology, standardized by EuroMRD, performs well to reproducibly detect MRD status across CLL patients at the threshold of 10-4. These studies serve as an example for the validation of personalized, patient-specific quantitative clinical assays for use as a primary endpoint in clinical trials. The authors would like to acknowledge the valuable work of the following people who contributed to this work: M. Brüggemann (UKSH Kiel), R Raja, C. Cox, W. Darbonne, R. Desai, and K. Trunzer. Disclosures Langerak: 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.; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). van Dongen:BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics.; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge.Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; 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.. Royalty income for EuroClonality-BIOMED-2 Consortium; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences)., Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL.. Ray:Genentech, Inc.: Employment. Punnoose:Genentech, Inc.: Employment. Kim:Genentech, Inc.: Employment. Haberberger:Genentech, Inc.: Employment. Bernaards:Roche: Employment. Zhu:Genentech, Inc.: Employment. Lewin-Koh:Genentech, Inc.: Employment. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Introduction The standard primary endpoint in clinical trials of chronic lymphocytic leukemia (CLL) is progression-free survival (PFS). Given the increasingly long follow up required to detect differences in PFS between treatment arms in the era of more efficient therapeutics, valid surrogate endpoints are urgently needed to reduce clinical trial duration, thereby accelerating drug development, reducing costs and allowing patients (pts) earlier access to novel treatment options. Pts with CLL who achieve levels of minimal residual disease (MRD) of <1 clonal cell/10.000 leukocytes in peripheral blood (PB) as determined by multicolor flow cytometry or allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) at the end of initial treatment are considered MRD negative, and have been shown to experience significantly improved PFS. This analysis aimed to support the evaluation of MRD response at the end of treatment as a surrogate endpoint for PFS in CLL, based on a retrospective analysis of 3 multicenter, randomized, Phase 3 clinical trials. Methods MRD was prospectively assessed in PB of treatment-naive pts with CLL that participated in the German CLL Study Group CLL8, CLL10 and CLL11 multicenter, randomized, open-label, Phase 3 clinical trials, which included induction treatment followed by observation. The primary endpoint of each study was investigator-assessed PFS. MRD was quantified by 4-color flow cytometry in CLL8 and CLL10, and ASO RQ-PCR in CLL11. Both methods had a detection threshold of 1 CLL cell/10,000 leukocytes. Pts who had no MRD result but had disease progression or died shortly after 6 treatment cycles (within 90 [CLL8 and CLL10] or 56 [CLL11] days of last dose) were included and classed as MRD positive. A meta-regression model was developed to predict treatment effect on PFS using treatment effect on MRD. Log ratio was selected as the most reliable representation of MRD response based on the best model fit. To ensure no systemic bias with pt selection, demographic characteristics and efficacy results for the pt population used in the MRD analysis were compared with the respective intent-to-treat populations for each study. Results PB MRD levels at the end of treatment (CLL8 and CLL10, 75-195 days after last dose; CLL11, 56-190 days after last dose) were assessed in 393, 336, and 474 pts from CLL8, CLL10 and CLL11, respectively. PFS events occurred in 24% to 51% of pts assessed for MRD assigned to the experimental arm and in 34% to 67% assigned to active control treatment. Key efficacy data for the 3 trials are shown in Table 1. To fit a meta-regression model, each study was split into subgroups according to region (CLL8), country (CLL11) or randomly (CLL10). For each subgroup, the PFS hazard ratio (HR) was plotted against the ratio of MRD response rates (MRD negative rate in experimental arm to MRD negative rate in control arm, on a log-scale), and a regression line fitted to reflect the relationship between the two parameters (Figure 1). Circle size represents the weighting of each subgroup to the overall model; least variability in PFS HR have the largest circles. Clustering of circles by study reflects the overall treatment effect (for both MRD and PFS) in the studies. A statistically significant relationship between improved MRD response rates and reduction in the risk of disease progression or death was observed (for each unit increase in log of the ratio of MRD response rates, the log of PFS HR decreases by -0.299; 95% CI, -0.441 to -0.157; p=0.0004). Predictions based on this meta-regression model suggest that risk of progression or death decreases as the ratio of MRD response rates (MRD relative risk: MRD-negative rate in experimental arm/MRD-negative rate in control arm) increases (Table 2); i.e. a larger difference in MRD-response rates leads to lower PFS HR. Conclusion A surrogate endpoint (MRD) should not only provide prognostic value for the specific clinical outcome (PFS), but also evidence that treatment effect on the surrogate endpoint (MRD) reliably predicts treatment effect on the clinical outcome (PFS). Results of the meta-regression model show a significant association between treatment effect on MRD and treatment effect on PFS with regard to chemoimmunotherapy. The findings also suggest that treatment effect on PFS can be predicted based on treatment effect on MRD response. This model supports the use of MRD as a surrogate for PFS in pts with CLL. Disclosures Dimier: Roche: Employment. Delmar:F. Hoffmann-La Roche, Ltd.: Employment, Equity Ownership. Ward:F. Hoffmann-La Roche Ltd: Employment. Morariu-Zamfir:F. Hoffmann-La Roche Ltd: Employment. Fingerle-Rowson:Roche: Employment, Equity Ownership. Fischer:Roche: Other: Travel Grants. Eichhorst:Mundipharma: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy; Roche: Consultancy, Research Funding, Speakers Bureau. Goede:Mundipharma: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; GSK: Honoraria; Bristol-Myers Squibb: Honoraria. van Dongen:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. Royalty income for EuroClonality-BIOMED-2 Consortium.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics. Royalty income for EuroFlow Consortium.; BD Biosciences: Other: Educational Lectures and Educational Workshops (+ related travelling costs). Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. Provided by the Laboratory of Medical Immunology, Erasums MC, Patents & Royalties; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Böttcher:Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Beckton Dickinson: Honoraria. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. ; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Board; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Board, Research Funding.

Background: Measurable Residual Disease (MRD) assessments are gaining increasing acceptance as a prognostic factor for tailoring treatment in hematological malignancies. Acute Myeloid Leukemia (AML) is a heterogeneous disease with high relapse rates and presents a high unmet need for effective treatment options. Measurement of residual disease after therapy reflects a combination of all resistance mechanisms and is currently used for guiding treatment options. Study Design: In this study, we aimed to validate an AML-MRD assay by multiparameter flow cytometry (MFC) methodology. This is a 4-tube, 8-parameter assay designed to incorporate cell differentiation (CD) markers for identification of a diverse group (covering roughly 90% of patients, Cloos et al, 2018) of Leukemia Associated Immunophenotypes (LAIPs) to accurately identify both native phenotypes and phenotype shifts after drug treatment. These CD markers were selected based on extensive investigation of many markers and in line with the consensus recommendations from European Leukemia Network AML working party (Schuurhuis et al, 2018), while specimen testing and interpretation principles were performed in accordance with Cloos et al, 2018. The assay validation focused on evaluation of sensitivity (MRD cut point and LOD), precision and accuracy as key criteria for evaluating assay performance utilizing primary patient specimens and AML cell lines representing different LAIPs. The results were orthogonally verified in a blinded manner by morphologic assessment at Navigate and by the MRD-team at VUMC Amsterdam. Results: Two experimental approaches were adopted to evaluate analytical and functional sensitivity (clinical applicability) of the assay. Results indicated analytical sensitivity (LOD) as low as 0.01% LAIPs of total WBC and functional sensitivity (LOQ) of 0.1% (MRD cut point). Excellent repeatability and reproducibility (less than 20% CV) was observed across instruments, operators and independent measurements (n = 75). The frequencies of AML blasts detected by MFC and morphological examination were highly concordant (Spearman r = 0.95, P value &lt; 0.001, n = 24). LAIPs deduced across nine patient specimens by the Navigate laboratory were independently confirmed by the MRD-team at VUMC Amsterdam. Conclusion: In summary, based on the use of consensus markers recommended by ELN for reliable capture of a broad group of LAIPs in AML patients and verification of key assay performance characteristics, we believe this comprehensive MFC based AML MRD assay is fit-for-purpose for accurately assessing measurable residual disease. Following clinical trial validation, MRD might be used as a surrogate endpoint for approval of emerging agents. Disclosures Marques Ramos: Novartis: Current Employment. Larson:BMS, Bioline, Celgene, Juno, Janssen: Research Funding; TORL Biotherapeutics: Current equity holder in private company. Sarikonda:Novartis: Current Employment.

Abstract Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disorder. Precision therapies for AML have been developed that target specific driver mutations. The efficacies of these therapies are variable, making it critical to determine successful therapies prior to patient relapse. For patients achieving a first complete remission, minimum residual disease (MRD) is an important prognostic factor, as MRD may provide a powerful and timely tool to evaluate therapeutic efficacy. There is a growing demand that new and promising drugs are approved as quickly as possible and accelerated approvals will require biomarker surrogate endpoints, such as MRD, rather than long-term survival endpoints. We have developed a sensitive NGS gene panel (MyMRD®), which identifies pathogenic variants in AML. The MyMRD panel targets single nucleotide variants (SNVs), insertions and deletions (indels) in coding exons of hotspots of 21 genes (ASXL1 BRAF CALR CEBPA CSF3R DNMT3A FLT3 IDH1 IDH2 JAK2 KIT KRAS MPL NPM1 NRAS PTPN11 RUNX1 SF3B1 SRSF2 TP53 ZRSR2), and structural variants of potential genomic breakpoint hotspots within 3 somatic gene fusion partners (CBFB-MYH11 KMT2A RUNX1-RUNX1T1). This 23 gene targeted panel can identify driver mutations that cause relapse in >90% of all AML patients, as well as common drivers in other myeloid neoplasms and myelodysplasic syndromes. MyMRD panel validation included determining the limit of blank (LoB), limit of detection (LoD), and linearity. Validation samples were generated using known variant containing DNA from cell lines and clinical samples diluted into NA12878 ("Genome in a Bottle") DNA. The average background, crossover, and carryover rates were determined to be 0.017%, 0.023%, and 0.016%, respectively. The LoB was determined to be 0.13% from calculating each of expected variants using the 95th percentile of all negative samples. Overall, we established an LoD of 0.5% for >95% of the targeted SNV and indel sites in the assay with lower LoDs for specific mutations of interest, such as 0.17% for FLT3 TKD and 0.34% for NPM1. The LoD for structure variants was determined to be 1.8%. The assay shows strong linearity with R2 = 0.969 - 0.994 of 11 selected targets in the entire range (0.17% - 50%) of variant allele frequencies (VAFs) tested. Linearity and LoD determined from clinical samples containing SNVs and indels were consistent with conclusions obtained from contrived cell line samples. Cell-free DNA (cfDNA) isolated from plasma is becoming a readily available source ofcirculating tumor DNA for testing. A similar molecular profile has been observed from cfDNA to that of bone-marrow tumor cells from multiple myeloma patients. Therefore, we investigated whether the MyMRD assay, developed for genomic DNA analysis, could be applied to cfDNA to assess variants at a level comparable to testing of genomic DNA. To overcome the limitation of cfDNA availability, DNA fragments with size similar to cfDNA (140-170bp), were generated from contrived cell line DNA to assess the LoD and linearity of assay. The cfDNA assay also showed strong linearity (R2=0.975 - 0.998) in the range of VAFs (0.1- 20%) tested. With cfDNA input of 25ng, the LoD was established to be 0.5% for targeted SNV sites, and 1% for targeted indel sites. The MyMRD cfDNA assay detected variants at the same LoD for SNVs and a slightly higher LoD for indels. Additional clinical samples were tested with the MyMRD assay. Variants detected were verified with capillary electrophoresis and/or amplicon-based NGS assays. The results showed 100% concordance with the MyMRD panel assay. The MyMRD gene panel is a sensitive and reliable assay that provides monitoring of MRD using genomic DNA and cfDNA. The assay is shown to detect clinically important driver variants and to have excellent linearity and LoD for targeted sites. The assay provides an accurate method for detecting mutations in multiple targets in patients and can be used to stratify patients for therapy and clinical trials, and may provide the sensitivity necessary to serve as a surrogate endpoint assay for targeted therapies in AML. Disclosures Xie: Invivoscribe Inc.: Employment. Chamberlain:Invivoscribe Inc.: Employment. Carson:Invivoscribe Inc.: Employment. Atkinson:Invivoscribe Inc.: Employment. McClain:Invivoscribe Inc.: Employment. Sprague:Invivoscribe, Inc.: Employment. Kiya:Invivoscribe Inc.: Employment. Xia:Invivoscribe Inc.: Employment. Huang:Invivoscribe Inc.: Employment. Patay:Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Blankfard:Invivoscribe Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership.

Abstract Background: Expression of p190 BCR-ABL mRNA is generally considered to be confined to patients with acute lymphoid or more rarely myeloid leukemias, whereas p210 BCR-ABL mRNA is the hallmark of CML. In reality it is not uncommon the presence of p190 m-RNA in p210 CML in chronic phase, due to alternative or missplicing1. Its presence seems to have no impact on prognosis in the pre-TKI era, although it may be expression of genomic instability. Aim: Primary object of this study was to investigate if the co-expression might influence the rate of early outcome surrogate endpoints such as such as early complete cytogenetic response in patients treated with imatinib. The secondary endpoint was the evaluation of failure free survival (FFS) measured from the start of imatinib to the date of any of the following events: progression to accelerated or blastic phase, death for any cause and switch to nilotinib/dasatinib for resistance or intolerance. Methods: Were evaluated patients with CML in chronic phase treated with imatinib at our institution. We excluded cases with less than one year of treatment and/or treated with other TKIs or conventional chemotherapy.The fusion transcripts BCR-ABL were evaluated at diagnosis in peripheral blood by NESTED-PCR2 and the cytogenetic response were evaluated in bone marrow cells with G-banding technique and fluorescent in situ hybridization (FISH)3. The patients were divided into two groups, "double transcripts" (DT) and "single transcript" (ST). All patients received imatinib 400 mg/die. Results: A total of 56 patients were analyzed. The median age of patients was 58 years (range 28-80 years) and 35 (62%) were male. Twenty patients (36%) were DT and thirty-six (64 %) ST. The distribution according to Sokal score was: 7 (35%), 8 (40%) and 5 (25%) patients for low, intermediate and high risk in the DT, whereas 18 (50%), 15 (42%) and 3 (8%) low, intermediate and high risk in ST, respectively. The complete cytogenetic response at 3 months was achieved in 2 patients with DT and 7 patients with ST (10% vs 19% p 0.35), at 6-month complete cytogenetic response was achieved in 8 patients with DT and 27 patients with ST (40% vs 75% p 0.01) (Table 1). After median follow-up of 1966 days, the FFS was significantly different between the DT and ST (55% vs 5 % p< 0.001) (figure 1), 11 patients in the DT group and 5 patients in ST group had shift to TKI 2¡ generation (55% vs 14% p 0.001) and 4 patients in DT group not achieved complete cytogenetic response. Summary/Conclusion: In our study the co-expression of p190 and p210 BCR-ABL transcripts influences the early cytogenetic response to imatinib and suggesting the need for a larger validation study Reference 1). van Rhee F, Hochhaus A, Lin F, Melo JV, Goldman JM, Cross NC. : "p190 BCR-ABL mRNA is expressed at low levels in p210-positive chronic myeloid and acute lymphoblastic leukemias."Blood. 1996 15;87:5213-7. 2). Hermans A, Selleri L, Gow J, Wiedemann L, Grosveld G: "Molecular analysis of the Philadelphia translocation in chronic myelocytic and acute lymphocytic leukemia." Leukemia 2:628,1988. 3) Landstrom AP, Tefferi A.: "Fluorescent in situ hybridization in the diagnosis, prognosis, and treatment monitoring of chronic myeloid leukaemia".Leuk Lymphoma.2006;47:397-402. Figure 1: Failure Free Survival: Figure 1:. Failure Free Survival: Table 1 : Complete Cytogenetic response: Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. CLL has a varied clinical course. Risk assessment and patient counseling relies on clinical parameters and validated biomarkers. Genetic biomarkers, as exemplified in the CLL FISH panel, allow for CLL patient risk stratification. Nonetheless, a subset of patients with apparently favorable lesions (del13q14 or normal FISH results) progresses rapidly and a fraction of patients with unfavorable findings (del17p or del11q) progress at less than predicted rate. Method: We studied 117 previously untreated CLL patients enrolled into a prospective study at the University of Michigan. We obtained unbiased, high-density, genome-wide measurements of sub-chromosomal copy number changes in highly purified DNA from sorted CD19+ cells and buccal cells using the Affymetrix 50K SNP-array platform. A genomic complexity score was derived and correlated with the validated surrogate endpoint time to first therapy (TTFT), a measure of disease aggressiveness. Genomic complexity was measured using two complementary methods: i) visual inspection of dChipSNP-generated heatmap displays for copy-number estimates for all patients and for all chromosomes. Two of the authors independently reviewed dChipSNP-based copy number displays and visually scored lesions that were i) at least 8 consecutive SNP positions in length and ii) either all blue (indicating less than 2N copy estimates=losses) or all red (indicating greater than 2N copy estimates=gains). Sensitivities and specificities for the visual lesion calling method were calculated against the clinical FISH data for del17p, del13q14, trisomy 12 and del11q as the gold standard. Sensitivities and specificities for (SNM/PDO) were [(94%/93%) and (93%/91%)] for del13q14, [(88%/88%) and (98%/98%)] for trisomy 12, [(100%/100%) and (99%/99%)] for del17p, and [(100%/100%) and (99%/100%)] for del11q, and ii) algorithmic detection of gains and losses using search methods optimized for detection using FISH-based del13q14 lesions as the gold standard. We devised a sliding window algorithm that scores a lesion as “copy loss” when a SNP window of 8 consecutive SNPs has at least 6 SNPs with a copy number estimate of 1.32 or less (the 8/6/1.32 rule). For validation, an approximately 5 Mb-long physical window was selected, overlapping with all described del(13q14) lesions. Defining an algorithmic del(13q14) call as any patient for whom the 8/6/1.32 rule identified at least one lesion within the 5Mb window, we achieved 81% sensitivity and 96% specificity for detecting or excluding 13q14 lesions as measured against the clinical FISH panel. Results: Within the group of 117 patients, 22(19%), 15(13%), 12(10%) and 10(9%) had visual complexity scores of equal to or greater than 2.5, 3, 3.5 or 4, respectively: a previously unanticipated degree of genomic complexity in CLL.In univariate analysis, CLL patients with four or more sub-chromosomal genomic lesions had a median TTFT of 15.8 months, whereas the median TTFT for all other patients was not reached in our study (two-sided p<10−4). In bivariate analysis, presence of genomic complexity defined a high-risk group of patients within the IgVH unmutated subgroup. Patients with unmutated IgVH/high complexity score had a TTFT of 16.5 months versus 95.6 months for unmutated IgVH/low complexity patients (p=0.02). Finally, high genomic complexity resulted in strong trends towards shorter TTFT for patients within other established risk groups.

The selection of the primary “endpoint” is a very important step in the design of clinical trials. Typically, the goal of a clinical trial is to assess the effect of treatment on this endpoint. It often happens, that the most sensitive and relevant clinical endpoint, which will be called the “true” endpoint, might be difficult to assess and to overcome this problem, a solution is to replace the true endpoint with another one, which is measured earlier, more conveniently or more frequently. Such “replacement” endpoint is termed “surrogate” and has the purpose of evaluating the effect of a specific treatment for a specific disease. Once a candidate surrogate is identified according to some specific properties, several formal methods are available for the validation, depending on the number of the trials performed. The first formal statistical approach dates back to 1989, when Prentice proposed a definition of surrogate endpoint and four criteria to validate it. The most important criterion among these is called “The Prentice’s Criterion”, which implies that “the full effect of treatment upon the true endpoint is captured by the surrogate”. More recently, a meta-analytic approach to the validation of a surrogate endpoint was proposed for a multi-trial context (Burzykowki, 2005). It consists in estimating associations at two different levels: the association between the surrogate and the clinical endpoint, called the “individual-level association”, and the association between the effect of treatment on the surrogate and on the clinical endpoint, called “trial-level association”. A good surrogate is one that has biological/clinical plausibility and is shown, statistically, to have strong individual-level and trial-level associations with the true endpoint. Many extensions of this meta-analytic approach exist to evaluate surrogacy when the candidate surrogate and the true clinical endpoint are not continuous or have different nature, e.g. both failure time endpoints or binary/ordinal surrogate and failure time endpoints. The motivating clinical question was the evaluation of Minimal Residual Disease (MRD) as a candidate surrogate endpoint in childhood acute lymphoblastic leukaemia (ALL). MRD, that quantifies the small numbers of leukemic cells circulating in the patient, has not yet been formally validated as a surrogate endpoint, whilst it is a well-established prognostic biomarker in ALL. The challenge has now evolved to the qualification of early MRD as an efficacy-response biomarker in the assessment of new drugs for the treatment of ALL. The main goal of the present work was to assess by the Prentice criteria and by the meta-analytic approach whether MRD, evaluated at the end of the induction treatment, can be considered a surrogate for Event Free Survival (EFS) in childhood B-lineage ALL patients treated with Dexamethasone or Prednisone in large collaborative randomized trials conducted in Europe and USA. From our analyses, MRD resulted a poor surrogate for EFS at the unit level, i.e. it does not permit reliable prediction of treatment effects on EFS but there is a considerable prognostic association at the individual level, after adjusting for treatment, i.e. the (global) odds of surviving event free is higher with negative/lower MRD. A secondary aim was to evaluate if EFS is a surrogate for Overall Survival (OS) and we found that EFS cannot be accepted as a surrogate for OS, although it is a good predictor of OS. Finally, as methods on the validation of a continuous surrogate for a failure time endpoint are missing, a proposal was made here. In line with the meta-analytic framework, a copula based approach was implemented and translated in a SAS macro.