Abstract: Introduction:With the advent of new potent therapies for chronic lymphocytic leukemia (CLL) minimal residual disease (MRD) detection becomes increasingly important to assess remission depth. While molecular MRD detection for CLL remains laborious and time consuming flow cytometry is a fast, economic and sensitive method in detecting low frequencies of CLL cells. The usefulness of the antigens CD81, CD5, CD20, CD43 and CD79b has been previously described for this purpose. ROR-1 has recently been identified as a signature gene in CLL and mantle cell lymphoma. The potential utility of ROR-1 in flow cytometric minimal residual cell analysis has not been evaluated yet. Methods: 10 normal samples and 77 remnants of randomly selected samples from diagnosed patients undergoing CLL therapy were analyzed by flow cytometry. A customized dry formulation of an antibody panel was used, comprising antibodies directed against CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 (DuraClone RE CLB). Linearity, repeatability and inter-operator variability of data analysis of the method were examined. B cell populations comprising at least 50 positive events (46 normal B cell populations, 25 CLL populations, paired and unpaired) were analyzed for their expression profile as assessed by respective mean fluorescence intensities of the antibody labels within classified populations. The expression profiles were subject to supervised discrimination analysis (DA). Results: Between124,000 and 2,122,000 (683,000 ± 450,000) CD45+ events were acquired from the 87 samples. The background of cells with a CLL-like phenotype in the normal samples was determined as 〈 0.001% of CD45+ events. Linearity was confirmed in the range from 1% to 0.0025%. The Repeatability analysis and the inter-operator variability showed concordance with typical Poisson distribution characteristics. The 46 populations with a typical normal B cell phenotype ranged from 0.014% to 9.592% with an average of 2.45% ± 2.75 of CD45+ events. The 25 populations with a classical or non-classical CLL phenotype ranged from 0.007% to 5.459% with an average of 1.41% ± 1.65 of CD45+ events. Posterior discrimination analysis revealed 100% correct discrimination for CLL populations and 96% correct discrimination for normal populations when relying on ROR-1 expression alone in CD19+CD45+ B cells. This result was only surpassed by the complete antibody combination (100% / 100%) but not by any other of the markers, neither in single use nor in combination Conclusion: The 8-color dry flow cytometry panel comprising CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 demonstrated sensitive, linear and specific detection of residual CLL cells in a relevant low range of frequency. ROR-1 revealed to be a highly discriminative marker in the analysis of residual CLL cells by flow cytometry. Utilizing this flow cytometry approach, MRD detection showing sensitivity comparable to molecular techniques can be achieved in CLL. Disclosures Hallek: AbbVIe: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria; Glaxo-SmithKline: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Research Funding, Speakers Bureau. Kreuzer:Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Introduction: Complex karyotype (CKT), defined as the presence of ≥3 chromosomal aberrations, is emerging as an adverse prognostic factor in chronic lymphocytic leukemia (CLL) (Thompson et. al., Cancer 2015; Herling et. al., Blood, 2016). Idelalisib (IDELA) is a selective PI3Kd inhibitor approved for use in patients (pts) with relapsed CLL. A phase 3 randomized study of rituximab (R) plus IDELA or placebo (PBO) in pts with relapsed CLL and comorbidities (NCT1539512) demonstrated superior progression-free survival (PFS) and overall survival (OS) in the IDELA arm (Furman et. al. NEJM 2014). This report examines the effect of CKT on long-term outcome in that study. Methods: Key eligibility criteria included CLL requiring therapy after progression within 24 months of last therapy, and being unfit to receive cytotoxic therapy. Pts were randomized to receive IDELA at 150 BID (n = 110) or PBO (n = 110) in combination with R at 375 mg/m2 (first dose) and then 500 mg/m2 q2 weeks x 4, followed by q4 weeks x 3 (8 doses total). After progression, prior PBO+R pts could enroll into an extension study to receive IDELA at 150 mg BID. The primary endpoint of PFS was assessed by an IRC using standard criteria (Hallek et. al. Blood 2008; Blood 2012; Cheson et. al. J Clin Oncol 2012). After the first prespecified interim analysis, with median IDELA exposure time of 3.8 months, the study was stopped due to overwhelming difference in efficacy and pts on PBO arm were offered open-label IDELA on the extension study. Baseline peripheral blood samples for karyotype analysis, TP53 mutation (mut), and FISH for del(17p) were analyzed by central labs. Metaphase spreads were produced after IL-2/CpG-stimulated cell cultivation and karyotypes were analyzed per ISCN 2013 nomenclature. Results: Based on geography, two labs received a total of 283 screening blood samples and prepared metaphase spreads, with one lab completing the karyotypic analyses. The overall karyotypic success rate was 65%, but was 98% for samples initially processed in one of the labs. Karyotypes were successfully performed in 127 of 220 randomized pts. The median follow-up for karyotyped IDELA and PBO pts was 21.4 months (range 0.3-39.4) and 12.0 months (range 0.2-35.8), respectively. CKT and non-CKT were found in 26 and 39 IDELA pts and 24 and 38 PBO pts, respectively. Overall, 16 IDELA pts and 15 PBO pts had del(17p)/TP53mut along with CKT. Within the IDELA group: the overall response rate (ORR) was 80.8% (95% CI 60.6-93.4) with CKT and 89.7% (95% CI 75.8-97.1) with non-CKT; the median PFS was 20.9 months in CKT vs 19.4 in non-CKT, HR = 1.18 (p = 0.63); the med OS was NR vs NR in CKT vs non-CKT, HR = 1.78 (95%CI 0.69-4.64; p = 0.23). The presence or absence of del(17p)/TP53mut and CKT status did not significantly affect PFS or OS in pts randomized to IDELA (Figure 1). Prior to crossover, the median PFS for IDELA vs PBO pts with CKT was NR vs 3.7 months, HR = 0.16 (p 〈 0.001) and for those with non-CKT was 19.4 vs 6.5 months, HR = 0.10 (p 〈 0.001). Median OS for IDELA vs PBO pts with CKT was NR vs 9.2 months, HR = 0.31 (p = 0.005) and for those with non-CKT was NR vs NR, HR = 0.47 (p = 0.065). Conclusion:In this first systematic evaluation of CKT in relapsed CLL, CKT was found in 44% of 185 successfully karyotyped screened patients, including 37% of those randomized on study. This post hoc exploratory analysis demonstrates the lack of any adverse prognostic effect of CKT on IDELA-treated patients with respect to PFS or OS, and there was no significant difference among subsets of these pts with and without del(17p)/TP53mut. As previously reported in the entire study intention-to-treat population, a survival and PFS advantage was imparted by the addition of IDELA to R in pts with CKT. Detailed analyses including other clinical and prognostic parameters will be presented. Figure Progression-Free Survival and Overall Survival in Patients Randomized to Idelalisib Figure. Progression-Free Survival and Overall Survival in Patients Randomized to Idelalisib Figure Figure. Disclosures Kreuzer: Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau. Furman:Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau; Genentech: Consultancy. Stilgenbauer:Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding. Dubowy:Gilead Sciences: Employment, Equity Ownership. Kim:Gilead Sciences: Employment, Equity Ownership. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Pettitt:Celgene: Speakers Bureau; Infinity: Research Funding; Gilead: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau. Hallek:Gilead Sciences: Research Funding, Speakers Bureau.