In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

BACKGROUND: The WHO and iwCLL diagnostic criteria for CLL rely on morphology and immunophenotype based on the co-expression of CD19/CD5/CD23 on B-cells with weak CD20 and monoclonal sIg expression. These diagnostic criteria are likely to persist in the near future because there is no specific diagnostic molecular abnormality for CLL. The current criteria have some limitations affecting reproducibility, particularly flexibility in marker expression with many centres using a scoring system that permits absence of CD5 or CD23. Potentially informative new markers have been identified but there is no consensus yet on which should be routinely assessed. AIM: To identify reproducible criteria and to achieve a consensus on markers recommended for the diagnosis of CLL METHODS: ERIC/ESCCA members were invited to classify 35 flow-cytometry markers as being required or recommended for the diagnosis of CLL. Consensus was considered to be achieved if >75% of participants agreed on the marker classification. A diagnostic panel was identified by the steering committee and characteristics of component markers that could be reproducibly validated within an individual laboratory were identified. The proposed panel was assessed in 13 different centres. RESULTS: Responses were received from 154 members (100 laboratory staff, 14 clinicians and 36 from both laboratory and clinic) with a diagnostic workload >20 cases per week in 23/154 (15%), 5-20 in 82/154 (53%) and <5 cases per week in 49/154 (32%). The consensus minimum diagnostic panel should include : CD19, CD5, CD20, CD23, Kappa and Lambda. Participants recommended the following markers: CD38, CD45, CD79b, CD10, CD22, CD43, CD200, and FMC7. A minimum and recommended panel with reproducible criteria for component reagents were determined and the criteria were applied to 10,876 cases diagnosed with a B-LPD, of which 8120 were CD5+ B-LPD. Out of 5947 sent as a primary referrals for diagnosis, 4493 (75.6%) met the proposed diagnostic criteria for CLL, 821 (13.8%) did not and had a clear alternative diagnosis (e.g. MCL) and 633 (10.6%) would not be readily classified by flow cytometry if the proposed criteria were applied. Out of 2173 cases previously diagnosed as CLL at another centre, 2028 (93.3%) met the proposed diagnostic criteria, 19 (0.9%) had a clear alternative diagnosis while 126 (5.8%) did not meet the flow-cytometry criteria. CONCLUSIONS: We present flow-cytometry criteria for the diagnosis of CLL largely consistent with current practice. In addition, reproducible definitions of the required expression pattern and performance characteristics of reagents are provided. Prospective evaluation of the proposed criteria as well as a parallel project to facilitate definitive diagnosis of CD5+ B-LPD cases that do not meet the proposed criteria are underway. Figure 1. required and recommended markers for use in the diagnosis of CLL with reagent specification based on expression patterns in normal peripheral blood. Figure 1. required and recommended markers for use in the diagnosis of CLL with reagent specification based on expression patterns in normal peripheral blood. ‡ Weak expression = median fluorescence intensity at least 20% lower than median for normal peripheral blood B-cells, reference range determined within each laboratory, based on ICSH/ISLH/CLIA guidelines for reproducibility *consensus, not specifically validated Disclosures Rawstron: Abbvie: Honoraria; Pharmacyclics: Research Funding; Celgene: Honoraria; Roche: Honoraria; BD Biosciences: Patents & Royalties; Gilead: Honoraria, Research Funding. Cuneo:Roche: Speakers Bureau; Gilead: Speakers Bureau; Jannsen: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Mulligan:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding. Hillmen:Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Hallek:Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:AbbVie: Consultancy; Janssen: Consultancy; Roche: Consultancy, Research Funding; Adaptive: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; GSK: Research Funding; Acerta Pharma BV: Research Funding; Pharmacyclics: Consultancy.

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as “required” or “recommended” for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate “required” markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5–20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for “required” diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. “Recommended” markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as “required” for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus “recommended” panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society

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