Peter Gambell

Persistence and Efficacy of Second Generation CAR T Cell Against the LeY Antigen in Acute Myeloid Leukemia

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.

Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen.

We have evaluated the carbohydrate antigen LewisY (LeY) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate LeY expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the LeY antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-γ secretion and cell lysis of LeY-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting LeY-positive malignant cells with T cells expressing a chimeric receptor recognizing LeY was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with LeY-positive myeloma or AML.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

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.

Extracorporeal photopheresis for the treatment of Sézary syndrome using a novel treatment protocol

Various studies have reported that extracorporeal photopheresis is effective in producing meaningful responses in patients with Sézary syndrome. A single-center, 5-year retrospective analysis was performed on our patients with Sézary syndrome who received extracorporeal photopheresis using a novel protocol. Thirteen patients were treated with extracorporeal photopheresis consistently for a minimum of 2 months. All patients received a modified protocol of one treatment per week for 6 sessions, one session every 2 weeks for 6 sessions, and then one session per month. The overall response rate was 62%: two patients achieved a complete response and 6 patients achieved a partial response. The median time to response was 10 months. The 2- and 4-year predicted overall survivals were 82%. This study was limited by its retrospective nature and small sample size. Response and survival compare favorably with those of previous studies. Our modified treatment protocol appears to produce outcomes similar to the two-day protocol.

Validation of cell-based fluorescence assays: Practice guidelines from the ICSH and ICCS – part IV – postanalytic considerations

Flow cytometry and other technologies of cell‐based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so‐called home brew assays performed in clinical laboratories. The absence of well‐defined guidelines for validation of cell‐based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell‐based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS ‐ Part IV ‐ Postanalytic considerations.

Peripheral Blood CD34+ Cell Enumeration as a Predictor of Apheresis Yield: An Analysis of More Than 1,000 Collections

The role of the peripheral blood (PB) CD34(+) cell count in predicting the CD34(+) cell yield in hematopoietic progenitor cell apheresis collections is well established. However, sometimes unexpectedly poor CD34(+) cell yields are obtained. To determine the effect, if any, of a range of factors on the ability of the PB CD34(+) count to predict collection CD34(+) cell count, we performed a retrospective analysis on consecutive hematopoietic progenitor cell apheresis collections between 2004 and 2008. Factors investigated included mobilization regimen, PB white blood cell count, body weight, and disease. After exclusion of collections involving apheresis complications, a total of 1,225 PB CD34(+) cell results with corresponding collection CD34(+) cell results from 458 patients were analyzed. Although differences in the median PB CD34(+) cell counts and collection CD34(+) cell counts were seen between mobilized collections with chemotherapy plus granulocyte colony-stimulating factor and those with granulocyte colony-stimulating factor alone, the predictive capability of the PB CD34(+) cell count for the collection CD34(+) cell yield remained similar. Although poorer collection efficiencies were observed in the myelodysplastic syndrome/myeloproliferative disorder diagnostic subgroup, our findings confirm that PB CD34(+) cell analysis remains a powerful and irreplaceable tool for predicting hematopoietic progenitor cell apheresis CD34(+) cell yield.

Pegfilgrastim compared with filgrastim for cytokine-alone mobilization of autologous haematopoietic stem and progenitor cells

Haematopoietic stem and progenitor cells (HSPC) mobilization, using cytokine-alone, is a well-tolerated regimen with predictable mobilization kinetics. Single-dose pegfilgrastim mobilizes HSPC efficiently; however, there is surprisingly little comparative data on its use without chemotherapy for HSPC mobilization. Pegfilgrastim-alone and filgrastim-alone mobilization regimens were compared in 52 patients with haematological malignancy. Pegfilgrastim 12 mg (n=20) or 6 mg (n=2) was administered Day 1 (D1) in 22 patients (lymphoma n=17; myeloma n=5). Thirty historical controls (lymphoma n=18; myeloma n=12) received filgrastim 10 mcg/kg daily from D1. Peripheral blood (PB) CD34+ counts reached threshold (⩾5 × 106/L) and apheresis commenced on D4(4–5) and D4(4–6). Median PB CD34+ cell count on D1 of apheresis was similar (26.0 × 106/L (2.5–125.0 × 106/L) and 16.2 × 106/L (2.6–50.7 × 106/L); P=0.06), for pegfilgrastim and filgrastim groups, respectively. Target yield (⩾2 × 106 per kg CD34+ cells) was collected in 20/22 (91%) pegfilgrastim patients and 24/30 (80%) in the filgrastim group (P=0.44), in a similar median number of aphereses (3(1–4) versus 3(2–6), respectively; P=0.85). A higher proportion of pegfilgrastim patients tended to yield ⩾4 × 106 per kg CD34+ cells; 16/22 (73%) versus 14/30 (47%) filgrastim patients (P=0.09). One pegfilgrastim patient developed hyperleukocytosis that resolved without incident. Pegfilgrastim-alone is a simple, well-tolerated, and attractive option for outpatient-based HSPC mobilization with similar mobilization kinetics and efficacy to regular filgrastim.

Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

BackgroundHigh purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL).ResultsWe adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 – 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 – 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis.ConclusionThis study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project

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.

A Risk-Adapted Protocol for Delayed Administration of Filgrastim After High-Dose Chemotherapy and Autologous Stem Cell Transplantation

INTRODUCTION The routine use of recombinant human granulocyte-colony stimulating factor (rhG-CSF) after high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) is associated with increased costs. We prospectively explored a strategy that used prophylactic delayed filgrastim only in patients with risk factors. PATIENTS AND METHODS This sequential cohort analysis compared the outcomes of consecutive patients, treated on the risk-adapted protocol (RAP) (risk factors: prior febrile neutropenia; age >60 years; and CD34+ cell infused dose of <2 × 10(6/)/kg), who received filgrastim from day +6 after auto-SCT with a historical cohort (historical day-1 cohort [HD1]), who received filgrastim from day +1. RESULTS Eighty-two patients were treated in the RAP cohort and compared with 115 patients in the HD1 cohort. There were no differences in median age (55 years) or median CD34+ cell dose (5.21 × 10(6)/kg [range, 2-62.2 × 10(6)/kg] vs. 5.24 × 10(6)/kg [range, 2.4-29.8 × 10(6)/kg]). Filgrastim was used for 6 fewer days in the RAP cohort (median 5 days [range, 0-11 days] vs. 11 days [range, 9-47 days]). There was a small absolute but significant difference in median time to neutrophil recovery in the HD1 cohort for the whole group, 10 days (range, 8-46 days) vs. 11 days (range, 9-22 days) (P = .03) and in patients with myeloma; 10 days (range, 9-14 days) vs. 11 days (range, 9-18 days) (P < .0001) as compared to the RAP cohort. There was no difference in median inpatient duration, 13 days (range, 10-26 days) vs. 12 days (range, 1-38 days) (P = .22) and 3-year survival (79% vs. 83% [P = .43]) between HD1 and RAP cohorts respectively. CONCLUSIONS The use of a RAP to identify patients likely to benefit from prophylactic filgrastim is safe and results in cost savings. Patients with myeloma benefit from earlier introduction of filgrastim in terms of neutrophil recovery; this disease-specific observation is an important consideration for future studies.