Olga Stehlíková

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.

Rituximab maintenance versus observation alone in patients with chronic lymphocytic leukaemia who respond to first-line or second-line rituximab-containing chemoimmunotherapy: final results of the AGMT CLL-8a Mabtenance randomised trial

BACKGROUND In many patients with chronic lymphocytic leukaemia requiring treatment, induction therapy with rituximab plus chemotherapy improves outcomes compared with chemotherapy alone. In this study we aimed to investigate the potential of rituximab maintenance therapy to prolong disease control in patients who respond to rituximab-containing induction regimens. METHODS In this randomised, international, multicentre, open-label, phase 3 clinical trial, we enrolled patients who had achieved a complete response (CR), CR with incomplete bone marrow recovery (CRi), or partial response (PR) to first-line or second-line rituximab-containing chemoimmunotherapy and randomly assigned them in a 1:1 ratio (central block randomisation in the electronic case report form system) to either intravenous rituximab 375 mg/m(2) every 3 months, or observation alone, for 2 years. Stratification was by country, line of treatment, type of chemotherapy added to the rituximab backbone, and degree of remission following induction. The primary endpoint was progression-free survival. Efficacy analysis was done in the intention-to-treat population. This is the final, event-triggered analysis. Final analysis was triggered by the occurrence of 92 events. This trial is registered with ClinicalTrials.gov, number NCT01118234. FINDINGS Between April 1, 2010, and Dec 23, 2013, 134 patients were randomised to rituximab and 129 to observation alone. Median observation times were 33·4 months (IQR 25·7-42·8) for the rituximab group and 34·0 months (25·4-41·9) for the observation group. Progression-free survival was significantly longer in the rituximab maintenance group (47·0 months, IQR 28·5-incalculable) than with observation alone (35·5 months, 95% CI 25·7-46·3; hazard ratio [HR] 0·50, 95% CI 0·33-0·75, p=0·00077). The incidence of grade 3-4 haematological toxicities other than neutropenia was similar in the two treatment groups. Grade 3-4 neutropenia occurred in 28 (21%) patients in the rituximab group and 14 (11%) patients in the observation group. Apart from neutropenia, the most common grade 3-4 adverse events were upper (five vs one [1%] patient in the observation group) and lower (three [2%] vs one [1%]) respiratory tract infection, pneumonia (nine [7%] vs two [2%]), thrombopenia (four [3%] vs four [3%]), neoplasms (five [4%] vs four [3%]), and eye disorders (four [3%] vs two [2%]). The overall incidence of infections of all grades was higher among rituximab recipients (88 [66%] vs 65 [50%]). INTERPRETATION Rituximab maintenance therapy prolongs progression-free survival in patients achieving at least a PR to induction with rituximab plus chemotherapy, and the treatment is well tolerated overall. Although it is associated with an increase in infections, there is no excess in infection mortality, suggesting that remission maintenance with rituximab is an effective and safe option in the management of chronic lymphocytic leukaemia in early treatment phases. FUNDING Arbeitsgemeinschaft Medikamentöse Tumortherapie gemeinnützige GmbH (AGMT), Roche.

Intra‐ and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high‐sensitivity paroxysmal nocturnal hemoglobinuria testing

Sutherland et al. recently published the Practical Guidelines for high‐sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing.

Ofatumumab added to dexamethasone in patients with relapsed or refractory chronic lymphocytic leukemia: Results from a phase II study

The treatment of relapsed/refractory chronic lymphocytic leukemia (CLL) remains a challenging clinical issue. An important treatment option is the use of high‐dose corticosteroids. The purpose of this clinical trial was to determine the efficacy and toxicity of an ofatumumab–dexamethasone (O‐Dex) combination in relapsed or refractory CLL. The trial was an open‐label, multicenter, nonrandomized, Phase II study. The O‐Dex regimen consisted of intravenous ofatumumab (Cycle 1: 300 mg on day 1, 2,000 mg on days 8, 15, and 22; Cycles 2–6: 1,000 mg on days 1, 8, 15, and 22) and oral dexamethasone (40 mg on days 1–4 and 15–18; Cycles 1–6). The O‐Dex regimen was given until best response, or a maximum of six cycles. Thirty‐three patients (pts) were recruited. Twenty‐four (73%) pts completed at least three cycles of therapy. The remaining nine pts were prematurely discontinued owing to Grade 3/4 infections (seven pts), disease progression (one pt), or uncontrollable diabetes mellitus (one pt). Overall response rates/complete remissions (ORR/CR) were achieved in 22/5 pts (67/15%). The median progression‐free survival (PFS) was 10 months. In pts with p53 defects (n = 8), ORR/CR were achieved in 5/2 pts (63/25%) with a median PFS of 10.5 months. The median overall survival (OS) was 34 months. The Grades 3–5 infectious toxicity in 33% of pts represented the most frequent side effect during the treatment period. In conclusion, the O‐Dex regimen shows a relatively high ORR and CR with promising findings for PFS and OS. The study was registered at www.clinicaltrials.gov (NCT01310101). Am. J. Hematol. 90:417–421, 2015.

Autocrine signaling by Wnt-5a deregulates chemotaxis of leukemic cells and predicts clinical outcome in chronic lymphocytic leukemia.

Purpose: ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. Experimental Design: B lymphocytes from the peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B, and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction, and downstream signaling were analyzed by immunoprecipitation and Western blotting. Migration capacity of primary CLL cells was analyzed by the Transwell migration assay. Results: By analyzing the expression in 137 previously untreated CLL patients, we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time to first treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. Conclusions: These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation. Clin Cancer Res; 22(2); 459–69. ©2015 AACR.

Lenalidomide proved effective in multisystem Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a rare idiopathic disease with diverse clinical manifestations ranging from a single osteolytic lesion to generalized disease. Various treatment regimens have been proposed for the multisystem type, however, with inconsistent outcomes. Herein we are the first to report on a therapy effect of a lenalidomide-based regimen in a patient with repeatedly relapsed aggressive form of multisystem LCH.

Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time‐consuming and require experienced laboratory staff.

Lenalidomide: a new treatment option for Castleman disease

A male born in 1961, aged 46, was referred to our department for evaluation of splenomegaly and generalized lymphadenopathy aff ecting the cervical, axillary, mediastinal, retroperitoneal and inguinal regions. Laboratory data revealed an increased erythrocyte sedimentation rate (16 mm/h and 30 mm/2 h) and C-reactive protein level (35.4 mg/L). Total protein and full blood counts as well as renal and hepatic profi les were within normal ranges, and microbiological screening revealed no infectious etiology. Other fi ndings, including radiological examinations and bone marrow biopsy, showed no further pathologies. Th e patient ’ s other medical history was signifi cant for arterial hypertension, chronic infl ammatory demyelinating polyneuropathy and mild right hemi-paresis with expressive language disorder. Based on lymph node biopsies from retroperitoneal and both inguinal regions, the patient was diagnosed with the plasma-cell variant of CD. Th e presence of a large pelvic mass compressing adjacent structures indicated the patient for therapy initiation. During fi rst-line treatment (R-CHOP: rituximab 375 mg/m 2 , cyclophosphamide 750 mg/m 2 , doxorubicin 50 mg/m 2 and vincristine 2 mg intravenously on day 1; prednisone 100 mg perorally on days 1 – 5 in a 21-day cycle, three cycles in total, 12/2008 – 2/2009), clinical progression of the disease was evident (gastrointestinal symptoms, swollen hands and feet with signs of vasculitis), and there was no radiological response on a restaging computed tomography (CT) examination after 3 months.

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.

ROR1-based immunomagnetic protocol allows efficient separation of CLL and healthy B cells.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia among adults in the Western world. This lymphoproliferative disorder is defined by the presence of at least 5 9 106 clonal lymphocytes/ml peripheral blood (Hallek,2015). The biggest clinical challenge is the fact that CLL eradication by any available therapy, apart from allogeneic stem cell transplantation, fails to cure the disease, inducing only temporary disease remission, i.e., when the disease does not manifest clinically but CLL cells still persist in the body. During this phase a population of CLL cells often slowly re-expands to trigger CLL relapse. The relapse is often based on the poorly understood but clinically important process of therapy-driven selection of more aggressive CLL subclones (Malcikova et al, 2015).