Loic Ysebaert

L-selectin controls trafficking of chronic lymphocytic leukemia cells in lymph node high endothelial venules in vivo

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Lymph nodes (LNs) are sites of malignant proliferation and LN enlargement is associated with poor prognosis in the clinics. The LN microenvironment is believed to favor disease progression by promoting CLL cell growth and drug resistance. A better understanding of the mechanisms regulating trafficking of CLL cells to LNs is thus urgently needed. Here, we studied the first step of CLL cell migration to LNs, their interaction with high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation in lymphoid organs. We observed that the density of HEV blood vessels was increased in CLL LNs and that CD20(+) CLL cells accumulated within HEV pockets, suggesting intense trafficking. We used intravital imaging to visualize the behavior of human CLL cells within the mouse LN microcirculation, and discovered that CLL cells bind to HEVs in vivo via a multistep adhesion cascade, which involves rolling, sticking, and crawling of the leukemic cells on the endothelium. Functional analyses revealed that the lymphocyte homing receptor L-selectin (CD62L) is the key factor controlling the binding of CLL cells to HEV walls in vivo. Interestingly, L-selectin expression was decreased on CLL cells from patients treated with idelalisib, a phosphoinositide-3-kinase δ inhibitor recently approved for CLL therapy. Interference with L-selectin-mediated trafficking in HEVs could represent a novel strategy to block dissemination of CLL cells to LNs and increase the efficacy of conventional therapy.

Clinical activity of a new regimen combining gemcitabine and alemtuzumab in high‐risk relapsed/refractory chronic lymphocytic leukemia patients

Optimal treatment strategies are lacking in relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Gemcitabine has shown activity and acceptable safety profile in B‐cell lymphomas. We present a retrospective case review of gemcitabine and alemtuzumab, every 21 d (for up to six courses) in 27 community‐based patients with high‐risk R/R CLL. Median age was 70 yr (44–83 yr), 55% patients had Binet stage C, deletion 17p (del(17p)) and/or deletion 11q (del(11q)) were found in 65% and 27%, bulky disease in 55.5%, and fludarabine‐refractoriness in 48% of cases, respectively. Overall response rate was 63% (29.6% clinical CR and 33.4% PR). At a median follow‐up of 31 months, median PFS and OS were 15.4 and 24 months. In multivariate analysis, median OS is influenced by prior lines of treatment = 3 and bulky disease. Combination of alemtuzumab and gemcitabine appears to be an active, easy to administrate treatment in routine practice, high‐risk R/R CLL patients.

Cancer stem cells of differentiated B-cell malignancies: models and consequences.

The concept of cancer stem cells has revolutionized our current vision of cancer development and was validated in solid tumors and cancers of the primitive hematopoietic compartment. Proof of the principle is still lacking, however, in malignancies of differentiated B-cells. We review here the current literature, which nevertheless suggests hierarchical organizations of the tumor clone for mostly incurable B-cell cancers such as multiple myeloma, lymphomas and B-chronic lymphocytic leukemia. We propose two models accounting for cancer stem cells in these contexts: a “top-to-bottom” clonal hierarchy from memory B-cells and a “bottom-to-top” model of clonal reprogramming. Selection pressure on the growing tumor can drive such reprogramming and increase its genetic diversity.

T cells in CLL: lost in migration

In this issue of Blood , Ramsay et al[1][1] unravel a new mechanism of immune subversion induced by chronic lymphocytic leukemia (CLL) cells to perturb chemokine-oriented migration of both CD4+ and CD8+ T cells, and they propose an original cereblon-dependent effect of the immunomodulatory drug

Abstract 2852: Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients.

Introduction : we and others have already demonstrated the superiority of obinutuzumab (GA101) over rituximab (RTX) in in vitro depletion assays against chronic lymphocytic leukemia (CLL) cells. It has been recently suggested that complex karyotype (3 or more chromosomal abnormalities) and recurrent somatic mutations of the TP53, NOTCH1 and SF3B1 genes, influenced treatment-free and overall survivals in CLL patients. We address the question whether these factors may also influence the efficacy of anti-CD20 directed immunotherapy. Aim: To assess in vitro pre-clinical activity of RTX and GA101 against CLL cells in freshly isolated PBMCs, and correlate depletion efficacy with modern and classical (FISH, IgVH mutational status, age, gender, Binet stage, b2-microglobulin, bulky adenopathies>5cm) prognostic parameters in 95 CLL patients. Moreover, CD20 antigen density was assessed in 24 patients. Methods: PBMCs were isolated from blood samples by Ficoll gradient centrifugation. Antibody-mediated B cell depletions were determined by enumerating trypan blue negative, flow cytometrically CD19-positive B lymphocytes after treatment with 10 μg/ml of anti-CD20 antibodies. The Quantibrite® kit was used to determine CD20 density, measured as quantity of antibodies bound per cell (CD20-ABC). The Mann-Whitney test was used to compare median depletion according to relevant clinical and biological data. Results: patients characteristics were as follows: 69% males, median age 66y (36% >70y), advanced Binet stage B/C in 39% (31.7% had NCI2008 criteria for treatment), 22.6% had lymph nodes>5cm (56% had b2-microglobulin>3.5mg/l). Molecular and cytogenetics studies were as follows: unmutated IgVH in 42.7%, del13q/tri12/del11q/del17p=25.6%/22%/20%/5.8% respectively, complex karyotype (CK) was found in 20.8% of cases, and somatic mutation of TP53/NOTCH1/SF3B1 in 11.1%/12.6/4.8% of patients. The median B-CLL depletion in freshly isolated PBMC fraction due to treatment with were 61% for GA101 vs 21.5% for RTX (n=95, p 50% in 64% of cases. As shown in Figure 1, in all categories of patients, GA101 was superior to RTX. This was particularly noticed in the patients with TP53 deletion and/or mutation (n=7). Median CD20-ABC was 8100 in 24 patients, who were categorized as high or low CD20 expression based on that median. Low CD20-ABC patients had significantly less antibody-induced B-cell depletion, 12.8% (vs 25%, p=0.37) for RTX, and 42.5% (vs 67.5%, p=0.048) for GA101. CD20-ABC was not found to be correlated with clinicobiological parameters described above. Conclusions: according to modern prognostic parameters, GA101 confirms its higher efficacy than RTX (three-fold), and deserves further combination with chemotherapy or non-chemotherapy entities in CLL management. Citation Format: Loic Ysebaert, Emilie Laprevotte, Christian Klein, Jean-Jacques Fournie, Anne Quillet-Mary. Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2013-2852