Lu/BCAM-mediated cell adhesion as biological marker of JAK2V617F activity in erythrocytes of polycythemia vera patients
Authors: Maria De Grandis, Bruno Cassinat, Jean‐Jacques Kiladjian, Christine Chomienne, Wassim El Nemer
Journal: American Journal of Hematology (2015)

Polycythemia vera (PV) is a myeloproliferative neoplasm characterized in most cases by the V617F activating mutation in the tyrosine kinase JAK2. PV patients exhibit increased red cell mass and high risk of thrombosis, a major cause of mortality and morbidity in this disease 1. In biomedical diagnostics, the JAK2V617F mutation is usually detected by allele specific PCR-based assays performed on DNA or RNA extracted from granulocytes or whole blood. The presence of the mutation is a major criterion for PV diagnosis and it may be a prognostic marker because the burden of circulating mutated alleles (%V617F) was shown to be significantly higher in patients who developed vascular complications and in PV patients developing secondary myelofibrosis 2. The %V617F can also be used to monitor minimal residual disease and to evaluate treatment efficacy, especially in patients treated with interferon-α 3. These assays are DNA-based analyses giving indirect estimations of the tyrosine kinase activity that is supposed to be the cause of the disease and its complications. JAK2V617F mutation primarily affects the proliferation of the red cell lineage but its impact on the phenotype of circulating RBCs is poorly documented. Our group showed increased adhesion of PV RBCs to endothelial cells mediated by erythroid Lu/BCAM and endothelial laminin α5 chain 4. Recently, we showed that this abnormal adhesion stems from JAK2V617F-mediated Lu/BCAM activation through a Rap1/Akt signaling pathway 5. Blocking this pathway by JAK2 specific inhibitors led to significant decrease of PV RBC adhesion 5. Here, we extended our findings by a quantitative analysis to determine the relationship between the rate of RBC adhesion to laminin and the JAK2V617F allele burden. We first asked whether variation between patients in RBC adhesion was related to Lu/BCAM expression on the surface of PV RBCs. Indeed, Lu/BCAM erythroid expression is heterogeneous within the same individual, including a variable surface expression level and a proportion of Lu/BCAM-negative RBCs. We performed adhesion assays with blood samples from 17 JAK2V617F-positive PV patients, with no cytotoxic antiproliferative treatment, and determined the number of RBCs adhering to laminin at 3 dyn/cm2. The adhesion values were variable, ranging from 14 to 1021 RBCs/mm2 (Supporting Information Table S1). The percentage of Lu/BCAM-positive RBCs as assessed by flow cytometry ranged from 38% to 90%, and the expression level, estimated by the mean fluorescence intensity (MFI), from 1249 to 3153 (Supporting Information Table S1). The number of adherent RBCs was plotted against the percentage of Lu/BCAM-positive RBCs or the MFI of each blood sample. We found no correlation between the number of adherent RBCs and the percentage of Lu/BCAM-positive RBCs (r = 0.468 and P = 0.058 at 3 dyn/cm2, Fig. 1A), and between RBC adhesion and Lu/BCAM MFI values (r = 0.345 and P = 0.174 at 3 dyn/cm2, Fig. 1B). These results indicated that Lu/BCAM expression alone did not account for the variability of PV RBC adhesion, in accordance with the role of Lu/BCAM phosphorylation in the adhesion of PV RBCs to laminin 5. PV RBC adhesion to laminin as a function of Lu/BCAM expression level or JAK2V617F allele burden. RBC adhesion at 3 dyn/cm2 as a function of Lu/BCAM expression level on the surface of RBCs from 17 PV patients, represented as (A) the percentage of circulating RBCs expressing Lu/BCAM and (B) Lu/BCAM mean fluorescence intensity (MFI). The same adhesion measurements are represented as a function of the JAK2V617F percentage (C). D: Typical microscopic images of RBC adhesion of PV patients with comparable percentages of Lu/BCAM-positive RBCs but different JAK2V617F percentages. Because Lu/BCAM phosphorylation is driven by a JAK2V617F-dependent pathway in PV RBCs 5, we asked whether RBC adhesion was influenced by JAK2V617F expression level. To date the most commonly used tool for evaluating the mutational load in one individual is by measuring the mutant allele burden in blood cell DNA. The JAK2V617F allele burden was determined for the 17 PV patients using an allele specific quantitative PCR (Mutaquant®, Qiagen) and ranged from 20 to 68% (Supporting Information Table S1). RBC adhesion values were plotted against the percentage of JAK2V617F. Statistical analysis showed a significant correlation between RBC adhesion and JAK2V617F percentage (r = 0.571 and P = 0.016, Fig. 1C) that was improved when the interval of Lu/BCAM-positive RBCs was restricted to 65–90% (r = 0.849 and P = 0.0009, n = 11 patients), indicating that for equivalent Lu/BCAM expression, higher JAK2V617F levels would drive higher RBC adhesion to laminin (Fig. 1D). As RBCs are enucleated, it is not possible to estimate the level of JAK2V617F using DNA or RNA based PCR assays. However, clonogenic assays showed that patients with higher JAK2V617F mutant allele burden in blood DNA carried more mutated erythroid progenitor cells 6. Our study revealed RBC adhesion as a biological marker to estimate the V617F mutational load at the protein level in PV RBCs; although indirect, this marker has the potential of informing about the functional level of JAK2V617F in the circulation. This functional assay should be evaluated in a prospective study because it is a rapid and routinely manageable tool to estimate the mutant kinase activity in PV RBCs, which may help assessing the disease progression and the risk of evolution, in particular of thrombotic events in which abnormal adhesion of JAK2V617F-positive RBCs to endothelium could play a role. Furthermore, such a tool may be important to monitor the efficacy of JAK2 inhibitors in PV patients during the very early stages of their treatment by assessing the effect of these inhibitors on JAK2V617F activity in RBCs very shortly after their administration giving important information on their pharmacokinetics in the blood circulation. A European patent application (n° EP13305131.8) was filed for this study, with the international extension n° PCT/EP2014/052153. M. De Grandis performed research, analyzed data, and wrote the article; B. Cassinat, J.J. Kiladjian, and C. Chomienne provided patient blood samples, biological, and clinical information, commented, and edited the article; W. El Nemer designed research, analyzed data, and wrote the article. The authors thank Ms. Eliane Vera at the Centre National de Référence pour les Groupes Sanguins for the management of red blood cell samples. They thank Mr Julien Picot and Mr Sylvain Bigot of the BioFACy-lity platform at the Institut National de la Transfusion Sanguine for flow cytometry analyses. Maria De Grandis,1,2,3,4 Bruno Cassinat,5 Jean-Jacques Kiladjian,6,7,8 Christine Chomienne,5,8 and Wassim El Nemer,1,2,3,4* 1Inserm, UMR_S1134, Paris, France; 2Université Paris Diderot, Sorbonne Paris Cité, Paris, France; 3Institut National De La Transfusion Sanguine, Paris, France; 4Laboratory of Excellence GR-Ex, France; 5Assistance Publique–Hôpitaux De Paris, Hôpital Saint-Louis, Service De Biologie Cellulaire, Paris, France; 6Assistance Publique–Hôpitaux De Paris, Hôpital Saint-Louis, Centre D'investigations Cliniques, Paris, France; 7INSERM CIC 9504, Paris, France; 8Université Paris Diderot, Sorbonne Paris Cité, IUH, UMRS, Paris, France Additional Supporting Information may be found in the online version of this article. Supporting Information Table 1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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