How to Improve the Definition of Chronic Lymphocytic Leukemia Outcome Using a Simple Flow Cytometric Score Based on CD49d and Homing Marker Expression
Authors: Giudice Valentina , Rocco Monia , Pezzullo Luca , Villani Giancarlo , Rosamilio Rosa , Marino Luigi , Annunziata Silvana , Fontana Raffaele , Ferrara Idalucia , Serio Bianca , Selleri Carmine 
Published: October 2019

Abstract
The identification of new molecular markers in Chronic Lymphocytic Leukemia (CLL) allowed to better define prognosis and clinical outcome. The actual staging systems could estimate the prognosis, but not the rapidity of disease evolution. Neither the identification of new molecular markers did allow to foresee the evolution and clinical response, because discordant findings were mostly reported. The aim of the present study was (1) to confirm the independent prognostic role of CD49d as a single marker in CLL patients, (2) to investigate the relationship between CD49d and other well-established CLL-membrane predictor markers (CD5, CD11c, CD20 and CD38) or clinical staging systems and (3) to evaluate the role of an immunophenotypic score based on the flow-cytometric detection of CD5, CD11c, CD20, CD38 and CD49d in the work up of CLL staging.
Heparinized whole blood was collected from 68 CLL patients for immunophenotyping using the following antibodies: anti kappa, anti-lambda, CD5, CD11c, CD19, CD20, CD23, CD38, CD45, CD49d. A scoring system was elaborated combining 5 membrane markers: CD5, CD11c, CD20, CD38 and CD49d. Antigens were divided in two groups, favorable (CD5 and CD20) and unfavorable (CD11c, CD38 and CD49d) prognostic markers, and the cut-off of positivity was chosen according to the literature (30% for CD5, CD11c, CD20 and CD38, and 45% for CD49d). A value of "0" or "1" ("2" only for CD49d positivity) was assigned according to antigen expression. Finally, we defined a favorable phenotype when the sum of all the cytometric features was equal or less than 2, conversely the unfavorable phenotype was defined for a sum equal or greater than 3 (between 3 and 6).
Flow cytometric analysis showed high CD49d expression in CD19+ cells in 47% of patients (n=32), and high CD38 expression in 44% of subjects (n=30), simultaneously expressed in 28% of patients (n=19). The 19% (n=13) of all CLL patients were CD5-, and interestingly the 85% of them showed higher expression of CD49d. Linear correlation was found between CD49d and CD38 (r2=0.08772, p=0.0142), and between CD49d and CD20 expression (r2=0.2490, p<0.0001). For CD5, the opposite tendency was registered (r2=0.1944, p=0.0002). Strong negative correlation between higher CD49d expression and total lymphocyte count was found (Pearson r = -0.3577, r2=0.1279, p=0.0068), but not for hemoglobin level and platelet count. The statistical power of each parameter of the score was also calculated by Chi-square test and all markers displayed a statistically significant weight (p<0.0001). After assessed the prognostic power of each marker, we applied the score to staged patients. Forty patients (59%) had a Favorable Phenotype and 28 of them (70%) an early stage disease; in this group of patients, only 7.5% (n=3) showed CD49d high expression. The overall response rate (ORR) was of 58%. The other 28 patients (41%) showed an Unfavorable Phenotype and 21 of them (75%) had an early stage disease. In this arm, all subjects carried>45% of CD49d positive cells. Four patients with Unfavorable Phenotype received chemotherapy with an ORR of 25%. Furthermore, a small population (n=16) of our CLL cohort was also studied for genetic abnormalities using FISH technique. According to FISH analysis, 25% of studied patients were classifies as very low-risk and, interestingly, no one of them showed an Unfavorable Phenotype (only one patient carried CD49d as unique negative marker). In our cohort, 50% of patients were low-risk with no genetic abnormalities or +12, but 63% of them showed an Unfavorable Phenotype with high CD49d and CD38 expression in 100% and 60% of cases, respectively.
Our data confirm the independent negative prognostic role of CD49d and suggest a stronger prognostic power compared to CD38 in the definition of CLL outcome, because of its complex activity as homing marker, signaling receptor and anti-apoptotic molecule. Thus, the prognostic significance of CD49d may be enhanced when considered in comparison with other established markers, as CD11c and CD38. In conclusion, our results propose the use of the CD49d marker in combination with other B-cell membrane antigens as an additional tool for routine diagnosis and risk-stratification of CLL patients. Identification of high-risk phenotype with a simple scoring method could improve the treatment of these patients, who could take advantage of the most recent molecular targeting therapies.

Disclosures
No relevant conflicts of interest to declare.