CD52 Expression in Adult Acute Lymphoblastic Leukemia (ALL): Quantitative Flow Cytometry Provides New Insights.
Authors: Lozanski Gerard , Sanford Ben , Yu Daohai , Pearson Rebecca , Edwards Colin , Byrd John C. , Larson Richard A. , Bloomfield Clara D. , Stock Wendy 
Published: October 2019

Abstract
We recently tested the feasibility of incorporating Alemtuzumab, a humanized anti-CD52 monoclonal antibody, into frontline therapy of adult ALL (CALGB 10102) in an attempt to eradicate minimal residual disease (MRD). We have previously reported that CD52 expression occurs in 68% of newly diagnosed ALL. This assessment was based upon the use of a qualitative flow cytometric assay performed on all pre-treatment cases in a central CALGB reference laboratory. Cases with > 10% CD52 expression on lymphoblasts relative to an isotype control were considered CD52 positive and eligible to receive Alemtuzumab during post-remission therapy. To better characterize the CD52 expression level on lymphoblasts and to obtain insights for the design of future subset specific therapies in adult ALL, we developed a quantitative assay to measure CD52 antigen density on lymphoblasts using custom PE conjugated clinical grade Alemtuzumab on 29 cases of precursor B-cell (pre-B) ALL and 9 cases of precursor T-cell (pre-T) ALL. In this assay, CD52 is expressed in arbitrary units of antibody bound per cell (ABC). We also measured CD52 levels on residual normal B and T lymphocytes in every specimen to calculate a normalized ratio (NR) of lymphoblast CD52ABC/ normal lymphocytes CD52 ABC. The results are tabulated below:
CD52 Expression Cell Type Precursor B-Cell ALL (n=29) median (range) Precursor T Cell ALL (n=9) median (range) CD52 ABC units Blasts 27658 (2042–206952) 10222 (3784–35337) CD52 ABC units Normal B Lymphocytes 125475 (59872–282245) 146478 (68098–291878) CD52 ABC units Normal T Lymphocytes 64142 (27406–141635) 78720 (22412–144350) CD52 NR Blasts/Normal B Lymphocytes 0.19 (0.02–0.88) 0.06 (0.02–0.16) CD52 NR Blasts/Normal T Lymphocytes 0.38 (0.04–2.16) 0.12 (0.07–0.26)
Pre-B lymphoblasts express significantly lower levels of CD52 antigen than normal B lymphocytes (p<0.0001). Similarly, Pre-T lymphoblasts express significantly lower levels of CD52 antigen than the normal T lymphocytes (p<0.0001). Moreover, lymphoblasts in pre-T ALL show a near-significant lower expression of CD52 than pre-B lymphoblasts (two-sided p=0.05). Calculation of the NR confirms these observations. We have also begun to investigate CD52 expression by cytogenetic subset. Of note, we have determined that Philadelphia chromosome positive (Ph+) lymphoblasts were CD52+ in 26/28 (93%) cases studied; quantification of CD52 expression in Ph+ cases and in other common cytogenetic subsets is in progress. Correlation of CD52 expression with Alemtuzumab plasma levels and with development of opportunistic infections is underway. In conclusion, these data are the first to demonstrate that lymphoblasts from patients with untreated ALL express lower levels of CD52 antigen than residual normal lymphocytes present at the time of diagnosis. These findings may lead to improved selection of patients most likely to benefit from administration of Alemtuzumab and may have important implications for optimizing dosing and scheduling of Alemtuzumab during ALL therapy.