Flow Cytometric Screening of Novel Antigen Expression in T- ALL

The distinction in antigen expression between normal T cells and T-ALL is currently limited by a narrow breadth of clinically useful antibodies. Our study aimed to enhance the diagnostic utility of flow cytometry in T-ALL diagnosis and minimal residual disease (MRD) detection by identifying novel aberrant antigen expression in T-ALL.

A high throughput flow cytometric screening method containing 242 antibodies (Lyoplate, Becton Dickinson) was used to evaluate 3 normal peripheral blood specimens and 9 diagnostic paediatric T-ALL cases. An LSRII (Becton Dickinson) was used for all flow acquisition, and Woodlist software for all analysis. The initial screening panel comprised a 9 colour assay (1 detection and 8 gating fluorochromes). All Lyoplate antibodies were analysed for differences in median fluorescence intensity between normal T cells and T-ALL populations. Subsequently, 9 specific novel antibodies were chosen for expanded testing in prospectively enrolled paediatric T-ALL cases on Children's Oncology Group protocols, using 3 additional tubes. A 9 color flow cytometric assay was designed with the following backbone antibody combination: CD3 PE-TR, CD16+CD56 PE-Cy5, CD5 PE-Cy7, CD7 V450, CD45 KO. Additionally, in each tube one of the following combinations was utilized: CD226 FITC, CD184 PE, CD229 APC or CD6 FITC, CD53 PE, CD200 APC or CD244 FITC, CD165 PE, CD27 APC. Median fluorescence intensites were compared between T-ALL populations and normal donor T cells, and the ratio of abnormal T-ALL: normal donor T cells calculated for each antibody.

A total of 59 patients with T-ALL were analyzed median age 8 (2–29) with 38 males, including 6 early thymic precursor (ETP) cases by St Jude's criteria. In total, 94 episodes were included: 59 pretreatment, and 35 post treatment cases at Day 29. In the T-ALL, CD27 expression was significantly reduced with a median blast/normal T cell ratio of 0.54 at diagnosis, and 0.52 post treatment. 48/53 (91%) patients had a reduced ratio <0.75 at diagnosis and 18/31 (58%) post treatment. Additionally CD6, CD226 and CD200 showed a mild reduction at diagnosis of 0.75, 0.81 and 0.84 respectively, with similar ratios seen with these 3 antibodies post treatment. The ETP cases showed a significant reduction in CD27 expression, median ratio 0.44, while reduced expression was also seen with CD6 (0.72), CD165 & CD184 (0.77), CD53 (0.78) and CD226 (0.80). There was no difference in the ratios calculated when using internal patient T cells versus normal donor T cell expression levels.

We have demonstrated that expression levels of CD27 are consistently reduced in the majority of T-ALL and ETP patients at diagnosis and expression levels are stable at post treatment assessment. This marker has potential utility in clinical practice for diagnosis and MRD assessment. A spectrum of additional antibodies also demonstrated reduced expression, especially in ETP cases, however further patient recruitment and evaluation is required for more definitive evaluation of the utility of these antigens.

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No relevant conflicts of interest to declare.