Refined Immunophenotypic Criteria Using CD184 and CD165 Improves Sensitivity of Identification of ETP T-ALL
Authors: Singh Monica , Westerman David , Winter Stuart S. , Dunsmore Kimberly P. , Teachey David T. , Shraim Rawan , Pölönen Petri , Mullighan Charles G. , Wood Brent L. 
Published: December 2024

The early thymic precursor (ETP) immunophenotype identifies high-risk T Lymphoblastic Leukemia (T-ALL). Detection of ETP by flow cytometry relies on the following criteria: CD5 negative/1 log lower than mature T cells, &lt;5% of CD1a and CD8 and &gt; 25% of one or more stem cell or myeloid antigens (CD34, CD117, HLA DR, CD13, CD33) on T-lymphoblasts. Subsequently, a subset of T-ALL cases were recognized to fulfill all but the CD5 criteria for ETP and termed Near ETP. Near ETP showed levels of residual disease and outcomes similar but not identical to ETP, suggesting a heterogenous composition or a stage of maturation intermediate between ETP and Non-ETP. Comprehensive sequencing studies, including RNA seq confirmed the former, but clinical RNAseq is impractical for routine clinical use. This study identifies an immunophenotypic approach to ETP recognition using novel flow markers.
To identify antigens suitable for subclassification of T-ALL, we performed high-throughput flow cytometric screening assay using LyoPlates (Becton-Dickinson) containing antibodies against 242 unique antigenic specificities. We tested mononuclear cells obtained from 3 normal peripheral blood donors and 9 children with T-ALL. T-ALL samples were frozen residual pretreatment material from the Children's Oncology Group (COG) trial AALL0434 and covered a range of immunophenotypes seen in this disorder including ETP (N=2), Near ETP (N=2) and Not ETP (N=5) types. From this dataset, promising novel antigens showing differential expression between mature T cells and T-ALL were identified, including CD6, CD27, CD53, CD165, CD184, CD200, CD226, CD229, and CD244. To efficiently evaluate these, a 3-tube, 9-color flow cytometric assay was designed with a 6-antibody backbone combination including CD45 KO, CD3 PE-TR, CD5 PE-Cy7, CD7 V450, CD16 PE-Cy5 and CD56 PE-Cy5 supplemented by one of the three antibody combinations: CD226 FITC, CD184 PE, CD229 APC or CD6 FITC, CD53 PE, CD200 APC or CD244 FITC, CD165 PE, CD27 APC. Using this panel, 121 consecutive pretreatment T-ALL samples from COG AALL0434 were tested in parallel with the standard COG MRD and ETP classification assays. Median fluorescence intensities (MFIs) were determined for all antigens tested on both leukemic and normal T cells using Woodlist 3.0. A majority of the samples (N=99) subsequently underwent comprehensive molecular characterization, allowing comparison of immunophenotypes between newly defined molecular subtypes.
Pretreatment cases included 7 ETP (7%), 20 Near ETP (20%) and 72 Not ETP (73%). On these the molecular subgroups identified were, ETP like (22), TAL1 DP-like (21), TAL1 αβ-like (17), TLX3 (14), NKX2-1 (4), MLLT10 (4), TLX1 (3), KMT2A (3), TME-enriched (3), HOXA9 TCR (2) LMO2 γδ-like (2), NUP98 (1), NKX2-5 (1), SPI1 (1), and NUP214 (1). Comparison between immunophenotype and molecular profile showed all 7 ETP cases to be ETP-like, but so were 10/20 (50%) Near ETP and 5/72 (7%) Not ETP. CD5 did not meet the ETP criteria in 15/22 (68%) ETP-like cases, demonstrating that the current immunophenotypic criteria using CD5 are unable to identify most of the ETP-like cases. Using our augmented flow panel, subsequent analysis of the ETP-like cases uniformly showed low to absent expression of CD184 and CD165. In addition, there was absence of CD1a and all but 2 cases (9%) showed presence of stem cell antigens. However, 14/77 (18%) non-ETP-like cases also showed this low to absent pattern of CD184 and CD165 expression, inclusive of the following genotypes: TLX3 (5/14), TLX1 (1/3), TME-enriched (2/3), TAL1 αβ-like (2/17), LMO2 γδ-like (1/2), NUP98 (1/1), NKX2-5 (1/1) and SPI1 (1/1). Application of the absent CD1a and presence of stem cell ETP criteria to these cases excluded most (64%), leaving only TXL3 (3), TME-enriched (1) and NUP98 (1). Other common but less uniform features of ETP-like cases were increased expression of CD244, absence of CD27 and absent to low CD200. Our augmented flow panel resulted in 91% sensitivity, 94% specificity, 80% PPV and 97% NPV for identification of ETP-like cases.
We conclude that our modified flow cytometric profile; absent CD165 and CD184 (substituting absence of CD5), absent CD1a and presence of 1 or more stem cell antigens will lead to easy, reproducible and practical classification of 91% of ETP like cases, with potential to advance ETP-directed therapies in future clinical trials.
This investigator-initiated trial was supported by Sandoz Inc.