Pb1710: an extended flow cytometry panel as a screening tool for ph-like all

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Acute lymphoblastic leukemia (ALL) comprises multiple entities with distinct molecular and clinical features. The latest WHO classification distinguishes several new ALL subtypes, including Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) - a high-risk subtype characterized by a kinase-activated gene expression profile resulting from multiple diverse genetic alterations. Although Ph-like ALL has been described more than a decade ago and multiple clinical trials have shown that targeted therapy may overcome adverse risk, the identification of Ph-like ALL patients, due to its challenging and not standardized nature, is still not routinely performed outside clinical trials. Due to its rapid turnaround time, cost-effectiveness, and wide availability of both the technology and diagnosticians experienced in immunophenotyping in virtually all hematologic departments, flow cytometry is a perfect candidate for the development of a screening test for Ph-like ALL. The overexpression of cytokine receptor-like factor-2 (CRLF2) has been shown to correlate with CRLF2 rearrangements and has been suggested as a screening tool for the identification of CRLF2-rearranged Ph-like ALL, comprising approximately 50% of Ph-like ALL cases. However, it does not allow to distinguish between CRLF2-overexpressing Ph-like and non–Ph-like ALL, and its use is limited to CRLF2 rearrangements only. To date there is no cytometric method to screen for the remaining half of Ph-like ALL cases. Aims: The study aims to evaluate the expression of a panel of surface and cytoplasmic proteins in ALL blast cells as a surrogate marker of kinase activation enabling routine screening for Ph-like ALL using widely available, multicolor flow cytometry. Methods: Candidate surface and intracellular markers were selected based on the analysis of published expression profiles of ALL patients. The extended panel of surface and intracellular markers included both standard and candidate immunophenotypic markers, i.e. CD45, CD3, CD19, CD34, HLA-DR, phospho-TYR, phospho-STAT3, phospho-STAT5, phospho-CRKL, CRLF2 CD127, CD115, CD25, SOCS1, BLNK, CD97, CD99, CD140A, CD140B, and viability stains (FVS780 and/or 7AAD). For candidate markers, the results were expressed as mean fluorescence intensity (MFI) (for phospho-STAT3, -STAT5 or -CRKL MFI) or as a ratio between respective MFI observed in CD19+ blasts and CD3+ cells that served as internal control (other markers). Clinical data, including classification to ALL subtype, were retrospectively obtained from medical records. For all B-other cases, panel RNA sequencing was performed to allow for classification of new ALL subtypes including Ph-like ALL. Results: Development cohort consisted of 158 bone marrow samples obtained at diagnosis from adult and pediatric ALL patients, including various molecular subtypes of the disease. Except for CRLF2, none of the single markers alone allowed to distinguish new subtypes of ALL. However, multiparameter statistical analysis of the data allowed to build a Ph-like ALL predictor for the identification of Ph-like ALL cases. Prospective analysis of validation cohort patients is ongoing. Summary/Conclusion: We propose a cytometric panel coupled with a statistical model that is capable to identify Ph-like ALL cases at diagnosis in routine clinical settings. Keywords: ALL, Flow cytometry, Acute lymphoblastic leukemia