Introduction / Objective: Philadelphia chromosome–like B-cell acute lymphoblastic leukemia (Ph-like B-ALL), also referred to as BCR::ABL1-like B-ALL, is a genetic subtype of B-ALL that more frequently affects children, young adults, and individuals of Hispanic origin. It is associated with poor prognosis. Ph-like B-ALL is defined by a gene expression profile similar to that of BCR::ABL1-positive (Ph+) B-ALL but lacks the t(9;22)(q34;q11.2)/BCR::ABL1 rearrangement. Instead, it presents with various genetic alterations that activate kinase signaling pathways, including ABL-class fusions, EPOR/JAK rearrangements, JAK-STAT pathway mutations, RAS mutations, or other undefined kinase-activating lesions. The CRLF2 gene, located on chromosomes Xp22.3 and Yp11.3, is overexpressed in approximately 50% of Ph-like B-ALL cases. CRLF2 expression can be assessed by PCR, fluorescence in situ hybridization (FISH), or flow cytometry (FC). Recently, new monoclonal antibodies have been introduced into flow cytometry panels to help predict underlying genetic or molecular alterations.This retrospective study aimed to evaluate CRLF2 antigen expression by flow cytometry and compare it with FISH analysis in patients diagnosed with B-ALL.

Materials and Methods: A total of 39 diagnostic samples (35 bone marrow and 4 peripheral blood) from B-ALL patients were analyzed between January 2022 and June 2025. The flow cytometry panel included: CD15-FITC / CD13-PE / CD33-PC5.5 / CD34-PC7 / CRLF2-APC / CD19-APC-A700 / CD25-Pacific Blue / CD45-Krome Orange.

CRLF2 expression was evaluated based on the percentage of positive cells and fluorescence intensity, using Navios and/or DxFLEX cytometers (Beckman Coulter) and analyzed with Kaluza software.

CRLF2 rearrangements were assessed using FISH with the CRLF2 breakapart probe (Xp22.33/Yp11.32) (Cytocell).

Results: Median age was 24 (5 – 59) years, 69,2% male. CRLF2 overexpression by flow cytometry was observed in 5 patients (14.3%), all of whom had CRLF2 rearrangements confirmed by FISH. Among these cases, three showed strong CD10 expression, and two exhibited aberrant expression of CD13 and CD33. In the remaining 34 cases (85.7%), CRLF2 expression was negative by FC, and no rearrangement was detected by FISH—showing 100% concordance between the two techniques at diagnosis.

In three patients with CRLF2 overexpression, subsequent samples were analyzed for measurable residual disease (MRD), with CRLF2 positivity detected by FC at levels of 0.03%, 0.001%, and 0.005%, respectively. In these cases, FISH results were negative, demonstrating that flow cytometry was more sensitive for MRD detection.

Discussion: The integration of immunophenotypic and genotypic characterization enables the identification of specific B-ALL subtypes with distinct prognostic and therapeutic implications. Flow cytometric detection of CRLF2 is a rapid and sensitive method for molecular screening and MRD monitoring. In addition to its key role in initial diagnosis, FC is a valuable screening strategy to identify cases with relevant genetic alterations, guiding prioritization of confirmatory molecular tests such as FISH and PCR.

Conclusion: Flow cytometry showed high concordance with FISH in detecting CRLF2 overexpression at diagnosis and demonstrated greater sensitivity for MRD detection. It is an efficient, rapid, and cost-effective tool for screening Ph-like B-ALL cases with CRLF2 overexpression, contributing both to diagnosis and disease monitoring.

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2025
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