CYTOGENETIC PROFILING OF ACUTE MYELOID LEUKEMIA IN ADULTS: INTEGRATING ASSOCIATION BETWEEN NEXT-GENERATION FISH, CYTOGENETICS, AND FLOW CYTOMETRY

Abstract

Background: The accurate diagnosis and risk stratification of acute myeloid leukemia (AML) are fundamentally reliant on a comprehensive assessment of its genetic and immunophenotypic landscape. While conventional karyotyping, fluorescence in situ hybridization (FISH), and multiparameter flow cytometry (MFC) are standard, their integration into a unified diagnostic model remains underexplored, particularly with the advent of Next-Generation FISH (NG-FISH) technologies. Aims & Objectives: This study aimed to establish an integrated diagnostic framework for adult AML by evaluating the synergy between conventional cytogenetics, NG-FISH, and MFC. The primary objectives were to determine the concordance and incremental diagnostic yield of NG-FISH compared to karyotyping and to identify significant associations between specific cytogenetic abnormalities and immunophenotypic profiles. Methodology: In this prospective, multicenter study conducted in Lahore (three tertiary care Hospitals) and Lodhran (one tertiary care hospital), 43 newly diagnosed adult AML patients were enrolled over 12 months (January 2025 – January 2026). The sample size was calculated using G*Power software. Diagnostic bone marrow samples underwent simultaneous triple-platform analysis: conventional G-banded karyotyping, targeted NG-FISH using an automated system with an AML-specific probe panel, and comprehensive immunophenotyping by 8-color MFC. Data were integrated and analyzed statistically to assess concordance and genotype-phenotype correlations. Results & Findings: Conventional cytogenetics detected abnormalities in 72.1% of cases. NG-FISH demonstrated an incremental diagnostic yield of 9.3%, identifying cryptic KMT2A and CBFB rearrangements in patients with a normal karyotype, thereby reclassifying their European LeukemiaNet (ELN) 2022 risk category. Strong immunophenotypic associations were identified: t(8;21) was significantly correlated with CD19+/CD56+ expression (p=0.003), and aberrant CD7 expression was a marker of adverse-risk cytogenetics (p=0.009). The integrated approach using all three modalities refined the initial risk stratification for 11.6% of the cohort. Conclusion: The strategic integration of conventional cytogenetics, NG-FISH, and MFC significantly enhances diagnostic precision in adult AML. NG-FISH provides critical detection of occult abnormalities, while MFC reveals phenotypic signatures predictive of genetic risk. This multimodal model mitigates the limitations of single-platform analysis, leading to more accurate risk stratification and informing timely, appropriate therapeutic decision-making.