Bridging conventional and molecular cytogenetics: Evaluating concordance of karyotyping and FISH in pediatric B-ALL and implications for post-induction MRD assessment, experience from Pakistan Free

Background/Objectives Cytogenetic and molecular genetics play a crucial role in the diagnosis of B-lymphoblastic leukemia (B-ALL). These genetic alterations are decisive in risk stratification and chemotherapy protocols. Conventional karyotyping and fluorescence in situ hybridization (FISH) are widely used diagnostic tools. Despite their complementary nature, discrepancies between these techniques can pose challenges, particularly when predicting post-induction minimal residual disease (MRD). This study aims to predict the gap between karyotyping and FISH by evaluating their concordance and investigating how combined cytogenetic findings correlate with post-induction MRD status.

Methods A retrospective study was conducted in the Cytogenetics laboratory of Indus Hospital, Karachi. All newly diagnosed patients of B-ALL (1-17 years) from January 2021 to September 2024. Cytogenetic including karyotyping by G-banding and interphase FISH for BCR-ABL1 (Dual color dual fusion proble), ETV6-RUNX1 (Dual color dual fusion probe), and KMT2A gene rearrangement (Break apart probe) were done on a semi-automated system. Post-induction MRD was done by 8-color flow cytometry using post-induction day 35 bone marrow aspirate. Data extracted from electronic medical records of the hospital after having IRB approval.

Results This study included 976 patients, with a median age of 7.00 years (IQR: 5.00–11.00) with M/F ratio 1.5:1. In the BCR-ABL1 analysis, the most frequent finding was gain of BCR1 followed by BCR/ABL1 positivity with a concordance rate of 61% and 76% respectively when compared with karyotype. The ETV-RUNX1 analysis revealed that the most frequent abnormality was RUNX1 gain followed by the ETV-RUNX1 fusion with a concordance rate 27% for both. KMT2A gain was observed in 8.73% of cases, with a concordance rate of 72.4% followed by KMT2A rearrangement. All negative cases were concordant across both FISH and karyotype. Statistical analysis confirmed a significant association between FISH findings and karyotype (p = < 0.001), with Cramer's V values of 0.61, 0.793, and 0.812 for BCR-ABL1, KMT2A, and ETV-RUNX1, respectively. Post-treatment minimal residual disease (MRD) was detected in 27% of the cohort. A detailed analysis revealed MRD positivity rates are highly associated with specific genetic alterations like BCR-ABL1.

Conclusion In this study, FISH and karyotyping demonstrated moderate to strong concordance for key cytogenetic abnormalities. Post-induction MRD revealed strong associations with BCR-ABL1 and KMT2A abnormalities, highlighting their potential as predictors of residual disease risk. Our findings underscore the complementary role of FISH and karyotyping in B-ALL diagnosis and risk assessment. These conventional techniques still play crucial role in resource constrained settings where advance techniques like gene sequencing is not available.