Exploring Treatment-Driven Subclonal Evolution of Prognostic and Predictive Triple Biomarkers: Dual Gene Fusions and Chimeric RNA Variants in Subtypes of Acute Myeloid Leukemia Patients with KMT2A Rearrangement

Introduction

Chromosomal rearrangements (CR) initiate leukemogenesis in approximately 50% of acute myeloid leukemia (AML) patients. Chromosomal abnormalities can give rise to fusion proteins, significantly contributing to the disease's ontogenesis and its recurrence or refractory nature. However, limited targeted therapies exist due to a lack of accurate molecular and genetic biomarkers of refractory mechanisms during treatment.

Methods

We investigated the pathological landscape of treatment resistance and relapse in 16 CR-AML patients by monitoring cytogenetic, RNA sequencing, and genome-wide changes among newly diagnosed, refractory, and relapsed AML cases. Bone marrow samples from seven patients were collected for chromosome analysis using GTG-banding, and a total of twenty metaphase cells were analyzed for each case using the ASI imaging system. The KMT2A gene rearrangements were tested on the bone marrow samples with FISH probes, analyzing a total of 200-500 interphase nuclei. Bone marrow mononuclear cells were isolated from blood samples of 2 patients, and high-quality total RNA was isolated. RNA sequencing was performed and after filtering, multiple candidate gene fusions were acquired for further analysis.

Additionally, RNA was isolated from patient samples for quantitative real-time PCR analysis, to detect DNA breakpoints of t(6;11) translocations. Sequence alignments were analyzed with BLAST and BLAT. Statistical analyses were performed using GraphPad Prism.

Results

FISH analysis revealed KMT2A (MLL gene, 11q23)/AFDN (AF6, 6q27) rearrangements in AML patients. RNA sequencing identified an unknown gene fusion, CCDC32 (15q15.1)/CBX3 (7p15.2), in both newly diagnosed and relapsed samples, a finding not previously reported in KMT2A/AFDN-rearranged AML. Quantitative qPCR analyses demonstrated that this gene fusion significantly affected the expression of the wild-type CCDC32, essential for embryonic development, and CBX3, an oncogene for solid tumors, particularly during relapse.

Further investigation confirmed the existence of triple biomarkers-KMT2A/AFDN rearrangement, CCDC32/CBX3 gene fusion, and chimeric RNA variants-in a 21-year-old male patient with rapid relapsed/refractory AML. Notably, among the 16 patients studied, two patients initially exhibited the KMT2A/AFDN gene fusion. However, one patient did not show this fusion upon relapse, whereas the other retained the KMT2A/AFDN fusion during both diagnosis and relapse, identifiable only by PCR and Sanger sequencing. Interestingly, the CCDC32/CBX3 gene fusion persisted in both patients throughout the diagnostic and relapse phases, suggesting a potential role in differential treatment resistance.

Conclusion

Our findings highlight the dynamic nature of genetic alterations in AML, particularly the persistence and evolution of prognostic and predictive biomarkers. The study identified triple biomarkers-KRAS mutation, dual fusions (KMT2A/AFDN and CCDC32/CBX3), and chimeric RNA variants-that may contribute to subclonal evolution and poor clinical outcomes. These results underscore the importance of continuous genetic monitoring to understand the mechanisms underlying treatment resistance and relapse in AML patients, potentially guiding more effective therapeutic strategies.

Kelly:Gilead: Consultancy; Servier: Consultancy; AstraZeneca: Speakers Bureau; Janssen: Speakers Bureau; Ispen: Speakers Bureau; Karyoplasm: Speakers Bureau; Eli Lilly: Speakers Bureau. Abdel-Azim:Adaptive: Other: Speaking/teaching , Advisory Committee and Research Funding; Vertex: Other: Advisory Committee; J&J: Other: Advisory Committee.