Investigations on Clinical Validation of mutated ANKRD36 as a Novel Molecular Biomarker for Monitoring Early Progression and Timely Therapeutic Interventions in Blast Crisis CML

Background Chronic Myeloid Leukemia (CML) is initiated in the bone marrow due to the chromosomal translocation t(9;22), resulting in the fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have transformed fatal CML into an almost curable disease. However, TKIs lose efficacy during disease progression, and the mechanism of CML progression remains to be fully understood. Additionally, common molecular biomarkers for CML progression are lacking. Our studies previously detected ANKRD36 (c.1183\_1184 delGC and c.1187\_1188 dupTT) associated exclusively with advanced phase CML. However, clinical validation of this finding was pending. Therefore, this study aimed to clinically validate mutated ANKRD36 as a novel biomarker of CML progression. Materials and Methods The study enrolled 124 patients in all phases of CML, recruited from Mayo Hospital and Hameed Latif Hospital in Lahore, Punjab, between January 2020 and September 2023. All response criteria were adopted from the European LeukemiaNet guideline 2020. Informed consent was obtained from all study subjects. The study was approved by scientific and ethical review committees of all participating centers. Sanger sequencing was employed to detect ANKRD36 mutations in CML patients in accelerated phase (AP) (n=11) and blast crisis (BC) (n=10), with chronic-phase CML (CP-CML) patients as controls (n=103). Samples were processed using Big Dye Terminator Cycle Sequencing Ready Reaction kits and sequenced using ABI Prism 3730 Genetic Analyzer, and sequencing using forward and reverse primers for ANKRD36. Data was analyzed using SPSS version 26. Results During our study, 17% of CML patients progressed to advanced phases AP-CML n= 11 (8.9%) and BC-CML n=10 (8.1%). The chronic- and advanced-phase patients showed significant difference with respect to male-to-female ratio, hemoglobin level, WBC count, and platelet count. Sanger sequencing detected ANKRD36 mutations c.1183\_1184 delGC and c.1187\_1185 dupTT exclusively in all AP- and BC-CML patients but in none of the CP-CML patients. Nevertheless, mutations status was not associated with male-to-female ratio, hemoglobin level, WBC count, and platelet count, which makes ANKRD32 as an independent predictor of early and terminal disease progression in CML. Conclusions The study confirms ANKRD36 as a novel genomic biomarker for early and late CML progression. Further prospective studies should be carried out in this regard. ANKRD36, although fully uncharacterized in humans, shows the highest expression in bone marrow, particularly myeloid cells and correlation with high WBC count. Functional integrated genomic studies are recommended to further explore the role of ANKRD36 in the biology and pathogenesis of CML. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul-Aziz City for Science and Technology, Kingdom of Saudi Arabia, Grant Number 14-Med-2817-02. The study was approved by King Abdullah International Medical Research Centre (KAIMRC), National Guard Health Affairs, Saudi Arabia, although no research funding was provided (project # RA17/002/A). This study was partially supported by the College of Medicine, Research Centre, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia. All authors have read and have consented to the acknowledgment. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study adhered to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participating patients. The approval of study protocols was obtained from Scientific Committees and Ethical Review Boards (ERBs) of King Abdullah International Medical Research Center (KAIMRC); King Saud bin Abdulaziz University for Health Sciences (KSAU-HS), Hayatabad Medical Complex (HMC), Mayo Hospital, and Hameed Latif Hospital in Lahore, Punjab, Pakistan. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors