Evaluation of TP53 and IGHV clinical molecular prognostic testing for patients with Chronic Lymphocytic Leukaemia at Tygerberg Hospital
Introduction: Chronic lymphocytic leukaemia (CLL) is a monoclonal, lymphoproliferative malignancy with variable clinical outcomes and treatment responses, largely influenced by genetic and molecular factors. Among these, genomic DNA sequence variants in the tumour suppressor gene p53 (TP53) and mutational status of the immunoglobulin heavy chain variable region (IGHV) gene have emerged as critical molecular prognostic biomarkers with significant therapeutic implications for risk stratification and treatment selection. This study aimed to optimise, validate, and verify polymerase chain reaction (PCR)-based molecular assays for the detection of TP53 gene sequence variants and determination of IGHV mutational status in CLL patients at Tygerberg Hospital (TBH). Material & Methods: TP53 variant analysis was determined via bi-directional Sanger Sequencing of PCR-amplified complementary DNA (cDNA) obtained from reverse transcription of extracted total ribonucleic acid (RNA), targeting exons 2 to 11 of the entire coding region. IGHV mutational status was assessed using a two-step approach, firstly clonality was determined via fragment analysis by capillary electrophoresis of framework regions of the heavy chain variable region (FR1, FR2, FR3 VH), followed by bi-directional Sanger sequencing of clonal samples from genomic DNA (gDNA). A total of 44 participants were included in the evaluation according to National Health Laboratory Service (NHLS) validation guidelines. Results: The TP53 variant analysis assay had been successfully validated. Four variants were identified: c.108G>A (p.Pro36=), c.136T>C (p.Ser46Pro) and c.215C>G (p.Pro72Arg) in exon 4 and c.639A>G (p.Arg213=) in exon 6. Among 20 participants analysed, 25% carried c.108G>A (5/20), 5% carried c.136T>C (1/20), 50% carried c.215C>G (10/20) and 5% had both c.215C>G and c.639A>G (1/20) while 15% of participants had no variants (3/20). All variants were classified as benign on ClinVar except the c.136T>C variant which was classified as variant of uncertain significance (VUS). Only one variant was located within the deoxyribonucleic acid (DNA)-binding domain (exons 5 to 8), a known hotspot region. The IGHV clonality and mutational analysis assay had been successfully verified and validated. Fragment analysis results indicated that 70% of participants (21/30) had single fluorescent peaks in FR1, FR2 and FR3 VH regions, indicating monoclonal rearrangements. Of these 21 participants, subsequent sequencing revealed 33% participants were classified as mutated IGHV (M-CLL) (7/21; mean homology = 91.48%); 57% of participants were classified as unmutated IGHV (UM-CLL) (12/21; mean homology= 99.56%) and remaining 10% were borderline IGHV (BL-CLL) (2/21; mean homology = 97.55%). Discussion & Conclusion: In conclusion, we successfully developed and validated TP53 and IGHV mutational status assay and verified a IGHV clonality assay. Despite the technical challenges encountered, future implementation of these assays will enhance risk stratification, treatment selection, and prognostic assessment of CLL patients at TBH, aligning local molecular diagnostic capabilities with international practices and contributing to improved patient outcomes in the South African healthcare context.
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