13 genes panel validation to assess minimal residual disease through urinary tumor DNA in muscle-invasive bladder cancer patients

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with a 5-year mortality rate of 40–50%. Standard therapy includes neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Unfortunately, almost 40% of the patients experience a disease recurrence after RC, due to the persistence of micro-metastatic disease (minimal residual disease, MRD). In those cases, markers predicting MRD persistence would be essential to enhance individual patient outcomes, as preoperatively determined histopathological variables are still insufficient to predict pCR. We here validated an urinary liquid biopsy test to be used for a non-invasive MRD assessment. Our panel consist of 13 genes mutated at a rate > 7% in MIBC cases, including: KRAS, HRAS, PIK3CA, TP53, TERT, FGFR3, ERCC2, ERBB2, ATM, RB1, AKT1, CDKN1A and CDKN2A. To validate this panel, 20 MIBC treatment-naïve patients candidate to RC were enrolled and compared to 10 healthy donors. All patients provided samples (paired blood, urine and tissue) prior to RC. FFPE DNA, circulating tumor DNA (ctDNA) and urinary tumor DNA (utDNA) have been extracted through Maxwell RSC system from tissue, blood and urine samples. FFPE DNA, ctDNA and utDNA quantity and quality were assessed before sequencing, performed using Ion Torrent NGS technology. 18/20 tumor tissue samples showed detectable mutations. The most frequently mutated genes in tissue samples were TERT (12/20; 60.0%) and TP53 (9/20; 45.0%). Mutations occurred less frequently (<20.0%) in the other 11 genes. We found that 88.9% of mutations found in tumor tissue were also identified in utDNA, whereas all the mutations found in utDNA were also detected in paired tumor tissues (concordance rate 90%). Conversely, the concordance between paired tissues and plasma samples was 40.5%. In MIBC mutant DNA is detected more frequently in urine compared to plasma. These preliminary data pave the way for deeper investigations to define whether a non-invasive MRD assessment can avoid needless and risky surgical procedures, allowing the prediction of the MRD status, personalizing therapeutic choice and ultimately improving patients’ quality of life.