In Vivo Cascade Cyclic Amplification Strategy for Detecting the Relative Expression of MYCN mRNA in Neuroblastoma

MYCN gene copy number in neuroblastoma tissue sections is a well-established prognostic parameter used to guide clinical risk stratification. Nevertheless, the majority of neuroblastoma cases are identified at late stages, when tumors are bulky and unresectable, precluding safe tissue sampling through surgery or core biopsy and thereby compromising the accuracy of molecular risk assessment. To address this critical diagnostic challenge, we have developed a nonbiopsy fluorescent signal conversion strategy capable of detecting MYCN amplification status without relying on tissue biopsies. The core of this method is a DNA-based biosensor based on cascade cyclic amplification (CCA), which undergoes conformational activation upon specific hybridization with intracellular MYCN or NAGK mRNA, releasing tunable fluorescent signals. The normalized fluorescence intensity ratio (referred to as the M/N ratio) quantitatively reflects the MYCN copy number status. This CCA system integrates two orthogonal functional modules-MYCN-CCA and NAGK-CCA-both exploiting elevated cytoplasmic APE1 activity in pathologically altered neuroblastoma cells as a tumor-specific molecular switch and endogenous enzymatic amplifier. APE1-mediated interactions trigger structural changes that drive the dissociation of fluorophore from quencher, generating stable and quantifiable fluorescent signals. Analytical validation demonstrates exceptional sensitivity, with detection limits of 0.52 aM for MYCN mRNA and 0.65 aM for NAGK mRNA. Importantly, the CCA platform enables precise quantification of the M/N ratio in both cell line-derived and patient-derived xenograft models, and allows rapid in situ evaluation in frozen sections of clinical neuroblastoma samples. Collectively, this work introduces a biopsy-independent detection scheme for MYCN amplification, which holds promise for broad deployment in precision medicine and routine diagnostics.