cf-DNA and EVs: Biomarkers for early detection of second primary malignancies in patients with heritable retinoblastoma

Patients with heritable retinoblastoma, a tumour predisposition syndrome caused by heritable pathogenic variants of the RB1 gene, have a high risk to develop a Second Primary Malignancy (SPM). We develop a non-invasive test for early detection of SPMs in Rb-survivors. The test exploits the fact that most SPMs are initiated by RB1 allele loss that results in an allelic imbalance. We extract EVs and cfDNA from blood samples from children with Rb and Rb-survivors with and without SPM. DNA released by tumour cells is expected to result in a skewed ratio of RB1 alleles in cfDNA. The allelic ratio at the RB1 locus is detected by analysis of linked SNP variants in heterozygous individuals. EVs are analyzed to identify EV characteristics of diagnostic relevance. Quantification of cfDNA by Droplet Digital PCR showed that the analysis of only one SNP may not be sufficient for sensitive detection of skewed allelic ratios. Parallel analysis of multiple SNPs by Deep Amplicon NGS will increase the power to detect skewed ratios. In view of the goal to offer a lifelong screening for early detection of SPMs analytical validity is of paramount importance.

Publication History

Article published online:
13 May 2020