Dados do Resumo

Título

Liquid biopsy as a tool for the detection of genetic alterations in retinoblastoma patients

Introdução

Retinoblastoma is a malignant pediatric intraocular tumor originating from retinal cells, driven by the biallelic loss of RB1. RB1 alterations are germline for 40% of the individuals, that exhibit bilateral predisposition, and sporadic for 60% are somatic mutations, which individuals usually present unilateral disease. Liquid biopsy represents a promising alternative for detection and monitoring the disease, as traditional biopsy is often avoided due to seeding potential.

Objetivo

We characterized genetic alterations in either germline and/or circulating tumor DNA in patients with retinoblastoma at the time of diagnosis.

Métodos

This study, approved by CEP (CAAE 22737219.1.0000.5376), included 14 patients with retinoblastoma from Boldrini Children’s Hospital. Blood samples were collected and centrifuged to isolate germline cells and plasma. Both germline DNA and cfDNA were analyzed for 4 patients; 1 was bilateral, and 3 were unilateral. Cell free DNA (cfDNA) was extracted from 1 mL of plasma using the QIAamp MinElute ccfDNA Mini Kit, and libraries were prepared using Nonacus Cell3 Target workflow and sequenced with 2 × 150 bp reads on NovaSeq X (Illumina). Germline DNA was extracted from leukocytes using the GenElut Mammalian Genomic DNA Miniprep Kit, with whole exome libraries prepared with “Illumina DNA prep with enrichment” and sequenced using 2 × 150 bp reads on NextSeq550 (Illumina).

Resultados

Six patients (42.9%) had bilateral and 8 (57.1%) patients had unilateral retinoblastoma. All 6 bilateral cases presented germline RB1 mutations, confirming hereditary disease, with one patient also developing a second cancer in the liver, a leiomyosarcoma that also carries TP53 mutation. One patient with unilateral disease and without family history presented RB1 germline mutation. Median cfDNA plasma concentration was 0,38 ng at diagnosis. Of the 8 patients with unilateral disease, 7 were evaluated for circulating tumor DNA (ctDNA). All cases showed RB1 mutations. Additional commonly altered genes found in the cfDNA included NF1 (66.7%), ATM (33.3%), among others.

Conclusões

The germline of patients with retinoblastoma revealed RB1 mutation for a case considered sporadic, showing the importance of sequencing germline DNA. Despite blood-ocular barrier and poor ctDNA blood release, we detect ctDNA with high-deep NGS in plasma showing that liquid biopsy holds potential for improving the diagnosis and monitoring of patients with retinoblastoma. However, further research is essential, as the disease's genetic landscape is beyond RB1 mutations.

Financiador do resumo

PRONON SIPAR 25000.012259/2019-42

Palavras Chave

Retinoblastoma; Liquid Biopsy; NGS