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Título

Implications of preanalytical factors in cfDNA detection in breast cancer patients

Introdução

Breast cancer (BC) is the leading cause of cancer-related death among women worldwide. Liquid biopsy (LB) comprises a minimally invasive technique that allows analysis of cell-free DNA (cfDNA) that reflects the genetic status of tumors and provides effective management of BC patients. However, the lack of BL standardization hampers its clinical implementation and the preanalytical factors may be relevant to the reliable results.

Objetivo

In this study, we aimed to examine the pre-analytical factors that influence the cfDNA detection, as well investigate the impact of demographic characteristics, clinicopathological aspects, and collection input materials on the cfDNA levels.

Métodos

This was a quantitative study conducted in the Oncology Department (UNACON) from Hospital Universitário João de Barros Barreto (HUJBB), Belém, Pará. A total of 25 adult females with confirmed breast carcinoma were selected through convenience sampling. The research is part of the broader "Liquid Biopsy in Breast Carcinoma as a Precision Medicine Tool'' project, approved by CEP and CONEP (Opinion: 3.847.758). Under consentiment, we collected 10 mL of total blood. The sample was centrifuged 13000 rpm per 10min at 4° C, we separate platelet poor plasma. We used MagMAX™ Cell-Free DNA Isolation Kit (Applied Biosystems) for cfDNA extraction. We evaluated the sample quality and concentration using Qubit 4. We used SPSS 19.0 for perform Spearman's correlation coefficients. We considered low correlations r< 0,02 and moderate r> 0,6, and p <0,05 were considered statistically significant.

Resultados

A total of twenty five breast cancer patients were included, with an average age of 55 (range 40 to 73). 68% had family history. The Estrogen Receptor (ER) averaged 47.92% and Progesterone Receptor (PR) averaged 14.94%. Menarche occurred around 13 years (range 10 to 16), roughly 60% were in menopause, 52% didn't use contraceptives. Luminal B subtype constituted 40%, triple negative 24%. Around 40% were T3N1M0/T2N0M0 in TNM classification. Spearman correlation analysis, at 95% confidence and 5% error, unveiled: weak positive correlation with cfDNA (p= 0,20- 0,60) in five variables (ki67, age of menarche, material used, time interval between collection and processing, blood quantity); moderate negative correlation in two (ER, Immunohistochemistry - IHQ); weak negative correlation in four (PR, TNM, contraceptive use, age); (family history, menopause).It is know observed higher Ki67 in rapidly dividing tumors, indicating more DNA release, particularly ctDNA, a vital tumor marker, in plasma. The optimal peripheral ctDNA collection method remains uncertain; certain devices, like needle-based (e.g., butterfly needles), show lower hemolysis, reducing genomic DNA contamination. A proper collection-processing gap is vital due to cfDNA's half-life, denoting the time for half of circulating cfDNA to degrade and be eliminated.

Conclusões

In short, the study emphasizes effects of pre-analytical factors on cfDNA, revealing possible blood collection issues for analysis. Larger studies are needed. Hormones, device, age, and blood amount affect cfDNA release. A better grasp of these factors can facilitate standardization and clinical integration of cfDNA as tool a liquid biopsy for breast cancer patients.

Palavras-chave

liquid biopsy, precision medicine, breast cancer

Financiador do resumo

Área

Estudo Clínico - Tumores de Mama