Dados do Resumo

Título

Validation of Histone H2A and Cystatin A as Diagnostic and Prognostic Biomarkers in Triple-Negative Breast Cancer

Introdução

This study, approved by the Human Research Ethics Committee of the Faculty of Medicine of São José do Rio Preto under protocol number 001-0004391/2019, addresses breast cancer, with an emphasis on the triple-negative subtype (TNBC) characterized by its unfavorable prognosis and limited therapeutic options. Using plasma-derived extracellular vesicles (EVs), we identified, through proteomic analysis, histone H2A (H2A) and cystatin A (CSTA) as potential biomarkers in TNBC. H2A, linked to DNA compaction, and CSTA, a protease inhibitor, were detected in EVs from patients, suggesting roles in tumor progression and resistance.

Objetivo

To validate the proteins histone H2A and cystatin A as diagnostic and prognostic biomarkers, confirming their expression in tumor tissue samples of TNBC by immunohistochemical assay.

Métodos

The H2A and CSTA proteins were selected for validation after isolation from EVs and proteomic analysis, as they stand out as potential biomarkers in TNBC. The breast tumor tissue samples were collected by the medical team at Hospital de Base de São José do Rio Preto and included in paraffin blocks, which were cut into 3-4 μm sections, prepared on silanized glass slides and taken to the oven for about 2h30min. Afterwards, deparaffinization was carried out in xylene, alcohol and distilled water, followed by antigen recovery in preheated citrate buffer (pH 6.0) for 30 minutes at 95 ºC. The slides were washed with PBS and treated with hydrogen peroxide to inhibit endogenous peroxidase for 15 minutes, followed by blocking of non-specific proteins. They were then incubated with anti-H2A (1:600) and anti-CSTA (1:300) antibodies overnight at 4 ºC. After washing, the HRP enzyme marker was added, followed by the DAB chromogen for 2 minutes. The slides were counterstained with Harris hematoxylin and dehydrated. Negative controls omitted antibodies; positive controls were appendix (H2A) and liver (CSTA). Expressions were quantified by Histoscore and Densitometry in Image J software.

Resultados

Nuclear and cytoplasmic markings of the H2A protein were obtained in 7 of the 8 TNBC samples, with Histoscore values ranging from 100-200. In one sample, no significant labeling was observed, resulting in a very low Histoscore. In the HER2+, Luminal A and Luminal B subtype samples, predominantly low-intensity cytoplasmic labeling was identified, with nuclear labeling being scarce and equally weak. In contrast, benign tumor samples showed nuclear and cytoplasmic markings, but with lower intensity than those observed in cancers. The CSTA protein showed cytoplasmic labeling in all 8 TNBC samples, with Histoscores between 90 and 200. In samples of the HER2+, Luminal A and Luminal B subtypes, and in benign tumors, the cytoplasmic markings were of strong intensity, similar to those observed in triple-negative tumors.

Conclusões

Overall, both proteins were detected in all tissue types. H2A showed reduced expression in the other breast cancer subtypes and in the benign tumor, while it was increased in TNBC. CSTA showed similar expression in all the tissues examined. The next stage of the study involves checking the expression of these proteins in blood samples isolated from EVs, to assess their potential as biomarkers in a broader, non-invasive context.

Financiador do resumo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - nº 2020/12970-8

Palavras Chave

Breast Neoplasms; H2A; biomarkers