RESUMEN
BACKGROUND: Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis. METHODS: We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis. RESULTS: Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level. CONCLUSIONS: Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients.
RESUMEN
BACKGROUND: Opting for or against the administration of adjuvant chemotherapy in therapeutic management of stage II colon cancer remains challenging. Several studies report few survival benefits for patients treated with adjuvant therapy and additionally revealing potential side effects of overtreatment, including unnecessary exposure to chemotherapy-induced toxicities and reduced quality of life. Predictive biomarkers are urgently needed. We, therefore, hypothesise that the spatial tissue composition of relapsed and non-relapsed colon cancer stage II patients reveals relevant biomarkers. METHODS: The spatial tissue composition of stage II colon cancer patients was examined by a novel spatial transcriptomics technology with sub-cellular resolution, namely in situ sequencing. A panel of 176 genes investigating specific cancer-associated processes such as apoptosis, proliferation, angiogenesis, stemness, oxidative stress, hypoxia, invasion and components of the tumour microenvironment was designed to examine differentially expressed genes in tissue of relapsed versus non-relapsed patients. Therefore, FFPE slides of 10 colon cancer stage II patients either classified as relapsed (5 patients) or non-relapsed (5 patients) were in situ sequenced and computationally analysed. RESULTS: We identified a tumour gene signature that enables the subclassification of tissue into neoplastic and non-neoplastic compartments based on spatial expression patterns obtained through in situ sequencing. We developed a computational tool called Genes-To-Count (GTC), which automates the quantification of in situ signals, accurately mapping their position onto the spatial tissue map and automatically identifies neoplastic and non-neoplastic tissue compartments. The GTC tool was used to quantify gene expression of biological processes upregulated within the neoplastic tissue in comparison to non-neoplastic tissue and within relapsed versus non-relapsed stage II colon patients. Three differentially expressed genes (FGFR2, MMP11 and OTOP2) in the neoplastic tissue compartments of relapsed patients in comparison to non-relapsed patients were identified predicting recurrence in stage II colon cancer. CONCLUSIONS: In depth spatial in situ sequencing showed potential to provide a deeper understanding of the underlying mechanisms involved in the recurrence of disease and revealed novel potential predictive biomarkers for disease relapse in colon cancer stage II patients. Our open-access GTC-tool allowed us to accurately capture the tumour compartment and quantify spatial gene expression in colon cancer tissue.