ABSTRACT
AIMS: We investigated key signalling pathways' activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence. METHODS: This is a retrospective case-control study of 18 patients with recurrent breast carcinoma with low and intermediate 21-gene RS (<25) and control group of 15 non-recurrent breast cancer patients. DNA and mRNA were extracted from tumour tissue. mRNA expression of genes involved in oestrogen receptor (ER), androgen receptor (AR), PI3K and MAPK signalling pathways was measured by real-time quantitative reverse transcription-qPCR (OncoSIGNal G4 test, InnoSIGN). Tumour mutational landscape was assessed by targeted DNA sequencing (Oncomine Precision Assay). RESULTS: There were no statistical differences between the groups' demographic and clinicopathological characteristics. PI3K pathway showed significantly higher activity in cases compared with controls (p=0.0014). Receiver operating characteristic curve analysis showed an area under the curve of 0.79 for PI3K pathway activity in the prediction of recurrent disease in low and intermediate 21-gene RS breast cancer. There was no difference in ER, AR and MAPK pathway activity. PIK3CA alterations were the most common driver mutations, but no difference was found between the groups (p=0.46) and no association with PI3K pathway activity (p=0.86). Higher Ki67 gene expression was associated with recurrences (p=0.042) CONCLUSION: Increased PI3K pathway activity, independent of PIK3CA mutations, may play a role in the recurrence of early-stage breast cancer with low and intermediate 21-gene RS. Pathway analysis can help to identify high-risk patients in this setting.
ABSTRACT
For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml), the novel non-enzymatic procedure (Polaris, 1-5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67%) and EasyMAG (58-79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.
