Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal-like breast cancer patients.

AIMS: To translate a clinical research finding into daily clinical practice requires well-controlled clinical trials. We have demonstrated the usage of absolute quantitation of Ki67 and cyclinD1 protein levels to improve prognosis of Luminal-like patients based on overall survival (OS) analysis of a cohort of 155 breast cancer specimens (cohort 1). However, this finding is considered the D level of evidence (LOE) to require subsequent validation before it may be used in daily clinical practice. To set the stage for future clinical trials, our findings were validated through OS analysis of an independent cohort (cohort 2) of 173 Luminal-like patients. METHODS: Both Ki67 and cyclinD1 levels were measured absolutely and quantitatively using the Quantitative Dot Blot (QDB) method in cohort 2. The proposed cutoffs for both biomarkers from cohort 1 were re-evaluated in cohort 2 and in the merged cohort of 1 and 2, respectively, through univariate, multivariate and Kaplan-Meier survival analysis. RESULTS: The proposed cutoffs of 2.31 nmol/g for Ki67 and 0.44 µmol/g for cyclinD1 were validated as effective cutoffs in cohort 2 and the merged cohort through OS analysis. The combined use of both biomarkers allowed us to identify patients with both biomarker levels below the cutoffs (59.3%) with10-year survival probability (SP) of 89%, in comparison to those above the cutoffs (8.3%) with 8 year SP of 28% through OS analysis in the merged cohort. CONCLUSIONS: This study validated our findings that absolute quantitation of Ki67 and cyclinD1 allows effective subtyping of luminal-like patients. It sets the stage for prospective or prospective-retrospective clinical studies.

Trimethylamine N-Oxide Promotes Abdominal Aortic Aneurysm Formation by Aggravating Aortic Smooth Muscle Cell Senescence in Mice.

Trimethylamine N-oxide (TMAO) has been linked to cardiovascular disease morbidity and mortality. However, the role of TMAO in the development of abdominal aortic aneurysms (AAAs) is not known. This study investigated the association between TMAO and AAA formation. TMAO and saline were added to the drinking water of angiotensin II (AngII)- and calcium chloride (CaCl2)-induced AAA model mice, respectively. After 4 weeks, the effects of TMAO on AAA development were determined by histology and immunohistology of aortic tissue. The in vitro effects of TMAO were also examined in mouse aortic smooth muscle cells (SMCs). The maximal aortic diameter, incidence of AAA, and degree of elastin degradation were significantly increased in TMAO-treated mice. TMAO also increased the accumulation of the senescence markers p21 and p16, as well as of reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9) in vivo and in vitro. TMAO promoted AAA development in mouse AAA models induced by AngII and CaCl2 by a mechanism involving cellular senescence.