The study focuses on proteins Securin, Pttg1IP and Separase, involved in
metaphase-anaphase transition of mitosis, and microRNAs -494, -205, -21 and
-126 in association with cell proliferation. The aim is to demonstrate the
expression patterns of the studied proteins and levels of microRNAs, and to
assess their applicability in predicting the outcome of patients diagnosed with
invasive breast cancer. In addition, the influence of fixation delay and time
on the staining results of fresh breast cancer tissue is studied.

The study comprises 447 invasive breast carcinomas and a total of 143
triple-negative breast carcinomas diagnosed in Central Hospital of Central
Finland during 1987 – 1997 and Turku University Hospital, Turku, Finland,
during 2005 – 2015 with complete clinical data and a maximum follow-up period
of 22 years. The tissue material was arranged in tissue microarrays. The tissue
sections were prepared by immunohistochemical and double and triple
immunofluorescence stainings for the investigation of the proteins. An
automated in situ hybridization method was developed for identifying the
studied miRNAs.

Securin, Pttg1IP and Separase showed divergent expression patterns in
normal breast epithelium as compared to breast carcinomas, and between specific
cancer subgroups. The studied proteins were associated with decreased
disease-specific outcome at a statistically significant level. In multivariate
analysis involving traditional prognostic features of breast cancer, Securin
and Separase were independently associated with higher breast cancer mortality
(HR 2.4, p <0.0001 and HR 1.8, p=0.002, respectively). In some breast cancer
subgroups abnormal levels of -494 and -205 were detected. In local disease
negative for microRNA-494 significantly higher breast cancer mortality was
observed (HR 8.5, p=0.04). Also multivariate analysis involving traditional
prognostic features of breast cancer suggested independent prognostic value for
microRNA-494 (HR 2.1, p=0.04). Variations in tissue fixation and processing
were tested in sets of breast specimen revealing the profound impact of
pre-analytical incidents on detecting proteins and microRNAs histochemically.