Optimization of immunofluorescence protocols for detection of biomarkers in colorectal and breast cancer tissues

Cancer remains an undetermined question for modern medicine. Every year millions of people ranging from children to adult die since the modern treatment is unable to meet the challenge. Research must continue in the area of new biomarkers for tumors. Molecular biology has evolved during last years; however, this knowledge has not been applied into the medicine. Biological findings should be used to improve diagnostics and treatment modalities. In this thesis, human formalin-fixed paraffin embedded colorectal and breast cancer samples were used to optimize the double immunofluorescence staining protocol. Also, immunohistochemistry was performed in order to visualize expression patterns of each biomarker. Concerning double immunofluorescence, feasibility of primary antibodies raised in different and same host species was also tested. Finally, established methods for simultaneous multicolor immunofluorescence imaging of formalin-fixed paraffin embedded specimens were applied for the detection of pairs of potential biomarkers of colorectal cancer (EGFR, pmTOR, pAKT, Vimentin, Cytokeratin Pan, Ezrin, E-cadherin) and breast cancer (Securin, PTTG1IP, Cleaved caspase 3, ki67).Siirretty Doriast

Histological detection and prognostic value of regulators of metaphase-anaphase transition in breast cancer

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

Proliferation-associated miRNAs-494, -205, -21 and -126 detected by in situ hybridization: expression and prognostic potential in breast carcinoma patients

PurposeTo visualize by in situ hybridization (ISH) the levels of a set of proliferation-associated miRNAs and to evaluate their impact and clinical applicability in prognostication of invasive breast carcinoma.MethodsTissue specimen from breast carcinoma patients were investigated for miRNAs-494, -205, -21 and -126. Prognostic associations for levels of miRNAs were analyzed based on complete clinical data and up to 22.5-year follow-up of the patient material (n = 285). For detection of the miRNAs, an automated sensitive protocol applying in situ hybridization was developed.ResultsMiRNA-494 indicated prognostic value for patients with invasive breast carcinoma. Among node-negative disease reduced level of miRNA-494 predicted 8.5-fold risk of breast cancer death (p = 0.04). Altered levels and expression patterns of the studied miRNAs were observed in breast carcinomas as compared to benign breast tissue.ConclusionsThe present paper reports for the first time on the prognostic value of miRNA-494 in invasive breast cancer. Particularly, detection of miRNA-494 could benefit patients with node-negative breast cancer in identifying subgroups with aggressive disease. Based on our experience, the developed automatic ISH method to visualize altered levels of miRNAs-494, -205, -21 and -126 could be applied to routine pathology diagnostics providing that conditions of tissue treatment, especially fixation delays, are managed.</div

PTTG1-interacting protein (PTTG1IP/PBF) predicts breast cancer survival

Background: PTTG1-interacting protein (PTTG1IP) is an oncogenic protein, which participates in metaphase-anaphase transition of the cell cycle through activation of securin (PTTG1). PTTG1IP promotes the shift of securin from the cell cytoplasm to the nucleus, allowing the interaction between separase and securin. PTTG1IP overexpression has been previously observed in malignant disease, e.g. in breast carcinoma. However, the prognostic value of PTTG1IP in breast carcinoma patients has not previously been revealed. Methods: A total of 497 breast carcinoma patients with up to 22-year follow-up were analysed for PTTG1IP and securin immunoexpression. The results were evaluated for correlations with the clinical prognosticators and patient survival. Results: In our material, negative PTTG1IP immunoexpression predicted a 1.5-fold risk of breast cancer death (p = 0.02). However, adding securin immunoexpression to the analysis indicated an even stronger and independent prognostic power in the patient material (HR = 2.5, p < 0.0001). The subcellular location of securin was found with potential prognostic value also among the triple-negative breast carcinomas (n = 96, p = 0.052). Conclusions: PTTG1IP-negativity alone and in combination with high securin immunoexpression indicates a high risk of breast cancer death, resulting in up to 14-year survival difference in our material.peerReviewe