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

PURPOSE: To 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. METHODS: Tissue 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. RESULTS: MiRNA-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. CONCLUSIONS: The 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.

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.

Separase is a marker for prognosis and mitotic activity in breast cancer.

BACKGROUND: Cancer cell proliferation is a critical feature in classifying and predicting the outcome of breast carcinoma. Separase has a central role in cell cycle progression in unleashing sister-chromatids at anaphase onset. Abnormally functioning separase is known to lead to chromosomal instability. METHODS: The study comprises 349 breast carcinoma patients treated in Central Hospital of Central Finland. The prognostic value, role as a proliferation marker and regulatory interactions of separase are evaluated by immunohistochemical and double- and triple-immunofluorescence (IF) detections based on complete clinical data and >22-year follow-up of the patient material. RESULTS: In our material, abnormal separase expression predicted doubled risk of breast cancer death (P<0.001). Up to 11.3-year survival difference was observed when comparing patients with and without separase expressing cancer cell mitoses. Particularly, abnormal separase expression predicted impaired survival for luminal breast carcinoma (P<0.001, respectively). In multivariate analyses, abnormal separase expression showed independent prognostic value. The complex inhibitory interactions involving securin and cyclin B1 were investigated in double- and triple-IFs and revealed patient subgroups with aberrant regulation and expression patterns of separase. CONCLUSIONS: In our experience, separase is a promising and clinically applicable proliferation marker. Separase expression shows strong and independent prognostic value and could be developed into a biomarker for treatment decisions in breast carcinoma, particularly defining prognostic subgroups among luminal carcinomas.

The Expression of Cohesin Subunit SA2 Predicts Breast Cancer Survival.

Cohesin is one of the main regulators of sister chromatid separation during the metaphase/anaphase transition. It is a multiprotein complex consisting of 4 core subunits, one of those being the SA2 subunit. SA2 plays the final role in dismantling the cohesion complex from the sister chromatids and also functions in DNA double-strand break repair and gene regulation. There is increasing evidence regarding the involvement of both overexpression and underexpression of cohesin in cancer. Here, we present expression patterns of SA2 in different types of human breast tissue, and the prognostic analysis in the material from breast cancer patients with long-term follow-up. SA2 immunoexpression was evaluated in benign, precancerous, and malignant breast tissue, and was classified into low-intensity or high-intensity groups. The DNA content was determined by image cytometry on breast cancer cell imprints. Prognostic analyses were based on 445 breast cancer patients with upto 20 years' follow-up. SA2 immunoexpression was equally high in both benign and precancerous breast tissue. Instead, 72% of the invasive breast cancers showed deficient SA2 expression. These patients were also associated with an unfavorable outcome as indicated by a 1.6-fold risk of breast cancer death (P=0.0208). The majority (75%) of the patients with low SA2 expression were alive 6.0 years after the diagnosis, whereas the majority of the patients with high SA2 expression survived 17.6 years after the diagnosis. No statistically significant association could be detected between SA2 immunoexpression and DNA aneuploidy. Our results and previous literature indicate that decreased SA2 immunoexpression is associated with malignant breast disease and a particularly unfavorable course of disease.

Low cdc27 and high securin expression predict short survival for breast cancer patients.

Cell cycle regulators cdc27 and securin participate in control of the mitotic checkpoint and survey the mitotic spindle to maintain chromosomal integrity. This is achieved by their functions in metaphase-anaphase transition, DNA damage repair, enhancement of mitotic arrest and apoptosis. We report on the roles of cdc27 and securin in aneuploidy and prognosis of breast cancer. The study comprises 429 breast cancer patients with up to 22 years of follow-up. DNA content was determined by image cytometry, and immunopositivity for cdc27 and securin was based on tissue microarrays. An inverse association between cdc27 and securin expression was observed in both image cytometric and immunohistochemical analyses. Low cdc27 and high securin expression identified patients with significant difference in disease outcome. Cdc27 and securin immunoexpression identified patients at risk of early cancer death within five years from diagnosis. In multivariate analysis, the combination of cdc27 and securin immunohistochemistry was the strongest predictor of cancer death after lymph node status. We demonstrate, for the first time in human breast cancer, the prognostic value of cdc27 and securin immunohistochemistry. Cdc27 and securin appear promising biomarkers for applications in predicting disease progression, prognostication of individual patients and potential in anti-mitotic drug development.

Securin predicts aneuploidy and survival in breast cancer.

AIMS: Securin is known to participate in maintaining chromosomal integrity during the cell cycle through regulation of metaphase-anaphase transition, DNA damage repair, and apoptosis. The aim of this study was to investigate the role of securin in aneuploidy and prognosis in human breast cancer. METHODS AND RESULTS: The study was based on 603 breast cancer patients with up to 20 years of follow-up. DNA content was determined by image cytometry on cell imprints, and securin immunohistochemistry was performed on tissue microarrays of breast cancer tissue. We show, for the first time in human breast cancer, that high-level securin expression predicts abnormal DNA content, with up to 9.8-fold odds for aneuploid DNA content (P = 0.0007). Securin also shows strong independent prognostic value for disease-specific survival, with a significant difference in survival time between patients with low-level and high-level securin expression. CONCLUSIONS: The main result of the present study is the association of aneuploidy and securin expression. According to our results, securin immunohistochemistry is also a potential new prognosticator for treatment decisions concerning breast cancer patients.

Protein and gene expression of estrogen receptor alpha and nuclear morphology of two breast cancer cell lines after different fixation methods.

We assessed morphology and ERalpha protein and gene expression of two breast cancer cell lines after three different fixatives: neutral-buffered 10% formaldehyde, LN-FIX and FineFIX and varying fixation times. We found that the cell morphology was best preserved in cells fixed with LN-FIX. Two commercial fixatives used in this study shrank cells less than formalin. In immunohistochemical assay samples were stained with two different ERalpha antibodies, clone 1D5 and clone SP1. All tested fixatives were suitable for immunohistochemistry. Staining was more intensive and the number of stained cells was larger with the clone 1D5 than with the clone SP1. Our gene expression analysis showed that formalin and LN-FIX preserve the ERalpha better than FineFIX, which is advertised to be optimal for molecular analysis. Our study suggests that tissues fixed with formalin are suitable also for molecular biology assays. This makes possible to research formalin-fixed paraffin-embedded archival tissues also with molecular techniques.

Her-2/neu oncogene amplification and protein over-expression in interval and screen-detected breast cancers.

BACKGROUND: Studies of Her-2/neu protein expression in interval and screen-detected breast cancers have been conflicting. Therefore we assessed the Her-2/neu amplification status in these groups by using in situ hybridization. PATIENTS AND METHODS: The Her-2/neu oncogene amplification and protein over-expression were compared in 79 screen-detected and 39 interval breast carcinomas, using a novel and well-documented chromogenic in situ hybridization (CISH) method and immuhistochemistry. RESULTS: There were 33% CISH-positive cases in the interval cancer group and only 4% CISH-positive cases in the screen-detected group. The odds ratio for interval cancer was 14.1 (95% CI 2.2 to 91.0) in the CISH-positive tumours compared with CISH-negative tumours after adjustment for nodal status, tumour size and histological grade. CONCLUSION: Our results indicate, for the first time, that there is a clear-cut difference in Her-2/neu amplification between screen-detected and interval breast carcinomas. The amplification appears to be a rare event in screen-detected cancers but distinctly frequent in interval cancers when compared to cancers described in earlier studies.