Quantum Dots-based Immunofluorescent Imaging And Quantitative Detection Of TOP2A、EGFR And Collagen Ⅳ And Identification Of Their Value In Triple Negative Breast Cancer

Breast cancer is the most common malignancy of women, the incidence of which rises every year and become the leading cause of cancer death in females worldwide. The classification of breast cancer has been more and more precise, and people pay more attention on the molecular classification. Triple negative breast cancer (TNBC), as a special subtype of breast cancer, is often aggressive and associated with a higher mortality than the other subtypes of breast cancer. TNBC is characterised by high cell proliferation, poor cellular differentiation, and relative lack of therapeutic options and poor prognosis. However, the mechanism of invasion and metastasis of TNBC is still unclear, and there is no effective biomarker, which can precisely predict the prognosis of TNBC.Quantum dot-based nanotechnology has demonstrated a promising future in preclinical and clinical research of breast cancer owing to its excellent optical and chemical properties. With the unique optical properties, the utilization of quantum dot-based nanotechnology has been expanded into a wide variety of attractive biomedical applications for cancer diagnosis, monitoring, pathogenesis, treatment, molecular pathology and heterogeneity in combination with cancer biomarkers. As the development of quantum dot-based immunofluorescent imaging and quantitative analytical system, quantum dot-based nanotechnology has expanded into single color and double color imaging and quantitave detection of multiple biomarkers in malignant tumor.Take the advantage of quantum dot-based nanotechnology, we explored the possibility of detection of TOP2A, EGFR and collagen IV in TNBC tissues using quantum dot-immunohistochemistry (IHC), and compared with traditional IHC. Through the detection of these three biomarkers, we attempted to find the effective biomarker which can reflect the sensitivity of cytotoxic chemotherapeutic drugs in TNBC tissues. Through integrating the molecules expressed in both cancer cells and the ECM, we explored the invasion and metastasis mechanism of TNBC preliminarily. We opened up a new field for predicting the clinical outcome in TNBC through integrating the molecules expressed in both cancer cells and the ECM. Through quantum dot-based immunofluorescent imaging and quantitative analytical system, we calculated the EGFR/collagen IV ratio and seek predictive and independent prognostic indicator in TNBC. As the increasing understanding of TNBC biological behaviors, we may realize personalized medicine actually for TNBC in the near future.This study was divided into two parts as follow:Part one:Quantum dot-based immunofluorescent imaging and quantitative analytical system:a new method to detect TOP2A protein in triple negative breast cancerTopoisomerase Ⅱ alpha (TOP2A) is a key enzyme in DNA replication and a target of various cytotoxic agents including anthracyclines. Previous studies evaluating the predictive and prognostic value of TOP2A in breast cancer are contradictory, likely secondary to the use of both different detection methods and different cutoff thresholds for positive status. Our previous studies confirmed the advantages of quantum dots-based nanotechnology for quantitative analysis of biomarkers compared with conventional immunohistochemistry (IHC). This study was designed to assess TOP2A by quantum dot-based immunofluorescent imaging and quantitative analytical system (QD-IIQAS); and investigate the relationship between TOP2A expression and major clinical traits in triple negative breast cancer (TNBC). TOP2A expression in 145 TNBC specimens was detected by IHC and QD-IIQAS. The comparisons between the two methods were made. The relationship between TOP2A expression and major clinical traits in TNBC was estimated. The same antigen localization, high correlation of staining rates (r=0.79), and high agreement of measurements (k=0.763) of TOP2A expression (cutoff:45%) in TNBC were found by QD-IIQAS and conventional IHC. TOP2A was significantly higher in larger tumors (p=0.002), higher grade tumors (p=0.005) and lymph node positive patients (p<0.001). QD-IIQAS was an easier and more accurate method for detecting and assessing TOP2A. Our study provides a good foundation for future studies exploring both the relationship between TOP2A expression and response to anthracyclines and the prognostic value of different level of TOP2A in TNBC patients.Part two:Quantum dot-based in situ simultaneous molecular imaging and quantitative analysis of EGFR and collagen IV and identification of their prognostic value in triple-negative breast cancerTriple-negative breast cancer (TNBC) is a unique breast cancer subtype with high heterogeneity and poor prognosis. Currently, the treatment effect of TNBC has reached a bottleneck, rendering new breakthroughs difficult. Cancer invasion is not an entirely cell-autonomous process, requiring the cells to transmigrate across the surrounding extracellular matrix (ECM) barriers. Developing a new system that integrates key constituents in the tumor microenvironment with pivotal cancer cell molecules is essential for in-depth investigation of the mechanism of invasion in TNBC. We describe a computer-aided algorithm developed using quantum dot (QD)-based multiplex molecular imaging of TNBC tissues. We performed in situ simultaneous imaging and quantitative detection of epidermal growth factor receptor (EGFR), expressed in the TNBC cell membrane, and collagen IV, the major extracellular matrix (ECM) constituent; calculated the EGFR/collagen IV ratio; and investigated the prognostic value of the EGFR/collagen IV ratio in TNBC. We simultaneously imaged and quantitatively detected EGFR and collagen IV in the TNBC samples. In all patients, quantitative determination showed a statistically significant negative correlation between EGFR and collagen Ⅳ. The 5-year disease-free survival (5-DFS) of the high and low EGFR/collagen IV ratio subgroups was significantly different. The EGFR/collagen IV ratio was predictive and was an independent prognostic indicator in TNBC. Compared to EGFR expression, the EGFR/collagen IV ratio had greater prognostic value for 5-DFS. Our findings open up a new avenue for predicting the clinical outcome in TNBC from the perspective of integrating molecules expressed in both cancer cells and the ECM.