Quantum Dots-based Multispectral Imaging And Computer Image Recognition Technique To Establish Ki67 Evaluation System In Breast Cancer

Breast cancer is the most common malignant tumor and the leading cause of death for females worldwide. Compared with North American and European countries, breast cancer shows features of younger onset age, higher proportion of negative hormone receptors, and lower incidence in developing countries including China. However, those studies have been mainly conducted in socioeconomically developed coastal areas of China, there have been no large-scale clinical studies from central China, a vast region of China’s hinterland with lower socioeconomic developments. The present studies have demonstrated that recurrence and metastasis are the main causes of cancer death, which are mainly due to high cancer cells proliferation and migration. As a specifical label marker, Ki67 antigen is closely associated with prognosis of breast cancer. While the current manual Ki67 measurement method is highly experience-based, subjective, time-consuming, intra- and inter-observer variations, significantly limiting its clinical application and generalization.Computer image recognition (CIR) technique can overcome shortcomings of manual method by efficienly and objectively recognizing, extracting and analyzing high-throughput information hiding in histological image. Poor image contrast, complex texture and many background interferences in the traditional Ki67 immunohistological (IHC) staining image make it is difficult for CIR technique to recognize Ki67 positive/negative cancer cells and stromal cancer cells only based on the features of cell nuclei such as color, shape and size. Therefore, the key for establishing Ki67 evaluation system is to develop a new imaging method and optimize the CIR technique.Quantum dots (QDs) are novel fluorescent nanoparticles with excellent properties, such as high fluorescence intensity, strong resistance to photobleaching, wide excitation spectrum, narrow emission spectrum, size-tunable emission wavelength and simultaneous multiple fluorescent colors under single source excitation. Therefore, QDs based in situ and simultaneous imaging is applied to study co-expression, space-time relationship and interaction of multiple targets. As a novel imaging technique, multispectral imaging can transform black-white or color RGB images with low contrast into spectral images with high color dimension. By unmixing procedure, multiple targets spectrum are unmixed into individual images to remove background auto-fluorescence from the sample and significantly increase the signal-to-noise ratio of the image to enhance the image quality and sharpness, and analysis information of multiple targets imaging.Based on analysis on breast cancer clinico-pathological features, this study aimed at establishing a breast cancer Ki67 evaluation system by combining QDs-based multiple imaging and multispectral imaging to label multiple targets associated with Ki67 expression in breast cancer. Additionally, the CIR technique was developed to recognize and analyze co-expression, space-time relationship and interaction of Ki67 associated multiple molecules. This study was divided into four parts, as the following:Part 1. Analysis of cinico-pathological characteristics for breast cancer in Wuhan city and surrounding areaBackground Breast cancer is the most common malignant cancer of globe female. Analysis on clinico-pathological features of breast cancer is very important for breast cancer screening, diagnosis and treatment. This study aimed at analyzing clinico-pathological features of three tertiary-referral hospital in Wuhan city to provide assistances for breast cancer screening, diagnosis and treatment.Methods Clinico-pathological information on breast cancer including age distributions, surgery patterns, histological types, TNM stages, and hormone receptor status from three tertiary referral hospitals in Wuhan city was collected and analyzed to explore characteristics of breast cancer in central China.Result From January 1995 to December 2012,2,525 cases of breast cancer with complete clinico-pathological information were collected. Analysis on age distribution indicated that the peak incidence is 45-49 years with a median age 50 years in Wuhan city. Modified radical mastectomy, radical mastectomy, breast-conserving surgery and simple mastectomy accounted for 71.8%(1,812/2,525),18.0% (454/2,525),5.2%(131/2,525) and 2.6% (66/2,525), respectively. Proportions of breast-conserving surgery in age< 44-year group (68/132,51.5%) and simple mastectomy in age> 60-year group (57/89,64.0%) were higher than other age groups. Invasive ductal carcinoma not otherwise specified is the most frequent pathological type (81.0%,1,982/2,446). TNM 0-1, II and III were accounted for 28.0% (682/2,441)^ 48.4%(1,180/2,441) and 23.7% (578/2,441), respectively. Distribution of N stage showed that NO accounted for 54.5%(1,345/2,471), and proportion of NO rose from 51.1%(157/307) in 30-39-year age group to 64.3%(110/171) in≥70-year age group, by an average increase of 3.3% in each age group (P=0.029). Breast cancer with positive estrogen receptor accounted for 53.0%(1,107/2,112). Compared with developed countries, this study showed earlier age patients, lower proportion of breast-conserving surgery, and higher proportion of negative estrogen receptor were existed in Wuhan area of China central.Conclusion Clinico-pathological characteristics in Wuhan city and surrounding area showed earlier incidence age, lower proportion of breast-conserving surgery and positive estrogen receptor patients than developed countries and areas. Additional classification systems should be developed to guide grading of early breast cancer more accurately, especially for NO patients. Invasive ductal carcinoma is the focus for intensive research.Part 2. Relationship between morphological parameters of breast invasive ductal carcinoma nests and prognosis of patients based on computer image recognition techniqueBackground The invasive behaviors of cancer nests are the main reflects of IDC’s malignant biological behavior. This study aimed at establishing a CIR method to explore the relationship between mathematical parameters of cancer nests and prognosis of breast cancer.Methods In present work, the CIR method was developed to automatically extract morphological parameters of IDC cancer nests which were specifically stained with cytokeratin (CK) by IHC method. The relationship between cancer nests’ parameters and patients’disease free survival (DFS) was studied.Results The developed CIR method successfully extracted 8 morphological parameters of cancer nests. The Kaplan-Meier analysis showed 7 of them were associated with patients’ DFS. Three nagetive morphological parameters (number, circularity and total perimeter) with area under curve> 0.5 and 4 positive mathematical parameters (average area, average perimeter, total area/total perimeter, average (area/perimeter)) with area under curve< 0.5 in ROC analysis were combined into integrated parameter 1 and integrated parameter 2, respectively. Multivariate analysis showed that integrated parameter 1 (P=0.040) was independent prognostic factor of patients’DFS. The hazard risk (HR) of integrated parameter 1 was 1.454 (95% CI [1.017-2.078]), higher than that of N stage (HR 1.396,95% CI [1.125-1.733]) and hormone receptor status (HR 0.575,95% CI [0.353-0.936]), but lower than that of histological grading (HR 3.370,95%CI [1.125-5.364]) and T stage (HR 1.610, 95% CI [1.026-2.527]).Conclusions This study indicated that analysis on morphological parameters of cancer nests extracted by CIR technique is helpful to predict the prognostic information of early stage breast cancer patients without lymph node metastasis.Part 3. Quantum dots-based multispectral imaging improve Ki67 evaluation in breast cancer and its prognostic significanceBackground As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer prognosis. Although IHC staining-based manual method is the current standard to evaluate Ki67, variations in analytical practice seriously limit its clinical application. This study was aimed to develop a quantum dots (QDs)-based quantitative multispectral imaging technique to analysis Ki67 expression and its prognostic significance.Methods A QDs-based in situ multispectral fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic CK as green signal. Both Ki67 and CK signals were automatically separated and quantified by professional spectrum analysis software. This technique was applied to tissue microarrays from 240 breast cancer patients. Both Ki67 and CK values, and Ki67/CK ratio were obtained for each patient, and their prognostic value on 5-year disease free survival was assessed.Results This method simultaneously stains nuclear Ki67 and cytoplasmic CK with clear signal contrast, making it easy for signal separation and quantification. The total fluorescent signal intensities of both Ki67 sum and CK sum were obtained, and Ki67/CK ratio calculated. Ki67 sum and Ki67/CK ratio were each attributed into two grades by X-tile software based on the best P value principle. Multivariate analysis showed Ki67 grade (P-0.047) and Ki67/CK grade (P=0.004) were independent prognostic factors. Furthermore, area under curve (AUC) of ROC analysis for Ki67/CK grade (AUC:0.683,95%CI:0.613-0.752) was higher than Ki67 grade (AUC:0.665,95%CI:0.596-0.734) and HER-2 gene (AUC:0.586,95%CI: 0.510-0.661), but lower than N stage (AUC:0.760,95%CI:0.696-0.823) and histological grade (AUC:0.756,95%CI:0.692-0.820) on predicting the risk for recurrence.Conclusions A QDs-based quantitative and in situ multispectral imaging on Ki67 and CK was developed to improve Ki67 assessment in breast cancer, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.Part 4. Multispectral imaging and computer image recognition technique establish an automatic method to evaluate tumor Ki67 indexBackground As a widely used proliferative marker, Ki67 has important impacts on breast cancer prognosis. However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index based on traditional IHC staining. This study is aimed at establishing QDs-based double imaging and organic dye imaging to automatically count breast cancer Ki67 index by developing a CIR method.Methods A QDs-based double imaging and organic dye imaging technique was developed, which stained nuclear Ki67 as red signal by QDs-655, cytoplasmic CK as yellow signal by QDs-585, and cell nucleus as blue signal by organic dye 4’,6-diamidino-2-phenylindole (DAPI). Ki67, CK and DAPI signals were automatically separated by professional CRi Nuance spectrum analysis software. An automatic CIR technique was developed to count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 (C-Ki67), visual Ki67 (V-Ki67), and marked Ki67 (M-Ki67) indexes were performed to access their Ki67 counting performances.Results The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67≤10%(low grade),10%< Ki67<50%(moderate grade), and Ki67> 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades.Conclusion Based on QDs-based double imaging and organic dye imaging on breast cancer tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index actually and objectively, which is helpful to diagnosis and analyze cancer associated molecules.