title: Quantum dots-based quantitative and in situ multiple imaging on ki67 and cytokeratin to improve ki67 assessment in breast cancer.
abstract: BACKGROUND
  As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer (BC) prognosis. Although immunohistochemical staining is the current standard method, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study was to develop a fluorescent spectrum-based quantitative analysis of Ki67 expression by quantum-dots (QDs) multiple imaging technique.
  
  
  METHODS
  A QDs-based in situ multiple fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic cytokeratin (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 BC 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 multiple imaging on Ki67 and CK was developed to improve Ki67 assessment in BC, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.
authors: Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan, Hubei, China.
authors: Jing Ping
authors: JP
authors: Yuan
authors: Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan, Hubei, China.
authors: Lin Wei
authors: LW
authors: Wang
authors: School of Computer, Wuhan University, Wuhan, Hubei, China.
authors: Ai Ping
authors: AP
authors: Qu
authors: Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan, Hubei, China.
authors: Jia Mei
authors: JM
authors: Chen
authors: Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan, Hubei, China.
authors: Qing Ming
authors: QM
authors: Xiang
authors: Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan, Hubei, China.
authors: Chuang
authors: C
authors: Chen
authors: Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan, Hubei, China.
authors: Sheng-Rong
authors: SR
authors: Sun
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, Hubei, China.
authors: Dai-Wen
authors: DW
authors: Pang
authors: School of Computer, Wuhan University, Wuhan, Hubei, China.
authors: Juan
authors: J
authors: Liu
authors: Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, Wuhan, Hubei, China.
authors: Yan
authors: Y
authors: Li
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000401:mortality / Q000473:pathology; D003116:Color; D005260:Female; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000235:genetics*; D019394:Ki-67 Antigen / Q000235:genetics*; D008875:Middle Aged; D015999:Multivariate Analysis; D009367:Neoplasm Staging; D061848:Optical Imaging / Q000379:methods* / Q000592:standards; D011379:Prognosis; D045663:Quantum Dots / Q000737:chemistry*; D012372:ROC Curve; D012984:Software; D016019:Survival Analysis; D046888:Tissue Array Analysis
chemical_list: D014408:Biomarkers, Tumor; D019394:Ki-67 Antigen; D007633:Keratins