新肿瘤标志物SP70在液体活检中的应用 潘世扬

McAbs library
Fast screen platform
Protein purification system
/region_name="RhoGEF" /note="Guanine nucleotide exchange factor for Rho/Rac/Cdc42-like GTPases; Also called Dbl-homologous (DH) domain.
基于肿瘤特异性抗原SP70的 循环肿瘤细胞检测技术
（SP70 for Liquid Biopsy ）
SP70 ( NJ001 Specific Antigen ) A Novel Biomarker for NSCLC
生物标志与精准医疗
SP70: a novel biomarker for NSCLC
• • • • • • • • •
Molecular Diagnostics
Circulating Tumor DNA DNA Methylation—RASSFIA, APC, B4GALT1, KIF1A, MCAM, RarB, PAK3, SSBP2, TIMP-3 Messenger RNA—CK7, EGFR, RUNX3, SCCA, SFTPB, TGFBR3, TRGC2, TRBV9 MicroRNA—miR-15b, -21, -27b, -126, -210, -486-5p, -574-5p, -1254, -let-7d, has-miR-328, hasmiR-26a, has-miR-383, has-miR-92a, msa-miR-662 Proteomics—CA19-9, CA125, cAMP, CEA, chromogranin, CYFRA 21-1, cystatin A, fibroblast growth factor, macrophage migration inhibitory factor, NSE, NNMT, SAA, thymosin ß4, ubiquitin Autoantibodies—14-3-3 theta, annexin I, C-MYC, CAGE, GBU4-5, HER2, MUC1, NY-ESO-1, p53, RPSA Breath Tests—3-p microsatellite signature, endothelin, COX-2, DNA methylation, survivin Bronchoalveolar Lavage Samples—angiopoietin 2, DNA methylation Sputum—5p15, CEP 6, epidermal growth factor, MYC, RASSFIA
SP70
NJ001
How to identify the center of target (the key biomarker)?
Proposed Lung Cancer Biomarkers
Research efforts to uncover biomarkers of early-stage lung cancer have been extensive. Numerous biomarkers in a variety of body fluids have been proposed.
The Crucial Validation Step
• Key considerations include validating the proposed marker in clinically appropriate and large enough populations, establishing its costeffectiveness, addressing practical issues about sample collection, handling, and processing, and determining the best use of the test—as part of a screening or diagnostic strategy.
A. The quarter 2-DE map of SPC-A1 cells B. Western blot detection for the target protein spot recognized by NJ001
SP70 expression in tissues
Discovery of SP70
A B
Fig1 direct inmumofluorescence assay（×400）
fluorescence
SP70 antigen located in cytoplasm and on cell membrane
C D
Discovery of SP70
Only non-small cell lung cancer cell lines express SP70 antigen!
Fig3. SP70 specific ELISA result of raw antigens from cultured cell lines
Discovery of SP70
Only NSCLC tissues express SP70! Reveals very strong signal!
Benign Lung Disease Adjacent normal tissue
ADC, lung adenocarcinoma; SCC, squamous cell lung cancer. -, +: negative, ++,+++: positive. ＊ ADC vs SCC；※ NSCLC vs Benign Lung Disease ；△ ADC vs Adjacent normal tissue
Table 2 Correlations of the NJ001 specific antigen expression with clinicopathological characteristics of lung adenocarcinoma • The relationship between tumor differentiation and NJ001 specific antigen expression was statistically significant (P =0.017). This antigen expression was also statistically associated with lymph node metastasis in lung adenocarcinoma (P < 0.001).
SP70 expression in tissues
Table 1 Expression of NJ001specific antigen in different tissues
Groups ADC NSCLC SCC Number 183 72 46 73 NJ001specific antigen 0 0 8 36 + 28 15 34 31 ++ 81 37 4 5 +++ 74 20 0 1 < 0.001※ < 0.001△ P Value 0.288＊
Liquid biopsy
CTC DETECTION
CellSearch Technique
2004, approved by FDA ，2012approved by CFDA
Liquid biopsy
Disadvantage： Non-specific not sensitive too complicated Instability A good tumor biomarker is needed!
Discovery of SP70
2-DE / Western Blot
A
KDa 250 100 75 50 PH3
B
PH3
LOCUS： CAB70682 648 aa linear PRI 26-JUL-2016 DEFINITION: hypothetical protein, partial [Homo sapiens].
新肿瘤标志物SP70在液体活检中的应用
潘世扬
MD/PhD
江苏省人民医院检验学部/南京医科大学检验学系
液体活检的概念
相对于传统的活检而言，液体活检主要 是以患者的体液作为肿瘤活检的样本，针 对体液中的 • 循环肿瘤细胞（ CTC ） • 循环肿瘤DNA （ctDNA） • 外泌体（Exosome）
Liquid biopsy
•
The positive rares of NJ001specific antigen in ADC, SCC, benign lung disease and adjacent normal tissue were 84.1%, 79.2% , 8.7% and 8.2%.
SP70 expression in tissues
DETECTION TARGET: 1. circulating tumor cell（CTC）
1869, Ashworth, Aus 2. circulating tumor DNA（cell free tumor DNA, cfDNA） 1940’s
CTC is from live tumor lesion, and ctDNA presents the dead tumor cell from tumor lesion or CTC
组织芯片
NJ001
affinity chromatography
SP70
differential expression ELISA
Pan S, Wang F, Huang P, Xu T, Zhang L, et al. (2012) , PLoS ONE 7(3): e33009. doi:10.1371/journal.pone.0033009
Adapted from: Cancer J 2011;17:3-10.
How to identify the center of target (the key biomarker)?
1. 发现 Discovery 2. 评估 Qualification 3. 确认 Verification 4. 检测方法学优化 Assay Optimization 5. 临床验证 Clinical Validation 6. 产品上市 Commercialization
• •
中国排名前十肿瘤病种的发病率和死亡率（总体），死亡率占比84.27% 每年新发肿瘤病例约为312万例，8550人/天，全国6人/分钟被诊断为癌症
1. 2012中国肿瘤登记年报，赫捷/陈万青主编， 全国肿瘤防治研究办公室
Liquid Biopsy needs more effective tumor biomarkers
•Biblioteka Baidu
SP70 expression in tissues
Prognostic value of the NJ001 specific antigen
• The overall survival rate of 102 lung adenocarcinoma patients with high (3+, n=38) expression of the antigen was significantly lower than that with low (2+, n=47) and negative (1+, n=17) expression.
Fig4. Photomicrographs of immunohistochemistry staining with NJ001 (×200).
(A) NSCLC lung adenocarcinoma; (B) NSCLC squamous lung cancer; (C) SCLC; (D) Breast carcinoma; (E) Gastric cancer; (F) Colon cancer; (G) Ovarian cancer; (H) Liver cancer; (I) Pulmonary pseudotumor; (J) Adjacent nontumourous lung tissues.