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1 Osteopontin CB

2 Osteopontin OPN non-small cell lung cancer NSCLC OPN 35 NSCLC PCR RT-PCR OPN RNA 662 OPN NSCLC RT-PCR OPN 80% NSCLC OPN 59.6% 25.2% P<0.001 OPN OPN % 49.7% P % 75.5% P OPN OR OPN Osteopontin 2

3 ABSTRACT Over-expression of Osteopontin in non-small cell lung cancers: its clinical significance HU Zhi, XIAO Ting, LIN Dongmei, GUO Suping, ZHANG Ziqiang, DI Xuebing, CHENG Shujun, GAO Yanning Cancer Institute/Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, Objective Data obtained from a differentially expressed cdna library constructed previously in this laboratory demonstrated that the extracellular matrix molecule Osteopontin (OPN) was one of the most considerably over-expressed genes in non-small cell lung cancers (NSCLCs). The purpose of the present study is to explore expressive status of OPN in a large scale tissue sample of NSCLCs, and estimate its significance in progression of the malignant disease. Methods RT-PCR was performed with the tumor and adjacent normal tissues of 35 patients with NSCLC, for the transcriptional levels of OPN. To determine the expression of OPN protein in the tumor tissues, immunohistochemical (IHC) staining was subsequently carried out on paraffin-embedded sections, in manner of tissue microarrays containing 662 samples, derived from NSCLC patients. The correlation between the expressive levels of OPN and clinical characteristics was then analyzed by statistical process. Results Comparing with the paired normal lung tissue, high level RNA of OPN was detected in 80.0% (28/35) of the tumor tissues of NSCLC by RT-PCR, which validated the information contributed previously by our differentially expressed cdna library. The 3

4 results of IHC analysis showed that positively stained OPN protein was observed in 59.6% (331/555) of the tumor tissues, which is remarkably higher than that (25.2%, 27/107) detected in the normal control tissues (P<0.001). Among the NSCLCs investigated, over-expressed OPN was more frequently found in squamous cell carcinomas (SCCs) than in adenocarcinomas. A further analysis on the SCCs demonstrated that the rates of over-expressed OPN were significantly different between the primary tumors with and without lymphatic metastases (68.6% vs. 49.7%, P=0.001), but similar in the primary tumors and their corresponding metastases in lymph node (68.6% vs. 75.5%, P=0.171). Conclusions Expression of OPN protein was distinctly increased in NSCLCs, particularly in SCCs. OPN over-expression was considerably correlated with lymph node invasion and aggrandized the risk of tumor metastasis (OR=2.212). The resulting data suggest that the OPN facilitates the progression of NSCLCs. Keywords Osteopontin; non-small cell lung cancers 4

5 Osteopontin OPN OPN OPN OPN OPN OPN OPN non-small cell lung cancer NSCLC cdna RT-PCR mrna OPN OPN RT-PCR 25 squamous cell carcinoma, SCC 8 adenocarcinoma, ADC 2 adenosquamous carcinoma, ASC UICC TNM 5

6 1.1.3 OPN Santa Cruz Biotechnology SC SP9003 Oligo 6.0 RT-PCR OPN 5 CTG CCA GCA ACC GAA GTT T3 OPN 5 ACC ATT CAA CTC CTC GCT TTC3 PCR 183bp RNA RNA TRIzol TM Invetrogen RNA DEPC -70ºC RT-PCR RNA A 5µg RNA 1µl 10mM dntp 0.5µg Oligo(dT) µg 10µl 65ºC 5 B 2 µl 10 buffer 1µl 0.1M dtt 1µl RNAse Inhibitor 9µl 25ºC 10 1 µl 5U SuperScript II Invetrogen A B 42ºC 60 70ºC 15 cdna -20ºC PCR 1µl cdna 4µl 5 buffer 1µl 10mM dntp 0.5µl 0.5µl 0.5U Taq 20µl PCR 95ºC ºC 40 54ºC 30 72ºC 30 72ºC OPN SP OPN % 2+ 5% SPSS 11.5 SPSS Inc. Chicago Pearson OPN OR odds risk OR 95% confidence interval CI OPN NSCLC p<0.05 6

7 OPN OPN RT-PCR 35 NSCLC OPN β-actin % OPN mrna OPN RT-PCR OPN 35 RT-PCR NSCLC OPN OPN 2 OPN OPN OPN % NSCLC OPN 59.6% % P <0.001 NSCLC SCC OPN 60.0% ADC 32.4% % 7 18 SCC OPN 49.7% 68.6% 2 P OR % CI OPN 75.5% P OPN OPN C-myc H-ras OPN mrna Ras 7

8 OPN Adwan OPN MDA-MB-231 OPN OPN RGD- RGD- Integrin αvβ3 αvβ5 αvβ1 CD44 RT-PCR OPN RNA cdna 662 OPN NSCLC P < OPN SCC 60.0% ADC 32.4% NSCLC 38.8% Zhang OPN 16 SCC 68.8% 24 ADC 20.8% Shijubo OPN 32 SCC 55 ADC 31.2% 54.5% OPN OPN 2 OPN OR OPN Ras OPN OPN Ras OPN OPN Ras Ras OPN SCC OPN Ras ADC Ras OPN Tcf-4 Sp1 Myc Oct-1 OPN OPN 8

9 1. Ding Q, Stewart J Jr, Prince CW, et al. Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain: differences in integrin signaling during cell adhesion to osteopontin versus vitronectin. Cancer Res. 2002, 62: Denhardt DT, Mistretta D, Chambers AF, et al. Transcriptional regulation of osteopontin and the metastatic phenotype: evidence for a Ras-activated enhancer in the human OPN promoter. Clin Exp Metastasis. 2003, 20: Liaw L, Birk DE, Ballas CB, et al. Altered wound healing in mice lacking a functional osteopontin gene (sppl). J Clin Invest. 1998, 101: Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res. 2004, 10(pt1): TPX Rittling SR and Novick KE. Osteopontin expression in mammary gland development and tumorigenesis. Cell Growth Differ. 1997, 8: Behrend EI, Craig AM, Wilson SM, et al. Reduced malignancy of ras-transformed NIH 3T3 cells expressing antisense osteopontin RNA. Cancer Res. 1994, 54:

10 8. Rudland PS, Platt-Higgins A, El-Tanani M, et al. Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res. 2002, 62: Agrawal D, Chen T, Irby R, et al. Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling. J Natl Cancer Inst. 2002, 94: Adwan H, Bauerle TJ, Berger MR. Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther. 2003, 11: Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem. 2004, 279: Zhang J, Takahashi K, Takahashi F, et al. Differential osteopontin expression in lung cancer. Cancer Lett. 2001, 171: Shijubo N, Uede T, Kon S, et al. Vascular endothelial growth factor and osteopontin in stage I lung adenocarcinoma. Am J Respir Crit Care Med 1999, 160: Wu Y, Denhardt DT, Rittling SR. Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br J Cancer. 2000, 83: Denhardt DT. Commentary re: Q-T Le et al, identification of osteopontin as a prognostic marker for Head and Neck Squamous Cell Carcinomas. clin cancer res, 9: 31-32, Clin Cancer Res. 2003, 9:

11 1 OPN OPN n % χ p<0.001 a SCC ADC p<0.001 b p c SCC a b SCC ADC c SCC 11

12 2 OPN OPN n % χ p a + * * p b * a + OR= % CI b + 12

13 1 RT-PCR Osteopontin RNA M 100bp DNA T N OPN 183bp β-actin 228bp 13

14 2 OPN A B C OPN 14

分级和气管是否受侵是影响预后的独立危险因素关键词状腺肿瘤 ; 癌, 乳头状 ; 预后 Clinical outcomes of 600 papillary thyroid carcinoma patients ZHANG Zong-min,

分级和气管是否受侵是影响预后的独立危险因素关键词状腺肿瘤 ; 癌, 乳头状 ; 预后 Clinical outcomes of 600 papillary thyroid carcinoma patients ZHANG Zong-min, 移性鳞癌患者的临床资料, 比较不同治疗手段的效果 50 例患者男性 44 例, 女性 6 例, 年龄 29~73 岁, 中位年龄 52.5 岁 ; 临床分期 :N2c 占 64% (32/50),N3 占 36% (18/50) 30 例患者行双颈淋巴结清扫术 50 例患者均行放疗, 剂量为 50~66