Salivary Transcriptome Diagnostics for Oral Cancer Detection Yang Li, 1 Maie A. R. St. John, 2 Xiaofeng Zhou, 1 Yong Kim, 1 Uttam Sinha, 6 Richard C. K. Jordan, 7 David Eisele, 8 Elliot Abemayor, 2 David Elashoff, 3 No-Hee Park, 1,4,5 and David T. Wong 1,4,5 1 School of Dentistry and Dental Research Institute, 2 School of Medicine, 3 School of Public Health, 4 Jonsson Comprehensive Cancer Center, and 5 Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California; 6 School of Medicine, University of Southern California, Los Angeles, California; and 7 School of Dentistry, and 8 Department of Otolaryngology-Head & Neck Surgery, University of California-San Francisco, San Francisco, California ABSTRACT Purpose: Oral fluid (saliva) meets the demand for non- invasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical ap- proach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease. Experimental Design: Unstimulated saliva was collected from patients (n  32) with primary T1/T2 OSCC and normal subjects (n  32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microar- rays were applied for profiling human salivary transcrip- tome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models. Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05). Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n  32) and controls (n  32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combina- tions of these biomarkers yielded sensitivity (91%) and spec- ificity (91%) in distinguishing OSCC from the controls. Conclusions: The utility of salivary transcriptome diag- nostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be ex- ploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveil- lance. INTRODUCTION More than 1.3 million new cancer cases are expected to be diagnosed in 2004 in the United States (1). Cancer will cause approximately 563,700 deaths of Americans this year, killing one person every minute. These numbers have been steadily increasing over the past 10 years, despite advances in cancer treatment. Moreover, for some cancers such as oral cavity cancer, the overall 5-year survival rates have not improved in the past several decades, remaining low at 30 to 50% (2, 3). A critical factor in the lack of prognostic improvement is the fact that a significant proportion of cancers initially are asymptom- atic lesions and are not diagnosed or treated until they reach an advanced stage. Early detection of cancer is the most effective means to reduce death from this disease. The genetic aberrations of cancer cells lead to altered gene expression patterns, which can be identified long before the resulting cancer phenotypes are manifested. Changes that arise exclusively or preferentially in cancer, compared with normal tissue of the same origin, can be used as molecular biomarkers (4). Accurately identified, biomarkers may provide new avenues and constitute major targets for cancer early detection and cancer risk assessment. A variety of nucleic acid-based biomar- kers have been demonstrated as novel and powerful tools for the detection of cancers (5–7). However, most of these markers have been identified either in cancer cell lines or in biopsy specimens from late invasive and metastatic cancers. We are still limited in our ability to detect cancer in its earliest stages with biomarkers. Moreover, the invasive nature of a biopsy makes it unsuitable for cancer screening in high-risk popula- tions. This suggests an imperative need for developing new diagnostic tools that would improve early detection. The iden- tification of molecular markers in bodily fluids that would predict the development of cancer in its earliest stage or in precancerous stage would constitute such a tool. It has been shown that identical mutation present in the primary tumor can be identified in the bodily fluids tested from affected patients (8). Cancer-related nucleic acids in blood, Received 6/16/04; revised 8/12/04; accepted 8/18/04. Grant support: USPHS grants UO1 DE15018 and RO1 DE15970 and UCLA Jonsson Comprehensive Cancer Center grant (D. Wong); USPHS grant T32 DE07296-07 and Cancer Research Foundation of American fellowship (X. Zhou). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: David T. Wong, UCLA School of Dentistry, Dental Research Institute, 73– 017 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095. Phone: (310) 206-3048; Fax: (310) 825-0921; E- mail: dtww@ucla.edu. ©2004 American Association for Cancer Research. 8442 Vol. 10, 8442– 8450, December 15, 2004 Clinical Cancer Research Research. on June 12, 2020. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from