Profiling Microdissected Epithelium and Stroma to Model Genomic Signatures for Cervical Carcinogenesis Accommodating for Covariates David Gius, 1 Margo C. Funk, 2 Eric Y. Chuang, 1 Sheng Feng, 3 Phyllis C. Huettner, 4 Loan Nguyen, 2 C. Matthew Bradbury, 1 Mark Mishra, 1 Shuping Gao, 1 Barbara M. Buttin, 2 David E. Cohn, 2 Matthew A. Powell, 2 Neil S. Horowitz, 2 Bradford P. Whitcomb, 2 and Janet S. Rader 2 1 RadiationOncologyBranch,CenterforCancerResearch,NationalCancerInstitute,NIH,Bethesda,Marylandand 2 DivisionofGynecologic Oncology,DepartmentofObstetricsandGynecology; 3 DivisionofBiostatistics;and 4 LaurenV.AckermanLaboratoryofSurgicalPathology, Washington University School of Medicine, St. Louis, Missouri Abstract This study is the first comprehensive, integrated approach to examine grade-specific changes in gene expression along the entireneoplasticspectrumofcervicalintraepithelialneoplasia (CIN) in the process of cervical carcinogenesis. This was accomplished by identifying gene expression signatures of disease progression using cDNA microarrays to analyze RNA fromlaser-capturedmicrodissectedepitheliumandunderlying stroma from normal cervix, graded CINs, cancer, and patient- matchednormalcervicaltissues.Aseparatesetofsampleswere subsequentlyvalidatedusingalinearmixedmodelthatisideal to control for interpatient gene expression profile variation, such as age and race. These validated genes were ultimately usedtoproposeagenomicallybasedmodeloftheearlyevents in cervical neoplastic transformation. In this model, the CIN 1 transition coincides with a proproliferative/immunosuppres- siongenesignatureintheepitheliumthatprobablyrepresents theepithelialresponsetohumanpapillomavirusinfection.The CIN 2 transition coincides with a proangiogenic signature, suggestingacooperativesignalinginteractionbetweenstroma andtumorcells.Finally,theCIN3andsquamouscellcarcinoma antigen transition coincide with a proinvasive gene signature that may be a response to epithelial tumor cell overcrowding. Thisworkstronglysuggeststhatpremalignantcellsexperience a series of microenvironmental stresses at the epithelium/ stromacellinterfacethatmustbeovercometoprogressintoa transformedphenotypeandidentifiestheorderoftheseevents in vivo and their association with specific CIN transitions. [CancerRes2007;67(15):7113–23] Introduction Cervical malignancies present a unique opportunity to profile neoplastic transformation. The histologic transition from normal epithelial cells to preinvasive cervical intraepithelial neoplasia (CIN 1–3) and finally to squamous cell carcinoma antigen (SCCA) have been well characterized. Histologically, CIN 1 consists of immature basal-type cells involving the lower third of the epithelium.InCIN2,theseimmaturebasal-typecellsinvolvemore thanthelowerthird,whereasCIN3involvesthefullthicknessofthe epithelium.Inaddition,higherCINgradesexhibitnuclearcrowding, pleomorphism, loss of cell polarity, and increased mitotic activity (1). These transitions seem to be well conserved and, as such, provide an intriguing system to use genomics to identify the early events in cervical cell transformation. The underlying stromal composition may vary slightly beneath the basement membrane; however,itisoverwhelminglycharacterizedbyfibroblasts.Complex molecular cross-talk between the epithelium and stroma has been implicatedasakeycomponentofneoplastictransformation,tumor invasion,andmetastasis.Therefore,toconstructaneffectivemodel for CIN progression in vivo , it is imperative to separately examine the different microenvironments involved in carcinogenesis. Multidisciplinary studies have unequivocally implicated human papillomavirus (HPV) as the etiologic agent of neoplastic cervical lesions, with HPV DNA being found in 99.7% of invasive cervical carcinomas (2). Even so, most HPV infections are transient and spontaneously cleared by the host immune system. Thus, few women infected with HPV develop CIN 3 and fewer still progress to invasive carcinoma (3), suggesting that HPV does not act alone in the development of cervical cancer. Persistence of HPV infec- tionandprogressiontocancervarybyageandrace:Womenover 30 years show a much higher persistence of HPV than younger women (4, 5), and progression to CIN 3 may vary by race (6). To address interpatient variability, tissue heterogeneity, and experimentalreproducibility,weusedlaser-capturemicrodissection (LCM) on 130 prospectively collected samples to isolate epithelial cells and stromal fibroblasts within 3 mm of the basement membrane from normal cervix, graded CINs, cancer, and patient compartment–matched ‘‘normal’’ cervical tissues. Microarray hybridization was done using only excellent quality RNA with triplicate replication per sample and a single universal reference acrossallsamples.Weidentifiedspecificmarkersintheepithelium and stromal compartments showing a progressive change in gene expression (increased or decreased) from viral cytopathic to inva- sive cancer and markers with a sharp change at CIN 3 and inva- sivecancer.Validationonaseparatesetofsampleswasimproved by using a linear mixed model accommodating for age and race and showed several intriguing findings. Both preneoplastic epithe- lial cells and the surrounding fibroblasts induce the expression of proangiogenic factors, suggesting a potential cooperative com- munication between these differing cell types at the transition Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). D. Gius and M.C. Funk contributed equally to this work. Current address for E.Y. Chuang: Biomedical Engineering Group, Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan. Requests for reprints: David Gius, Radiation Oncology Branch, National Cancer Institute,NIH,9000RockvillePike,Bethesda,MD20892.Phone:301-496-5457;Fax:301- 480-5439; E-mail: giusd@mail.nih.gov and Janet S. Rader, Departments of Obstetrics and Gynecology and Genetics, Washington University School of Medicine, 4911 Barnes-JewishHospitalPlazaBox8064,St.Louis,MO63110.Phone:314-362-3181;Fax: 314-362-2893; E-mail: raderj@wustl.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0260 www.aacrjournals.org 7113 Cancer Res 2007; 67: (15). August 1, 2007 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/67/15/7113/2865018/7113.pdf by guest on 02 October 2023