Abstract PR10: Intra- and intertumor heterogeneity in epithelial ovarian cancer: Consequences for biomarker-dependent stratification of therapies
Authors: O'Donnell Rachel L. , Woodhouse Laura C. , McCormick Aiste , Kaufmann Angelika , Dixon Michelle , Mukhopadhyay Asima , Curtin Nicola J. , Edmondson Richard J. 
Published: August 2014

Abstract
Introduction: Ovarian cancer is recognized as a heterogeneous disease but is currently treated with a single treatment strategy. Improved outcomes will require stratified use of novel cytotoxic agents exploiting the molecular pathology of the tumor but this is dependent upon the availability of reliable biomarkers. Most research testing the efficacy of biomarker stratified treatment use a single tumor sample, assuming the biomarker profile to be homogeneous. The reliability of such biomarkers may however be complicated by intra-tumor heterogeneity (ITH).
The aim of this study was to evaluate the extent of ITH within a series of epithelial ovarian cancers using homologous recombination (HR) DNA repair status as a marker, given that this has been shown to predict sensitivity to PARP inhibitors ex vivo and cisplatin in vivo.
Methods: Primary cultures (PCO) were generated from ascitic fluid and solid tumor taken from a variety of intra-abdominal sites. Cultures were characterized based upon their morphology and using an established panel of immunofluorescent-labeled antibodies. Results were cross-referenced with formal pathological examination of FFPE tissue and cytology of ascites.
The functional status of HR was determined for each culture by quantification of nuclear Rad51 focus formation following induction of DNA DSB. Standard growth inhibition (SRB) assays were used to calculate the GI50 for cisplatin.
Results: PCO cultures from ascites with matched solid tumor were cultured from 19 patients. Solid tumor was sampled from multiple intra-abdominal regions in ten patients. In the remaining nine patients solid tumor was taken from a single site from the bulk of the pelvic tumor.
Inter- and intra-tumor heterogeneity was seen between the expression of epithelial and ovarian markers (EpCAM, cytokeratin, CA125, MOC-31 and vimentin). PCO samples taken from eight patients showed homogeneous staining patterns between all subcultures. Eleven patients however demonstrated variation in the staining patterns between subcultures from ascites and solid tumor or between solid tumors from varying sites. This heterogeneity was not reflected in the histological classification of the tumors with only 2/ 11 heterogeneous patients being classified as mixed histological subtype upon formal pathological examination.
Variability in the HR status between ascites and solid tumor subcultures was seen in 9/19 (47%) patients. Functional heterogeneity was seen even in patients with homogeneous expression of markers and homogeneous histological subtype. The remaining 10 patients showed agreement in HR status in ascitic and all solid subcultures. When two or more solid tumor samples from anatomically distinct sites were assessed in the same patient there was variability between cultures in 7/9 patients.
The sensitivity of PCO subcultures to cisplatin, as hypothesized, correlated with HR status with a GI50 of 265.6μM in HR competent cultures and 6.27μM in defective cultures, p&lt;0.0001.
Conclusions: Inter- and intra-tumor heterogeneity exists that cannot be detected using traditional histological classification. The success of stratification for novel therapeutic agents on the basis of biomarker testing is dependent upon the sample analyzed being representative of the tumor as a whole. This study suggests that a single sample cannot be assumed to be representative in terms of morphology, expression of markers or more importantly, in its DNA repair functional status and therefore subsequent sensitivity to therapy.
Accepting that functional ITH exists and tumor subpopulations are variably responsive to both therapeutic agents suggests that despite response to therapy in some subpopulations, resistant subpopulations may survive, proliferate, and result in disease relapse. This is not necessarily a failure of treatment but it does suggests that alternative treatment strategies in heterogeneous tumors may be more successful.
This abstract is also presented as Poster B53.
Citation Format: Rachel L. O'Donnell, Laura C. Woodhouse, Aiste McCormick, Angelika Kaufmann, Michelle Dixon, Asima Mukhopadhyay, Nicola J. Curtin, Richard J. Edmondson. Intra- and intertumor heterogeneity in epithelial ovarian cancer: Consequences for biomarker-dependent stratification of therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR10.