Heterogeneity Of Epithelial Ovarian Cancer Research Articles

Morphological and molecular heterogeneity of epithelial ovarian cancer: Therapeutic implications

Ovarian epithelial cancer (OEC) is the most lethal gynecologic malignancy. Despite current chemotherapeutic and surgical options, this high lethality can be attributed to multiple factors, including late-stage presentation. In order to optimize OEC treatment, it is important to highlight that it is composed of five main subtypes: high-grade serous ovarian carcinoma (HGSOC), low-grade serous ovarian carcinoma (LGSOC), endometrioid ovarian carcinoma (EOC), ovarian clear cell carcinoma (CCOC), and mucinous ovarian carcinoma (MOC). These subtypes differ in their precursor lesions, as well as in epidemiological, morphological, molecular and clinical features. OEC is one of the tumours in which most pathogenic germline mutations have been identified. Accordingly, up to 20% OC show alterations in BRCA1/2 genes, and also, although with a lower frequency, in other low penetrance genes associated with homologous recombination deficiency (HRD), mismatch repair genes (Lynch syndrome) and TP53. The most important prognostic factor is the 2014 FIGO staging, while older age is also associated with worse survival. HGSOC in all stages and CCC and MOC in advanced stages have the worse prognosis among histological types. Molecular markers have emerged as prognostic factors, particularly mutations in BRCA1/2, which are associated with a better outcome. Regarding treatment, whereas a proportion of HGSOC is sensible to platinum-based treatment and PARP inhibitors due to HRD, the rest of the histological types are relatively chemoresistant. New treatments based in specific molecular alterations are being tested in different histological types. In addition, immunotherapy could be an option, especially for EOC carrying mismatch repair deficiency or POLE mutations.

Identification of proteins associated with paclitaxel resistance of epithelial ovarian cancer using iTRAQ-based proteomics.

Chemotherapy is an important adjuvant therapy for epithelial ovarian cancer (EOC). The main cause of chemotherapy failure in EOC is paclitaxel resistance. The present study aimed to identify novel biomarkers to predict chemosensitivity to paclitaxel and improve our understanding of the molecular mechanisms underlying paclitaxel resistance in EOC. In the present study, the heterogeneity of EOC was evaluated by adenosine triphosphate-tumor chemosensitivity assay (ATP-TCA) in vitro. Fresh samples were collected from 54 EOC cases during cytoreductive surgery. Tumor cells were isolated, cultured, and tested for sensitivity to paclitaxel. Proteins that were differentially expressed between paclitaxel-resistant tissues and paclitaxel-sensitive tissues were identified via isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. Two upregulated proteins, plexin domain containing 2 (Plxdc2) and cytokeratin 7 (CK7), were selected to verify the iTRAQ method using western blot analysis in EOC tissues with different chemosensitivities (sensitive, weakly sensitive and resistant). There was notable heterogeneity of chemosensitivity in the EOC specimens. Highly to mildly-differentiated or early-stage (I/II) EOC specimens had decreased sensitivity to paclitaxel compared with specimens with low differentiation (P<0.05) or an advanced stage (III; P<0.05), respectively. A total of 496 significantly differentially expressed proteins, including 263 that were downregulated (P<0.05) and 233 that were upregulated (P<0.05) in paclitaxel-resistant tissues compared with paclitaxel-sensitive tissues, were identified using iTRAQ in combination with LC-MS/MS. The expression levels of two proteins associated with paclitaxel resistance, Plxdc2 and CK7, were further validated by western blotting, which revealed that they were upregulated in the paclitaxel-resistant tissues. The present study determined candidate proteins associated with paclitaxel resistance in EOC. Plxdc2 and CK7 may be potential makers for distinguishing patients with paclitaxel-resistant EOC from those with paclitaxel-sensitive EOC.

Abstract MIP-051: EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS

Abstract INTRODUCTION: Ovarian cancer is the most lethal gynecologic malignancy. Initial treatment with platinum and taxane drugs are effective, however inevitably these patients will recur and become resistant to cytotoxic therapies. Thus, new alternatives for durable treatments need to be identified. One approach is to explore the heterogeneity of epithelial ovarian cancer and identify the subpopulation of cells that are resistant to treatment. It has been hypothesized, that cancer stem cells (CSCs) can survive chemotherapy and have enhanced tumor-initiating capabilities. The AKT-PI3K-mTOR pathway is well studied and has been shown to be critical for tumor cell survival. This pathway closely regulates both mTOR complexes (mTORC1 and mTORC2). mTORC1 regulates protein synthesis and autophagy whereas mTORC2 regulates cell motility. mTORC1 and 2 ultimately affect cell proliferation, survival and angiogenesis, all inherently important in tumorgenesis. OBJECTIVES: Rapalogs are specific and potent inhibitors of mTORC1 but have no effect of mTORC2. We investigate the efficacy of dual mTORC1/2 inhibitors and its effect on the CSC population. METHODS: Carboplatin resistant ovarian cancer cell line OVCAR3 were cultured and treated with dimethyl sulfoxide (DMSO), carboplatin, or INK128, a potent dual inhibitor of mTORC1/2. Using cell surface markers CD133 and CD44, CSCs were quantified by flow cytometry. OVCAR3 spheroids were cultured as a surrogate for CSCs. Colony formation-survival assays were performed with spheroids treated with the above mentioned conditions. Analysis was performed using ArrayScan technology. Doxycyline inducible silencing cell lines derived from OVCAR3 were generated; Non-silenced (Nsi), Raptor (Sh-mTORC1) and Rictor (Sh-mTORC2). Colony formation assays using spheroids from silenced lineages were performed to evaluate growth and survival. Western blot analysis was performed to confirm silencing of targets, and to evaluate protein expression in CSCs. RESULTS: 4.87% of the untreated OVCAR3 population were CSCs. INK128 treatment of the cells confered a 2 fold increase of CD44+/CD133+ cells, presumably CSCs, P&amp;lt;0.005 and no difference between DMSO and carboplatin treated cells (NS). INK128 treated cells formed fewer colonies in comparison to DMSO (mean number of colonies 36.3 + 23.8 vs. 83.3 + 32.1, P&amp;lt;0.001) and smaller colonies (mean number of cells in a colony 5260 + 835.8 vs 6907 + 702.4 P &amp;lt;0.0002). Similar populations of CD44+/CD133+ were isolated in Nsi and Raptor (NS), and there were no difference in the number or size of colonies. In comparison to Nsi, Rictor resulted in 50% decrease in CD44+/CD133+ cells, (P &amp;lt;0.002) and fewer colonies (1.6+1.3 vs 10+7.2, p&amp;lt; 0.03). There was no difference in colony size. CSCs expressed Oct4, Sox2 and phosphorylated 4E-BP1 confirms activation of AKT. CONCLUSIONS: The PI3K/AKT/mTOR pathway appears to have a role in the biogenesis of platinum resistant ovarian cancer cells. The inhibition of mTORC1/2 delays growth of OVCAR3 cells, and may have a role in activating in stem like CSCs. However, it does not appear to selectively inhibit CD133+/CD44+ cells. These findings suggest that targeting CSCs may lead to advances in ovarian cancer treatment. Citation Format: Jing-Yi Chern MD, Melissa Frey MD, Fernanda Musa MD, Amandine Alard PhD, Stephanie V. Blank, MD, Robert Schneider, PhD. EFFICACY OF MTORC1/2 INHIBITION ON OVARIAN CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-051.

Targeting the mitogen-activated protein kinase pathway in low-grade serous carcinoma of the ovary.

Until recently, there has been little change in the management of epithelial ovarian cancer with the majority of women receiving identical systemic therapy, regardless of histological subtype. The heterogeneity of epithelial ovarian cancer is now well established, with distinct subtypes characterized by specific molecular alterations and patterns of clinical behavior. Low-grade serous carcinoma is a rare subtype associated with an indolent biological behavior and inherent resistance to chemotherapy. The mitogen-activated protein kinase pathway plays a prominent role in the pathogenesis of low-grade serous carcinoma, and provides an attractive target for novel therapeutic agents. Selumetinib, a MEK1/2 inhibitor, demonstrates promising efficacy in women with relapsed low-grade serous carcinoma, and further trials of MEK-inhibition are underway. Translational research will be essential to identify predictive biomarkers for this treatment approach.

GRHL2-miR-200-ZEB1 maintains the epithelial status of ovarian cancer through transcriptional regulation and histone modification.

Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor—Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.

Genetic and epigenetic heterogeneity of epithelial ovarian cancer and the clinical implications for molecular targeted therapy.

Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy, and tumoural heterogeneity (TH) has been blamed for treatment failure. The genomic and epigenomic atlas of EOC varies significantly with tumour histotype, grade, stage, sensitivity to chemotherapy and prognosis. Rapidly accumulating knowledge about the genetic and epigenetic events that control TH in EOC has facilitated the development of molecular‐targeted therapy. Poly (ADP‐ribose) polymerase (PARP) inhibitors, designed to target homologous recombination, are poised to change how breast cancer susceptibility gene (BRCA)‐related ovarian cancer is treated. Epigenetic treatment regimens being tested in clinical or preclinical studies could provide promising novel treatment approaches and hope for improving patient survival.

Abstract POSTER-BIOL-1301: The receptor tyrosine kinase AXL modulates oncogenic signaling and epithelial mesenchymal transition in epithelial ovarian cancer

Abstract Metastasis in epithelial ovarian cancer (EOC) is lethal, and results into a 5-year-survival rate lower than 30%. In EOC, tumor cells undergo Epithelial-Mesenchymal Transition (EMT) to attain the invasive and migratory potential crucial for metastasis. The analysis of molecular heterogeneity in EOC also demonstrated the important role of EMT. Our group has previously classified EOC tumors and cell lines into 5 distinct molecular subtypes based on their global gene expression patterns. Among these subtypes, the Mesenchymal subtype is believed to be driven by EMT. Receptor Tyrosine Kinases (RTKs), which play a pivotal role in proliferation and survival pathways, have been strongly indicated to mediate signals in EMT. Upon interrogating the kinome expression patterns by determining the top 10 ranked kinases in each molecular subtype, the RTK AXL was revealed to have a significantly high ranking in the Mesenchymal subtype. The purpose of this study is to elucidate the role of AXL in oncogenic signaling and EMT in EOC. Western Blotting of total Axl protein in a panel of 40 EOC cell lines revealed a distinctive pattern among the 5 molecular subtypes. Upon ligand activation by Gas6 in Axl-expressing cell lines, pERK and pAkt, two signaling molecules downstream of Axl, displayed a unique temporal signaling pattern specific to the Mesenchymal subtype. This resulted into an implication of the preferential sensitivity to Axl-specific inhibitors in the Mesenchymal subtype. The IC-50 of R428, one small molecule inhibitor specific to Axl, was significantly lower in the Mesenchymal cell lines compared to the other subtypes (Average of 2.91 μM versus 9.38 μM; p-value=0.01) Stable clones were generated by using shRNAs to knockdown AXL in Mesenchymal EOC lines. shAXL SKOV3 cells demonstrated a reversal of EMT evident by restoration of Ecadherin. siRNA mediated transient silencing of AXL showed a reduction in cell motility with decreased total displacement and velocity measured over 12 hours (31.98 μm compared to 44.59 μm, p-value=0.01; and 17.35 μm/second compared to 23.15 μm/second, pvalue= 0.03) measured by using single cell tracking. Survival analysis showed that AXL over expression correlated positively with poor overall survival of EOC in both univariate (HR=2.2 p-value=6.46e−11), and multivariate analysis (HR=1.513 p-value=0.0012). In conclusion, AXL is significantly over-expressed and displays a unique signaling pattern in the Mesenchymal subtype of EOC. Knocking down AXL in the Mesenchymal cell lines affects their EMT phenotype and migratory properties. AXL is an independent prognostic factor for EOC and is a clinically relevant target that is potentially involved in cross talks between RTK oncogenic signaling and EMT. Citation Format: Jane Antony, Tuan Zea Tan, Andrew Paterson, Chiara Recchi, Hani Gabra, Jean Paul Thiery, Ruby Yun-Ju Huang. The receptor tyrosine kinase AXL modulates oncogenic signaling and epithelial mesenchymal transition in epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1301.

Abstract PR10: Intra- and intertumor heterogeneity in epithelial ovarian cancer: Consequences for biomarker-dependent stratification of therapies

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&amp;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.

Molecular Signatures of Epithelial Ovarian Cancer: Analysis of Associations with Tumor Characteristics and Epidemiologic Risk Factors

Six gene expression subtypes of invasive epithelial ovarian cancer were recently defined using microarrays by Tothill and colleagues. The Cancer Genome Atlas (TCGA) project subsequently replicated these subtypes and identified a signature predictive of survival in high-grade serous (HGS) cancers. We previously validated these signatures for use in formalin-fixed paraffin-embedded tissues. The aim of the present study was to determine whether these signatures are associated with specific ovarian cancer risk factors, which would add to the evidence that they reflect the heterogeneous etiology of this disease. We modeled signature-specific tumor characteristics and epidemiologic risk factor relationships using multiple regression and multivariate response multiple regression models in 193 patients from a case-control study of epithelial ovarian cancer. We observed associations between the Tothill gene expression subtype signatures and both age at diagnosis (P = 0.0008) and race (P = 0.008). Although most established epidemiologic risk factors were not associated with molecular signatures, there was an association between breast feeding (P = 0.024) and first-degree family history of breast or ovarian cancer (P = 0.034) among the 106 HGS cases. Some of the above associations were validated using gene expression microarray data from the TCGA project. Weak associations were seen with age at menarche and duration of oral contraceptive use and the TCGA survival signature. These data support the potential for genomic characterization to elucidate the etiologic heterogeneity of epithelial ovarian cancer. This study suggests that molecular signatures may augment the ability to define etiologic subtypes of epithelial ovarian cancer.

Platinum-Sensitive Recurrence in Ovarian Cancer: The Role of Tumor Microenvironment

Despite several advances in the understanding of ovarian cancer pathobiology, in terms of driver genetic alterations in high-grade serous cancer, histologic heterogeneity of epithelial ovarian cancer, cell-of-origin for ovarian cancer, the survival rate from ovarian cancer is disappointingly low when compared to that of breast or prostate cancer. One of the factors contributing to the poor survival rate from ovarian cancer is the development of chemotherapy resistance following several rounds of chemotherapy. Although unicellular drug resistance mechanisms contribute to chemotherapy resistance, tumor microenvironment and the extracellular matrix (ECM), in particular, is emerging as a significant determinant of a tumor’s response to chemotherapy. In this review, we discuss the potential role of the tumor microenvironment in ovarian cancer recurrence and resistance to chemotherapy. Finally, we propose an alternative view of platinum-sensitive recurrence to describe a potential role of the ECM in the process.

Cytokine analysis as a tool to understand tumour–host interaction in ovarian cancer

Epithelial ovarian cancer (EOC) is an immunogenic tumour and exploits many suppressive ways to escape immune eradication. EOC is known to spread primarily by tumour cell implantations in peritoneal cavity. Therefore, ascites may be an ideal fluid compartment to unravel the immune status of the peritoneal cavity.We analysed the expression of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α, TNF-β, TGF-β and CCL22 in ovarian cancer ascites, representing immune activating and suppressing cytokines.We observed high expression of pro-inflammatory cytokines IL-6, IL-8 and immune suppressive cytokines IL-10, CCL22 and TGF-β in most samples whereas Th1 (IL-12p70, IFN-γ) and Th2 (IL-4, IL-5) cytokines were only detectable in 13% of the samples. TGF-β was only detected in latent form, questioning its immune suppressive role. CCL22 was in similar levels present in early stage compared to advanced stage tumours. At advanced stage, we observed a negative correlation with CCL22 levels and Th1/2 cytokine expression. We found a positive correlation between IL-6 concentration in ascites and residual disease after debulking. Additionally, IL-6 levels were remarkably higher at recurrence compared to primary advanced disease, which opens an opportunity for inhibition of IL-6 expression in the prevention of recurrence. Despite the heterogeneity of EOC and the complexity of cytokine functions, our results show that cytokine analysis in ascites may aid in understanding tumour–host interaction in EOC.

ASPM and microcephalin expression in epithelial ovarian cancer correlates with tumour grade and survival.

Background:The clinico-pathological and molecular heterogeneity of epithelial ovarian cancer (EOC) complicates its early diagnosis and successful treatment. Highly aneuploid tumours and the presence of ascitic fluids are hallmarks of EOC. Two microcephaly-associated proteins, abnormal spindle-like microcephaly-associated protein (ASPM) and microcephalin, are involved in mitosis and DNA damage repair. Their expression is deregulated at the RNA level in EOC. Here, ASPM and microcephalin protein expression in primary cultures established from the ascites of patients with EOC was determined and correlated with clinical data to assess their suitability as biomarkers.Methods:Five established ovarian cancer cell lines, cells derived from two benign ovarian ascites samples and 40 primary cultures of EOC derived from ovarian ascites samples were analysed by protein slot blotting and/or immunofluorescence to determine ASPM and microcephalin protein levels and their cellular localisation. Results were correlated with clinico-pathological data.Results:A statistically significant correlation was identified for ASPM localisation and tumour grade, with high levels of cytoplasmic ASPM correlating with grade 1 tumours. Conversely, cytoplasmic microcephalin was only identified in high-grade tumours. Furthermore, low levels of nuclear microcephalin correlated with reduced patient survival.Conclusion:Our results suggest that ASPM and microcephalin have the potential to be biomarkers in ovarian cancer.

The heterogeneity of epithelial ovarian cancer

Epithelial ovarian cancer is heterogeneous and comprises 4 major rare subtypes—mucinous, clear cell, low‐grade serous, and endometrioid—all distinct from the more common high‐grade serous carcinoma. Women with uncommon histotypes should be triaged to separate clinical trials.

Genetic intra-tumour heterogeneity in epithelial ovarian cancer and its implications for molecular diagnosis of tumours

Genetic intra-tumour heterogeneity in epithelial ovarian cancer and its implications for molecular diagnosis of tumours

Novel functional proteomics approach to defining ovarian cancer molecular heterogeneity

5502 Background: A number of clinicopathologic risk factors are used for survival prediction and clinical decision-making in epithelial ovarian cancer (EOC). Information from novel technologies such as gene arrays has not had an impact on patient management. We studied EOC protein signaling profiles to determine if their addition to accepted clinicopathologic factors improves their accuracy in predicting individual patient outcomes. Methods: We applied a novel functional proteomics technology, reverse phase protein array (RPPA), to quantify expression and activation of 42 steroid and kinase signaling pathway proteins in 106 high-grade EOCs from patients with stages 1–4 tumors managed with surgery and platinum-based chemotherapy. Cox regression analysis and a novel committee modeling approach were used to study the impact of functional proteomics on patient outcomes. Results: In a Cox model using only clinical variables, stage and residual disease were significantly related to overall survival. By adding the proteins to the clinical Cox model, two proteins that were significantly associated with overall survival on univariate analysis (phosphorylated-MAPK (p-MAPK; log rank p = 0.0047) and progesterone receptor (PR; log rank p = 0.027)) remained significant at the alpha=0.10 level (z-test p-values 0.074 and 0.034, respectively, when treated as binary variables according to martingale residual plots); as a result, these two proteins added to the predictive accuracy of the clinical survival model. However, using the novel committee modeling approach in test and validation EOC sets, a closest neighbor metric was applied to successfully define distinct proteins groups, each composed of nine proteins, that are predictive of specific survival times in patients with EOC. This granular approach to modeling is particularly suited to defining the molecular heterogeneity of EOC. Conclusions: EOC is a complex process with significant individual variability. Using novel approaches to functional proteomic study and statistical modeling, our striking finding is that distinct combinations of steroid and kinase signaling proteins are predictive markers of specific survival times in EOC. No significant financial relationships to disclose.

Genetic intra‐tumour heterogeneity in epithelial ovarian cancer and its implications for molecular diagnosis of tumours

Genetic analysis of solid tumours using DNA or cDNA expression microarrays may enable individualized treatment based on the profiles of genetic changes that are identified from each patient. This could result in better response to adjuvant chemotherapy and, consequently, improved clinical outcome. So far, most research studies that have tested the efficacy of such an approach have sampled only single areas of neoplastic tissue from tumours; this assumes that the genetic profile within solid tumours is homogeneous throughout. The aim of this study was to evaluate the extent of genetic intra-tumour heterogeneity (ITH) within a series of epithelial ovarian cancers. Several different regions (five to eight regions) of tumour tissue from 16 grade 3, serous epithelial ovarian cancers were analysed for genetic alterations using a combination of microsatellite analysis and single nucleotide polymorphism (SNP) analysis, in order to establish the extent of ITH. Maximum parsimony tree analysis was applied to the genetic data from each tumour to evaluate the clonal relationship between different regions within tumours. Extensive ITH was identified within all ovarian cancers using both microsatellite and SNP analysis. Evolutionary analysis of microsatellite data suggested that the origin of all tumours was monoclonal, but that subsequent clonal divergence created mixed populations of genetically distinct cells within the tumour. SNP analysis suggested that ITH was not restricted to random genetic changes, but affected genes that have an important functional role in ovarian cancer development. The frequent occurrence of ITH within epithelial ovarian cancers may have implications for the interpretation of genetic data generated from emerging technologies such as DNA and mRNA expression microarrays, and their use in the clinical management of patients with ovarian cancer. The basis of genetic ITH and the possible implications for molecular approaches to clinical diagnosis of ovarian cancers may apply to other tumour types.