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Gross genomic alterations and gene expression profiles of high- grade serous carcinoma of the ovary with… Pradhan, Manohar; Risberg, Björn Å; Tropé, Claes G; van de Rijn, Matt; Gilks, C B; Lee, Cheng-Han Sep 15, 2010

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RESEARCH ARTICLE Open AccessGross genomic alterations and gene expressionprofiles of high- grade serous carcinoma of theovary with and without BRCA1 inactivationManohar Pradhan1, Björn Å Risberg1,2, Claes G Tropé3,4, Matt van de Rijn5, C Blake Gilks6*, Cheng-Han Lee6AbstractBackground: BRCA1 gene inactivation causes chromosomal instability, leading to rapid accumulation ofchromosomal rearrangements and mutations. The loss of BRCA1 function due to either germline/somatic mutationor epigenetic silencing is observed in most high-grade serous carcinomas of the ovary.Methods: DNA ploidy and gene expression profile were used in order to compare gross genomic alteration andgene expression pattern between cases with BRCA1 loss through mutation, BRCA1 epigenetic loss, and no BRCA1loss in cases of high-grade serous carcinoma with known BRCA1 and BRCA 2 status.Results: Using image cytometry and oligonucleotide microarrays, we analyzed DNA ploidy, S-phase fraction andgene expression profile of 28 consecutive cases of ovarian high-grade serous adenocarcinomas, which included 8tumor samples with BRCA1 somatic or germline mutation, 9 samples with promoter hypermethylation of BRCA1,and 11 samples with no BRCA1 loss. None had BRCA2 mutations. The prevalence of aneuploidy and tetraploidywas not statistically different in the three groups with different BRCA1 status. The gene expression profiles werealso very similar between the groups, with only two genes showing significant differential expression whencomparison was made between the group with BRCA1 mutation and the group with no demonstrable BRCA1 loss.There were no genes showing significant differences in expression when the group with BRCA1 loss throughepigenetic silencing was compared to either of the other two groups.Conclusions: In this series of 28 high-grade serous carcinomas, gross genomic alteration characterized byaneuploidy did not correlate with BRCA1 status. In addition, the gene expression profiles of the tumors showednegligible differences between the three defined groups based on BRCA1 status. This suggests that all ovarianhigh-grade serous carcinomas arise through oncogenic mechanisms that result in chromosomal instability,irrespective of BRCA status; the molecular abnormalities underlying this in the BRCA intact tumors remainsunknown.BackgroundIn the western world, ovarian cancer is the leading causeof death among patients with gynecological cancers [1].High-grade serous carcinoma accounts for 70% of allovarian cancers, and a disproportionate number ofdeaths as these tumors are more likely to present withadvanced stage disease [2]. Germ line mutations ofBRCA1 or BRCA2 genes predispose primarily to high-grade serous carcinoma of the ovary and approximately16% of high-grade serous carcinoma is associated withgerm line BRCA gene mutation [3]. BRCA1 gene inacti-vation is caused either through mutation or epigeneticsilencing by promoter hypermethylation, in contrast toBRCA 2 gene where promoter hypermethylation doesnot significantly contribute to loss of function [4].Operating as tumor suppressor genes, the primaryfunction of BRCA genes is to preserve the structuraland numerical stability of chromosomes during cell divi-sion [5]. The proteins are expressed in the dividing cellsand located in the nucleus. BRCA1, by forming a multi-protein complex [6], senses double strand DNA breaks* Correspondence: Blake.Gilks@vch.ca6Department of Pathology and Laboratory Medicine, University of BritishColumbia and Vancouver General Hospital, Vancouver, British Columbia,CanadaFull list of author information is available at the end of the articlePradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493© 2010 Pradhan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.and recruits molecules that repair the breaks by error-free homologous recombination [7,8]. BRCA2, on theother hand, functions as a specific mediator of the inter-actions leading to homologous recombination [9]. Inabsence of functional BRCA1 or BRCA2, double standDNA breaks are repaired by error-prone non-homolo-gous end joining mechanism leading to further muta-tions and genomic instability [10]. According to thechromosomal instability model for the pathogenesis ofBRCA-associated cancers, genetic alterations causingloss of cell-cycle checkpoints and chromosomal instabil-ity are crucial during oncogenesis [11,12]. Chromosomalinstability can be assessed by degrees of aneuploidy [13]and DNA ploidy related parameters [14]. Gross genomicalteration evidenced by aneuploidy is usually the resultof chromosomal instability [15].Earlier, by analyzing BRCA1 mutation, expression andpromoter hypermethylation, we proposed a potentialsubclassification of high-grade serous adenocarcinomasinto three groups: BRCA 1 loss through mutation,BRCA1 epigenetic loss and no BRCA loss [16]. Thera-peutically, the subclassification might be useful fortumors susceptible to targeted treatment with inhibitorsof poly (ADP-ribose) polymerase (PARP1) [17]. In orderto determine associations between BRCA1 loss andgross genomic alteration, tumor proliferation rate andgene expression profile, we have evaluated DNA ploidyand S-phase fraction by high-resolution image cytometryand gene expression profile using oligonucleotide micro-arrays, in a cohort of high-grade serous carcinomas withdefined BRCA1 and BRCA2 status.MethodsTumors and patientsSamples from the patients with ovarian carcinoma fromJanuary 2004 to September 2005 were collected at theVancouver General Hospital in Vancouver, Canada. Thediagnosis of high-grade serous carcinoma was mademorphologically and these cases are a subset of thosepreviously reported [16]. Ethical approval was obtainedfrom the University of British Columbia Ethics Board(#H02-61375 and #H03-70606).DNA ploidy analysis by image cytometryImage cytometric DNA ploidy analysis was performed inthe cohort as described previously [18]. Briefly, using 50micron sections of paraffin embedded tissue, a mono-layer was prepared and stained with Feulgen method.The images of the nuclei were captured and integratedoptical density of individual nuclei was measured usingthe Fairfield DNA ploidy system. Histograms, madefrom the integrated optical density, were classified usingestablished criteria [18]. The S-phase fraction wasmanually calculated by multiplying the number ofchannels between mid-G0/G1 and mid-G2/M peaks (C)by the mean number of registrations per channel in aneven part of the S-phase region (M) and the productwas subsequently was divided by the total number ofnuclei between the beginning of G0/G1 and the end ofG2/M peak (N) expressed in percentage (CxMx100/N)[19]. In the tumors with aneuploid peaks, the S-phasefraction of aneuploid subpopulation was estimated. TheS-phase fraction was divided into high and low by usingthe median as a cutoff point. The DNA index and coef-ficient of variation (CV) of the peaks were alsoregistered.Oligonucleotide microarrays for gene expression profileThe Human Exonic Evidence Based Oligonucleotidemicroarrays (HEEBO, Stanford) were used to studythe global gene expression profiles. Frozen tumorswere available for all except two cases of serous carci-nomas with no demonstrable BRCA1 loss (case num-ber 208 and 273). Prior to RNA extraction, frozensection analysis was performed and all tumor sampleswere confirmed to contain viable and representativetumor with no contaminating normal tissue structures.Specimens were subsequently homogenized in Trizolreagent (Invitrogen, Carlsbad, CA, USA) and totalRNA was extracted and reverse transcribed intocDNA using a mixture of oligo dT (Operon, HPLCpurified) and random hexamer (Amersham, Cat 27-2166-01) primers with incorporation of amino allyl-dUTP (Ambion 8439). Cy3 and Cy5 dyes (AmershamRPN 5661) were used for indirect labeling of thecDNA from reference RNA (Stratagene, Universalhuman reference RNA, Cat 740000) and cDNA fromtumor specimens respectively. After hybridization andwashing, microarrays were scanned on a GenePix 4000microarray scanner and fluorescence ratios (tumor/reference) were calculated using GenePix software. Toensure that the measured signals reflect true readings,only spots with a ratio of signal over background of atleast 1.5 in the Cy5 or 1.5 in the Cy3 channel wereincluded. Genes were filtered retaining only thosewhose expression levels differed by at least 4-fold(with respect to the series average in expression levelfor individual genes) in at least 3 samples and thosewith > 70% available good data. Gene centering wasapplied to the expression values across this series oftumors. The filtered dataset contain a total of 1603genes (Additional file 1). A less stringent gene filteringcriteria (2-fold difference in 3 samples) was also usedin an attempt to identify genes that display moresubtle variations between the three BRCA1-statusdefined groups (6843 genes, Additional file 2). Thecomplete gene array dataset is available through theaccompanying website (http://smd.stanford.edu/).Pradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 2 of 8BRCA1 and BRCA2 status of the tumorsAccording to the genetic status of BRCA1 gene, wedivided 28 consecutive cases of high- grade serous carci-noma of the ovary into three groups: BRCA1 lossthrough mutation, BRCA1 epigenetic loss and noBRCA1 loss by analyzing BRCA1 mutation, immuno-expression and promoter hypermethylation. The techni-ques used for evaluating the BRCA mutations, loss ofheterozygosity and microsatellite instability at both loci,mRNA level of BRCA1, immunoexpression of BRCA1and BRCA1 promoter hypermethylation of the tumorsand results obtained have previously been described[16]. Briefly, BRCA1 loss through mutation is defined asgermline or somatic mutation with low level of BRCA1RNA and less than 1% nuclei positive for BRCA1 byimmunohistochemistry. Cases with epigenetic BRCA1loss show ≥4% fully methylated molecules, low level ofBRCA1 RNA and less than 1% nuclei positive forBRCA1 in immunohistochemistry, with no BRCA1 orBRCA2 mutations. The no BRCA1 loss cases showedmore than 1% nuclei positive for BRCA1 in immunohis-tochemistry and average RNA expression, and lackedeither BRCA1 or BRCA2 mutations (somatic or germ-line) [16]. None of these tumors have BRCA2 mutations.Statistical analysisStatistical analysis was performed using SPSS version 16.Fishers exact test was used to assess the associationbetween the variables. Statistical significance wasreached at p < 0.05. For gene expression profile data,unsupervised hierarchical clustering analysis and signifi-cance analysis of microarrays (SAM) were performed asdescribed previously [20,21] and a false-discovery rate(FDR) of less than 5% was considered significant in theSAM analysis for the current study.ResultsGross genomic alteration by DNA ploidyDNA ploidy analysis was performed in 28 BRCA1 andBRCA 2 defined cases using an image cytometricmethod. The mean coefficient of variation of the diploidpeaks was 3.31 (range 1.17-5.16) and that of aneuploidpeaks was 3.36 (range 2.02-5.86). The mean number ofnuclei analyzed was 901. Aneuploidy and tetraploidywere detected in 12 (42.9) and 7 (25%) samples respec-tively. The prevalence of aneuploidy and tetraploidy wasnot statistically different in the samples with BRCA 1loss through mutation, BRCA 1 epigenetic loss and noBRCA loss (Table 1). The DNA index of all the aneu-ploid tumors was ≥ 1.4 (Figure 1) and in 2 samples itwas more than 2.1. Three tumors with microsatelliteinstability [16] were diploid, including two with epige-netic loss (number 344 and 345) and one with BRCA1mutation (number 223).S-phaseThe proliferation fraction of the tumors was evaluatedmanually from the histograms. S-phase fraction wasdivided into two groups, low and high, using mediancutoff value 8.41. Even though BRCA1 loss throughmutation shows low S-phase compared to others, thefrequency of low and high S-phase in subgroups ofBRCA1 status was not statistically different (Table 2).Gene expression profileGlobal gene expression profiles could be analyzed for 26of the 28 BRCA1 defined cases (8 with BRCA1 muta-tion, 9 with BRCA1 epigenetic loss through promoterhypermethylation and 9 with no demonstrable BRCA1loss). Hierarchical clustering analysis showed no clearseparation of the three BRCA1-defined groups based onthe expression profiles of the filtered gene set (Figure 2).The gene expression levels of the three BRCA1-definedgroups were directly compared to each other by SAManalysis. As shown in Table 3, only a small number ofdifferentially expressed genes were identified by SAMcomparison between the three BRCA1-defined groupswith a FDR < 5% (list of genes with a FDR < 20%shown in Additional file 3). Comparisons between thegroup with BRCA1 mutation versus the group withBRCA1 epigenetic loss and between the group withBRCA1 epigenetic loss versus the group with no BRCA1loss showed no genes with significant differential expres-sion between the groups. Two genes (CKMT1B andKIAA1324) were found to be significantly up-regulatedin the group with BRCA1 mutation compared to thegroup with no BRCA1 loss (FDR < 5%). No additionalgenes were identified to be differentially expressed (FDR< 5%) between the three groups using the less strin-gently filtered dataset though there was a trend forBRCA1 and a gene that is positively regulated byBRCA1, AREG [22], to be expressed more highly in thegroup with no demonstrable BRCA1 abnormality com-pared to the other groups (Additional file 4). The sametrend in BRCA1 expression was also observed by qRT-PCR analysis as reported by us previously [16]. However,the expression levels of BRCA1 and AREG were not suf-ficiently homogeneous and distinct in each group for thedifference to be identified as being statistically signifi-cant. In the case of BRCA1, this may reflect the con-founding effect of the presence of normal cells in thesesamples, which do have BRCA1 mRNA, even thoughthe tumor cells may lack expression.DiscussionWe found that the gross genomic alteration and geneexpression profiles were similar in high- grade serouscarcinoma of the ovary with BRCA1 loss through muta-tion, BRCA1 epigenetic loss and no evidence of BRCA1Pradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 3 of 8loss. There is mounting evidence that BRCA1 plays acritical role in maintaining the genomic stability of cells[23]. Mouse embryonic fibroblasts carrying targeteddeletion of BRCA1 gene were defective in a G2-Mcheck point leading to multiple spindle poles within asingle cell resulting unequal segregation of chromo-somes, abnormal nuclear division and aneuploidy [5].The mechanism of the genetic instability is caused byTable 1 BRCA1 status in diploid, aneuploid and tetraploid tumorsBRCA1 status DNA ploidy diagnosisDiploid(%) Aneuploid(%) Tetraploid(%) Total(%) p valueBRCA1 loss through mutation 2 (25) 3 (37.5) 3 (37.5) 8 (28.6)BRCA1 epigenetic loss 3 (33.3) 4 (44.4) 2 (22.2) 9 (32.1)No BRCA1 loss 4 (36.4) 5 (45.5) 2 (18.2) 11(39.3) 0.96Figure 1 Similar histology and histograms from high-grade serous carcinoma of the ovary with different BRCA1 status. (a1, a2) BRCA1loss due to genetic mutation. (b1, b2) BRCA1 loss through epigenetic promoter hypermethylation. (c1, c2) no BRCA1 loss.Pradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 4 of 8the failure of homologous DNA recombination, one ofthe pathways for the repair of double-stranded DNAbreaks during DNA replication. In this process, thedamaged strand is repaired using intact, homologoussequence as a template [7,8]. BRCA1 acts at the DNAdamaged site as a recruiter of molecules that sense andrepair DNA break and an effector of response to DNAdamage during homologous recombination process [6].In absence of BRCA1 function, the repair is through analternate pathway, nonhomologous end joining, which iserror-prone and mutagenic leading to genetic instabilityand aneuploidy [11].In breast carcinoma, the total number of genomicchanges, as determined by cytogenetics, was found to bealmost two times higher in tumors with BRCA1 muta-tion than in control group [24]. In ovarian tumors,increased clonal chromosomal aberrations was observedin BRCA mutated tumors, compared to BRCA non-mutant tumors [25]. In that series, all BRCA positivetumors were serous carcinoma and the BRCA non-mutant tumors were of different histologic types. It iswell known that the morphologically defined ovariancarcinomas are distinct diseases with different molecularevents during oncogenesis [12], and it seems likely thatthis may have confounded the findings. In order toaddress this, we analyzed a series consisting of onlyhigh-grade serous carcinomas, excluding other subtypes(including genomically stable low-grade serous carcino-mas) [19,26]. Furthermore, we have separately evaluatedthe tumors with BRCA1 loss due to mutation and dueto epigenetic silencing due to a reported difference inprognosis [27]. In this series of 28 cases, we haveobserved that there is no significant difference in thedistribution of aneuploidy and tetraploidy in the threesubgroups. This indicates that BRCA1 inactivation isnot the only mechanism for the development of aneu-ploidy in high-grade serous carcinoma of the ovary.Importantly, none of these cases had BRCA2 mutationsthat could account for chromosomal instability. In addi-tion, all aneuploid tumors had DNA index > 1.4 indicat-ing genomic unstable tumors [13]. Therefore, the resultsindicate a second currently unknown mechanism thatleads to aneuploidy in ovarian serous cancer.We observed lower S-phase in the group of tumorswith BRCA1 loss through mutation compared to theother groups. The S-phase of BRCA mutant mouseembryonic fibroblast cells was significantly lower thanthe control cells as determined by flow cytometry[5].However, ovarian carcinomas with BRCA germ linemutation had higher proliferation fraction than sporadictumors as measured by Ki 67 [28].BRCA1 and BRCA2 mutated ovarian tumors have dif-ferent gene expression profiles, however, the geneexpression profile of sporadic ovarian tumor overlapswith both [29]. In this study, we found essentially nodifferences in gene expression profile based on BRCA1status. Only two differentially expressed genes, out ofthousands examined, were identified in one of the pair-wise comparisons. This is in keeping with an earlierfinding made by Tone et al on a smaller series of 13high-grade serous carcinomas (of either ovarian or tubalorigin), where highly overlapping gene expression pro-files were observed between cases with known BRCA1/2mutation and/or family history and cases with unknownfamiliar status [30]. These genes show no functionalrelationship to each other or to the genes known to beinvolved in BRCA function and this finding is mostprobably due to chance alone. While the relative smallsample sizes (n = 8~9) of the different BRCA1-definedgroups examined here may contributes to the paucity ofTable 2 BRCA1 status in tumors with low and highS-phase fraction (median cut off 8.4%)BRCA1 status S-phaseLow (%) High (%) Total (%) p valueBRCA1 loss through mutation 6 (75) 2 (25) 8 (28.6)BRCA1 epigenetic loss 3 (33.3) 6 (66.7) 9 (32.1)No BRCA1 loss 5 (45.5) 6 (54.5) 11 (39.3) 0.1Figure 2 Unsupervised hierarchical clustering of high-gradeserous carcinomas of the ovary with different BRCA1 status.Based on the expression profiles with 1603 filtered genes, there wasno tendency for tumors to cluster based on BRCA1 status (BRCA1loss through mutation, BRCA1 epigenetic loss through promoterhypermethylation, and no demonstrable BRCA1 loss). The length ofthe dendrogram arms is inversely proportional to the relatedness ofgene expression between cases.Pradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 5 of 8consistent differences identified, it does represent thelargest series examined to date and a larger number ofdifferentially expressed genes can usually be identifiedbetween different tumor types with similar sample sizes[31,32]. Therefore, the paucity of differences observedbetween these groups of serous carcinomas with differ-ent BRCA1 status is likely a reflection of intra-groupnon-uniformity and inter-group overlap in the geneexpression patterns. In addition, we recently showedthat these groups of ovarian carcinoma classified basedon BRCA1 status also show near-identical miRNAexpression profiles [33]. This absence of distinct pat-terns of mRNA or miRNA expression in groups withdifferent BRCA1 status may reflect the rapid divergencein tumors once they acquire chromosomal instability, sothat every individual tumor is sufficiently unique thatclustering analysis identifies no patterns. As such, theirgene expression profiles irrespective of BRCA1 status allshow significant dysregulation/difference from that ofthe putative tissues of origin in normal ovarian surfaceepithelium and normal fallopian tube as demonstratedpreviously [30,34]. This rapid divergence can alsoexplain the dearth of differentially expressed genes onsupervised (SAM) analysis, as some consistent abnorm-alities would have to occur within each group for thereto be differences in gene expression. What this doesindicate, however, is that the same abnormality, chro-mosomal instability, appears to be present in all groupsof high-grade serous carcinoma analyzed, irrespective ofBRCA1 status. While chromosomal instability can beaccounted for in the BRCA1 mutant and BRCA1 epi-genetically silenced groups, it will be important to iden-tify the mechanism in the large group of tumors thatlack BRCA1 or BRCA2 abnormalities and these mayinvolve BRCA1/2-related mechanism(s) or non-BRCA1/2 related mechanism(s). PARP inhibitors have beenshown to have activity in tumors with mutations ofBRCA1[35]. PARP inhibitors target base excision repairmechanisms in the cell [36]. In cells that lack BRCA1 orBRCA2, homologous repair of double-stranded DNA isdefective and the single strand breaks that cannot berepaired because of PARP inhibition are converted todouble strand breaks in dividing cells; in the absence ofBRCA proteins the double strand breaks are repaired bynon-homologous mechanisms, such as non-homologousend joining, which is lethal to the cell [17,37]. ThusPARP inhibition can specifically target cells lackingBRCA, while sparing normal cells. It remains to be seenwhether PARP inhibitors will be active in high-gradeserous carcinomas with either BRCA1 epigenetic silen-cing or no evidence of BRCA1 loss, although it is con-ceivable that such cells may lack homologous repairfunctions.ConclusionsThere was no relationship between gross genomicalteration, detected by high resolution DNA image ana-lysis, and BRCA1 inactivation in high-grade serous car-cinoma of the ovary. Gene expression profile analysissimilarly revealed no significant differences betweenthese groups. This raises two questions. What is themechanism underlying genomic instability and develop-ment of aneuploidy in ovarian high-grade serous carci-nomas that lack BRCA1 and BRCA2 abnormalities? Willthese tumors, which do have genomic instability, be sen-sitive to therapy targeted at cells lacking in DNA repaircapability, such as PARP inhibitors?Additional materialAdditional file 1: This is an excel file containing the full data for1603 genes that met the filtering criteria.Additional file 2: This is an excel file containing the full data for6843 genes that met a less stringent gene filtering criteria.Additional file 3: This is a table showing all the differentiallyexpressed genes identified by significance analysis of microarray(SAM) between the different BRCA1-defined groups with a false-discovery rate (FDR) of < 20%.Additional file 4: Expression levels of BRCA1 and AREG for all cases.Expression level is depicted compared to the mean of all samples, withgreen indicating lower than mean expression level and red indicatinghigher than mean expression level. Black indicates expression at themean for the entire group. Gray indicates missing data.AcknowledgementsWe thank Signe Eastgate and Erika Thorbjørnsen for their skillful technicalassistance in DNA ploidy analysis. Financial support: Radiumhospitaletslegater, and an unrestricted educational grant from sanofi aventis to CBG.Author details1Division of Pathology, Oslo University Hospital, Oslo, Norway. 2Institute forMedical Informatics, Oslo University Hospital, Oslo, Norway. 3Department ofGynecologic Oncology, Oslo University Hospital, Oslo, Norway. 4FacultyDivision the Norwegian Radium Hospital, University of Oslo, Oslo, Norway.5Department of Pathology, Stanford University Medical Center, Stanford, CA,Table 3 Significance analysis of microarrays (SAM) comparisons between groups with different BRCA1 status(FDR < 5%)Comparisons Upregulated genes Downregulated genesBRCA1 loss through mutation vs. BRCA1 epigenetic loss none noneBRCA1 loss through mutation vs. no BRCA1 loss CKMT1B, KIAA1324 noneBRCA1 epigenetic loss vs. no BRCA1 loss none nonePradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 6 of 8USA. 6Department of Pathology and Laboratory Medicine, University ofBritish Columbia and Vancouver General Hospital, Vancouver, BritishColumbia, Canada.Authors’ contributionsMP classified the DNA ploidy histograms and drafted the manuscript. BR,CGT, CBG, MvdR helped in designing the study and drafting the manuscript.CHL performed oligonucleotide microarray experiments for gene expressionprofile and helped in drafting manuscript. All authors read and approvedthe final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 26 April 2010 Accepted: 15 September 2010Published: 15 September 2010References1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics, 2009. CACancer J Clin 2009, 59:225-249.2. Köbel M, Kalloger SE, Huntsman DG, Santos J, Swenerton KD, Seidman JD,Gilks CB: Differences in Tumor Type in Low versus High Stage OvarianCarcinomas. 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BMC Cancer 2010 10:493.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitPradhan et al. BMC Cancer 2010, 10:493http://www.biomedcentral.com/1471-2407/10/493Page 8 of 8


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