UBC Faculty Research and Publications

Intratumoral heterogeneity in a minority of ovarian low-grade serous carcinomas Tone, Alicia A; McConechy, Melissa K; Yang, Winnie; Ding, Jiarui; Yip, Stephen; Kong, Esther; Wong, Kwong-Kwok; Gershenson, David M; Mackay, Helen; Shah, Sohrab; Gilks, Blake; Tinker, Anna V; Clarke, Blaise; McAlpine, Jessica N; Huntsman, David Dec 18, 2014

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-12885_2014_Article_5198.pdf [ 1.42MB ]
JSON: 52383-1.0223862.json
JSON-LD: 52383-1.0223862-ld.json
RDF/XML (Pretty): 52383-1.0223862-rdf.xml
RDF/JSON: 52383-1.0223862-rdf.json
Turtle: 52383-1.0223862-turtle.txt
N-Triples: 52383-1.0223862-rdf-ntriples.txt
Original Record: 52383-1.0223862-source.json
Full Text

Full Text

RESEARCH ARTICLEIntratumoral heterogeneitovarian low-grade serous2,ay1,Tone et al. BMC Cancer 2014, 14:982http://www.biomedcentral.com/1471-2407/14/982similar to HGSC patients; this is highlighted by a recentCanadaFull list of author information is available at the end of the articleBackgroundIn comparison to the more commonly occurring high-gradeserous carcinomas (HGSC), ovarian low-grade serouscarcinomas (LGSC) are characterized by a younger age atonset, lower mitotic rate and longer median overall survival[1-6]. Whereas the vast majority (80%) of patients withHGSC are responsive to platinum-based chemotherapy,patients with LGSC are highly resistant to treatment in theneoadjuvant, adjuvant and recurrent setting, with responserates of 4-5% [1,7,8]. Women diagnosed with LGSCtypically experience multiple recurrences over a protractedclinical course before ultimately dying of their disease, withan associated 10-year survival rate of <50% [2]. Thissuggests that despite having a less aggressive clinicalcourse, women with LGSC have a poor long-term prognosis* Correspondence: dhuntsma@bccancer.bc.ca†Equal contributors1Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, BC, Canada2BC Cancer Agency, Room 3427, 600 West 10th Avenue, Vancouver, BC,AbstractBackground: Ovarian low-grade serous carcinoma (LGSC) has fewer mutations than ovarian high-grade serouscarcinoma (HGSC) and a less aggressive clinical course. However, an overwhelming majority of LGSC patients do notrespond to conventional chemotherapy resulting in a poor long-term prognosis comparable to women diagnosedwith HGSC. KRAS and BRAF mutations are common in LGSC, leading to clinical trials targeting the MAPK pathway.We assessed the stability of targetable somatic mutations over space and/or time in LGSC, with a view to informstratified treatment strategies and clinical trial design.Methods: Eleven LGSC cases with primary and recurrent paired samples were identified (stage IIB-IV). Tumor DNA wasisolated from 1–4 formalin-fixed paraffin-embedded tumor blocks from both the primary and recurrence (n = 37 tumorand n = 7 normal samples). Mutational analysis was performed using the Ion Torrent AmpliSeqTM Cancer Panel, withtargeted validation using Fluidigm-MiSeq, Sanger sequencing and/or Raindance Raindrop digital PCR.Results: KRAS (3/11), BRAF (2/11) and/or NRAS (1/11) mutations were identified in five unique cases. A novel,non-synonymous mutation in SMAD4 was observed in one case. No somatic mutations were detected in theremaining six cases. In two cases with a single matched primary and recurrent sample, two KRAS hotspot mutations(G12V, G12R) were both stable over time. In three cases with multiple samplings from both the primary andrecurrent surgery some mutations (NRAS Q61R, BRAF V600E, SMAD4 R361G) were stable across all samples, whileothers (KRAS G12V, BRAF G469V) were unstable.Conclusions: Overall, the majority of cases with detectable somatic mutations showed mutational stability over spaceand time while one of five cases showed both temporal and spatial mutational instability in presumed drivers ofdisease. Investigation of additional cases is required to confirm whether mutational heterogeneity in a minority ofLGSC is a general phenomenon that should be factored into the design of clinical trials and stratified treatment forthis patient population.Keywords: Heterogeneity, Low-grade cancer, Ovarian serous carcinoma, Mutation, KRAS, BRAF, NRAS, SMAD4Alicia A Tone1,2,3†, Melissa K McConechy1,2†, Winnie YangKwong-Kwok Wong5, David M Gershenson5, Helen MackBlaise Clarke7, Jessica N McAlpine2,8 and David Huntsman© 2014 Tone et al.; licensee BioMed Central. TCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open Accessy in a minority ofcarcinomasJiarui Ding4, Stephen Yip1,2, Esther Kong2,6, Sohrab Shah4, Blake Gilks1, Anna V Tinker2,2*his is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Tone et al. BMC Cancer 2014, 14:982 Page 2 of 13http://www.biomedcentral.com/1471-2407/14/982study reporting a similar hazard ratio for death in LGSCand HGSC patients with measurable residual disease afteradjusting for additional variables [9].In an effort to identify potential molecular targets,limited mutational studies in primary or recurrentLGSC samples have revealed an overall low mutationfrequency, with exome sequencing by Jones et al.showing an average of 10 validated somatic mutations(or 7.5 somatic non-synonymous or splice site mutations)per tumor [10]. The mitogen-activated kinase (MAPK)pathway is most frequently mutated [11], with 19-35% ofcases containing a KRAS mutation and 2-33% containinga BRAF mutation [3,10,12-14]. KRAS and BRAF muta-tions are also frequently detected in serous borderlinetumors (SBT), the histologic precursor to invasive LGSC[5,6,11,15-17].The prevalence of KRAS/BRAF mutations in LGSChas resulted in clinical trials of inhibitors of MAP kinasekinase (MEK1/2), which lies immediately downstream ofBRAF and upstream of ERK1/2 in the MAPK pathway[18,19]. Previous studies have reported profound growthinhibition and apoptosis in ovarian cancer cells withmutated but not wildtype KRAS or BRAF upon treatmentwith CI-1040 [20] in tissue culture and xenograft studies[19,21], suggesting that mutation status predicts sensitivityto MEK inhibition. A recent phase II study of selumetinib,another small molecular inhibitor of MEK1/2, in womenwith recurrent ovarian/peritoneal LGSC has shown anobjective 15% response rate despite heavy pre-treatment;however patient response does not appear to be correlatedwith KRAS/BRAF mutation status [18]. The mutationstatus of the patients in this trial was based solely ona single sample of LGSC; most were obtained fromthe primary tumor and a small percentage were obtainedfrom the recurrent tumor. In this study we aimed to assessthe stability of targetable mutations over space and/ortime by targeted sequence analysis of one or more tumorsamples from both the primary and recurrence, to informfuture clinical trial design. Herein we report our findingsof mutational stability in the majority of cases, as well asremarkable instability in one case of ovarian LGSC, inpresumed drivers of disease KRAS and BRAF. If validatedin more cases this could impact clinical trial design forthis patient population in the future.MethodsStudy casesA total of 11 cases of LGSC with matched primary and re-current samples available were identified from the UniversityHealth Network in Toronto, Ontario (“UHN”, n = 3), MDAnderson Cancer Center in Houston, Texas (“MDACC”,n = 3) and the BC Cancer Agency in Vancouver, BritishColumbia (“BCCA”, n = 5). The stage breakdown included:IIB (n = 1), IIIB (n = 3), IIIC (n = 6) and IV (n = 1). Researchethics approval was obtained from each site (UBC BCCAResearch Ethics Board, University Health Network ResearchEthics Board and The University of Texas MD AndersonCancer Center Institutional Review Board). All patientsprovided written informed consent to have their tissuesamples used for research purposes, including genomicstudies. Written informed consent was obtained fromevery patient for publication of the specific clinical detailsincluded within this research article and any accompany-ing images. However, potentially identifying informationsuch as date of diagnosis have been removed to protectprivacy. Upon inclusion in the study, all formalin-fixedparaffin-embedded (FFPE) sections chosen for sequenceanalysis were subjected to secondary pathologic review(B.G.). Two of eleven cases originally presented as a SBT,and recurred as invasive LGSC. Nine patients receivedadjuvant treatment following diagnosis, including six treatedwith combined carboplatin/paclitaxel.A total of 37 tumor samples (from either FFPE blocks[BCCA] or unstained sections [MDACC/UHN]) wereincluded for analysis. At least one sampling from both theprimary and recurrent setting were included for each case,with H&E-guided macrodissection used to isolate tumorfrom adjacent stromal cells. Tumor cellularity achievedfollowing macrodissection was estimated at a median of80% (range 50-95%) and was comparable among samplesobtained from the same case. Normal samples wereavailable for 7 cases (6 matched normal tissue, 1 buffycoat). We were also able to obtain a fresh blood samplefrom one BCCA study patient for extraction of circulatingtumor DNA (“ctDNA”). Summary information for allstudy cases is included in Table 1, with more detailedinformation on tumor sites and normal samples used inAdditional file 1 and case images in Additional files 2, 3, 4,5, 6, 7, 8, 9, 10, 11 and 12.DNA extractionTumor and normal DNA was extracted from FFPEblocks/unstained sections (see Additional file 13 forsupplemental methods). For extraction of ctDNA, wholeblood was collected in EDTA tubes then centrifuged at2,500 rpm for 15 min. Plasma was then stored at -80C in1-2 mL aliquots, followed by extraction of plasma ctDNAusing the Qiagen Circulating Nucleic Acid kit as permanufacturer’s protocol. ctDNA was then eluted in 30uLof Buffer AVE.Ion torrent AmpliSeq cancer hotspot sequencingThe Ion Torrent AmpliSeqTM Cancer Hotspot PanelVersion 1 (Life Technologies, Grand Island/NY/USA)[22] was used to prepare sequencing libraries from alltumor DNA, normal DNA and plasma ctDNA as permanufacturer’s protocols (see Additional file 13 for meth-odological details and Additional file 14 for a comprehensiveInararplaarlatiartoardiTone et al. BMC Cancer 2014, 14:982 Page 3 of 13http://www.biomedcentral.com/1471-2407/14/982list of genes and mutations included on the AmpliSeqpanel). A list of predicted variants was generated for eachsample using the built-in AmpliSeq Cancer Variant Callerfollowing each run. Upon completion of all samples,we performed a separate bioinformatics analysis. Sequencereads were aligned to the human reference sequence(UCSC hg19) using the BWA SW algorithm (v0.6.1) [23]with default parameters. Single nucleotide variants (SNV)Table 1 Summary of study casesCase Source Age # TumorsamplesStage Primary tumorsamplesLGSC-2 UHN 57 2 IIIC 2-P* CLGSC-3 UHN 51 2 IIIC 3-P* CLGSC-4 UHN 66 3 IIIB 4-PLGSC-5 MDACC 51 2 IIIC 5-P CarboLGSC-6 MDACC 41 2 IIIC 6-P CLGSC-8 MDACC 33 2 IIIC 8-P CispLGSC-9** BCCA 51 6 IIIB 9-P1, P2, P3*LGSC-10 BCCA 57 8 IV 10-P1, P2, P3, P4* CLGSC-11** BCCA 62 2 IIIC 11-P*LGSC-12 BCCA 57 6 IIB 12-P1, P2, P3, P4* ELGSC-13 BCCA 58 2 IIIB 13-P* CMean(Total)53.1 3.4 (37)*normal sample also available; **initial diagnosis of SBT; ***time in months sinceAbbreviations: AWD = alive with disease, DOD = dead of disease.were called using mutationSeq [24], a feature-basedmethod to filter out technical artifacts. We used a variantprobability threshold of 0.5 to nominate SNVs. Variantspredicted in the pooled normal data were considered asgermline mutations and were removed.Variants were selected for targeted validation by MiSeqin all samples according to the following selection criteria:[1] either somatic (variant not found in correspondingnormal sample if available) or predicted somatic (for caseswith no corresponding normal – variant not found indbSNP or in normal samples from other cases), [2]non-synonymous and [3] predicted functional impactaccording to MutationAssessor [25], a method to predictthe functional impact of missense mutations on proteinproducts based on evolutionary conservation of aminoacid residues in multi-sequence alignment of homologousprotein sequences.Fluidigm-MiSeq targeted sequencing validationVariants identified by Ion Torrent AmpliSeq sequencingresults were verified by validation sequencing using theFluidigm 48X48 AccessArray amplification (Fluidigm, SanFrancisco/CA/USA) coupled with the Illumina MiSeqpersonal sequencer (Illumina Inc, San Diego/CA/USA)(see Additional file 13). Sequence reads were aligned usingthe mem algorithm of BWA v0.7.4 [26] to a referencedatabase containing only the targeted loci. We inferredthe presence/absence of the targeted variants using aBinomial exact test. In the context of this analysis, asomatic mutation was considered to be “validated” if: [1]both tumor and normal data had a minimum of 50 readscovering the targeted position, [2] the Binomial exacttervening Tx/s Recurrent samples(***Time to recurrence)***Time to/Statuslast followupboplatin/paclitaxel 2-R (46 mo) 86 mo / AWDboplatin/paclitaxel 3-R (17 mo) 19 mo / AWDCarboplatin 4-R1 (25 mo), 4-R2 (45 mo) 60 mo / DODtin/paclitaxel, letrozole 5-R (37 mo) 53 mo / DODboplatin/paclitaxel 6-R (24 mo) 87 mo / DODn/cyclophos-phamide 8-R (7 mo) 12 mo / DODNo treatment 9-R1, R2, R3 (100 mo) 141 mo / DODboplatin/paclitaxel,radiation10-R1, R2, R3, R4 (45 mo) 62 mo / DODNo treatment 11-R (156 mo) 180 mo / DODposide, tamoxifen,anastrozole12-R1, R2 (18 mo) 53 mo / DODboplatin/paclitaxel 13-R (46 mo) 59 mo / DODagnosis.test result (Benjamini Hochberg adjusted p-value) forthe tumor was <0.01, [3] the Binomial exact test result(Benjamini Hochberg adjusted p-value) for the normalwas > =0.01, and [4] the proportion of reads indicating thevariant in the tumor was ≥5%. For the cases without anormal control, the validated variants also shown in thepooled normal data were considered as germline mutationsand were removed. All mutations were visually confirmedusing the Integrative Genomics Viewer (Broad Institute,Cambridge/MA/USA).Sanger sequencingSanger sequencing was used to confirm select high allelicfraction mutations, using methods previously described[27]. Primer sequences are listed in Additional file 15.Raindance raindrop digital PCR assayCustom TaqMan SNP Genotyping assays (Life Technologies,CA/USA) were used as primer/probes (40X) to confirmlow allelic fraction mutations using the RaindanceRaindrop digital PCR assay (Raindance, Billerica/MA/USA).Sequences for primers are shown in Additional file 16.Digital PCR assays were performed as per manufacturer’sprotocols (see Additional file 13).Definition of true positive mutationsOnly those mutations that were detected by at least twoindependent technologies were considered “true positives”and were included in intra-patient comparisons.ResultsOverall mutational landscape of LGSC study casesFollowing initial screening by Ion Torrent and validationby MiSeq, Sanger and/or digital PCR, very few “truepositive” mutations were observed overall, with only 7mutations detected among 5 of 11 cases. No somaticmutations were observed in cases LGSC-2, LGSC-4,LGSC-5 and LGSC-13, whereas predicted mutations incases LGSC-6 and LGSC-8 were only detected by one ofthe sequencing platforms used. True positives includedwas alive with disease. Sequencing analysis discovered aKRAS hotspot mutation (chr12:25,398,284C >A, G12V) ata similar allelic fraction of ~50% (range 48-53%) inthe primary and recurrent samples, suggesting that thiswas a stable feature in this tumor (see Additional file 19for confirmation by Sanger).LGSC-11 is from a 62 year old patient diagnosed withstage IIIC SBT of the left ovary, with ovarian surfaceinvolvement and non-invasive implants. This patientreceived no additional treatment, recurred with metastaticLGSC 13 years later and died of disease 15 years post-diagnosis. The tumor was found to have a KRAS hotspotmutation (chr12:25,398,285C >G, G12R) at a similar allelicfraction in the primary (SBT, 57%) and recurrent (LGSC,44%) sample by both Ion Torrent and MiSeq.ioninTone et al. BMC Cancer 2014, 14:982 Page 4 of 13http://www.biomedcentral.com/1471-2407/14/982KRAS mutations in 3 cases (n = 2 G12V and n = 1G12R), BRAF mutations in 2 cases (n = 1 V600E andn = 1 G469V), and NRAS (Q61R) and SMAD4 (R361G)mutations in one case each. The average allelic fraction ofeach of these mutations in individual samples as deter-mined by Ion Torrent and MiSeq is shown in Figure 1(see Additional file 17 and Additional file 18 for data fromeach platform). Mutational patterns over time/space willbe discussed for individual cases in the following sections.Mutational stability over timeTwo cases with one primary and recurrent sample each(LGSC-3 and LGSC-11) were used to assess temporalstability of confirmed somatic mutations (see Figure 2A-Bfor overview of clinical course).LGSC-3 is from a 51 year old patient diagnosedwith bilateral ovarian LGSC with extensive extra-ovarianinvolvement, stage IIIC (described in Additional file 1).This patient received adjuvant carboplatin/paclitaxel priorto disease recurrence 17 months after primary diagnosis.At last follow-up (19 months post-diagnosis) the patientBRAF V600EBRAF G469VKRAS G12VKRAS G12RNRAS Q61RSMAD4 R361GFigure 1 Average allele fraction of confirmed somatic mutations byin a specific tumor sample (listed at bottom) is indicated by a colored boxaverage allele fraction as detected by Ion Torrent and MiSeq. Correspondingdescribed mutations.Mutational stability over space and timeMultiple samplings from both the primary and recurrentsetting from three cases (LGSC-9, LGSC-10 and LGSC-12)were used to assess the spatial and temporal stability offeatures (see Figure 2C-E for overview of clinical course).LGSC-9 is from a 51 year old patient diagnosedwith stage IIIB SBT of the right ovary with non-invasive implants. No additional treatment was givenafter primary surgery. More than 8 years (100 months)following initial diagnosis, there was tumor recurrenceinvolving the ovary and rectosigmoid, demonstrating ma-lignant transformation to LGSC with borderline features.This was treated by complete surgical resection. A secondrecurrence (sigmoid mass) of LGSC occurred 23 monthslater. At this time she was treated with anastrozole (a non-steroidal aromatase-inhibitor [28]), and died of disease141 months following initial diagnosis. Sequencing ana-lysis revealed a somatic, non-synonymous mutation inNRAS (chr1:115,256,529 T > C, Q61R) at a comparableallele fraction (mean of 50%, range 40-73%) in all sixMutation Frequency0.00-0.050.05-0.150.15-0.250.25-0.350.35-0.450.45-0.550.55-0.650.65-0.750.75-1.00torrent and MiSeq. The presence of a specific mutation (listed on left)the corresponding position, with the shade of the box reflecting thenormal samples are not shown, as these were all negative for theSCno62 urGof Tone et al. BMC Cancer 2014, 14:982 Page 5 of 13http://www.biomedcentral.com/1471-2407/14/982ALGSC-3 Diagnosis(LGSC, 51 years) 17 months Carboplatin/paclitaxel Recurrence 2 months Alive with  disease BLGDiag(SBT, Rec(LDead tumor samples assessed, including 3 samplings fromthe original SBT and 3 from the first recurrence ofLGSC (2 from rectosigmoid and 1 from the left pelvicsidewall). The same mutation was also observed at alower fraction (5%) in a fresh ctDNA sample obtainedfollowing the second recurrence. The stability of thismutation among all 7 samples was confirmed by digitalPCR (Figure 3/Additional file 20).LGSC-12 is from a patient diagnosed with stage IIBLGSC at the age of 57. Her disease was distributedthroughout the pelvis with implants on the rectosigmoidcolon. Following diagnosis, this patient was treated withetoposide (topoisomerase inhibitor), tamoxifen (estro-gen receptor inhibitor) and anastrozole (non-steroidalaromatase inhibitor), before recurring 18 months laterwith LGSC involving the abdominal wall. She died ofdisease 53 months following her original LGSC diagno-sis. Of note, this patient had a documented history ofDLGSC-12 Diagnosis(LGSC, 57 years) 18 months Etoposide, tamoxifen, anastrozole Recurrence 35 months Dead of disease ELGSCDiagn(LGSC, 57Carboplatin/paclitaxel, radiation, anastrozole, etoposide RecurrDead of dRadiation,          pegylated          liposomal       doxorubicin, gemcitabine Figure 2 Overview of clinical course for patients with true positive mLGSC-12 (D) and LGSC-10 (E) are shown, with treatment at each step displ-11 sisyears) 13 years rence SC) 2 years disease CLGSC-9 Diagnosis(SBT, 51 years) 100 months 1st Recurrence (LGSC) 23 months 2nd Recurrence (LGSC) Anastrozole 18 months SBT 36 years prior to her diagnosis with LGSC; howevertissue samples were not available for analysis. Sequen-cing of 4 primary LGSC samples (including 2 from thepelvic tumor, 1 from the rectosigmoid tumor and 1 froma peri-aortic tumor nodule) and 2 recurrent LGSCsamples (both from the abdominal wall tumor) revealedsomatic non-synonymous mutations in both BRAF(chr7:140,453,136A > T, V600E) and SMAD4 (chr18:48,591,918C >G, R361G). Both of these mutations had anallelic fraction of 31-55% in all samples (BRAF median51%, range 37-55%; SMAD4 median 49%, range 31-51%),suggesting that they were both stable over space andtime (see Additional file 19 for confirmation by Sanger inselect samples).LGSC-10 is from a patient diagnosed with bilateralovarian LGSC with extensive extra-ovarian involvement(stage IV) at the age of 57. Adjuvant treatment included6 cycles of carboplatin/paclitaxel, radiation, anastrozoleDead of disease -10 osis years) 45 months ence 17 months isease utations. The clinical course for LGSC-3 (A), LGSC-11 (B), LGSC-9 (C),ayed on the left and time indicated on the right.Tone et al. BMC Cancer 2014, 14:982 Page 6 of 13http://www.biomedcentral.com/1471-2407/14/982and etoposide. This patient recurred with LGSC 45 monthslater at which point she was treated with radiation,liposomal doxorubicin chemotherapy and gemcitabineFigure 3 Stability of nras q61r mutation in multiple tumor samplingsNRAS Q61R mutation in tumor samples from the original SBT (Sample 9P1-panels) by the Raindance Raindrop digital PCR assay are shown. Mutation sthe second LGSC recurrence (Sample 9CTDNA), corresponding normal (Samtype (‘WT’) and mutant (‘MUT’) population are circled in each panel, with thWT +MUT droplets). Consistent with Ion Torrent and MiSeq, the NRAS Q61sample, and was not detected in the corresponding normal.before dying of her disease 62 months following initialdiagnosis. Unlike cases LGSC-9 and LGSC-12, sequencingof 4 primary and 4 recurrent samples revealed extensiveover space and time and circulating tumor DNA. Detection of the9P3, top panels) and first LGSC recurrence (Sample 9R1-9R3, middletatus was also determined in the ctDNA sample obtained followingple 9 N) and non template control (NTC) (bottom panels). The wilde % MUT indicated in the top right corner (MUT drops/total ofR mutation was observed in all 6 tumor samples and the ctDNAmutational variability. As shown by digital PCR in Figure 4(and Additional file 20), 2 of 4 specimens from theprimary setting, both from the right ovary, containeda KRAS G12V hotspot mutation (22-31% allele fraction),while the specimen from the left ovary contained alow level (3-7%) BRAF mutation (chr7:140,481,402C >A, G469V). Neither of these mutations were detected inthe remaining specimen from the primary surgery (vaginalseptal tumor) or any of the recurrent samples (including 3from a right lower quadrant subcutaneous nodule and 1r bcorthinn o, wTone et al. BMC Cancer 2014, 14:982 Page 7 of 13http://www.biomedcentral.com/1471-2407/14/982Figure 4 Instability of KRAS G12V AND BRAF G469V mutations oveconfirm KRAS and BRAF mutation status in all 8 tumor samples and the(Sample 10P1-10P4) shown on the left and a representative sample from(Sample 10 N) shown on the right. The relative location of each samplesurgery colored in green and those from the recurrent surgery colored iwild type (‘WT’) and mutant (‘MUT’) population are circled in each panelWT +MUT droplets). The KRAS G12V mutation was detected in Samples 10in Sample 10P3. All remaining samples were negative for KRAS G12V, BRAFoth space and time. Raindance Raindrop digital PCR was used toresponding normal, with the four samples from the primary surgerye recurrent surgery (Sample 10R1) and the corresponding normalthe patient is shown in the bottom right, with those from the primaryrange (courtesy of Vicky Earle, UBC graphics). Similar to Figure 3, theith the % MUT indicated in the top right corner (MUT drops/total ofP1 and 10P2, while the BRAF G469V mutation was exclusively detectedG469V and NRAS Q61R (not shown).from an umbilical margin large nodule). This was nota reflection of tumor purity, as all mutation-negativespecimens had comparable tumor cellularity by histo-pathologic assessment (80-95%) and identical allelefractions of common SNPs in FGFR3 and PDGFRA(≥99%, data not shown).Overall trends in mutational stabilityAs shown in Table 2, four of five cases with true positivemutations were stable over time and/or space, includingtwo cases that originally presented as SBT and recurredas an invasive LGSC. In contrast, one case showedinstability of KRAS and BRAF over both space andTone et al. BMC Cancer 2014, 14:982 Page 8 of 13http://www.biomedcentral.com/1471-2407/14/982time. Overall, mutations in NRAS and SMAD4 werestable in one case each, while genes mutated in more thanone study case (KRAS and BRAF) showed differentpatterns of stability/instability for distinct variants(BRAF V600E vs. G469V, KRAS G12R vs. G12V) andeven for the same variant (KRAS G12V).DiscussionAmong the 11 cases of LGSC sequenced in our study,only 7 confirmed somatic mutations were identified in5 cases from a targeted hotspot panel of 46 cancer-associated genes. This low mutation rate is consistentwith the detection of only 10 mutations per tumor byexome sequencing by Jones et al. [10], and furthersuggests that few mutational events are required toachieve malignancy. The frequency of mutations inLGSC is much lower than in other subtypes of ovariancarcinoma such as HGSC (n = 61 mutations/tumor byexome sequencing) [29] and clear cell carcinoma (n = 34mutations/tumor by exome sequencing) [30]. This likelysuggests that: [1] there is limited replication of precursorcells prior to initiation of tumorigenesis, [2] there are fewbottlenecks once initiation occurs, and [3] the ratio ofdriver to passenger mutations should be higher than inother tumor types [10]. Consequently, targeted agentsTable 2 Overall trends in stability over time and space forconfirmed somatic mutations in LGSC*Time Time and SpaceLGSC-3 LGSC-11 LGSC-9 LGSC-10 LGSC-12BRAF G469V UnstableBRAF V600E StableKRAS G12R StableKRAS G12V Stable UnstableNRAS Q61R StableSMAD47 R361G Stable*Only those mutations observed by two independent technologies(true positives) included.Note: not all cases included in table as no confirmed somatic mutations inLGSC-2, −4, −5 or −13; mutations in LGSC-6 and −8 only observed by eitherIon Torrent or MiSeq.would likely be particularly effective in women with LGSCif key mutations are shown to be stable.The most commonly reported drivers in LGSC areKRAS and BRAF. We detected a KRAS mutation inthree patients (including two stage IIIC and one stageIV) and a BRAF mutation in two patients (including onestage IIB and one stage IV). Previous studies havereported conflicting findings with respect to mutation ofKRAS/BRAF and disease stage, with the Jones study [10]detecting KRAS or BRAF mutations in 4/13 (31%) and3/13 (23%) of stage III LGSC patients respectively.Additional studies report BRAF mutations in only 3%[12] and 5% [13] of advanced stage LGSC. Grishamand Wong both reported that women with mutationsin KRAS and/or BRAF [12,13] experience a more favorableoutcome than women without these mutations. This posi-tive prognostic effect appears to be dominated by BRAFV600E mutations, with a lower incidence of stage III-IVdisease, enrichment for SBT rather than invasive LGSCand reduced requirement for systemic treatment amongwomen with this mutation [12,13]. Possible explanationsinclude reports that SBTs from women with BRAFmutations over-express genes with cell growth inhibitoryeffects [12] or that activating BRAF mutations inducecellular senescence and prevent progression to LGSC[12,31-33]. In our study we observed a trend for increasedmean overall survival in study patients with a MAPKpathway mutation (KRAS, BRAF, NRAS) compared topatients with wildtype status (92 months vs. 60 monthsrespectively; p = 0.23); however, this difference in out-come was largely influenced by the two cases originallypresenting as a SBT (143 and 183 months) and disappearedwhen these cases were excluded from the analysis.The mutational status of NRAS, member of the MAPKpathway, showed stability over multiple different tumorsites and over a span of 8 years between original diagnosiswith SBT and recurrence with an invasive LGSC (caseLGSC-9). The presence of this stable feature at a low levelin plasma ctDNA, obtained following a second recurrenceof LGSC, also clearly highlights the potential utility ofthis source for disease monitoring (i.e. tumor response,persistence or recurrence).SMAD4 mutational status in case LGSC-12 was alsoconsistent among 6 tumor samples from 4 different sitesin the primary and recurrence, and despite multipletreatment cycles. Although found to be unstable inanother case, all samples from LGSC-12 also contained aBRAF mutation at a similar allelic fraction. The observedSMAD4 mutation (chr18:48,591,918C >G, R361G) is at ahighly conserved genomic position among placentalmammals, and is situated within the C-terminus MH2domain of the SMAD4 protein. This domain mediatesprotein-protein interactions and provides functionalspecificity and selectivity. It was previously reportedTone et al. BMC Cancer 2014, 14:982 Page 9 of 13http://www.biomedcentral.com/1471-2407/14/982as the most frequent target of SMAD4 missense mutationsin human tumors, with a mutational hotspot correspondingto codons 330–370 [34]. Lassus et al. reported allelic loss atone or more loci at 18q12.3-q23 in 59% of ovarian serouscarcinomas (or 7.1% of grade 1 tumors), with lost orweak expression of SMAD4 protein in a subset ofthese tumors [35]. Mutations in SMAD4 have beenreported to frequently co-exist with KRAS mutationsin colorectal cancer [36], and studies in pancreaticcancer suggest that wildtype SMAD4 blocks progressionof KRAS G12D-initiated tumors [37]. In addition,mutation of KRAS, NRAS and BRAF [38-46], and lossof functional SMAD4 [47], have all been reported topredict resistance to anti-EGFR therapy. Unfortunately wewere unable to assess the impact of the SMAD4 R361Gmutation on protein expression by IHC in our samples,therefore we cannot comment on the utility of SMAD4mutation status as a predictive marker in women withLGSC without further study.In contrast to NRAS and SMAD4, mutations in KRASand BRAF were not stable in one patient (LGSC-10) inour study, despite traditionally being thought of as‘drivers’ of tumorigenesis. This is akin to our recentobservation that mutations in other key ‘drivers’ PIK3CAand CTNNB1 are only present in a subset of ovarianHGSC samples from the same patient [48]. These examplesclearly defy the concept of oncogene addiction, whichposits that the growth and survival of a tumor is dependenton a single dominant oncogene [49,50]. Our findings inLGSC-10 suggest that even at the time of primarydiagnosis three distinct tumors/clones were present(i.e. KRAS-mutation positive, BRAF-mutation positive andKRAS/BRAF-mutation negative). As neither KRAS norBRAF were mutated in any of the recurrent samples, adifferent, as yet unidentified, dominant gene or pathwayin the KRAS/BRAF-negative population was likely drivingdisease recurrence. One possibility is that we have misseda mutation in gene/s either directly or indirectly involvedin the MAPK pathway that is not included on the targetedpanel used to screen our samples. The KRAS and BRAFmutations were detected at an allelic fraction of 22-31% inthe right ovary and 3-7% in the left ovary respectively,hence the clonal population containing an undetecteddriver mutation could have already been present in someor all of the tumor samples at primary debulking; expan-sion/recurrence of this population could then explain theabsence of mutant KRAS/BRAF in the recurrent set-ting. In addition, mutations such as those in KRASand BRAF that occur early in the development of SBT/LGSC [17] may not be required and/or advantageous fortumor maintenance once additional alterations are ac-quired. This phenomenon has previously been describedin HGSC, in which secondary mutations in BRCA1/2restore protein function and result in acquired resistanceto treatment [51]; however, reversion of both a KRASand BRAF mutation in the current scenario seemshighly unlikely.Of potential interest, LGSC-10 was the only study casediagnosed with stage IV disease and the only patienttreated with radiation after primary diagnosis. While thepresence of mutational instability in the primary setting(prior to treatment) argues against a direct impact ofradiation, the possibility of instability exclusively in stageIV LGSC is an intriguing one that requires more study.To date, limited studies have reported on either temporalor spatial instability of BRAF/KRAS mutations in SBT andLGSC. Instability in KRAS mutation status was recentlydescribed in a subset of matched SBT-LGSC pairs(2/5 cases discordant) [52] and matched SBT-peritonealimplant pairs (3/37 discordant for KRAS, while 14/14 con-cordant for BRAF) [53]. A recent study by Heublein et al.[54] also noted instability in KRAS and BRAF in 2/5 casesof bilateral SBT. In one case, a KRAS G12V mutationwas detected in one ovary and a BRAF V600E mutationwas detected in the contralateral ovary, while the othercase contained a KRAS G12V and BRAF V600E mutationin one ovary and only a KRAS G12V mutation in the otherovary. This is consistent with our finding of spatial hetero-geneity in the primary setting in LGSC-10. Unfortunately,a detailed breakdown of disease stage in cases with dis-cordant vs. concordant sample pairs was not provided inany of these studies. Instability in KRAS has also been de-scribed for metastatic colorectal cancer [55,56]. Bossardet al. [55] reported several patterns of heterogeneity inKRAS mutation status in 22% of 18 colorectal carcin-omas studied. This included exclusive presence in theprimary tumor or metastatic site, presence in somemetastases but not others, varied status among differ-ent samplings from the same metastatic site, andpresence in the recurrent but not primary setting.Similarly, Otsuka et al. [56] reported the presence of aKRAS mutation in metastatic sites but not the primarycolorectal tumor in 1 of 9 patients studied; BRAFmutation status was concordant in all cases, in contrast towhat we observed.Our finding that mutations in genes such as KRAS orBRAF are not necessarily stable features could providean alternative explanation, in some patients, for the lackof correlation between response to selumetinib andKRAS/BRAF mutation status observed by Farley et al.[18]. Targeted sequencing (i.e. codon 599 of BRAF andcodons 12 and 13 of KRAS) using a single representativetumor sample from 34/52 (65%) patients revealed aBRAF and KRAS mutation in 2 (6%) and 14 (41%) casesrespectively. A similar proportion of mutation positivevs. negative cases responded to selumetinib treatment,leading the authors to postulate that its activity maynot depend on BRAF/KRAS mutational activation. TissueAdditional file 1: “Additional Information on Study Samples”.Provides more detailed information on pathologic diagnosis, DNATone et al. BMC Cancer 2014, 14:982 Page 10 of 13http://www.biomedcentral.com/1471-2407/14/982used for mutational analysis was obtained from the primarytumor in 82% of sequenced cases, metastatic tumor in 6%and recurrent or persistent tumor in 12% of cases. It istherefore possible that targetable mutations detected in theprimary tumor were not present in the metastatic or recur-rent tumor, or vice versa, leading to altered treatmentresponse. It is also possible that some of these patients hadundetected mutations in NRAS, a stable feature in ourstudy, which also predicts response to MEK inhibitors.It is important to recognize the limitations of ourstudy, most notably small sample size and use of a hot-spot targeted gene panel. Firstly, the small number ofcases used in this study (despite being a collaborationbetween three institutions) is illustrative of the challengein identifying primary-recurrent pairs for a rare tumortype such as LGSC. Confirmation of our findings in alarger cohort of LGSC will therefore require participa-tion by multiple institutions or establishment of a world-wide registry. Secondly, by limiting the sequencingdiscovery phase to a panel of hotspot mutations in 46genes, it is highly likely that we have missed additionalcase-specific mutations in our study population. How-ever, a closer look at the mutations discovered by Joneset al. through exome sequencing [10] revealed that onlyKRAS and BRAF were recurrently mutated in LGSC.This suggests that it is also unlikely that we have missedadditional recurrent drivers of disease, although patient-specific drivers outside the normal patterns of LGSCmay exist. Thirdly, we have not investigated potential al-ternative drivers of disease that may be important incases without identified somatic mutations, such as copynumber alterations, epigenetic changes or microRNAs.Singer et al. [57] previously reported a progressive in-crease in copy number alterations from SBT through toLGSC, most notably allelic imbalance of chromosomes1p, 5q, 8p, 18q and 22q. This was confirmed by Kuoet al. [58] who reported an increased chromosomal in-stability index in LGSC relative to SBT, suggesting thatamplifications, deletions and aneuploidy play a role inthe malignant transformation of SBT. Hemizygous dele-tion of chromosome 1p36 was especially enriched inLGSC samples; this region contains the microRNA miR-34a, which was found to have an anti-proliferative andpro-apoptotic effect in an LGSC cell line [58]. Finally,several groups have reported on differential methylationpatterns in SBT and LGSC [59-61], suggesting thatmethylation-induced transcriptional silencing of tumorsuppressor genes may play an undefined role in malig-nant transformation and progression and response tosystemic or targeted therapy.ConclusionsThe extent of intratumoral heterogeneity in kidney,breast, leukemia and ovarian cancers has recently beenquantity and estimated cellularity.Additional file 2: “LGSC-2 Case Images”. LGSC-2 is from a patientdiagnosed with bilateral ovarian LGSC (stage IIIC) at 57 years old(LGSC-2-P, top) and metastatic LGSC 46 months after primary diagnosis(LGSC-2-R, bottom; both 20X).Additional file 3: “LGSC-3 Case Images”. LGSC-3 is from a patientdiagnosed with bilateral ovarian LGSC (stage IIIC) at 51 years old(LGSC-3-P, top), and recurrent LGSC 17 months later (LGSC-3-R,bottom; both 20X).Additional file 4: “LGSC-4 Case Images”. LGSC-4 is from a patientdiagnosed with ovarian LGSC (IIIB) at the age of 66 (LGSC-4-P), followedby two separate recurrences 25 months (LGSC-4-R1) and 45 months(LGSC-4-R2) later (all 20X).Additional file 5: “LGSC-5 Case Images”. LGSC-5 is from a patientdiagnosed with LGSC (stage IIIC) at age 51 (LGSC-5-P, top) and recurrentLGSC 37 months later (LGSC-5-R, bottom; both images 100X).Additional file 6: “LGSC-6 Case Images”. LGSC-6 is from a patientdiagnosed with LGSC (stage IIIC) at 41 years old (LGSC-6-P, top) andrecurrent LGSC 24 months later (LGSC-6-R, bottom; both images 100X).Additional file 7: “LGSC-8 Case Images”. LGSC-8 is from a patientdiagnosed with metastatic LGSC (stage IIIC) at the age of 33 (LGSC-8-P,top), with disease recurrence 7 months later (LGSC-8-R, bottom; bothimages 100X).Additional file 8: “LGSC-9 Case Images”. LGSC-9 is from a patientdiagnosed with a serous borderline tumor (stage IIIB) at age 51 (LGSC-9-P1,LGSC-9-P2, LGSC-9-R3 shown in left panels). This patient received noadditional treatment after surgical resection and recurred with LGSC100 months later (LGSC-9-R1, LGSC-9-R2, LGSC-9-R3 shown in rightpanels; all images 20X).described [48,62-64]. Most papers have focused on high-grade cancers with many somatic mutations, and mostof the mutations described have no immediate clinicalrelevance. Herein we show that, in a cancer type knownto have a sparse mutational landscape [10], heterogen-eity in targetable mutations can be observed. While thevast majority of evaluable cases contained mutations thatwere detected in all samples, one case showed remark-able instability in hotspot mutations of presumed driversof disease, despite not receiving treatment that couldhave driven the specific evolution of (KRAS/BRAF) mu-tant clones. In addition, as we looked within a limitedmutational space, the possibility remains that moreunderlying heterogeneity may be revealed in more caseswith further study. Investigation of additional cases is re-quired to confirm whether a consistent minority ofLGSC cases show clinically relevant mutational hetero-geneity; this would necessitate a change in clinical trialdesign with contemporary samplings of a cancer re-quired to guide treatment decisions. Alternatively, if notfound to be a general phenomenon upon further study,confirmation of mutational status in a single samplewould be sufficient.Additional filesAdditional file 9: “LGSC-10 Case Images”. LGSC-10 is from a patientdiagnosed with bilateral ovarian LGSC (stage IV) at the age of 57by a 2-tier system: a gynecologic oncology group study. Cancer 2012,Tone et al. BMC Cancer 2014, 14:982 Page 11 of 13http://www.biomedcentral.com/1471-2407/14/982(LGSC-10-P1, LGSC-10-P2, LGSC-10-P3, LGSC-10-P4 shown in left panels),followed by disease recurrence 45 months later (LGSC-10-R1, LGSC-10-R2,LGSC-10-R3, LGSC-10-R4 shown in right panels; all images at 20X).Additional file 10: “LGSC-11 Case Images”. LGSC-11 is from a patientdiagnosed with a serous borderline tumor (stage IIIC) at 62 years(LGSC-11-P, top) followed by recurrence with LGSC 13 years later(LGSC-11-R, bottom; both images at 20X).Additional file 11: “LGSC-12 Case Images”. LGSC-12 is from a patientdiagnosed with LGSC (stage IIB) at the age of 57 (LGSC-12-P1, LGSC-12-P2,LGSC-12-P3, LGSC-12-P4 are shown). This patient was treated with etoposide,tamoxifen and anastrozole prior to recurrence with LGSC 18 months later(LGSC-12-R1, LGSC-12-R2 are shown; all images at 20X).Additional file 12: “LGSC-13 Case Images”. LGSC-13 is from a patientdiagnosed with LGSC (stage IIIB) at the age of 58 (LGSC-13-P, top),followed by recurrence with LGSC 46 months later (LGSC-13-R, bottom;both images 20X).Additional file 13: “Supplemental Methods”. Describes additionalmethodological details for DNA extraction, sequencing and digital PCR.Additional file 14: “Genes/Mutations on Ion Torrent AmpliSeqpanel v1”. Lists genes and hot spot mutations included on the IonTorrent AmpliSeq panel.Additional file 15: “Primer sequences for Sanger sequencing”. Listsprimer sequences used for validation of mutations by Sanger sequencing.Additional file 16: “Primer sequences for digital PCR”. Lists primersequences used for validation of mutations by digital PCR.Additional file 17: “Allele fraction of confirmed somatic mutationsby Ion Torrent and MiSeq”. The presence of a specific mutation (listed onleft) in a specific tumor sample (listed at bottom) is indicated by a coloredbox in the corresponding position, with the shade of the box reflecting theallelic fraction as detected by (A) Ion Torrent or (B) MiSeq. Correspondingnormal samples were all negative for the described mutations.Additional file 18: “Ion Torrent and MiSeq reads for true positivemutations”. Lists the variant reads, total reads and variant frequency bysample for both Ion Torrent and MiSeq.Additional file 19: “Stable KRAS, BRAF and SMAD4 mutations incases 3 and 12 by Sanger sequencing”. Detection of the KRAS G12Vmutation by Sanger sequencing in LGSC-3-P (A) and LGSC-3-R (B) areshown. Sanger sequencing also confirmed the presence of the BRAFV600E and the SMAD4 R361G mutation in LGSC-12-P1 (C and F respectively)and LGSC-12-R1 (D and G respectively), but not the corresponding normalsample LGSC-12-N (E and H respectively).Additional file 20: “Digital PCR results”. Lists the % mutant and %wildtype droplets corresponding to the digital PCR results shown inFigures 3 and 4.AbbreviationsBCCA: BC cancer agency; BRAF: V-raf murine sarcoma viral oncogenehomolog B1; ctDNA: Circulating tumor DNA; DNA: Deoxyribonucleic acid;ERK: Extracellular signal-regulated kinase; FFPE: Formalin-fixed paraffin-embedded; FGFR3: Fibroblast growth factor receptor 3; HGSC: High-gradeserous carcinoma; KRAS: Kirsten rat sarcoma viral oncogene homolog;LGSC: Low-grade serous carcinoma; MAPK: Mitogen-activated kinase;MDACC: MD Anderson cancer centre; MEK: MAP kinase kinase;NRAS: Neuroblastoma RAS viral (v-ras) oncogene homolog; PCR: Polymerasechain reaction; PDGFRA: Platelet-derived growth factor receptor alpha;SBT: Serous borderline tumor; SMAD4: Mothers against decapentaplegichomolog 4; SNV: Single nucleotide variant; UHN: University health network.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAAT contributed to study design, data collection, analysis and interpretation,generation of figures, literature searches and writing of the manuscript. MMcontributed to data collection, data analysis, data interpretation, generationof figures and writing of the manuscript. WY contributed to data collectionand data analysis. JD performed data analysis and contributed to generation118(12):3087–3094.5. Malpica A, Deavers MT, Lu K, Bodurka DC, Atkinson EN, Gershenson DM,Silva EG: Grading ovarian serous carcinoma using a two-tier system.Am J Surg Pathol 2004, 28(4):496–504.6. Vang R, Shih Ie M, Kurman RJ: Ovarian low-grade and high-grade serouscarcinoma: pathogenesis, clinicopathologic and molecular biologicfeatures, and diagnostic problems. Adv Anat Pathol 2009, 16(5):267–282.7. Schmeler KM, Sun CC, Bodurka DC, Deavers MT, Malpica A, Coleman RL,References1. Gershenson DM, Sun CC, Lu KH, Coleman RL, Sood AK, Malpica A, Deavers MT,Silva EG, Bodurka DC: Clinical behavior of stage II-IV low-grade serouscarcinoma of the ovary. Obstet Gynecol 2006, 108(2):361–368.2. Schmeler KM, Gershenson DM: Low-grade serous ovarian cancer:a unique disease. Curr Oncol Rep 2008, 10(6):519–523.3. Diaz-Padilla I, Malpica AL, Minig L, Chiva LM, Gershenson DM, Gonzalez-Martin A: Ovarian low-grade serous carcinoma: a comprehensive update.Gynecol Oncol 2012, 126(2):279–285.4. Bodurka DC, Deavers MT, Tian C, Sun CC, Malpica A, Coleman RL, Lu KH,Sood AK, Birrer MJ, Ozols R, Baergen R, Emerson RE, Steinhoff M, BehmaramB, Rasty G, Gershenson DM: Reclassification of serous ovarian carcinomaAcknowledgementsWe would like to acknowledge our funding sources, including the BC CancerFoundation, VGH + UBC Hospital Foundation, Canadian Cancer SocietyResearch Institute Impact Grant led by D. Huntsman (Contextual genomics:The foundation for subtype specific approaches to ovarian cancer control) andThe University of Texas MD Anderson Cancer Centre Specialized Program ofResearch Excellence in Ovarian Cancer NIH grant # P50 CA08369.Author details1Department of Pathology and Laboratory Medicine, University of BritishColumbia, Vancouver, BC, Canada. 2BC Cancer Agency, Room 3427, 600 West10th Avenue, Vancouver, BC, Canada. 3Division of Gynecologic Oncology,Princess Margaret Cancer Centre, Toronto, ON, Canada. 4Department ofComputer Science, University of British Columbia, Vancouver, BC, Canada.5Department of Gynecologic Oncology & Reproductive Medicine, Universityof Texas MD Anderson Cancer Center, Houston, TX, USA. 6Division of MedicalOncology and Hematology, Princess Margaret Hospital, Toronto, ON, Canada.7Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON,Canada. 8Obstetrics and Gynecology, University of British Columbia,Vancouver, BC, Canada.Received: 22 July 2014 Accepted: 11 December 2014Published: 18 December 2014of figures and writing of the manuscript. SY and EK contributed to datacollection. KKW, DG, HM, BG, AVT, JM and BC all participated in theconceptualization of the study/study design and sample selection/acquisition. SS contributed to data analysis. DH participated in theconceptualization and design of the study, data interpretation, manuscriptpreparation and supervised the project. All authors read and approved thefinal manuscript.Authors’ informationAlicia A. Tone, PhDScientific Associate IIMelissa K. McConechy, BScDoctoral CandidateDavid Huntsman, MD, FRCPC, FCCMGDr. Chew Wei Memorial Professor of Gynaecologic OncologyUBC Professor, Departments of Pathology & Lab Medicine and Obstetrics &Gynaecology UBC Director of OvCaRe, Vancouver General Hospital, BCCancer AgencyUBC Medical Director, Centre for Translational and Applied Genomics, PHSALaboratoriesRamirez PT, Gershenson DM: Neoadjuvant chemotherapy for low-gradeserous carcinoma of the ovary or peritoneum. Gynecol Oncol 2008,108(3):510–514.Tone et al. BMC Cancer 2014, 14:982 Page 12 of 13http://www.biomedcentral.com/1471-2407/14/9828. Gershenson DM, Sun CC, Bodurka D, Coleman RL, Lu KH, Sood AK, Deavers M,Malpica AL, Kavanagh JJ: Recurrent low-grade serous ovarian carcinoma isrelatively chemoresistant. Gynecol Oncol 2009, 114(1):48–52.9. Fader AN, Java J, Ueda S, Bristow RE, Armstrong DK, Bookman MA,Gershenson DM: Survival in women with grade 1 serous ovariancarcinoma. Obstet Gynecol 2013, 122(2 Pt 1):225–232.10. Jones S, Wang TL, Kurman RJ, Nakayama K, Velculescu VE, Vogelstein B,Kinzler KW, Papadopoulos N, Shih Ie M: Low-grade serous carcinomasof the ovary contain very few point mutations. J Pathol 2012,226(3):413–420.11. Singer G, Oldt R III, Cohen Y, Wang BG, Sidransky D, Kurman RJ, Shih Ie M:Mutations in BRAF and KRAS characterize the development of low-gradeovarian serous carcinoma. J Natl Cancer Inst 2003, 95(6):484–486.12. Wong KK, Tsang YT, Deavers MT, Mok SC, Zu Z, Sun C, Malpica A, Wolf JK,Lu KH, Gershenson DM: BRAF mutation is rare in advanced-stage low-grade ovarian serous carcinomas. Am J Pathol 2010, 177(4):1611–1617.13. Grisham RN, Iyer G, Garg K, DeLair D, Hyman DM, Zhou Q, Iasonos A, BergerMF, Dao F, Spriggs DR, Levine DA, Aghajanian C, Solit DB: BRAF mutation isassociated with early stage disease and improved outcome in patientswith low-grade serous ovarian cancer. Cancer 2013, 119(3):548–554.14. Romero I, Sun CC, Wong KK, Bast RC Jr, Gershenson DM: Low-grade serouscarcinoma: new concepts and emerging therapies. Gynecol Oncol 2013,130(3):660–666.15. Staebler A, Heselmeyer-Haddad K, Bell K, Riopel M, Perlman E, Ried T,Kurman RJ: Micropapillary serous carcinoma of the ovary has distinctpatterns of chromosomal imbalances by comparative genomichybridization compared with atypical proliferative serous tumors andserous carcinomas. Hum Pathol 2002, 33(1):47–59.16. Crispens MA, Bodurka D, Deavers M, Lu K, Silva EG, Gershenson DM:Response and survival in patients with progressive or recurrent serousovarian tumors of low malignant potential. Obstet Gynecol 2002,99(1):3–10.17. Ho CL, Kurman RJ, Dehari R, Wang TL, Shih IM: Mutations of BRAF andKRAS precede the development of ovarian serous borderline tumors.Cancer Res 2004, 64(19):6915–6918.18. Farley J, Brady WE, Vathipadiekal V, Lankes HA, Coleman R, Morgan MA,Mannel R, Yamada SD, Mutch D, Rodgers WH, Birrer M, Gershenson DM:Selumetinib in women with recurrent low-grade serous carcinoma ofthe ovary or peritoneum: an open-label, single-arm, phase 2 study.Lancet Oncol 2013, 14(2):134–140.19. Nakayama N, Nakayama K, Yeasmin S, Ishibashi M, Katagiri A, Iida K,Fukumoto M, Miyazaki K: KRAS or BRAF mutation status is a usefulpredictor of sensitivity to MEK inhibition in ovarian cancer. Br J Cancer2008, 99(12):2020–2028.20. Allen LF, Sebolt-Leopold J, Meyer MB: CI-1040 (PD184352), a targetedsignal transduction inhibitor of MEK (MAPKK). Semin Oncol 2003,30(5 Suppl 16):105–116.21. Pohl G, Ho CL, Kurman RJ, Bristow R, Wang TL, Shih IM: Inactivation of themitogen-activated protein kinase pathway as a potential target-basedtherapy in ovarian serous tumors with KRAS or BRAF mutations. CancerRes 2005, 65(5):1994–2000.22. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH,Johnson K, Milgrew MJ, Edwards M, Hoon J, Simons JF, Marran D, Myers JW,Davidson JF, Branting A, Nobile JR, Puc BP, Light D, Clark TA, Huber M,Branciforte JT, Stoner IB, Cawley SE, Lyons M, Fu Y, Homer N, Sedova M,Miao X, Reed B et al: An integrated semiconductor device enablingnon-optical genome sequencing. Nature 2011, 475(7356):348–352.23. Li H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 2010, 26(5):589–595.24. Ding J, Bashashati A, Roth A, Oloumi A, Tse K, Zeng T, Haffari G, Hirst M,Marra MA, Condon A, Aparicio S, Shah SP: Feature-based classifiers forsomatic mutation detection in tumour-normal paired sequencing data.Bioinformatics 2012, 28(2):167–175.25. Reva B, Antipin Y, Sander C: Predicting the functional impact of proteinmutations: application to cancer genomics. Nucleic Acids Res 2011,39(17):e118.26. Li H: Aligning Sequence Reads, Clone Sequences and Assembly Contigs withbwa-mem. arXiv Preprint arXiv. 2013:1303.27. McConechy MK, Anglesio MS, Kalloger SE, Yang W, Senz J, Chow C, Heravi-Moussavi A, Morin GB, Mes-Masson AM, Carey MS, McAlpine JN, Kwon JS,Prentice LM, Boyd N, Shah SP, Gilks CB, Huntsman DG: Subtype-specificmutation of PPP2R1A in endometrial and ovarian carcinomas. J Pathol2011, 223(5):567–573.28. Geisler J: Differences between the non-steroidal aromatase inhibitorsanastrozole and letrozole–of clinical importance? Br J Cancer 2011,104(7):1059–1066.29. Cancer Genome Atlas Research Network: Integrated genomic analyses ofovarian carcinoma. Nature 2011, 474(7353):609–615.30. Jones S, Wang TL, Shih Ie M, Mao TL, Nakayama K, Roden R, Glas R, SlamonD, Diaz LA Jr, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N:Frequent mutations of chromatin remodeling gene ARID1A in ovarianclear cell carcinoma. Science 2010, 330(6001):228–231.31. Dhomen N, Reis-Filho JS, da Rocha Dias S, Hayward R, Savage K, Delmas V,Larue L, Pritchard C, Marais R: Oncogenic Braf induces melanocytesenescence and melanoma in mice. Cancer Cell 2009, 15(4):294–303.32. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR: Oncogenic BRAFinduces senescence and apoptosis through pathways mediated by thesecreted protein IGFBP7. Cell 2008, 132(3):363–374.33. Zeppernick F, Ardighieri L, Hannibal CG, Vang R, Junge J, Kjaer SK, Zhang R,Kurman RJ, Shih IM: BRAF mutation is associated with a specific cell typewith features suggestive of senescence in ovarian serous borderline(Atypical Proliferative) tumors. Am J Surg Pathol. 2014, 38(12):1603–1611.34. Iacobuzio-Donahue CA, Song J, Parmiagiani G, Yeo CJ, Hruban RH, Kern SE:Missense mutations of MADH4: characterization of the mutational hotspot and functional consequences in human tumors. Clin Cancer Res2004, 10(5):1597–1604.35. Lassus H, Salovaara R, Aaltonen LA, Butzow R: Allelic analysis of serousovarian carcinoma reveals two putative tumor suppressor loci at18q22-q23 distal to SMAD4, SMAD2, and DCC. Am J Pathol 2001,159(1):35–42.36. Sameer AS, Chowdri NA, Syeed N, Banday MZ, Shah ZA, Siddiqi MA:SMAD4–molecular gladiator of the TGF-beta signaling is trampledupon by mutational insufficiency in colorectal carcinoma of Kashmiripopulation: an analysis with relation to KRAS proto-oncogene. BMCCancer. 2010, 10:300.37. Bardeesy N, Cheng KH, Berger JH, Chu GC, Pahler J, Olson P, Hezel AF,Horner J, Lauwers GY, Hanahan D, DePinho RA: Smad4 is dispensable fornormal pancreas development yet critical in progression and tumorbiology of pancreas cancer. Genes Dev 2006, 20(22):3130–3146.38. Vakiani E, Solit DB: KRAS and BRAF: drug targets and predictivebiomarkers. J Pathol 2011, 223(2):219–229.39. Miller VA, Riely GJ, Zakowski MF, Li AR, Patel JD, Heelan RT, Kris MG,Sandler AB, Carbone DP, Tsao A, Herbst RS, Heller G, Ladanyi M, Pao W,Johnson DH: Molecular characteristics of bronchioloalveolar carcinomaand adenocarcinoma, bronchioloalveolar carcinoma subtype, predictresponse to erlotinib. J Clin Oncol 2008, 26(9):1472–1478.40. Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M, Zakowski MF, Heelan RT,Kris MG, Varmus HE: KRAS mutations and primary resistance of lungadenocarcinomas to gefitinib or erlotinib. PLoS Med 2005, 2(1):e17.41. Zhu CQ, da Cunha Santos G, Ding K, Sakurada A, Cutz JC, Liu N, Zhang T,Marrano P, Whitehead M, Squire JA, Kamel-Reid S, Seymour L, Shepherd FA,Tsao MS: Role of KRAS and EGFR as biomarkers of response to Erlotinibin national cancer institute of Canada clinical trials group study BR.21.J Clin Oncol 2008, 26(26):4268–4275.42. Hirsch FR, Varella-Garcia M, Cappuzzo F, McCoy J, Bemis L, Xavier AC,Dziadziuszko R, Gumerlock P, Chansky K, West H, Gazdar AF, Crino L,Gandara DR, Franklin WA, Bunn PA Jr: Combination of EGFR gene copynumber and protein expression predicts outcome for advancednon-small-cell lung cancer patients treated with gefitinib. Ann Oncol2007, 18(4):752–760.43. Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS,Ince WL, Janne PA, Januario T, Johnson DH, Klein P, Miller VA, Ostland MA,Ramies DA, Sebisanovic D, Stinson JA, Zhang YR, Seshagiri S, Hillan KJ:Mutations in the epidermal growth factor receptor and in KRAS arepredictive and prognostic indicators in patients with non-small-cell lungcancer treated with chemotherapy alone and in combination witherlotinib. J Clin Oncol 2005, 23(25):5900–5909.44. Janakiraman M, Vakiani E, Zeng Z, Pratilas CA, Taylor BS, Chitale D, HalilovicE, Wilson M, Huberman K, Ricarte Filho JC, Persaud Y, Levine DA, Fagin JA,Jhanwar SC, Mariadason JM, Lash A, Ladanyi M, Saltz LB, Heguy A, Paty PB,Solit DB: Genomic and biological characterization of exon 4 KRASmutations in human cancer. Cancer Res 2010, 70(14):5901–5911.64. Ding L, Ley TJ, Larson DE, Miller CA, Koboldt DC, Welch JS, Ritchey JK,Young MA, Lamprecht T, McLellan MD, McMichael JF, Wallis JW, Lu C, Shen D,Harris CC, Dooling DJ, Fulton RS, Fulton LL, Chen K, Schmidt H, Kalicki-Veizer J,Magrini VJ, Cook L, McGrath SD, Vickery TL, Wendl MC, Heath S, Watson MA,Link DC, Tomasson MH et al: Clonal evolution in relapsed acute myeloidleukaemia revealed by whole-genome sequencing. Nature 2012,481(7382):506–510.doi:10.1186/1471-2407-14-982Cite this article as: Tone et al.: Intratumoral heterogeneity in a minorityof ovarian low-grade serous carcinomas. BMC Cancer 2014 14:982.Tone et al. BMC Cancer 2014, 14:982 Page 13 of 13http://www.biomedcentral.com/1471-2407/14/98245. De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G,Kalogeras KT, Kotoula V, Papamichael D, Laurent-Puig P, Penault-Llorca F,Rougier P, Vincenzi B, Santini D, Tonini G, Cappuzzo F, Frattini M, Molinari F,Saletti P, De Dosso S, Martini M, Bardelli A, Siena S, Sartore-Bianchi A,Tabernero J, Macarulla T, Di Fiore F, Gangloff AO, Ciardiello F, Pfeiffer P et al:Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy ofcetuximab plus chemotherapy in chemotherapy-refractory metastaticcolorectal cancer: a retrospective consortium analysis. Lancet Oncol 2010,11(8):753–762.46. Loupakis F, Ruzzo A, Cremolini C, Vincenzi B, Salvatore L, Santini D, Masi G,Stasi I, Canestrari E, Rulli E, Floriani I, Bencardino K, Galluccio N, Catalano V,Tonini G, Magnani M, Fontanini G, Basolo F, Falcone A, Graziano F: KRAScodon 61, 146 and BRAF mutations predict resistance to cetuximab plusirinotecan in KRAS codon 12 and 13 wild-type metastatic colorectalcancer. Br J Cancer 2009, 101(4):715–721.47. Herman JM, Fan KY, Wild AT, Wood LD, Blackford AL, Donehower RC,Hidalgo M, Schulick RD, Edil BH, Choti MA, Hruban RH, Pawlik TM, Cameron JL,Laheru DA, Iacobuzio-Donahue CA, Wolfgang CL: Correlation of Smad4 statuswith outcomes in patients receiving erlotinib combined with adjuvantchemoradiation and chemotherapy after resection for pancreaticadenocarcinoma. Int J Radiat Oncol Biol Phys 2013, 87(3):458–459.48. Bashashati A, Ha G, Tone A, Ding J, Prentice LM, Roth A, Rosner J, Shumansky K,Kalloger S, Senz J, Yang W, McConechy M, Melnyk N, Anglesio M, Luk MT, Tse K,Zeng T, Moore R, Zhao Y, Marra MA, Gilks B, Yip S, Huntsman DG, McAlpine JN,Shah SP: Distinct evolutionary trajectories of primary high-grade serousovarian cancers revealed through spatial mutational profiling. J Pathol 2013,231(1):21–34.49. Weinstein IB: Cancer. Addiction to oncogenes–the Achilles heal of cancer.Science 2002, 297(5578):63–64.50. Torti D, Trusolino L: Oncogene addiction as a foundational rationale fortargeted anti-cancer therapy: promises and perils. EMBO Mol Med 2011,3(11):623–636.51. Dhillon KK, Swisher EM, Taniguchi T: Secondary mutations of BRCA1/2 anddrug resistance. Cancer Sci 2011, 102(4):663–669.52. Tsang YT, Deavers MT, Sun CC, Kwan SY, Kuo E, Malpica A, Mok SS,Gershenson DM, Wong KK: KRAS (but not BRAF) mutations in ovarianserous borderline tumor are associated with recurrent low-grade serouscarcinoma. J Pathol. 2013, 231(4):449–456.53. Ardighieri L, Zeppernick F, Hannibal CG, Vang R, Cope L, Junge J, Kjaer SK,Kurman RJ, Shih I-M: Mutational analysis of BRAF and KRAS in ovarianserous borderline (atypical proliferative) tumours and associatedperitoneal implants. J Pathol 2014, 232:16–22.54. Heublein S, Grasse K, Hessel H, Burges A, Lenhard M, Engel J, Kirchner T, Jeschke U,Mayr D: KRAS, BRAF genotyping reveals genetic heterogeneity of ovarianborderline tumors and associated implants. BMC Cancer. 2013, 13:483.55. Bossard C, Kury S, Jamet P, Senellart H, Airaud F, Ramee JF, Bezieau S,Matysiak-Budnik T, Laboisse CL, Mosnier JF: Delineation of the infrequentmosaicism of KRAS mutational status in metastatic colorectaladenocarcinomas. J Clin Pathol 2012, 65(5):466–469.56. Otsuka K, Satoyoshi R, Nanjo H, Miyazawa H, Abe Y, Tanaka M, Yamamoto Y,Shibata H: Acquired/intratumoral mutation of KRAS during metastaticprogression of colorectal carcinogenesis. Oncol Lett 2012, 3(3):649–653.57. Singer G, Kurman RJ, Chang HW, Cho SK, Shih IM: Diverse tumorigenicpathways in ovarian serous carcinoma. Am J Pathol 2002, 160(4):1223–1228.58. Kuo KT, Guan B, Feng Y, Mao TL, Chen X, Jinawath N, Wang Y, Kurman RJ,Shih Ie M, Wang TL: Analysis of DNA copy number alterations in ovarianserous tumors identifies new molecular genetic changes in low-gradeand high-grade carcinomas. Cancer Res 2009, 69(9):4036–4042.59. Zeller C, Dai W, Curry E, Siddiq A, Walley A, Masrour N, Kitsou-Mylona I,Anderson G, Ghaem-Maghami S, Brown R, El-Bahrawy M: The DNAmethylomes of serous borderline tumors reveal subgroups withmalignant- or benign-like profiles. Am J Pathol 2013, 182(3):668–677.60. Keita M, Wang ZQ, Pelletier JF, Bachvarova M, Plante M, Gregoire J, Renaud MC,Mes-Masson AM, Paquet ER, Bachvarov D: Global methylation profiling inserous ovarian cancer is indicative for distinct aberrant DNA methylationsignatures associated with tumor aggressiveness and disease progression.Gynecol Oncol 2013, 128(2):356–363.61. Shih Ie M, Chen L, Wang CC, Gu J, Davidson B, Cope L, Kurman RJ, Xuan J,Wang TL: Distinct DNA methylation profiles in ovarian serous neoplasmsand their implications in ovarian carcinogenesis. Am J Obstet Gynecol2010, 203(6):584. e1-22.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 redistribution62. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E,Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S,McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M,Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z,Downward J, Futreal PA, Swanton C: Intratumor heterogeneity and branchedevolution revealed by multiregion sequencing. N Engl J Med 2012,366(10):883–892.63. Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A, Delaney A,Gelmon K, Guliany R, Senz J, Steidl C, Holt RA, Jones S, Sun M, Leung G,Moore R, Severson T, Taylor GA, Teschendorff AE, Tse K, Turashvili G, VarholR, Warren RL, Watson P, Zhao Y, Caldas C, Huntsman D, Hirst M, Marra MA,Aparicio S: Mutational evolution in a lobular breast tumour profiled atsingle nucleotide resolution. Nature 2009, 461(7265):809–813.Submit your manuscript at www.biomedcentral.com/submit


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items