genomic landscape of colorectal mucosa and adenomas...eduardo vilar1,4,5,8 abstract the molecular...

Research Article

Genomic Landscape of Colorectal Mucosa andAdenomasEster Borras1, F. Anthony San Lucas2,3,4, Kyle Chang1,4, Ruoji Zhou1,4, Gita Masand1,Jerry Fowler2, Maureen E. Mork5, Y. Nancy You5,6, Melissa W. Taggart7,Florencia McAllister1,4,8, David A. Jones9, Gareth E. Davies10,Winfried Edelmann11,Erik A. Ehli10, Patrick M. Lynch5,12, Ernest T. Hawk1, Gabriel Capella13, Paul Scheet2,4, andEduardo Vilar1,4,5,8

Abstract

The molecular basis of the adenoma-to-carcinoma transitionhas been deduced using comparative analysis of genetic altera-tions observed through the sequential steps of intestinal car-cinogenesis. However, comprehensive genomic analyses ofadenomas and at-risk mucosa are still lacking. Therefore, ouraim was to characterize the genomic landscape of colonic at-risk mucosa and adenomas. We analyzed the mutation profileand copy number changes of 25 adenomas and adjacentmucosa from 12 familial adenomatous polyposis patientsusing whole-exome sequencing and validated allelic imbal-ances (AI) in 37 adenomas using SNP arrays. We assessed forevidence of clonality and performed estimations on the pro-portions of driver and passenger mutations using a systemsbiology approach. Adenomas had lower mutational rates thandid colorectal cancers and showed recurrent alterations in

known cancer driver genes (APC, KRAS, FBXW7, TCF7L2) andAIs in chromosomes 5, 7, and 13. Moreover, 80% of adenomashad somatic alterations in WNT pathway genes. Adenomasdisplayed evidence of multiclonality similar to stage I carcino-mas. Strong correlations between mutational rate and patientage were observed in at-risk mucosa and adenomas. Our dataindicate that at least 23% of somatic mutations are present inat-risk mucosa prior to adenoma initiation. The genomic pro-files of at-risk mucosa and adenomas illustrate the evolutionfrom normal tissue to carcinoma via greater resolution ofmolecular changes at the inflection point of premalignantlesions. Furthermore, substantial genomic variation existsin at-risk mucosa before adenoma formation, and deregulationof the WNT pathway is required to foster carcinogenesis.Cancer Prev Res; 9(6); 417–27. �2016 AACR.

IntroductionColorectal cancer is the third most common cancer diagnosed

in both men and women in the United States and the secondleading cause of cancer-related deaths when both sexes are com-bined (1). Colorectal cancer arises from adenomatous polyps,also known as adenomas, benign tumors with altered differen-tiation features found in 25%ofmenby age 50 years in theUnitedStates (2, 3). The critical transition from adenoma to carcinoma isfostered by the sequential acquisition of molecular events indriver genes and is known as the "adenoma-to-carcinoma" pro-gression model. This model was deduced from comparison ofgenetic alterations observed amongnormalmucosa, adenomas ofprogressively larger size, and carcinomas. The key somaticallyacquired genomic events include chromosomal gains (13 and 20)and losses (18q and 17p), as well as point mutations and smallinsertions/deletions in driver genes, such as APC, KRAS, and TP53(2). In fact, somaticmutations inAPChave beendescribed in 80%of colorectal adenomas and carcinomas (4). Moreover, germlinemutations in APC result in a genetic condition known as familialadenomatous polyposis (FAP), in which patients have a 100%lifetime riskof developing colorectal cancer,with the vastmajoritydeveloping carcinomas by age 35 years secondary to the devel-opment of hundreds of adenomas (2, 5). Although FAP is respon-sible for approximately only 1% of colorectal cancer diagnoses,this disease represents an attractive model for characterizing thegenomic profile of adenomas and investigating the initial eventsthat cooperate with APC loss in intestinal carcinogenesis. In fact,

1Department of Clinical Cancer Prevention,TheUniversityof TexasMDAnderson Cancer Center, Houston,Texas. 2Department of Epidemiol-ogy, The University of Texas MD Anderson Cancer Center, Houston,Texas. 3Department of Translational Molecular Pathology,The Univer-sity of Texas MDAnderson Cancer Center, Houston, Texas. 4GraduateSchool of Biomedical Sciences, The University of Texas MDAndersonCancerCenter, Houston,Texas. 5Clinical CancerGeneticsProgram,TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.6Department of Surgical Oncology,TheUniversityof TexasMDAnder-son Cancer Center, Houston, Texas. 7Department of Pathology, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.8Department of Gastrointestinal Medical Oncology, The University ofTexasMDAndersonCancerCenter,Houston,Texas. 9Immunobiology&Cancer Research Program, Oklahoma Medical Research Foundation,OklahomaCity,Oklahoma. 10Avera Institute forHumanGenetics, SiouxFalls, South Dakota. 11Department of Cell Biology, Albert EinsteinCollege of Medicine, Bronx, New York. 12Department of Gastroenter-ology,HepatologyandNutrition,TheUniversityof TexasMDAndersonCancer Center, Houston, Texas. 13Translational Research Laboratory,Catalan Institute of Oncology, Barcelona, Spain.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

E. Borras, F.A. San Lucas, K. Chang, P. Scheet, and E. Vilar contributed equally tothis article.

Corresponding Author: Eduardo Vilar, Clinical Cancer Prevention, The Univer-sity of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1360,Houston, TX 77230. Phone: 713-745-4929; Fax: 713-794-4403; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-16-0081

�2016 American Association for Cancer Research.

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FAP is the molecular model for 70% to 85% of colorectal cancersthat progress through alterations in the WNT pathway and whichare known as chromosomal instable tumors (2, 6). Whole-exomesequencing (WES) analyses of colorectal cancers have provided acomprehensive view of the genomic make-up of this disease andhave demonstrated the utility of this approach in identifyingnovel driver mutations (7, 8). Despite this wealth of information,however, there has been limited success in obtaining a compre-hensive genomic analysis of colorectal adenomas and APC-medi-ated carcinogenesis in at-risk mucosa.

Furthermore, large-scale genomic studies based on high-throughput technologies have systematically ignored the contri-bution of the adjacent surrounding mucosa to intestinal carcino-genesis by using it as the normal reference to establish the somaticstatus of alterations. This approach could potentially bias results,due to the fact that preexistingmutations in themucosa are carriedthrough the development of adenomas due to the fact that theyare secondary to exposure to carcinogens, aging, and other factors.Therefore, thesemutations have been regarded as germline eventsand ignored as importantmolecular changes. Interestingly, recentsystems biology analyses have determined that half of the somaticmutations identified in carcinomas originate prior to tumorinitiation (9), thus making the assessment of genetic variationin at-risk mucosa critical for a global understanding of intestinalcarcinogenesis.

In this study, we sought to characterize the mutational land-scape of colorectal adenomas to identify novel candidate genesthat cooperate withAPC in intestinal carcinogenesis, to determinethe presence of multiclonality in premalignant intestinal lesions,and to study the somatic variation accumulated in at-risk colonicmucosa using samples from FAP patients.

Materials and MethodsSubjects and samples

WES was performed in a total of 25 colorectal adenomas, 10adjacent mucosa, and 12 blood samples from 12 patients diag-nosed with FAP at the Catalan Institute of Oncology (Barcelona,Spain) and The University of Texas MD Anderson Cancer Center(Houston, TX). Furthermore, SNP arrays were performed on anadditional 37 colorectal andmatched blood samples from14 FAPpatients collected atMDAndersonCancerCenter (SupplementaryTable S1). Informed consent was obtained from all individuals,and the Institutional Review Boards of both institutions approvedthis study (MD Anderson protocol ID numbers: PA12-0327 andPA13-0178; Catalan Insitute of Oncology ID number: 30/06).Details on tissue preservation, nucleic extraction, and pathologiccharacteristics are provided in the Supplementary Methods (Sup-plementary Table S2).

Illumina sequencingSamples were sequenced on an IlluminaHiSeq 2000 sequencer

with 76-base paired end reads at the MD Anderson SequencingCore Facility. Germline DNA extracted from blood samples wasused as the genomic reference for the analysis of both adenomasand at-risk mucosa. A strategy for categorizing mutations into 5functional tiers was applied to somatic events. Validation ofexonic somatic mutations and indels detected in adenomas byusing Illumina sequencing was performed using Sanger sequenc-ing (see details in Supplementary Methods).

TCGA colorectal sample mutation callingTo minimize batch effects in our mutation comparisons

between FAP adenomas and The Cancer Genome Atlas (TCGA;ref. 7) colorectal cancer samples, we downloaded available WESdata for 107 carcinoma normal pairs from the TCGA project(stages I–V)where sampleswere sequencedon the IlluminaHiSeq2000 sequencer. We then analyzed these colorectal cancer exomesamples using the same bioinformatics pipelines that we used inthe analysis of our FAP samples to identify somatic point muta-tions and generate mutation reports that were applied to thecolorectal cancer exomes. Samples with�10mutations permega-base (Mb) were classified as hypermutated and those with

�10% allele frequency and segmented copy number to obtainclonality, purity, and ploidy calculations. Exomes of adenomasfrom our dataset and carcinomas from TCGA were used in thisassessment (see Supplementary Methods for details).

ResultsAssessment of the mutation landscape of adenomas by WES

We performed WES of 25 colorectal adenomas and matchedgermline DNA samples that served as the genomic reference toestablish the somatic mutational status. Samples were collectedfrom 12 patients diagnosed with FAP with clinically confirmedgermline APC alterations (Supplementary Tables S1 and S2).Exomes were sequenced to a mean depth of 65 reads, with86% of the targeted regions covered by at least 50 high-qualityreads (Supplementary Tables S7–11). In adenomas, we detected atotal of 237 indels (20 exonic), 10 splicing variants, and 2,067somatic mutations (1,067 exonic), with the somatic mutationsconsisting of 750 nonsilent, 317 silent, and 1,000 noncodingmutations (Fig. 1A; Supplementary Tables S8–11). The mostcommon base substitutions detected were C>T transitions(51%), similar to nonhypermutator colorectal cancers fromTCGA (Fig. 1B and Supplementary Fig. S1; Supplementary TableS12; refs. 7, 15). In addition, we analyzed mutational signaturesusing 6 substitution subtypes (i.e., C>A, C>G, C>T, T>A, T>C, andT>G) and their 50 and 30 bases adjacent to the mutation site,generating 96 combinations of mutations (Supplementary Fig.S2; Supplementary Table S13). Previous studies have identified atotal of 27 distinct mutation signatures across various types ofhuman cancers (16). Our analysis showed that adenomas wereclosely related to signature 1, which is the most frequentlyidentified among colorectal cancers as being associated with ageof diagnosis (16) and which mirrors the pattern displayed bynonhypermutator colorectal cancers (Supplementary Fig. S2).

The averagemedian allelic frequency of themutations detectedper adenoma was 15% (range 8%–25%), indicative of lowaberrant DNA proportions (Fig. 1C; Supplementary Table S9–11). On average, the mean somatic mutation frequency was 83mutations per adenoma (range 9–186), resulting in a meanmutation rate of 1.75 mutations/Mb (range 0.2–4.1; Fig. 1C andD; Supplementary Table S14). Theoverall adenomamutation ratewas compared with 107 colorectal cancers from the TCGA. Ade-nomas had lower mutation rates than carcinomas but did exhibitsome degree of overlap with nonhypermutator colorectal can-cers (1.75 mutations/Mb in adenomas compared with 4.26 innonhypermutator carcinomas and 50.88 for hypermutators,both P < 0.0001; Fig. 1D; Supplementary Tables S14 and S15).

Profiling of AIs in colorectal adenomas and stage I carcinomasWe used WES data to characterize chromosomal alterations

that result in somatic AIs. We detected AI in 14 adenomas (56%)of our samples (Fig. 2 and Supplementary Fig. S3; SupplementaryTable S2). We then compared the AI profiles in adenomas withthose for stage I nonhypermutated colorectal cancers from TCGA(7). Regions found to be aberrant in both adenomas and stage Icarcinomas are suggestive of events occurring early in coloniccarcinogenesis, such as loss of 5q, which we observed in 5 ade-nomas (20%), and amplifications of chromosomes 7 (20%),13 (16%), 19 (12%), and 20 (16%; Fig. 2; Supplementary TableS16). On the other hand, recurrent events found only in carci-nomas, such as deletions of 17p and 18q (observed in 60% and

70% of stage I colorectal cancers, respectively), are candidateevents for association with progression to carcinomas. We vali-dated the frequency of AIs using SNP array technology in anadditional cohort of 37 adenomas from 14 FAP patients. AIs inchromosomes 5 (11%), 7 (14%), and 13 (16%) were highlyfrequent, thus confirming the importance of these events in earlycarcinogenesis (Supplementary Table S17). Interestingly, we not-ed that adenomas with AI in 20q had a mutational rate higherthan the mean mutation rate in all adenomas (3.23 comparedwith 1.75, P ¼ 0.01) and similar to the rate observed in non-hypermutated colorectal cancers, thus supporting the idea thatthese adenomas have progressed further along the adenoma-to-carcinoma transition and also implicating genes in 20q for a rolein progression.

Detection of candidate genes related to intestinalcarcinogenesis

We developed a system that categorized somatic mutationsbased on their predicted deleterious effects to discover candidategenes related to APC-driven intestinal carcinogenesis (see Materi-als andMethods for details). A total of 100 exonicmutations (9%)were classified as damaging events, 35 (3%) as recurrent poten-tially damaging, 359 (33%) as potentially damaging, 129 (12%)as variants of unknown significance, and 474 (43%) as neutralevents (Supplementary Tables S11, S18 and S19; SupplementaryFig. S3). Damaging events resided in well-known cancer genes,such as APC, KRAS, FBXW7, TCF7L2, and BRCA2 (Fig. 3). Amongthe recurrent potentially damagingmutations, several novel genesemerged as candidates for intestinal carcinogenesis, such as ALKand CNOT3 (Fig. 3; Supplementary Tables S11, S18, and S19).Moreover, the following 11 genes harboring mutations classifiedas either damaging or recurrent potentially damaging were foundaltered in at least 2 adenomas: APC, ARID1A, CDC27, CNOT3,EWSR1, FBXW7, GNAQ, KRAS, PCMTD1, TCF7L2, and TTN(Supplementary Table S20; Supplementary Fig. S3). In addition,we annotated AIs to known copy number aberrant colorectalcancer genes and novel candidate genes from our somatic muta-tion analysis and found recurrent AIs in well-known genes such asAPC, EPHB6, MAP1B, MAP2K7, and MET and novel candidatessuch as CNOT3 (Fig. 3; Supplementary Table S21; refs. 7, 8).

Pathway analysis and detection of somatic second hit in APC incolonic adenomas

Integrated analysis of damaging mutations and AIs in adeno-mas revealed enrichment in the WNT, MAPK, VEGF, and ErbBpathways, thus suggesting them as future avenues for chemopre-vention (Supplementary Table S22). Among these pathways,WNT stood out as deregulated in 80% of all adenomas, with20 of 25 harboring somatic mutations in at least 1 gene (Sup-plementary Fig. S4). In fact, cooccurrence of mutations in morethan 1 gene of the pathway was observed in 7 adenomas (28%;Supplementary Table S23). The most frequently mutated gene inthe WNT pathway was APC (18 adenomas, 72%), harboring asomatic second hit either secondary to LOH of chromosome 5q(5 adenomas, 28%)or pointmutations (13 adenomas, 44%). Themajority of adenomas with somatic loss of 5q were detected in apatient with a germline mutation identified within the mutationcluster region (MCR) of APC (amino acids 1286–1514). Amongthe point mutations detected, 10 were nonsense and 3 were smallinsertions or deletions (Supplementary Fig. S5; SupplementaryTable S24). Four of the samples without somatic APC mutations

Genomic Profiling of Colorectal Mucosa and Adenomas

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lacked any alteration in other WNT signaling pathway genes(Supplementary Table S23) and were characterized by lowermutational rates compared with the 84% of adenomas withidentified somatic WNT pathway alterations (0.511 vs. 1.99mutations/Mb). Interestingly, in those samples harboring somat-ic APC mutations, the allelic frequency of the APC events washigher compared with the median allele frequency of the sample(Fig. 1C; Supplementary Tables S11, S16, S17, and S24).

Evidence of multiclonality in adenomasWe searched for evidence of clonality in our APC-driven ade-

nomasby visually inspecting thedistribution ofmutations rankedby allelic frequency. We observed 2 clusters of mutation allelicfrequencies in 5 samples, suggesting the presence of 1major cloneand other minor subclones (Fig. 4A and Supplementary Fig. S6).However, this type of analysis could be confounded by tumorpurity and copy number variation. To mitigate the effect ofvariations in these 2 factors, we applied computational tools totransform the frequency distribution ofmutations in each sample

and to infer the number of clones. We compared in silico estima-tions of purity, ploidy, and number of clones between adenomasand stage I colorectal cancer. The purity was significantly lower inadenomas comparedwith stage I colorectal cancers (0.35 vs. 0.58,P < 0.0001; Fig. 4B; Supplementary Table S25), as was the ploidy(1.93 vs. 2.61, P < 0.0001; Fig. 4C; Supplementary Table S25).Then, our analysis revealed the presence of multiclonality in 18(72%) of 25 adenomas, with at least 1 major and 1 minorsubclone per lesion. Moreover, more than 50% of the polypsfrom the same individual is estimated to have multiclonality.Interestingly, the number of clones estimated in adenomas wasnot significantly different from that for stage I CRC (1.72 vs. 2.06,P ¼ .165; Fig. 4D; Supplementary Table S25).

Assessment of the mutation landscape of at-risk colorectalmucosa by WES

To explore the contribution of the at-risk mucosa to intestinalAPC-driven carcinogenesis, we performedWES ofmatchedmuco-sa samples in 10 of the 12 patients previously analyzed using the

Figure 1.Exome analysis of colorectal adenomas. A, total number of mutations per adenoma classified in each of the categories displayed in the legend on the bottom.B, relative proportions of the 6 different possible base pair substitutions, as indicated in the legend on the bottom. C, median mutation frequencies. For eachsample, all the mutations are shown as circles indicating the allelic frequency of the respective mutant alleles. Median allele frequency, black solid bar; allelicfrequency of somatic APC mutations, red circle. The total number of somatic mutations in each adenoma sample is displayed in the lower graph (SNV, singlenucleotide variation). D, mean somatic mutation frequency observed in adenomas compared with colorectal cancers (CRC) from TCGA classified byhypermutator status. Averages are shown in red (� , P < 0.0001).

Figure 2.AIs detected in colorectal adenomas. A, integrative analysis of genomic changes detected in adenomas and nonhypermutated stage I colorectal cancers(CRC) from the colorectal TCGA dataset. AIs of the 22 autosomes are shown in shades of red for amplification, blue for deletion, and black for cn-LOH (adenomasonly). Results in carcinomas are shaded by degree of imbalance, with red shades for amplification and blue for deletion (darker: greater copy numberchange in sample). B, summary of AIs detected in adenomas and nonhypermutated stage I CRC from the colorectal TCGA dataset.






germline DNA calls as genomic reference and compared themutational profiles with their corresponding adenomas (Supple-mentary Tables S1 and S2). Colorectal mucosa exomes weresequenced to a mean depth of 62 reads, with 82% of the targetedregions covered by at least 50 high-quality reads (SupplementaryTable S7). We identified a total of 11 indels (4 exonic) and 268somatic mutations (115 exonic), with the somatic mutationsconsisting of 36 nonsilent, 78 silent, and 154 noncoding muta-tions (Fig. 5A; Supplementary Tables S26–28). The vast majority(86%) of exonic mutations was classified as neutral or variants ofunknown significance, and only a few (total of 6%) were iden-tified as damaging (Supplementary Tables S29 and S30). Themutations classified as damaging were detected in genes such asHDAC10, PLEKNH1, PHOX2B, CDC27, and PPP1CC, which havenot been previously described as related to intestinal carcinogen-esis, thus suggesting they are not leading to a known route ofinitiation for carcinogenesis.

The most common base substitutions detected were C>T transi-tions (37%), similar to adenomas andnonhypermutator colorectalcancers (Fig. 5B; Supplementary Table S31; ref. 15). Analysis of

mutation signatures showed that colorectal mucosas were closelyrelated to signature 5, which has been observed in 14.4% ofcolorectal cancers but has unknown etiology (Supplementary Fig.S2; Supplementary Table S32; ref. 16). In addition, the averagemedian allelic frequency of themutations detectedwas 10% (range5%–17%), which is lower than was observed for adenomas (Fig.5C; Supplementary Tables S27 and S28). The mean number ofsomatic mutations observed was 27 (range 8–90), resulting in amean mutation rate of 0.49 (range 0.1–1.5), significantly lowerthan what was observed for adenomas (0.49 vs. 1.75 mutations/Mb, P < 0.01; Fig. 5C and D; Supplementary Tables S14 and S33).Interestingly, 3 adenomas that hadmutational rates similar to theirmatchedmucosa did not harbor somatic events in APC or in othergenes involved in the WNT pathway (Supplementary Fig. S7A;Supplementary Tables S14, S23, and S33).

Proportion of driver and passenger mutations and the role ofaging in intestinal carcinogenesis

We analyzed different factors that could influence the muta-tional rate observed in adenomas, such as age (>40 vs.�40 years),

Figure 3.Genome-wide mutational landscapeand genomic changes in colorectaladenomas. A, mutational rate incolorectal adenomas. B, keyclinicopathologic characteristicsincluding sex (M, male; F, female),diagnosis of cancer (Cancer Dx),location of the germlineAPCmutation(Out15, mutations locate outside ofexon 15; In15, mutations located insideof exon 15), and type of germline APCmutation (Del, deletions; Sp, splicingmutations; Non, nonsense mutations).C, summary of mutations and AIs inselected genes. Each row is agene andeach column is a sample. Mutationsand AIs are colored by the type ofalteration as indicated in the legend.Left, total number of somaticalterations that targeted each geneand the percentage of individualsaffected; right, heatmap displayingfrequencies of carcinomas mutated ineach gene from TCGA dataset. CRC,colorectal cancer; SNS, singlenucleotide substitution.

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sex (male vs. female), and the type (tubular vs. tubulovillous),location (colon vs. rectum), and size (>10 vs. �10 mm) of theadenoma lesions and the type of somatic second hit in APC (LOHvs. point mutation). Age seemed to be the most significant factor(P < 0.0015), showing a positive correlation with the mutationalrate (R¼0.63,P¼ .005; Fig. 6A). Likewise, agewas correlatedwiththe mutational rate in at-risk mucosa (R¼ 0.74, P¼ .01; Fig. 6B),consistent with observations of mutations accumulating as indi-viduals age. Then, to study the relation between the occurrence ofdriver and passenger mutations, we recategorized the eventsobserved in both adenomas and at-risk mucosa into 2 categories:drivers (those events classified as damaging and recurrent poten-tially damaging) and passengers (the rest).We observed a positivecorrelation between the number of passengers and age in the at-riskmucosa samples (R¼ 0.74, P¼ .01; Supplementary Fig. S7B);

however, the lack of correlation between the number of passengerand driver events in the at-risk mucosa (R ¼ 0.23, P ¼ .5;Supplementary Fig. S7C) suggests that mutations classified asdrivers by bioinformatics tools in themucosa were not functionaland did not lead to an effective increase of passenger aberrations(Supplementary Tables S1 and S34). In those adenomas thatharbored second hits in APC, we observed a correlation betweenthe number of driver and passenger mutations (R ¼ 0.56, P ¼0.01; Fig. 6C) and between the number ofmutations and age (R¼0.53, P ¼ 0.02; Fig. 6D; Supplementary Tables S1 and S34).Moreover, comparison of the slope of the linear regressionbetween passenger mutations and age between at-risk mucosaand adenomas (0.94 vs. 1.36) demonstrated that adenomasharboring second hits in APC accumulatedmore passenger muta-tions (Fig. 6E), thus indicating that haploinsufiency in APC (i.e.,

Figure 4.Clonality analysis of colorectal adenomas. A, mutation counts detected by WES in 4 different adenomas, ordered by allelic frequency and provided asexamples presenting evidence of clonality. B and C, purity and ploidy of adenomas and stage I colorectal cancers (CRC) were estimated by the ABSOLUTEcomputational method (� , P < 0.0001). D, numbers of clones in adenomas were inferred by clustering cancer cell fraction of mutations estimated byABSOLUTE.






presence of one hit in APC in at-risk FAP normal mucosa) is notsufficient to foster carcinogenesis.

Finally, the average numbers of exonic mutations per samplewere 12 (range 1–56) in at-risk mucosa and 51 (range 2–101)in adenomas with a somatic hit in APC (Supplementary TableS30). On the basis of this data, we extrapolated that 23% of themutations existed before carcinogenesis started in a context ofgermline APC alterations. To further explore the relationship ofdrivers and passengers, we compared our results with estimatesproposed by Bozic and colleagues (17) using mathematicalmodels describing a logarithmic relation between drivers andaccumulation of passengers. We observed a significant corre-lation between estimated and observed numbers of passengersin adenomas (7.6 vs. 6.9 passenger mutations per driver,respectively, R ¼ 0.59, P ¼ 0.01; Fig. 6F; Supplementary TableS34), indicating that this model provides accurate estimatesbased on our data (17).

DiscussionWe have here provided the first comprehensive annotation of

the genomic landscape of APC-driven adenomas from FAPpatients using next-generation sequencing technologies. Our

classification system of mutation by tiers based on bioinformaticprediction tools helps to characterize a set of recurrent genesinvolved in early tumor progression. Somatic hits in APC, KRAS,or FBXW7were recurrently detected in our samples and classifiedinto tiers 1 and 2, which is consistent with the assignment of adriver status, thus validating our tier classification. In addition, wefound novel candidate genes that merit further investigation aspotential cooperators of APC deficiency in early stages of carci-nogenesis, including EWSR1, CNOT3, and PCMTD1, amongothers. Someof these genes havebeen shownpreviously to harborsomaticmutations in other cancer types. For example,CNOT3hasbeen reported in T-cell acute lymphoblastic leukemia, pancreaticcancer, and bladder cancer (18, 19) and ARID1A in ovarian andendometrial cancers (15). In addition, we observed recurrentmutations in TTN andCDC27 that have been previously reportedas spurious with respect to driver status, due to their genomic sizeor accumulation of systematic technical artifacts (20).

Our comparative analysis of genomic imbalances in adenomasand stage I carcinomas provides an opportunity to detect aberrantregions in both types of samples, suggestive of their importance inoccurring early in the development of colorectal cancer. Weobserved losses in 5q and events in chromosomes 7, 13, and20 in both adenomas and carcinomas. Loss of 5q is a common

Figure 5.Exome analysis of at-risk mucosa samples. A, total number of mutations per at-risk mucosa classified in each of the categories displayed in the legend on thebottom. B, relative proportions of the 6 different possible base pair substitutions. C, median mutation frequencies. For each sample, all the mutations areshown as circles indicating the allelic frequency of the respective mutant alleles. Median allele frequency, black solid bar. The total number of somatic mutationsin each sample is displayed in the graph below (SNV). D, mean somatic mutation frequency observed in the mucosa compared with adenomas. Averages areshown in red (� , P < 0.001). CRC, colorectal cancer.

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mechanism inactivating the tumor suppressor gene APC and isrecognized as an initiating event of intestinal carcinogenesis (2).Gains in chromosomes 7 and 13 have been associated with EGFRand MET amplifications and with CDX2 gains, respectively (12).Previous studies have suggested several genes located on chro-mosome 20 that could be involved in adenoma progression,including C20orf24, AURKA, RNPC1, ADRM1, and TCFL554(21, 22), thus supporting the idea that these adenomas havegained invasiveness (22).Wedidnot observe deletions of 17p and18q, typically regions deleted in colorectal cancer. Genes locatedin these regions could constitute candidate genes involvedin tumor progression, such as SMAD4 (chr18: 48556582–48611411). This observation is related to a recent observationthat loss of SMAD4 is associated with poor survival in colorectalcancer patients (23).

Finally, we identified AI in 56% of adenomas, a proportionlower than previously reported by others (24) and which couldbe explained by the use of a different technical approach, suchas comparative genomic hybridization array or by samplecharacteristics.

The gene most frequently mutated in our series was APC, forwhich 72% of the samples presented a somatic second hit, whichis in agreement with the frequency previously observed by othergroups (25).Wewere not able to detect a second hit inAPC or in agene involved in the WNT signaling pathway in 4 adenomas.

These adenomas presentedwith lowermutational rates comparedwith the rest that harbored somatic WNT pathway alterations andwere also similar to the mutational rates detected in normalmucosa samples, thus suggesting that these lesions were at a veryearly stage of carcinogenesis. In line with this hypothesis, these 4lesions would have a higher than the average percentage ofnormal mucosa, thus masking the presence of a somatic APCalteration. Higher depth in the sequencing would have helped toincrease the resolution and uncover an APCmutation in a sparsebut upcoming clones.

The majority of somatic APC alterations observed in ourcohort were truncating mutations localized in the MCR coupledwith germline events spread through other regions of the gene,thus highlighting the dependency between germline andsomatic APC events (25–27). Moreover, results from the path-way enrichment analysis indicated that deregulation of theWNT pathway was the most prominent derangement, whichis in line with the results of the TCGA analysis of colorectalcancer (7). Indeed, APC is an essential component of the WNTpathway, regulating b-catenin, migration, and polarity andhaving a central role in maintaining the stem cell fate of theintestinal crypt (28, 29). In addition, we detected cooccurrentmutations in more than 1 gene in other well-known WNTpathway genes, such as TCF7L2, FBXW7, ARID1A, CTNNB1,AXIN2, and SOX9 in 28% of adenomas. Interestingly, we did

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R = 0.70P < 0.0001

R = 0.74P = 0.01

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R = 0.53P = 0.02

Figure 6.Proportion of drivers and passengers. A, correlation analysis of mutation rate versus age in adenomas. B, correlation analysis of mutation rate versus age inat-risk mucosa samples. C, correlation between numbers of passenger mutations versus drivers in adenomas. D, correlation between age versus numbers ofpassenger mutations in adenomas. E, correlation between numbers of passenger mutations versus age in adenomas and at-risk mucosa. Correlation isshown in red for adenomas and in green for at-risk mucosa samples. F, correlation between numbers of passenger mutations observed in our set of adenomasamples versus estimations based on mathematical models.






not observe other major oncogenic pathways deregulated insignificant numbers of our adenomas, thus highlighting thealmost exclusive dependency on WNT pathway events at thisstage of carcinogenesis. Therefore, these findings further sup-port the development of therapeutic strategies targeting WNTinhibitors for colorectal cancer prevention through adenomainterception.

To further explore the dynamics of tumor growth, we usedcomputational tools to determine and characterize the presenceof multiclonality in colorectal adenomas. Our findings indi-cated the presence of multiple clones in 72% of adenomas. Thefact that we did not detect a significant difference betweenadenomas and stage I colorectal cancer in the number of clonessuggests that clonality originates very early in carcinogenesisand not from late clonal expansions. These observations couldsuggest that the expansion of an APC-driven clone constitutesthe founder progenitor of the tumor cell population early incarcinogenesis. The mutational profile of this founder clonecontains the catalog of "public" mutations that are thus sub-sequently present in all tumor cells and include those cooperat-ing with APC. The progeny of this major clone will then acquireadditional mutations that are the source of private low-fre-quency events that remain less abundant, as these minorsubclones are marginally distributed within the geography ofthe tumor mass. Our analysis is not adequate to observegeographic variations of genomic alterations in adenomas.However, it provides indirect support to this model of colo-rectal carcinogenesis (30).

To begin to understand the contribution of the at-risk muco-sa to colorectal carcinogenesis and to separate passenger muta-tions from those important in progression, we performed WESof at-risk mucosa samples. It has been proposed by others thatthe number of mutations in tumors of self-renewing tissues,such as the large intestine, should be positively correlated withthe age of the patient and that a large fraction (at least 50%) ofthe somatic mutations arises before tumor initiation (9). Ourdata confirmed this correlation between the number of muta-tions in at-risk mucosa (and adenomas) and age, even in thecontext of accelerated carcinogenesis due to the presence ofgermline APC mutation. Moreover, our sequencing results forat-risk mucosa have allowed us to calculate that 23% ofmutations in adenomas were present prior to acquisition ofthe first initiating somatic driver event, which is the loss of APC(second hit). A possible explanation for the discrepancybetween our calculations and the fraction reported previouslyby others (9) is that our samples already harbor a germline hitin APC, thus requiring only the acquisition of 1 somatic eventto fully initiate tumorigenesis. Therefore, the timeline foradenoma formation is much shorter in FAP adenomas, anda much lower number of somatic alterations will be present inthe at-risk mucosa prior to the acquisition of the tumor initi-ation event. This fact also suggests that only one hit in APC isnot sufficent to accelerate the accumulation of mutations in thenormal mucosa. Additional analysis comparing normal muco-sa samples from sporadic individuals (general population) andFAP patients will be necessary to investigate the timeline ofacquisition, role, and function of these somatic alterationsaccumulating in a normal tissue. Furthermore, we observedthe presence of a relatively lower proportion of driver events inat-risk mucosa compared with adenomas, and these eventswere identified in genes previously reported to be spurious

(20) or not related to colorectal carcinogenesis (such as CDC27,PLEKNH1, and PHOX2B), thus suggesting that these driversdetected in at-risk mucosa are not biologically effective or maybe "false positives" (as drivers). Only 2 genes found mutated inthe at-risk mucosa samples, WNT11 (31) and ARID1B (32),have been previously reported to play a role in colorectalcarcinogenesis, thus indicating that these mucosa samples havealready acquired a growth advantage.

In summary, we performed a comprehensive analysis of thegenomic landscape of colorectal adenomas and the first suchexamination of the surrounding mucosa as an independent at-risk tissue to assess the contribution of the accumulated somat-ic variation to carcinogenesis. Our data show that the majorityof recurrently mutated genes identified in adenomas areinvolved in the WNT pathway, confirming that APC loss issufficient for the early growth of adenomas (33) and suggestingthat future chemopreventive strategies should focus on devel-oping drugs that can effectively target this pathway. Finally,using in silico analyses, we identified the presence of clonality inadenomas and estimated that 23% of somatic mutations inAPC-driven adenomas were present before tumor initiation,thus highlighting the fact that the occurrence of at least 1 driverevent, in this case the acquisition of a somatic APC aberration(second hit in a tumor suppressor), is necessary to trigger theaccumulation of additional driver and passenger mutationsthat will ultimately propel carcinogenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: E. Borras, D.A. Jones, E.T. Hawk, E. VilarDevelopment of methodology: E. Borras, F.A. San Lucas, M.W. Taggart,G.E. Davies, P. Scheet, E. VilarAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Borras, F.A. San Lucas, R. Zhou, G. Masand,M.E. Mork, Y.N. You, M.W. Taggart, D.A. Jones, G.E. Davies, E.A. Ehli,P.M. Lynch, G. Capella, E. VilarAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Borras, F.A. San Lucas, K. Chang, J. Fowler,G. Capella, P. Scheet, E. VilarWriting, review, and/or revision of the manuscript: E. Borras, F.A. San Lucas,K. Chang, M.E. Mork, Y.N. You, M.W. Taggart, F. McAllister, D.A. Jones,G.E. Davies, W. Edelmann, E.A. Ehli, E.T. Hawk, G. Capella, P. Scheet, E. VilarAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F.A. San Lucas, K. Chang, M.E. Mork, Y.N. You,G.E. Davies, E.A. Ehli, E. VilarStudy supervision:, P. Scheet, E. VilarOther (proofreading): J. Fowler

AcknowledgmentsThe authors thank the patients and their families for their participation and

the staff of the Sequencing and Microarray Facility at The University of TexasMD Anderson Cancer Center for the generation of the exome sequencing data.The authors also thank the Clinical Cancer Prevention Research Core for theirassistance in obtaining the samples for this study.

Grant SupportThis work was supported by grants R03CA176788 (NIH/NCI), the MD

Anderson Cancer Center Institutional Research Grant (IRG) Program, and agift from the Feinberg Family (to E. Vilar); U01GM92666 and R01HG005859(NIH; to P. Scheet); the Janice Davis GordonMemorial Postdoctoral Fellowshipin Colorectal Cancer Prevention (Division of Cancer Prevention/MD AndersonCancer Center; to E. Borras); the Schissler Foundation Fellowship (The Uni-versity of Texas Graduate School of Biomedical Sciences) and Translational


Borras et al.




Molecular Pathology Fellowship (MD Anderson Cancer Center; to F.A. SanLucas); Cancer Prevention Educational Award (R25T CA057730, NIH/NCI; toK. Chang); SAF2012-33636 (Spanish Ministry of Economy and Competitive-ness) and the Scientific Foundation Asociaci�on Espa~nola Contra el C�ancer(to G. Capella); R01CA76329 (NIH/NCI; toW. Edelmann); and P30CA016672(NIH/NCI) to the MD Anderson Cancer Center Core Support Grant.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 21, 2016; accepted April 7, 2016; published OnlineFirstMay 24, 2016.

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2016;9:417-427. Published OnlineFirst May 24, 2016.Cancer Prev Res Ester Borras, F. Anthony San Lucas, Kyle Chang, et al. Genomic Landscape of Colorectal Mucosa and Adenomas

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