Integrated Genetic, Epigenetic, and Transcriptional Profiling Identifies Molecular Pathways in the Development of Laterally Spreading Tumors

Published OnlineFirst September 26, 2016; DOI: 10.1158/1541-7786.MCR-16-0175

Genomics Molecular Cancer Research Integrated Genetic, Epigenetic, and Transcriptional Proﬁling Identiﬁes Molecular Pathways in the Development of Laterally Spreading Tumors Luke B. Hesson1, Benedict Ng1, Peter Zarzour1, Sameer Srivastava1,2, Chau-To Kwok1, Deborah Packham1, Andrea C. Nunez1, Dominik Beck1, Regina Ryan1, Ashraf Dower1, Caroline E. Ford1, John E. Pimanda1, Mathew A. Sloane1, Nicholas J. Hawkins3, Michael J. Bourke4, Jason W.H. Wong1, and Robyn L. Ward1,5

Abstract

Laterally spreading tumors (LST) are colorectal adenomas that colorectal neoplasia (ANO5, MED12L, EPB41L4A, RGMB, develop into extremely large lesions with predominantly slow SLITRK1, SLITRK5, NRXN1, ANK2). Alterations to pathways com- progression to cancer, depending on lesion subtype. Comparing monly mutated in colorectal cancers, namely, the p53, PI3K, and and contrasting the molecular profiles of LSTs and colorectal TGFb pathways, were rare. Instead, LST-altered genes converged on cancers offers an opportunity to delineate key molecular alterations axonal guidance, Wnt, and actin cytoskeleton signaling. These that drive malignant transformation in the colorectum. In a dis- integrated omics data identify molecular features associated with covery cohort of 11 LSTs and paired normal mucosa, we performed noncancerous LSTs and highlight that mutation load, which is a comprehensive and unbiased screen of the genome, epigenome, relatively high in LSTs, is a poor predictor of invasive potential. and transcriptome followed by bioinformatics integration of these data and validation in an additional 84 large, benign colorectal Implications: The novel genetic, epigenetic, and transcriptional lesions. Mutation rates in LSTs were comparable with microsatel- changes associated with LST development reveal important insights lite-stable colorectal cancers (2.4 vs. 2.6 mutations per megabase); into why some adenomas do not progress to cancer. The finding however, copy number alterations were infrequent (averaging only that LSTs exhibit a mutational load similar to colorectal carcinomas 1.5 per LST). Frequent genetic, epigenetic, and transcriptional has implications for the validity of molecular biomarkers for alterations were identified in genes not previously implicated in assessing cancer risk. Mol Cancer Res; 14(12); 1–12. 2016 AACR.

Introduction grow laterally within the colonic mucosa (1). LSTs are an inter- esting subtype of adenoma because they grow to extremely large Colorectal carcinomas develop from benign intraepithelial sizes, often many centimeters in diameter, but rarely develop lesions known as adenomas. Laterally spreading tumors (LST) submucosal invasion (1). are a morphologically heterogeneous group of adenomas that Comprehensive profiling of colorectal carcinomas has shown that they often contain deranged genomes (2, 3) making it fi fi 1 dif cult to identify speci c culprits of malignant transformation. Adult Cancer Program, Lowy Cancer Research Centre and Prince of Wales The value of studying LSTs is that they rarely progress to cancer. Clinical School, UNSW Australia, Sydney, New South Wales, Australia. 2Depart- ment of Biotechnology, Motilal Nehru National Institute of Technology, Allaha- Therefore, tracking their molecular etiology and comparing this bad, India. 3School of Medical Sciences, UNSW Australia, Kensington, Sydney, with cancers offers the potential to understand why some ade- Australia. 4Department of Gastroenterology, Westmead Hospital, Sydney, New nomas do not become invasive. South Wales, Australia. 5Level 3 Brian Wilson Chancellery, The University of LSTs can be endoscopically characterized as either granular or Queensland, Brisbane, Queensland, Australia. nongranular on the basis of their surface morphology or by the Note: Supplementary data for this article are available at Molecular Cancer Paris classification system, which describes the macroscopic mor- Research Online (http://mcr.aacrjournals.org/). phology of different types of adenoma (1). Granular LSTs (G-LST) The datasets supporting the conclusions of this article are available in the NCBI consist of aggregates of mucosal nodules with an uneven surface, Gene Expression Omnibus repository (GSE77635, http://www.ncbi.nlm.nih. whereas nongranular LSTs (NG-LST) have a smooth even surface. gov/geo/query/acc.cgi?acc¼GSE77635). Although cancer rarely develops from LSTs, the risk of submucosal Corresponding Authors: Luke B. Hesson, Adult Cancer Program, Lowy Cancer invasion differs with morphology (4, 5). For example, in a Research Centre, UNSW Australia, Kensington, Sydney 2052, Australia. Phone: prospective, multicenter, observational study of all patients 61-2-93851457; Fax: 61-2-93851500; E-mail: [email protected]; and Robyn referred for endoscopic mucosal resection (EMR) of large LSTs L. Ward, Level 3, Brian Wilson Chancellery, The University of Queensland, (20 mm), invasion was found in 15.3% (15 of 98) of NG-LSTs Brisbane 4072, Australia. Phone: 61-7-33659044; E-mail: [email protected] but in only 3.2% (10 of 311) of G-LSTs (6). The molecular basis of doi: 10.1158/1541-7786.MCR-16-0175 the extensive growth of LSTs in the absence of cancer and the 2016 American Association for Cancer Research. increased invasive potential of NG-LSTs when compared with

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G-LSTs is unclear primarily because no studies have undertaken a tions and deletions (indels) were identified using Strelka and systematic exploration of the molecular characteristics of this GATK-HaplotypeCaller subtraction (17, 18). Only mutations subtype of adenoma. with 10 coverage, present in 10% of reads and detected on This study integrates multiple layers of "omics" data (genomic, 4 reads were considered as genuine. Only mutations predicted epigenomic, and transcriptomic) from LSTs to achieve 3 aims: (i) to be damaging to protein function (ANNOVAR; ref. 19) were to identify alterations in genes and pathways that coincide with considered when identifying candidate driver gene alterations. the development of LSTs; (ii) to compare genetic alterations between LSTs and colorectal carcinomas; and (iii) to identify Whole-genome sequencing analysis molecular differences between granular and nongranular LSTs, Whole-genome sequencing (WGS) data from 33 colorectal which differ in their malignant potential. carcinomas and normal mucosa from TCGA (2) were analyzed using the same approach described above for whole-exome Materials and Methods sequencing (WES). Tumors were categorized as follows: 19 microsatellite-stable (MSS), 8 microsatellite-instable (MSI), and 6 with Patients and tissue samples nonsynonymous mutations in the exonuclease domain of the A discovery cohort (7 G-LSTs, 4 NG-LSTs, n ¼ 11, Supplemen- POLE gene. tary Table S1 and Supplementary Fig. S1) of LSTs and paired normal mucosal tissue was obtained from 9 patients (6 males; mean age, 70 years; range, 51–87 years) using EMR. This included Copy number alterations 3 separate lesions (G1, G2, and NG) that were obtained from one A total of 22 LSTs were analyzed for copy number alterations individual (male, 70 years) during the same endoscopic proce- (10 discovery cohort and 12 validation cohort) using Illumina dure. Only non-polypoid (Paris IIa) components were used for Human610-Quad SNP arrays or Comparative Genomic Hybrid- genomic analyses. Validation cohort 1 (n ¼ 44, Supplementary ization (CGH) 720K whole-genome tiling array v3.0 (Nimble- Table S2) consisted of large (20 mm) LSTs with mixed Paris Gen, 05520860001). SNP array data were analyzed using classification and validation cohort 2 (n ¼ 40, Supplementary OncoSNP, as described previously (20). CGH data were analyzed > Table S2) consisted of 20 granular and 20 nongranular large using SignalMap (NimbleGen) with LogR ratio thresholds of 0.2 fi (20 mm) LSTs (Paris IIa or IIb). Histologic assessment indicated representing copy number gains (ampli cations/duplications)

all lesions showed no evidence of submucosal invasion. All and 0.2 representing copy number losses (deletions). Deletions cohorts were obtained from fresh tissues selected (by M.J. Bourke) at 5q were assessed at the microsatellite markers D5S346, from a previously described consecutively collected cohort D5S2495, and D5S617 using PCR and capillary electrophoresis obtained by EMR between 2009 and 2011 at Westmead Hospital on an ABI 3500 instrument (Applied Biosystems). (ethics approval 2009/6/4.6 and 11194; ref. 7). Sanger sequencing mutation analysis KRAS NRAS RNA-Seq The mutation status of (codons G12 and G13), BRAF RNA-Seq was performed as described previously (8) using the (codons G12 and G13), and (codon V600) were assessed BRAF HOMER package (9) and the trimmed mean of m-value method using pyrosequencing as described previously (21). codon KRAS NRAS (10). Signiﬁcant differential expression between LST and normal G469, codons D117 and A146 and, codon Q61 tissue was determined using DESeq (ref. 11; P 0.05). See mutations were assessed by sequencing. The mutation status of APC CTNNB1 Supplementary Methods for further details. and was assessed using sequencing as described previously (22).

DNA methylation analysis Real-time quantitative reverse transcriptase PCR – Methyl-CpG binding domain (MBD) protein DNA enrich- Quantitative reverse transcriptase PCR (qRT-PCR) was per- ment and high-throughput sequencing (MBD-Seq) were performed as described previously (23). Samples were analyzed in formed as described in Supplementary Methods. Methylation quadruplicate, and gene expression was normalized to GAPDH status was validated using combined bisulphite restriction anal- [NM_002046.5] gene. All primer sequences are available on fi ysis (COBRA) and single-molecule bisul te sequencing (12, 13). request. Methylation status of 413 colorectal carcinomas from The Cancer Genome Atlas (TCGA; ref. 2) was determined by calculating the Data analysis b mean value for probes within the promoter CpG island (probes c2 tests were used for categorical variables and one-way ANOVA outside the CpG island were excluded) and classifying tumors as for continuous variables. Statistical significance was defined at a b b < either methylated ( 0.25) or unmethylated ( 0.25). level of less than 0.05. Analyses were carried out using IBM SPSS software (version 22; SPSS Inc., Chicago). Gene set enrichment Whole-exome sequencing analysis (GSEA) was performed as described previously using Whole exomes were captured from 10 LSTs (lesion 5 was MSigDB (24). Ingenuity Pathway Analysis was performed as fi omitted because of insuf cient DNA) and normal mucosa sam- described in Supplementary Methods. ples using the Roche SeqCap EZ Choice HGSC (Catalogue number: 06465587001) and sequenced using the Illumina HiSeq 2500 platform (2 125 bp). Data were processed and aligned Results (hg19) using Picard MarkDuplicates (Broad Institute) and Bow- Mutation rates in LSTs are similar to those observed in MSS tie2 (14). Somatic single-nucleotide variants (SNV) were identi- colorectal carcinomas fied using the intersection of Strelka (15) and MuTect (16) Using WES, we identified a median of 110 (mean, 100; range, following comparison with normal mucosa. Somatic small inser- 3–155) exonic SNVs (synonymous and nonsynonymous) per

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lesion in our discovery cohort of LSTs (mean sequence coverage of Identification of genes altered in LSTs relative to paired normal 55 in normal tissues and 134 in LST tissues). This represented a mucosa median of 2.4 mutations per megabase. We compared this We followed the analytic strategy outlined in Supplementary mutation rate to that observed in 33 colorectal carcinomas from Fig. S3 to enrich for genes showing multiple damaging altera- TCGA (2) using the same bioinformatics approach. Surprisingly, tions. This involved only considering nonsynonymous SNVs we found a similar rate of mutations in MSS colorectal carcino- and indels that were predicted to be damaging to protein mas (median, 119 mutations; mean, 135; median, 2.6 mutations function and that were present in expressed genes, as deter- per megabase, Fig. 1A). Sanger sequencing of 10 SNVs and 2 mined by RNA-Seq analysis of corresponding normal mucosa. indels confirmed that mutation calls in LSTs were accurate This identified a median of 44 damaging mutations per LST (Supplementary Fig. S2). As expected, significantly higher muta- (mean, 39; range, 0–65, Fig. 3A). No nonsynonymous mutation rates were found in colorectal carcinomas with MSI (medi- tions were detected in one LST (specimen 82), despite a read an, 1,648; mean, 1,774; median, 35.9 mutations per megabase, coverage across exons that was comparable to the other LSTs P 0.05, t test) and in colorectal carcinomas containing muta- (55 in normal, 142 inLST).Next,weidentified which genes tions to the exonuclease domain of the POLE gene (median, were affected by the copy number alterations described above 6,078; mean, 6,852; median, 132 mutations per megabase, (validation cohort LSTs only). Six deletions that encompassed Fig. 1A). We compared the variant allele frequency (VAF), which a total of 807 separate genes on chromosomes 18p, 18q, 1p, measures the proportion of copies of a gene that contain a 6p, or 5q were detected in 5 LSTs (Fig. 2). The only recurrent specific mutation, and found that the median VAF of mutations deletion was at chromosome 5q (lesions 81 and 85) encom- in LSTs was significantly lower than the median VAF in colorectal passing the APC gene. Duplication of the whole of chromo- carcinomas without MSI (P 0.001, t test, Fig. 1B). CGH and somes 7, 8, 12, 13, 19, or 20 was detected in 4 LSTs; however, SNP array data showed that copy number alterations were rare, genes on these duplicated chromosomes were not considered with none detected in 3 LSTs and an average of only 1.5 whole further because of the large numbers of genes involved and the chromosome duplications or deletions per lesion (Fig. 2). To nonspecifi c nature of this type of copy number alteration. confirm that copy number alterations were rare in LSTs, we Finally, using MBD-Seq, we detected promoter CGI hyper- examined a further 12 lesions (7 G-LSTs, 5 NG-LSTs) from methylation at a median of 711 genes (mean, 804; range, validation cohort 1 using CGH and SNP arrays. Only 4 deletions 189–1,837) per LST. However, on average, only 28.5% of these and 12 whole chromosome duplications were detected across all aberrations correlated with loss of gene expression, defined as 12 lesions (mean, 1.6 copy number alterations per lesion, Fig. 2). >2-fold downregulation relative to paired normal mucosa Deletions of chromosome 1p, 5q, 14q, and 18 and duplications (ref. 8; hereafter referred to as epigenetic inactivation). of chromosome 7, 8, 13, 19, and 20 represent some of the most This corresponded to a median of 225 epigenetically inacti- common changes seen in colonic and rectal adenocarcinomas vated genes (mean, 225; range, 53–564, Fig. 3A) per LST and a (Fig. 2). Copy number changes that were specific to colorectal total of 1,389 separate genes across all 11 lesions profiled. carcinomas, yet lacking in LSTs, included deletions to 8p, 15q Comparison with methylation data from TCGA colonic and and the TP53 gene at 17p. Collectively, these data show that the rectal adenocarcinomas showed that most genes identified as number of mutations in LSTs is similar to colorectal carcinomas frequently epigenetically inactivated in LSTs were also fre- without MSI; however, mutations in LSTs are not compounded quently methylated in colorectal carcinomas (Supplementary by widespread copy number alterations. Fig. S4).

n.s. ABLST MSS MSI POLE 20,000 110 119 1,648 6,078 1.0 15,000 10,000 5,000

500 0.5 400 Compound mutations 300 200 100 Number of exonic SNVs

0 Variant allele frequency LST MSS MSI POLE 0 (n = 10) (n = 19) (n = 8) (n = 6)

Figure 1. LSTs contain a similar number of mutations as MSS colorectal carcinomas but fewer compound mutations. A, Number of SNVs (synonymous and nonsynonymous) within exonic DNA in LSTs and colorectal carcinomas is shown. Colorectal carcinomas are separated as MSS, MSI, and those with mutations in the exonuclease domain of the POLE gene (POLE). Median numbers of mutations are indicated at the top of the chart and by the red line. n.s. ¼ not signiﬁcant, t test. B, VAF of SNVs identiﬁed in each tumor is shown. Red lines indicate the median VAF of SNVs in each tumor.

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6 4 2 0 LSTs 2 (discovery) 4 (n = 10) 6 6 4 2 0 LSTs 2 (validation) Figure 2. 4 (n = 12) Copy number alterations are infrequent in LSTs. Each chart 6 indicates the location (x-axis) and frequency (y-axis) of copy 600 number alterations identiﬁed in the discovery cohort of LSTs 400 (n ¼ 10), the validation cohort (n ¼ 12), colorectal 200 adenocarcinomas (n ¼ 1,006), and rectal adenocarcinomas 0 COADs (n ¼ 338). Green indicates copy number gain, red indicates copy 200 (n = 1006) number loss. Data from LSTs were generated using CGH and SNP Frequency of CNAs (tumors) 400 arrays. Data from colorectal carcinomas were obtained from 600 TCGA (2). 300 200 100 0 READs 100 (n = 338) 200 300 21 43 65 87 109 11 12 13 15 17 19 21 14 16 1820 22

Copy number gain Copy number loss

Integration of genomic, epigenomic, and transcriptomic data MAPK (KRAS, BRAF) pathways; however, significantly fewer identifies novel genes with multiple alterations alterations were detected in the TGFb and p53 pathways (P ¼ Having identified genetic and epigenetic alterations, we next 0.039 and P ¼ 0.046, respectively) and the TP53 gene (P ¼ 0.019), integrated these data to identify genes with multiple alterations with a trend toward significantly fewer alterations in the PI3K across our discovery cohort. This involved identifying genes with pathway (P ¼ 0.069, Fisher exact test; Fig. 3C). To determine damaging mutations in more than one patient (recurrent) or whether mutations in these pathways were present in only a small those with multiple alterations (damaging mutation, epigenetic proportion of cells (subclonal), we identified driver gene muta- inactivation, deletion) in a single lesion and at least one alteration tions with a VAF < 0.1. Only 3 additional damaging mutations in in LSTs from other patients (see Supplementary Fig. S3 for a driver genes were identified: a p.R2871T SNV in ATM (VAF, summary of analytical approach). This identified a total of 22 0.064), a c.1494þ1G>A splice site variant in APC (VAF, 0.082), genes that included known colorectal carcinoma–related genes and a p.R95H SNV in ERBB4 (VAF, 0.092). These findings rein- such as APC, KRAS, SOX9, and BRAF (Fig. 3B). However, although force that colorectal carcinoma driver gene mutations are rare in these genes were mutated, deleted, or epigenetically inactivated in LSTs, aside from mutations to Wnt and MAPK genes. multiple LSTs, all (with the exception of APC, KRAS, BRAF, and SLC2A10) were mutated, deleted, or showed focal high-level Genomic, epigenomic, and transcriptomic changes in LSTs amplification in less than 10% of colorectal carcinomas converge on the axonal guidance, Wnt, and actin cytoskeleton (Fig. 3B). These included ANO5, MED12L, EPB41L4A, RGMB, signaling pathways NRXN1, SLITRK1, and ANK2. Many of the mutations in known We next investigated whether altered genes were enriched driver genes such as APC, BRAF, KRAS, and SOX9 showed VAF > within specific biologic pathways. We took all genes identified 0.25, which indicates that these are early clonal events (25). In as epigenetically inactivated or mutated in our discovery cohort addition, several genes identified as frequently altered in LSTs, (n ¼ 1,728), as well as those inactivated in G-LSTs (n ¼ 1,427) or such as ANO5, NRXN1, and SLITRK1, also showed VAF > 0.25 NG-LSTs (n ¼ 573), and performed Ingenuity Pathway Analysis (Supplementary Table S3) suggesting these were also early clonal (see Supplementary Methods). When all LSTs were considered, events in the development of these lesions. significant enrichments were observed in the axonal guidance, We next determined the frequency of alterations to 30 genes thyroid cancer, and Wnt/b-catenin signaling pathways (Fig. 4A). previously described as frequently altered by mutation or copy These pathways were targeted by recurrent alterations to genes number alterations during the development of colorectal carci- encoding ephrins (EPHA4, EPHA6, EPHA7), neurotrophic tyro- nomas (n ¼ 212; ref. 2; Fig. 3C). These genes separated into 5 sine kinase receptors (NTRK1-3), and Wnts (WNT7A). We also signaling pathways: Wnt (10 genes), TGFb (8 genes), PI3K (5 identified frequent alterations to a family of predicted axonal genes), MAPK (5 genes), and p53 (2 genes). In LSTs, frequent guidance genes known as the SLIT and NTRK-like family, namely, alterations were found in genes within the Wnt (APC, SOX9) and SLITRK1 (mutated in 3), SLITRK2 (epigenetically inactivated in

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A 142 3G15G2NG818285 Exonic SNVs 119 147 70 104 – 138 116 72 155 783 Exonic indels 7 2–1 3 10 5 217 0 Damaging mutations 53 51 20 38– 65 50 27 530 35 # of Separate deletions 00211 1 0 0001 # of Deleted genes 00203 392 1160 000 74 96 Epigenetically inactivated 275 564 340 263 331 171 70 53 225 79 101

900 800 Damaging mutations 700 Deletions 600 Epigenetically inactivated 500 400 Figure 3. 300

Integrated genomic view of genes Number of 200 altered genes altered frequently altered in LSTs and 100 comparison with colorectal carcinomas. 0 A, Summary of the number of alterations in the 11 LSTs examined and 15*32 4G1G2NG818285 the mechanism of alteration. Exonic SNVs include synonymous and nonsynonymous mutations. Damaging B Genes with multiple alterations C Comparison with mutations include those predicted to be CRC driver genes damaging to protein function as G NG % of determined using ANNOVAR. The G1 G2 NG CRCs LSTs number of separate deletions and the CRCs 142 3 5* 818285 (n = 212) (n = 10) total number of genes these deletions APC 76 encompassed are indicated. B, ANO5 Integrated genomic view of frequently 2 APC 76 8 altered genes and the mechanism of KRAS 42 FBXW7 17 1 alteration. For each gene, the TACR1 1 AMER1 11 2 percentage of colorectal carcinomas in ST8SIA5 3 ARID1A 9 1 n ¼ TCF7L2 TCGA ( 212) with alterations MED12L x 5 14 1 (nonsynonymous somatic mutations, EPB41L4A DKK1-4 12 0 Wnt homozygous deletions, or high-level 3 FZD10 CBLN2 3 1 focal ampliﬁcations) is indicated to the 0 AXIN2 8 0 right. C, Comparison of the genomic RGMB 1 CTNNB1 5 0 landscapes of colorectal carcinomas SLCO4C1 6 SOX9 5 2 and LSTs. Shown are the percentages of SOX9 5 LRP5 2 1 colorectal carcinomas in TCGA with NRXN1 9 ACVR2A 12 0 alterations in the Wnt, TGFb, PI3K, TGFBR2 SLITRK1 7 10 0 MAPK, and p53 pathways. The number SMAD4 14 1 of LSTs with damaging mutations and CCDC39 4 ACVR1B 8 0 β hemizygous deletions are indicated. No LRFN2 TGF * 4 SMAD3 4 0 homozygous deletions were detected MAPK4 2 SMAD2 8 0 in LSTs. Signiﬁcant differences in the KAZN TP53 3 TGFBR1 3 0 mutation frequencies of genes ( ) SLC2A10 PIK3CA and pathways (TGFb and p53) are 13 20 0 PTEN indicated with asterisks (P 0.05, RIOK2 3 6 0 IGF2 Fisher exact test). ASPM 7 3 0 PI3K PIK3R1 BRAF 10 4 0 ANK2 IRS2 1 0 9 KRAS 42 5 BRAF 10 2 Epigenetic inactivation ERBB3 7 0 Truncating mutation NRAS 10 1 MAPK ERBB2 7 0 Damaging missense mutation ERBB4 8 1 * TP53 52 1 Hemizygous deletion p53* ATM 12 0 x Potentially damaging missense mutation 0 100 10 No alteration detected #%

3), and SLITRK5 (epigenetically inactivated in 7 LSTs, Supple- each subtype may be different. When G-LSTs were considered mentary Fig. S5). However, the marked difference in the number separately, we identiﬁed enrichment of colorectal carcinoma of gene alterations in G-LSTs (n ¼ 1,728, mean 247 per lesion) metastasis signaling (Fig. 4B and D), whereas NG-LSTs showed versus NG-LSTs (n ¼ 573, mean 143 per lesion) raised the alterations to actin cytoskeletal and Rho GTPase signaling genes possibility that the predominant signaling pathways altered in (Fig. 4C and D). Alterations to axonal guidance genes were heavily

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AC0.5 B 0.5 0.5 All LSTs G-LSTs NG-LSTs (n =1,728) (n = 1,427) (n = 573) 0.4 2 0.4 0.4 2 0.3 0.3 0.3 3 1 3 1 Ratio Ratio Ratio 0.2 4 0.2 0.2 1

0.1 5 0.1 0.1 2 4 5 3 5 0 0 0 4 1357911 1357911 1357911 –log(P) –log(P) –log(P)

Canonical pathway PP# of Genes Canonical pathway # of Genes Canonical pathway P # of Genes 1. Axonal guidance signaling 1.2×10 –9 69 1. Axonal guidance signaling 2.4×10–9 61 1. Thyroid cancer signaling 1.9×10–3 5 2. Thyroid cancer signaling 7.0×10 –8 15 2. Thyroid cancer signaling 6.4×10–8 14 2. ERK5 Signaling 2.9×10–3 6 3. hESC Pluripotency (NANOG) 5.8×10 –5 21 3. hESC Pluripotency (NANOG) 4.6×10–6 21 3. Actin cytoskeleton signaling 3.8×10–3 12 4. Wnt/β-catenin signaling 3.0×10 –4 26 4. Wnt/β-catenin signaling 5.3×10–5 25 4. RhoGTPase signaling 6.9×10–3 12 5. GPCR Sign. (enteroendocrine)1.6×10 –4 16 5. CRC Metastasis signaling 1.8×10–3 27 5. PTEN Signaling 1.7×10–2 7

D β Only Axonal guidance Wnt/ -catenin CRC Metastasis Thyroid cancer Actin cytoskeleton Rho GTPase recurrent G NG G NG G NG G NG G NG G NG shown G1 G2 NG G1 G2 NG G1 G2 NG G1 G2 NG G1 G2 NG G1 G2 NG 12345* 818285 12345* 818285 12345* 818285 12345* 818285 12345* 818285 12345* 818285 KRAS APC APC KRAS APC WASF3 SEMA6D PPP2R2B KRAS NTRK3 TIAM1 SEPT5 BMP3 CDKN2A ADCY1 NTRK2 FGF9 MYLK NGFR TCF4 RHOF NTRK1 KRAS CDH4 NTRK3 WNT7A TCF4 TCF4 VAV2 RHOF EPHA7 SOX5 MMP16 BRAF MYH10 GNG4 ADAMTS1 CDH2 MMP25 RET MYLK GNB4 TUBB2B NR5A2 BRAF TP53 MATK CDH2 NTNG1 SOX9 GNB4 NTF3 FGF13 CDH7 NTRK1 FZD10 WNT7A NRAS FN1 ESR1 UNC5C SFRP2 MMP17 GDNF FGF14 GNAQ NTRK2 FRZB GNG4 BDNF VAV3 GNA14 ADAMTS8 WNT6 PRKACB NGF PDGFD CDH15 NTN1 SOX2 FZD10 RXRA NRAS NFKB2 GNB4 DKK2 WNT6 TCF7L2 PDGFA CDH11 UNC5D PPP2R2C ADCY8 TRIO WIPF1 DPYSL5 MAP4K1 TP53 BCAR1 CDH17 EPHA6 TP53 LRP5 ERK5 TTN GNG12 PDGFD LRP5 NRAS Signaling FGF4 FNBP1 SLIT1 WNT9B WNT9B G NG IQGAP2 ARHGAP4 UNC5A WNT9A WNT9A G1 G2 NG FGF11 EPHA4 GNAQ WNT3A 12345* 818285 GNG12 WNT7A WNT3A PRKAR2B KRAS GLI3 DKK1 JAK3 SGK1 TCF7L2 TUBB4A TCF7L2 NTRK1 Epigenetic inactivation Hemizygous deletion GNG4 WNT1 WNT1 SFN Key ROBO3 FNBP1 RPS6KA3 EFNA1 GNG12 GNAQ Damaging missense mutation SEMA7A NFKB2 NRAS PRKCB PRKAG2 NGF Truncating mutation No alteration detected

EFSignificantly upregulated (n = 303) G Canonical pathway P # of Genes CRC Metastasis signaling 8.49×10–5 11 Wnt/β-catenin signaling 7.18×10–3 7 Down in G & NG Up in G & NG Wnt/Ca+ pathway 7.42×10–3 4 n n ( = 458) ( = 303) Axonal Guidance signaling 1.33×10–2 12 Significantly downregulated (n = 458) pathway activated 4 Direction if 12345G1G2NG818285 Canonical pathway P # of Genes MMP7 3 DCC –2 MMP10 Tight junction signaling 2.08×10 8 MMP1 –2 2 MMP9 Rho GTPase signaling 4.98×10 9 LEF1 Direction if pathway activated MMP3

CRC MMP11 1 MMP12 12345G1G2NG818285 Metastasis NOS2 CLDN8 WNT5A IGSF5 CLDN23 0 SNAP25 MMP7 MYLK CDH3 CLDN4 LEF1 Tight –1 UBD ACTG2 β - AXIN2 junctions CLDN3 Wnt CD44 –2 catenin WNT5A DES MYLK PLCB4 RHOF

2+ CREB5 WASF3 –3 WNT5A GNA11 SLC9A1 Ca

Wnt PLCB1 Rho

Log10 average fold change in NG-LSTs ARHGEF18

GTPase ACTG2 –4 MMP7 RHOD –4 –3 –2 –1 0 1 2 3 4 DCC EPHB1 Log10 average fold change in G-LSTs MMP10 Log10 FC PRKCG –4 0 4.5 MMP9 SEMA7A MMP11

Axonal PLCB4

Guidance BMP4 WNT5A PLCB1 Log10 FC –4 0 4.5

Figure 4. Genetic, epigenetic, and transcriptional changes converge on axonal guidance, Wnt, and actin cytoskeletal genes. A–C, Bubble charts summarizing the canonical pathways that are significantly enriched for gene alterations (damaging somatic mutations or epigenetic inactivation), as identified by Ingenuity Pathway Analysis. The y-axis indicates the ratio of genes showing alterations within each pathway. Bubble size is proportional to the number of genes. Pathways highlighted in brown are described in the tables below each chart, which indicate the P value (right-tailed Fisher exact test) and the number of genes altered. D, genes altered in the pathways underlined in A–C and the mechanism by which they are altered. , WES not performed on LST 5. E, Significant gene expression changes in G-LSTs and NG-LSTs relative to paired normal mucosa (as determined by DESeq, see Supplementary Methods). F and G, Summary of Ingenuity Pathway Analysis of significantly upregulated (F) and downregulated (G) genes. Columns at the right of F and G the expected direction of gene expression change if the pathway is activated. Red, upregulated; green, downregulated.

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Gender Male Wnt Pathway MAPK Methylation Female a 5 1 Locaon Proximal e 2 7 1 Distal M2 APC ANO5 JA IIa GenderLocationParis Surfac HistotypeDysplasi CTNNB1 5q loss KRAS BRAF PEBP1IQGAP2WNT5ATMBIM1NTRK FLNC EPHA RHOF TIAM SLIT1 SCN3BSLITRKCNTN TFPI2 RASSF2 Paris IIa+Is Is IIc

Granular Surface Non-granular Mixed Tubular Histologic Tubulovillous type Sessile serrated Villous

Focal High-grade Dysplasia Low-grade Non

Wnt Pathway Actin cytoskeleton Axonal guidance MAPK Pathway Copy number analysis (CGH/SNP array)

86 91 93 97 98 98 100 100 100 74 63 66 77 Mutation/methylation 51 50 52 50 24 16 frequency (%) 5 008 14 0

APC KRAS BRAF FLNC JAM2 SLIT1 TFPI2 5q loss PEBP1 NTRK2 ANO5EPHA7 RHOF TIAM1 SCN3B CTNNB1 IQGAP2WNT5ATMBIM1 SLITRK5CNTN1 RASSF2

Figure 5. Validation of frequent genetic and epigenetic alterations. A, Genetic and epigenetic alterations in MAPK, Wnt, actin cytoskeletal, and axonal guidance genes in validation cohort 1 (n ¼ 44). Clinicopathologic features are indicated by the key in the left. Paris classification, which is an indicator of macroscopic morphology, as determined at endoscopy, is defined as IIa (flat elevated), IIa þ Is (flat elevated with one or more large nodules), Is (sessile and raised >2.5 mm relative to surrounding normal mucosa), and IIc (flat depressed). Methylation was determined using COBRA and bisulfite sequencing. Black, methylated; white, unmethylated; gray, not determined. The chart indicates the percentage mutation and methylation frequencies at each gene analyzed. Red boxes at the far right indicate which LSTs were analyzed using CGH and SNP arrays (see Fig. 2). biased toward epigenetic inactivation and were markedly less (20 mm in diameter) noninvasive LSTs (validation cohort 1). frequent in NG-LSTs when compared with G-LSTs (Fig. 4D). We also investigated the recurrence of 5q deletions, mutation of We reasoned that biologically relevant signaling pathways the APC, KRAS, BRAF, and CTNNB1 genes, and the hypermethy- would also show transcriptional changes. To this end, we used lation of 17 genes on the basis of their frequent occurrence in the DESeq (11) to identify 303 and 458 genes showing significant discovery cohort of LSTs or because they were connected with upregulation or downregulation relative to paired normal tissues axonal guidance, Wnt, or actin cytoskeletal signaling. Copy num- (Fig. 4E). Upregulated genes showed significant enrichment for ber analysis using microsatellite markers at 5q and sequencing colorectal carcinoma metastasis, Wnt, and axonal guidance sig- across the APC gene identified deletions or mutations in 83% of naling and again we observed changes to ephrins (EPHB1) and 41 informative lesions (Fig. 5). CTNNB1 mutations were found in Wnts (WNT5A) as well as matrix metalloproteinases (MMP1, 3, 7, 5% of 44 lesions, whereas KRAS and BRAF mutations occurred in 9, 10, 11, and 12), and cadherins (CDH3, Fig. 4E). Downregulated 51% of 43 and 16% of 44 lesions, respectively (Fig. 5). COBRA genes showed significant enrichment for tight junction and Rho and bisulfite sequencing confirmed frequent methylation GTPase signaling genes including claudins (CLDN3, 4, 8, and 23), (50%) of 13 of the 17 genes investigated including SLITRK5 MYLK, WASF3, and Rho's (RHOD, RHOF, Fig. 4E). Interestingly, (methylated in 98%) and ANO5 (methylated in 63%, Fig. 5, downregulation of genes in the Rho GTPase signaling pathway, Supplementary Fig. S5), 2 of the most frequently epigenetically which is consistent with the deactivation of this pathway, was inactivated genes identified in the discovery cohort. There was observed in all 11 LSTs (Fig. 4E). This demonstrates that while Rho no correlation between tumor location, Paris classification, mor- GTPase signaling is one of the most significantly enriched path- phology, histologic type or dysplasia, and mutation or methyl- ways for gene alterations in NG-LSTs, it is in fact similarly altered ation at the genes examined. across all 11 LSTs examined. To further investigate the importance of axonal guidance sig- Genetic, epigenetic, and transcriptional alterations correlate naling in the development of LSTs, we investigated gene expres- with LST morphology sion microarray data from a previous study of 25 LSTs and 17 A convergence of evidence suggested that the molecular land- polypoid adenomas (26). Gene Set Enrichment Analysis (GSEA) scape of granular and nongranular LSTs was likely to be different. confirmed that axonal guidance genes were significantly differ- For example, in our discovery cohort, epigenetic inactivation was entially expressed (>2-fold up- or downregulated) in LSTs relative significantly more frequent in G-LSTs (average, 288 genes; range, to normal mucosa (FDR q ¼ 0.006) but not in polypoid adeno- 70–564) compared with NG-LSTs (average, 115 genes; range 53– mas relative to normal mucosa (FDR q ¼ 0.115). 225; t test: P ¼ 0.03). In support of this, genes with recurrent epigenetic inactivation specifically in one morphological subtype Validation of genetic and epigenetic alterations were also more common in G-LSTs (315 genes) than in NG-LSTs To explore the broader significance of the alterations we had (9 genes, Supplementary Fig. S4 and Supplementary Table S4). identified, we investigated their prevalence in a cohort of 44 large Furthermore, pathway analysis of altered genes had revealed the

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enrichment of different signaling pathways in G-LSTs and NG- nificantly upregulated or downregulated in either G-LSTs or NG- LSTs (Fig. 4B and C). Therefore, we further investigated the LSTs (Fig. 6B). A small proportion of these genes (n ¼ 258) was molecular differences between G-LSTs and NG-LSTs. In a previous significantly upregulated in G-LSTs yet downregulated in NG-LSTs study, we had shown that in a homogeneous cohort of flat LSTs or vice versa (genes within gray-shaded boxes in Fig. 6B). We (Paris classification IIa or IIb), KRAS mutations at codons G12 and reasoned that these transcriptional changes represented the most G13 were significantly more frequent in G-LSTs than in NG-LSTs extreme differences in gene expression between the 2 morpho- (22). However, the detection of KRAS D117N (n ¼ 1) and A146V logic subtypes. The remaining 3,087 genes were upregulated or (n ¼ 1), NRAS Q61K (n ¼ 1) and BRAF G469V (n ¼ 1) mutations downregulated in both G-LSTs and NG-LSTs but to different in 4 of 10 LSTs in the discovery cohort prompted us to reinves- degrees. Hierarchical clustering showed that the 258 differentially tigate the relationship between morphology and mutations in expressed genes robustly grouped granular lesions separately these genes in a second validation cohort (validation cohort 2) from nongranular lesions and that the 3 LSTs from the same consisting of 20 G-LSTs and 20 NG-LSTs (Paris classification IIa or patient clustered together (Fig. 6C). Pathway analysis of these 258 IIb). KRAS mutations (at codons G12, G13, D117, or A146) differentially expressed genes showed enrichment for cAMP- occurred in 55% (22 of 40), NRAS mutations (at codons G12, mediated and CXCR4 signaling (Fig. 4D). Genes within the G13, or Q61) occurred in 0% (0 of 40), and BRAF mutations CXCR4 signaling pathway were mostly upregulated in NG-LSTs, (at codons G469 or V600) occurred in 5% (2 of 40) of lesions whereas the same genes were mostly downregulated in G-LSTs (Supplementary Fig. S6). Differentiation of lesions according to (Fig. 6D), which is consistent with the hyperactivation of CXCR4 surface morphology revealed a trend toward KRAS mutations in signaling specifically in NG-LSTs. Collectively, these data indicate G-LSTs (70%, 14 of 20) versus NG-LSTs (40%, 8 of 20, c2 test, P ¼ that the different morphologic subtypes of LSTs show distinct 0.055). Differentiation of lesions according to histologic type genetic, epigenetic, and transcriptional profiles at a subset of showed that mutation of KRAS was significantly more frequent in genes. tubulovillous LSTs (77%, 17 of 22) than in those with tubular architecture (28%, 5 of 18, c2 test, P ¼ 0.002). Next, we investigated transcriptional differences between G- Discussion LSTs and NG-LSTs. Principal component analysis (PCA) of gene In this study, multiple layers of genome-wide data were inte- expression showed that the discovery cohort of LSTs could be grated to generate molecular maps of LSTs. This showed that stratified on the basis of surface morphology (Fig. 6A) but not despite the low malignant risk of this subtype of adenoma they histologic type or tumor location. To further explore these tran- have an unexpectedly high mutation rate that is comparable with scriptional differences, we identified 3,345 genes that were sig- MSS cancers. These mutations are not compounded by copy

G-LSTs Altered in G or NG A B (n = 3,345) NG-LSTs Differentially expressed (n = 258) 250 4

200 in NG-LSTs, 3 in G-LSTs 150 Figure 6. 2 100 82 NG Transcriptomic changes correlate with G2 1 surface morphology. A, PCA of log2 fold 50 81 G1 changes in gene expression in each 0 85 0 lesion relative to paired normal mucosa. PC 2 1 –50 5 –1 LSTs group separately according to 3 ﬁ –100 4 2 morphologic subtype. B, Signi cant –2 gene expression changes in G-LSTs or –150 –3 in NG-LSTs, NG-LSTs relative to paired normal –200 in G-LSTs mucosa, as determined by DESeq. Log10 average fold change in NG-LSTs –250 –4 Quadrants shaded in gray highlight –250 –200 –150 –100 –500 50 100 150 200 250 –4 –3 –2 –10421 3 PC1 Log10 average fold change in G-LSTs genes that were upregulated in NG- LSTs yet downregulated in G-LSTs or vice versa (i.e., differentially expressed, G-LSTs n ¼ 258). C, hierarchical clustering of C n NG-LSTs D Differentially expressed (G vs. NG, n = 258) ( = 258 genes) LSTs using the log fold change in P 2 4 Canonical pathway # of Genes expression (relative to paired normal cAMP-mediated signaling 1.85x10–3 9 3 mucosa) of the 258 genes from B. D, CXCR4 Signaling 3.13x10–3 7

5 Direction if pathway activated Summary of Ingenuity Pathway 2 Analysis of the 258 differentially 1G1432 5G2NG818285 1 DUSP1 expressed genes. Columns at the right RGS2 GNAL of C, to E, Indicate the expected G2 AKAP7 CNR2 G1 CAMK1G direction of gene expression change if ADRA2A cAMP PTH1R the pathway is activated. Red, NG Signaling DRD1 upregulated; green, downregulated. 85 MAPK10 GNG2 CXCL12 81 GNAL EGR1 82 FOS

CXCR4 GNG4 Signaling Log2 FC –3 0 3 Log10 FC –2.25 0 2.75

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number alterations and target different genes when compared gaining compound alterations in important colorectal carcino- with mutations observed in colorectal carcinomas. LSTs frequent- ma–related genes. This is supported by previous reports that copy ly show alterations to genes in the axonal guidance, Wnt, and actin number alterations are rare in adenomas relative to colorectal cytoskeleton pathways but rarely show alterations to genes in the carcinomas (20). The absence of mutations in the PI3K, TGFb, and TGFb (SMAD2, SMAD3, ACVR1B, ACVR2A, TGFBR1, TGFBR2), p53 signaling pathways in LSTs shows that the mutation of PI3K (PIK3CA, PIK3R1, PTEN, IGF2, IRS2), and p53 (TP53 and PIK3CA, PTEN, SMAD2/3, or TP53 is also likely to be important ATM) pathways, which are known to be altered in colorectal in malignant progression. Previously, assessment of PIK3CA in carcinomas. These data are consistent with a model in which the LSTs detected mutations in 14% of 35 cases (30). Although Chang extensive growth of LSTs is driven by molecular alterations that and colleagues also described invasive cancer in 14% of these impart continued proliferation, such as APC, SOX9, KRAS, and LSTs, it was not clarified whether these were the same lesions with BRAF mutations, but are insufficient for malignant transforma- PIK3CA mutations. tion. Comparison of molecular alterations in the 2 morphologic A potential limitation of this study is the relatively small subtypes of LSTs, which differ in their invasive potential, identi- number of LSTs profiled in our discovery cohort. However, we fied subtle molecular differences, including a higher frequency purposefully chose to profile a small cohort of LSTs and normal of KRAS mutations and epigenetic inactivation in very low-risk tissues using multiple methods rather than acquire insufficient G-LSTs and the hyperactivation of CXCR4 signaling in NG-LSTs, detail from large numbers using only one method and with which by comparison are associated with an increased risk of inappropriate controls. This provided a much richer appreciation cancer. of the molecular landscape of LSTs. Our validation of genetic and Determining the molecular events that drives an adenoma to epigenetic alterations in 84 additional lesions confirms that many become cancerous was recently highlighted as 1 of 5 crucial of the alterations we identified are common in LSTs. While questions that need to be addressed to better understand how analysis of additional numbers will undoubtedly clarify the colorectal carcinoma develops (27). To address this, this study precise frequencies of molecular alterations in LSTs, it is already took a novel approach by specifically focusing on large (>20 mm clear that their underlying biology is different from colorectal in diameter) LSTs, which despite their size have a very low risk of carcinomas. progressing to cancer. At the outset, it was anticipated that the A summary of the genes and pathways identified as altered in mutation rate in LSTs would be low, as described previously for LSTs is provided in Fig. 7. The convergence of genetic, epigenetic, other types of benign lesions. For example, parathyroid adenomas and transcriptional alterations in the axonal guidance and actin contain an average of only 3.6 nonsynonymous mutations per cytoskeletal networks was a key finding of this study. GSEA and adenoma (28). The number of mutations in polypoid colorectal data from a previous study (26) confirmed that transcriptional adenomas has also been reported to be much lower than in changes in axonal guidance genes were significantly overrepre- colorectal carcinomas with an average mutation rate of 0.63 per sented in LSTs, but not in polypoid adenomas, indicating that this megabase or a median of 35.5 mutations (synonymous and pathway may be particularly important in LSTs. Axonal guidance nonsynonymous) per adenoma (mean, 40.8; range, 12–101; signaling is not specific to neuronal tissues and it has been linked ref. 29). The relatively high mutation load found in LSTs in this to the development of colorectal, breast, and pancreatic cancers study did not appear to be driven by alterations in any known (31–33). Alterations to axonal guidance signaling molecules can DNA repair pathway, and we have shown previously that LSTs do influence cancer cell migration and invasive through deregulation not exhibit MSI (22). However, this study also shows that a high of the actin cytoskeleton (33, 34), which we also found was mutation load is not necessarily a feature of all LSTs, as we were targeted by multiple alterations in LSTs. Extensive validation was unable to find a single nonsynonymous mutation in one lesion in performed on many of the genetic, epigenetic, and copy number our cohort despite high-coverage exome sequencing data. alterations identified in this study, including the MAPK (3 genes), The integration of multiple layers of genomic and epigenomic Wnt (3 genes), actin cytoskeletal (5 genes), and axonal guidance data from paired normal mucosa and neoplastic tissues was (5 genes) pathways. SLITRK5 is known to regulate neurite out- crucial in identifying genes with multiple alterations. Hyper- growth through regulation of the actin cytoskeleton (35, 36). Its methylation and loss of gene expression in tumors relative to epigenetic inactivation in the vast majority of lesions tested normal tissue are cardinal features of epigenetic inactivation, suggests this may be an early event in colorectal neoplasia. Several whereas simply assaying methylation neither precludes nor dic- additional members of the SLITRK (SLITRK1 and 2), SLIT (SLIT1), tates that the methylation is tumor-specific that it affects expres- and NTRK (NTRK1, 2, and 3) gene families were also recurrently sion of the linked gene, nor that the gene was expressed in relevant mutated or epigenetically inactivated in LSTs. RGMB (repulsive normal tissue from the same patient. We excluded hypermethy- axon guidance molecule family member B) is an axon guidance lated, mutated, or deleted genes not expressed (transcriptionally molecule that regulates SMAD and Wnt signaling through bone silent) in the corresponding normal tissue as well as hypermethy- morphogenetic protein (BMP) receptors (37). RGMB was deleted lated genes that did not show transcriptional silencing in the LST. and showed different frameshift mutations in 2 LSTs (specimens Finally, our operating logic was that genes that are most likely to 81 and 85) suggesting this gene is biallelically inactivated. be important would be altered by more than one mechanism. MED12L (mediator complex subunit 12-like) was mutated in all Consequently, this study has identified genes that are frequently 3 LSTs from the same patient (G1 and G2, p.V1694L; NG, p. altered by epigenetic inactivation, mutation, and/or copy number V2042M) and was epigenetically inactivated in 3 LSTs, with both alteration that may have been overlooked in previous studies. mutation and epigenetic inactivation in one LST. MED12L func- Occasionally, LSTs do progress to cancer (6), which raises the tions as a b-catenin–dependent transcriptional coactivator. The question what additional molecular alterations might drive this identified mutations flank the b-catenin–binding domain and progression. The low incidence of copy number alterations in LSTs could potentially impact on transcriptional coactivation of Wnt suggests that genomic structural changes would be important in target genes.

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Key Epigenetic inactivation Damaging missense mutation Truncating mutation

Hemizygous deletion Upregulation Downregulation

MMP7 ADAMTS1 MMPs MMP1 MMP3 MMP9 MMP10 BMP4 MMP11 FGF9 WNT7A BMPs EFNA5 FGFs WNTs BMP3 MMP12 FGF13 CLDN3 CDH2 WNT5A MMP16 FGF14 CLDN4 CDH3 MMP17 CLDN8 CDH4 MMP25 CLDN23 CDH7

UNC5 SLITRK5 RGMB DCC NTRKs EPHAs NRXN1 FGFR EGFR Claudins Cadherins FZD SLITRK1 ANO5 A/C/D NTRK1 EPHA6 NTRK2 EPHA7 NTRK3 EPHA4 VAV2 VAV3 APC AXIN PPP2R2B KRAS BRAF

RHOD RHOD CTNNB1 SOX9 RHOs RHOs RHOF RHOF

SOX5 MAPK

MYLK TIAM1 SEPT5 WASF3 SMADs MED12L Actin ACTG2 Myosin MYH10 ANK2 RAC

EPB41L4A ? Actin cytoskeleton Wnt Target genes ?

Axonal guidance Actin cytoskeleton Wnt Unknown

Figure 7. A summary of the major pathways and genes altered in LSTs. The mechanism of alteration is indicated beside each protein. Only genes altered in more than one patient are included.

By profiling both G-LSTs and NG-LSTs, we were able to explore In summary, this study represents the first comprehensive the molecular differences between these morphologic subtypes. genome-wide survey of the molecular landscape of LSTs. Our Our data support previous findings that both villosity and gran- approach of integrating 4 layers of omics data has allowed us to ular morphology are related (22) and that KRAS mutations are elucidate biologically relevant genes and pathways that would not associated with villosity (38). Two studies have shown that CGI otherwise have been evident. In doing this, we have discovered hypermethylation at several candidate genes was more frequent in novel genes associated with LST development and have gained G-LSTs than NG-LSTs, but that overall, CIMP is rare in LSTs (39, important insights into why some adenomas do not progress to 40). Our data delve far deeper than these previous reports by cancer. In the broader context, the high mutation load in LSTs integrating genome-wide gene expression and DNA methylation shows that adenomas with a low risk of progressing to cancer can data from each LST and its paired normal tissue. In addition to exhibit as many mutations as colorectal carcinomas. This provides showing that only a subset of hypermethylation events correlate new perspectives on the established notion that colorectal cancer with loss of gene expression, our study is the first to show that results from a progressive accumulation of mutations and has epigenetic inactivation is much more common in G-LSTs and the important implications for the validity of molecular biomarkers first to identify the specific genes (genome-wide) that contribute for assessing cancer risk. to epigenetic differences between the 2 morphological subtypes. Finally, we also show that the morphologic subtypes of LST can be fl distinguished on the basis of gene expression profile. We show Disclosure of Potential Con icts of Interest fl this globally but also identify significant differences in the expres- No potential con icts of interest were disclosed. sion of specific genes and pathways, most notably in CXCR4 signaling, which is involved in chemotaxis and cell migration Authors' Contributions (41). Therefore, it may be possible to distinguish G- and NG-LSTs Conception and design: L.B. Hesson, R.L. Ward and consequently invasive potential, by assessing the genetic, Development of methodology: L.B. Hesson, J.W.H. Wong, B. Ng, S. Srivastava, epigenetic, and transcriptional status of a panel of genes. M.A. Sloane

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Acquisition of data (provided animals, acquired and managed patients, Other (methylation analysis, mutation analysis, gene expression analysis, etc.): provided facilities, etc.): L.B. Hesson, B. Ng, S. Srivastava, P. Zarzour, L.B. Hesson, B. Ng, P. Zarzour, S. Srivastava, C.-T. Kwok, D. Packham, D. Beck, C.-T. Kwok, D. Packham, A.C. Nunez, A. Dower, M.A. Sloane, N.J. Hawkins, R, Ryan, A.C. Nunez, A. Dower, M.A. Sloane M.J. Bourke, R.L. Ward Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Grant Support computational analysis): L.B. Hesson, B. Ng, P. Zarzour, S. Srivastava, J.W.H. Wong is supported by a Future Fellowship (FT130100096) from the D. Packham, A.C. Nunez, D. Beck, A. Dower, C.E. Ford, J.E. Pimanda, Australian Research Council. M.A. Sloane, J.W.H. Wong, R.L. Ward The costs of publication of this article were defrayed in part by the payment of Writing, review, and/or revision of the manuscript: L.B. Hesson, J.E. Pimanda, page charges. This article must therefore be hereby marked advertisement in M.A. Sloane, J.W.H. Wong, R.L. Ward Administrative, technical, or material support (i.e., reporting or orga- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nizing data, constructing databases): N.J. Hawkins, M.J. Bourke, R.L. Ward Received May 19, 2016; revised August 15, 2016; accepted September 7, 2016; Study supervision: L.B. Hesson, M.A. Sloane, J.W.H. Wong, R.L. Ward published OnlineFirst September 26, 2016.

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OF12 Mol Cancer Res; 14(12) December 2016 Molecular Cancer Research

Integrated Genetic, Epigenetic, and Transcriptional Profiling Identifies Molecular Pathways in the Development of Laterally Spreading Tumors

Luke B. Hesson, Benedict Ng, Peter Zarzour, et al.

Mol Cancer Res Published OnlineFirst September 26, 2016.

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