High Prevalence of Sessile Serrated Adenomas With BRAF Mutations: A Prospective Study of Patients Undergoing Colonoscopy

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1 GASTROENTEROLOGY 2006;131: High Prevalence of Sessile Serrated Adenomas With BRAF Mutations: A Prospective Study of Patients Undergoing Colonoscopy KEVIN J. SPRING,* ZHEN ZHEN ZHAO, # ROZEMARY KARAMATIC,* MICHAEL D. WALSH, VICKI L. J. WHITEHALL,* TANYA PIKE,* LISA A. SIMMS,* JOANNE YOUNG, MICHAEL JAMES, # GRANT W. MONTGOMERY, # MARK APPLEYARD, DAVID HEWETT, KAZUTOMO TOGASHI, JEREMY R. JASS, and BARBARA A. LEGGETT* *Conjoint Gastroenterology Laboratory and Department of Gastroenterology and Hepatology, Royal Brisbane and Women s Hospital Research Foundation, and Molecular Epidemiology Laboratory and Molecular Cancer Epidemiology Laboratory, Queensland Institute of Medical Research, Herston, Brisbane, Australia; Division of Endoscopy and Department of Surgery, Jichi Medical University, Shimotsuke, Tochigi, Japan; and # Department of Pathology, McGill University, Montreal, Quebec, Canada See editorial on page Background & Aims: Sporadic colorectal cancers with a high degree of microsatellite instability are a clinically distinct subgroup with a high incidence of BRAF mutation and are widely considered to develop from serrated polyps. Previous studies of serrated polyps have been highly selected and largely retrospective. This prospective study examined the prevalence of sessile serrated adenomas and determined the incidence of BRAF and K-ras mutations in different types of polyps. Methods: An unselected consecutive series of 190 patients underwent magnifying chromoendoscopy. Polyp location, size, and histologic classification were recorded. All polyps were screened for BRAF V600E and K-ras codon 12 and 13 mutations. Results: Polyps were detected in 72% of patients. Most (60%) were adenomas (tubular adenomas, tubulovillous adenomas), followed by hyperplastic polyps (29%), sessile serrated adenomas (SSAs; 9%), traditional serrated adenomas (0.7%), and mixed polyps (1.7%). Adenomas were more prevalent in the proximal colon (73%), as were SSAs (75%), which tended to be large (64% 5 mm). The presence of at least one SSA was associated with increased polyp burden (5.0 vs 2.5; P.0001) and female sex (P.05). BRAF mutation was rare in adenomas (1/248 [0.4%]) but common in SSAs (78%), traditional serrated adenomas (66%), mixed polyps (57%), and microvesicular hyperplastic polyps (70%). K-ras mutations were significantly associated with goblet cell hyperplastic polyps and tubulovillous adenomas (P.001). Conclusions: The prevalence of SSAs is approximately 9% in patients undergoing colonoscopy. They are associated with BRAF mutation, proximal location, female sex, and presence of multiple polyps. These findings emphasize the importance of identifying and removing these lesions for endoscopic prevention of colorectal cancer. Colorectal cancer (CRC) is a heterogeneous disease that develops through diverse molecular pathways. The evolution of a large proportion of CRCs, which are primarily microsatellite stable, is consistent with the adenoma-carcinoma sequence proposed by Kinzler and Vogelstein in the late 1980s and involves the stepwise accumulation of multiple genetic mutations in genes such as APC, K-ras, and p53. 1 However, approximately 15% of sporadic CRCs exhibit both high levels of microsatellite instability (MSI-H) and methylation of multiple CpG islands, a phenomenon known as the CpG island methylator phenotype (CIMP). 2 Methylation induces the transcriptional silencing of MLH1, which leads to mismatch repair deficiency, as well as epigenetic silencing of other key genes such as p16. While CIMP is seen in most sporadic MSI-H CRCs, a subset of non MSI-H cancers without MLH1 methylation is also CIMP positive. 3 Morphologic and molecular heterogeneity are also evident in premalignant polyps, of which a proportion progress to CRC, and have important implications for prevention and screening strategies. It is gaining increasing acceptance that sporadic MSI-H CRCs evolve from serrated polyps and not from adenomas. 4 8 Serrated polyps include hyperplastic polyps, composed of goblet cell and microvesicular subtypes, traditional serrated adenomas (TSAs), and the more recently described sessile serrated adenomas (SSAs), which have also been described as sessile serrated polyps. 4,9 Traditional hyperplastic polyps are generally small and commonly found in the distal colon. Epithelial proliferation within these hyperplastic polyps is equivalent to that seen within normal epithelium. However, there is an apparent failure of apoptosis leading to the accumulation of epithelial cells, which are accommodated by infolding (serration) of the epithelial lining of the crypt. 10 Serrated adenomas are relatively rare lesions with serrated architecture reminiscent of hyperplastic polyps but with unequivocal dysplasia. SSAs have only recently been recognized and are characterized by some degree of abnormal proliferation, tend to be larger than hyperplastic polyps, and are more likely to be located in the proximal colon. 9 In addition, it has been shown that a high proportion (92%) of SSAs and TSAs demonstrate CIMP, thus strengthening the evidence that SSAs are the precursor lesions for sporadic MSI-H cancers and probably also for CIMP-positive, non MSI-H cancers. 11,12 It is well established that approximately 30% of CRCs and large adenomas harbor activating mutations in the K-ras onco- Abbreviations used in this paper: AS-PCR, allele-specific real-time polymerase chain reaction; CIMP, CpG island methylator phenotype; CRC, colorectal cancer; MP, mixed polyp; MSI-H, high degree of microsatellite instability; SSA, sessile serrated adenoma; TSA, traditional serrated adenoma by the AGA Institute /06/$32.00 doi: /j.gastro

2 November 2006 SERRATED POLYPS WITH BRAF MUTATIONS 1401 gene. 13,14 These mutations are also seen in approximately 20% of hyperplastic polyps. 15,16 The principal effectors of K-ras are the serine-threonine kinases of the Raf family and their downstream target, the mitogen-activated protein kinase cascade. In 2002, it was discovered that approximately 10% of all CRCs carry an activating mutation of the BRAF oncogene. 17 The vast majority (80%) of BRAF mutations in both CRC and other tumors are a T to A transversion at nucleotide 1796 converting a valine at amino acid 600 to glutamic acid (V600E). Not unexpectedly, BRAF and K-ras mutations are mutually exclusive, indicating a possible lack of survival advantage to cells activating 2 proteins in the same signaling cascade. Importantly, BRAF mutations show a striking association with sporadic CRCs exhibiting MSI-H and/or CIMP; in addition, BRAF mutations have been reported in hyperplastic polyps, TSAs, and SSAs 16,18 and significantly less frequently in adenomas. Thus, there is strong evidence that SSAs and/or polyps bearing BRAF mutations are the likely precursors of the subgroup of cancers that show MSI-H and/or CIMP. 11,16,18 To date, all studies of the occurrence of BRAF mutations in polyps have been conducted using highly selected retrospective series of specimens. 15,16,18 The present study aimed to determine the prevalence, characteristics, and mutation profile of colorectal polyps in a prospective series of patients undergoing colonoscopy for clinical indications. Chromoendoscopy with magnification was performed by a single expert operator so that the maximal number of polyps was identified, removed, and retrieved. These data are important in understanding the prevalence and distribution of the likely precursors of MSI-H and/or CIMP-positive cancers and therefore in the planning of colonoscopy surveillance to prevent the development of such cancers. Materials and Methods Subjects A consecutive unselected series of 190 patients underwent colonoscopy by a single operator (K.T.) over a 5-month period in 2003 at the Royal Brisbane and Women s Hospital. The aim was to identify, remove, and retrieve all polyps. Patients gave written informed consent, and the study was approved by the Human Research Ethics Committee of the Royal Brisbane and Women s Hospital. Colonoscopy was performed for standard clinical indications, and the only patients not offered participation in the study were those with familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, and hyperplastic polyposis. One patient met the criteria of hyperplastic polyposis and had previously been diagnosed with this condition and was therefore excluded. Of the 189 remaining patients included in the study, 2 patients with polyps had synchronous CRC. The indication for colonoscopy and whether the patient had a history of CRC in a first-degree relative was recorded. All colonoscopies were performed with Olympus high-resolution instruments (CF140Z, PCF 160 AL; Olympus America Inc., Melville, NY). To maximize polyp detection, total dye spraying of the proximal colon to the splenic flexure and targeted dye spraying of the distal colon were performed with 0.4% indigo carmine solution. The mean withdrawal time was 13.7 minutes, and the cecum was reached in all cases. The site of each polyp was noted. For the purposes of analysis, polyps from the cecum, ascending colon, and transverse colon were regarded as proximal and those from the descending colon, sigmoid colon, and rectum were regarded as distal. The size of each polyp was assessed by the colonoscopist using the open biopsy forceps technique. Histology All retrieved polyps were formalin fixed and paraffin embedded. H&E-stained sections were examined by a single pathologist (J.R.J.) and serrated polyps classified using the system as described in detail by Torlakovic et al. 19 Briefly, hyperplastic polyps were grouped as goblet cell and microvesicular types. The former deviated minimally from normal with only mild serration and prominent goblet cells alternating with absorptive-type columnar cells. The microvesicular variant showed increased serration, and the columnar cells were distended with small, apical vesicles. The remaining serrated polyps included SSAs, mixed polyps (MPs), and TSAs. SSAs were characterized by exaggerated serration, architectural changes including crypt dilatation and horizontally extending crypt bases, increased mucin production (both intracellular and intraluminal), increased proliferation, and mild nuclear changes falling short of dysplasia. MPs comprised nondysplastic hyperplastic polyps or SSAs and a dysplastic component that resembled either TSAs or conventional adenomas. TSAs were composed of epithelium that was unequivocally dysplastic or adenomatous throughout as well as showing a serrated architecture. Conventional adenomas were classified as either tubular adenomas or tubulovillous adenomas, with serrated epithelium not a feature of these polyps. Archival DNA Extraction Genomic DNA was extracted from formalin-fixed, paraffin-embedded sections using a Chelex-100 extraction method. 20 Briefly, 200 L of 0.5% Tween 20 in TE, ph 8.0, was added to two 15- m sections, heated to 90 C for 10 minutes, and then cooled to 55 C. Proteinase K (Promega, Madison, WI) was added to a final concentration of 400 g/ml, and the sections were allowed to digest for 3 hours at 55 C with gentle agitation. After digestion, 200 L 5% Chelex-100 (Bio-Rad, Hercules, CA) in TE, ph 8.0, was added, heated to 99 C for 10 minutes, and centrifuged at 10,500g for 15 minutes. Samples were placed on ice to allow the wax layer to harden before removal and then heated to 45 C. Chloroform (200 L) was added and gently agitated, followed by centrifugation at 10,500g for 15 minutes. Finally, the aqueous top layer containing genomic DNA was then removed for analysis. BRAF and K-ras Mutation Analysis BRAF (V600E) and K-ras codon 12 and 13 mutations were detected by a modified multiplex genotyping assay on a MassARRAY platform 21 (SequenomInc., San Diego, CA) and by allele-specific real-time polymerase chain reaction (AS-PCR). The MassARRAY system uses matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry designed for the detection of primer-extended PCR products. AS-PCR was used to directly compare and assess the sensitivity and specificity of the MassARRAY genotyping assay. The detection of BRAF and K-ras mutations by MassARRAY was performed as follows. BRAF and K-ras mutant alleles were amplified in a volume of 2.5 L containing 10 ng of genomic DNA, 0.1 U of HotStar Taq

3 1402 SPRING ET AL GASTROENTEROLOGY Vol. 131, No. 5 Table 1. PCR and Extension Primers for BRAF and K-ras MassARRAY Forward primer Bases added Primer mass Reverse primer Bases added Primer mass PCR reaction BRAF 5=-ACGTTGGATGGACTGAATATAAACTTGTGG-3= 5=-ACGTTGGATGAGGGATTTAAGCTTACCTGC-3= K-ras 5=-ACGTTGGATGGATGAGAAACGAGAGAAAGC-3= 5=-ACGTTGGATGTAGCTGTATCGTCAAGGCAC- 3= Primer extension BRAF WT (GTG) 5=-TAGGTGATTTTGGTCTAGCTACA-3= GTGA =-GGGACCCACTCCATCGAGATTT-3= CA V600E (GAG) 5=-TAGGTGATTTTGGTCTAGCTACA-3= GA =-GGGACCCACTCCATCGAGATTT-3= CTCTGTA K-ras codon 12 WT (GGT) 5=-TTGTGGTAGTTGGAGCT-3= GGTGGCGTA =-CACTCTTGCCTACGCCA-3= CCA G12D (GAT) 5=-TTGTGGTAGTTGGAGCT-3= GA =-CACTCTTGCCTACGCCA-3= TCA G12V (GTT) 5=-TTGTGGTAGTTGGAGCT-3= GTTGGCGTA =-CACTCTTGCCTACGCCA-3= A G12A (GCT) 5=-cccTGGTAGTTGGAGCTGC-3= TGGCGTA =-agCTCTTGCCTACGCCA-3= CA 6022 G12C (TGT) 5=-TTGTGGTAGTTGGAGCT-3= TGTGGCGTA =-CACTCTTGCCTACGCCA-3= CA G12S (AGT) 5=-TTGTGGTAGTTGGAGCT-3= A 5=-CACTCTTGCCTACGCCA-3= CTA G12R (CGT) 5=-ccGTGGTAGTTGGAGCTC-3= GTGGCGTA =-ggaTCTTGCCTACGCCACG-3= A 6062 K-ras codon 13 WT (GGC) 5=-TTGTGGTAGTTGGAGCT-3= GGTGGCGTA =-CACTCTTGCCTACGCCA-3= CCA G13D (GAC) 5=-TTGTGGTAGTTGGAGCT-3= GGTGA =-ggCACTCTTGCCTAC-3= GTCA polymerase (Qiagen, Valencia, CA), 100 mol/l of deoxynucleoside triphosphates, and 100 nmol of PCR primers in 1 standard buffer provided with the enzyme supplemented to 3 mmol/l MgCl 2. Cycling conditions were 15 minutes at 95 C, followed by 45 cycles of 20 seconds at 94 C, 30 seconds at 56 C, and 60 seconds at 72 C. Shrimp alkaline phosphatase was added to the PCR reaction as per standard procedures. Post-PCR primer extension was performed in a final 5- L extension reaction containing 1200 nmol/l each of forward and reverse extension primers; 10 mol/l each of dideoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxyadenosine triphosphate, and deoxycytidine triphosphate; and 0.08 U/ L ThermoSequenase (Sequenom) in 0.22 PCR buffer. The reactions were heated in an ABI-9700 thermocycler for 30 seconds at 94 C and then cycled 99 times for 5 seconds at 94 C, 15 seconds at 52 C, and 5 seconds at 72 C. The post-pcr reaction products were desalted by diluting samples with 15 L of water and 3 L of resin to optimize mass spectrometric analysis and then spotted onto a 384-element SpectroCHIP bioarray (Sequenom) and processed and analyzed in a Compact Mass Spectrometer using MassARRAY Workstation (version 3.3) software (Sequenom). PCR primers, extension primers, and expected primer mass (daltons) are shown in Table 1. The detection of BRAF (V600E) mutations by allele-specific amplification and melt curve analysis was performed using a RotorGene 3000 instrument (Corbett Research, New South Wales, Australia) as previously described. 22 In the case of K-ras, only the major codon 12 and 13 mutations (G12D, G12V, and G13D) were identified by AS-PCR. To detect K-ras mutations, allele-specific primers were designed to selectively amplify wildtype (G35), codon 12 (A35 and T35), and codon 13 (A38) mutant alleles. The primer sequences are as follows: K35G-F, 5=-CTTGTGGTAGTTGGAGCGGG-3=; K35A-F, 5=-CGCGC- CGGGCGCGGGCTTGTGGTAGTTGGAGCTGA-3=; K35T-F, 5=-CGCGCCGGGCGCGGGCTTGTGGTAGTTGGAGCTGT- 3=; K38G-F, 5=-GTGGTAGTTGGAGCGGGTGG-3=; K38A-F, 5=-CTTGTGGTAGTTGGAGCTGTGTGGTAGTTGGAGA- GGGTGA-3=; and 98T-R, 5=-CAAGATTTACCTCTATTGTTG- GAT-3=. Each K-ras mutant allele was amplified separately using 20 ng genomic DNA in a 15- L reaction mix containing 1 Platinum SYBR Green qpcr SuperMix UDG (Invitrogen, Carlsbad, CA); primers Kras35G-F (200 nmol/l), KrasWT-R (600 nmol/l), and Kras35A-F (200 nmol/l) or Kras35T-F (200 nmol/l) for G12D and G12V mutations; and primers Kras38G-F (200 nmol/l), Kras38A-F (200 nmol/l), and KrasWT-R (600 nmol/l) for G13D mutations. Amplification conditions consisted of 50 C for 2 minutes, 95 C for 2 minutes, and 40 cycles of 95 C for 15 seconds and 60 C for 35 seconds. After amplification, samples were subjected to a temperature ramp from 60 C to 99 C for the melting curve analysis. Statistical Analysis Differences in frequency were assessed by either 2-sided Fisher exact test or Student t test. P values.05 were considered significant. Results Demographic Features of Patients With Polyps Colorectal polyps were detected in 136 of the 189 patients (72%) included in this study. Indications for colonoscopy were as expected in this clinical series and are shown in Table 2. Patients with polyps were more likely to be having their colonoscopy for surveillance after a previous polyp (P.05) and less likely to be having colonoscopy due to general abdominal symptoms. Patients with polyps had a mean age of 61 years compared with a mean age of 53 years for those without polyps (P.001, t test). Patients with polyps were equally likely to be male (51% [70/136]) or female (49% [66/136]). However, patients presenting without polyps were more likely to be female (66% [35/53]; P.01). Patients with polyps tended to have a first-degree relative with a history of CRC (28% [38/136]) compared with those without polyps (17% [9/53]), although this did not reach statistical significance. Whereas 21% of patients (28/ 136) with polyps had previously had one or more polyps removed, only 6% of patients (3/53) without a polyp had a previous history of polyps (P.05).

4 November 2006 SERRATED POLYPS WITH BRAF MUTATIONS 1403 Table 2. Indications for Colonoscopy Indication for colonoscopy No. of patients without polyps (n 53) No. of patients with polyps (n 136) Abdominal symptoms 23 (43) 36 (26).035 Anemia 15 (28) 21 (15) NS Family history of colorectal cancer 4 (8) 17 (13) NS Hematochezia 5 (9) 26 (19) NS Surveillance after colorectal cancer 3 (6) 8 (6) NS Surveillance after colorectal polyps 3 (6) 28 (21).015 NOTE. All values are expressed as n (%) unless otherwise indicated. P value Morphologic and Anatomic Distribution of Polyps The vast majority (99%) of polyps removed were retrieved for analysis, and a total of 414 polyps were classified as neoplastic (by J.R.J.) as shown in Table 3. The majority (60%) of polyps were tubular or tubulovillous adenomas, and interestingly the greater proportion of these occurred in the proximal colon (73%). The second most common polyp type was hyperplastic, comprising 29% of all polyps and mostly small, distal lesions. There was no difference in anatomic distribution between the goblet cell rich and microvesicular subtypes of hyperplastic polyps. The newly recognized SSA made up 9% of all polyps. These were usually larger lesions in the proximal colon, with 64% (23/36) 5 mm and 75% (27/36) located in the proximal colon. Characteristics of Patients Bearing SSAs To identify risk factors associated with SSAs, we examined the characteristics of patients in whom these polyps occurred. A higher proportion of patients bearing SSAs were female (65% [17/26] female vs 35% [9/26] male) compared with all remaining patients (45% [50/110] female vs 55% [60/110] male; P.05). There was no significant variation in the age of these patients when compared with the remainder of the study population. They were more likely to have a first-degree relative with CRC, with 42% of patients (11/26) having such a history compared with all remaining patients (25% [27/110]), although this did not attain statistical significance. Whereas 19% (5/26) of these patients had a history of previous polyps or cancer, this was not significantly more common than the balance of patients with polyps (9% [10/110]). However, patients with at least one SSA tended to have a higher total number of polyps than patients with other polyp subtypes only. The average number of neoplastic polyps in patients with at least one SSA was 5.0 (130 polyps in 26 patients) compared with 2.5 (279 polyps in 110 patients) in those without SSAs (P.0001). Detection of BRAF and K-ras Mutations by Sequenom MassARRAY For the analysis of serrated polyps, we developed a high-throughput assay using the Sequenom MassARRAY platform to simultaneously detect both BRAF (V600E) mutations and all codon 12 and 13 K-ras mutations. We screened 414 polyp samples for BRAF and K-ras mutations and compared the results with AS-PCR and melt curve analysis of the same samples. All variants detected by MassARRAY were also detected by AS-PCR. Using MassARRAY, we detected 86 (21%) BRAF and 63 (15.2%) K-ras mutations, whereas 89 (21.5%) BRAF mutations were detected by AS-PCR. For K-ras mutations, only the 3 main codon 12 and 13 mutations (G12D, G12V, and G13D) were assayed by AS-PCR. Fifty-three K-ras mutations were detected by AS-PCR, as compared with 50 codon 12 and 13 (G12D, G12V, and G13D) mutations by MassARRAY. The high degree of concordance of BRAF and K-ras mutations (96.6% and 93.4%, respectively) detected by both techniques shows the specificity of the MassARRAY assay. Further, a greater number of mutations were identified by AS-PCR, indicating that this technique is slightly more sensitive than MassARRAY. Of all colorectal polyps screened for mutations, none had both BRAF and K-ras mutations. BRAF/K-ras Mutation and Polyp Subtype The frequency of mutations within the various polyp types is shown in Table 4. Interestingly, only one BRAF muta- Table 3. Polyp Characteristics Location Size (mm) Polyp type Number (n 414) Proximal Distal Serrated polyps Goblet cell rich 66 (16) 21 (32) 45 (68) 61 (92) 3 (5) 2 (3) Microvesicular 54 (13) 14 (26) 40 (74) 42 (78) 12 (22) 0 (0) SSA 36 (9) 27 (75) 9 (25) 13 (36) 17 (47) 6 (17) TSA 3 (0.7) 2 (66) 1 (33) 1 (33) 0 (0) 2 (66) MP 7 (1.7) 4 (57) 3 (43) 3 (43) 2 (29) 2 (29) Conventional adenomas Tubular adenoma 237 (57) 176 (74) 61 (26) 185 (78) 43 (18) 9 (4) Tubulovillous adenoma 11 (2.7) 6 (55) 5 (45) 0 (0) 5 (45) 6 (55) NOTE. All values are expressed as n (%).

5 1404 SPRING ET AL GASTROENTEROLOGY Vol. 131, No. 5 Table 4. BRAF and K-ras Mutation According to Polyp Type Polyp type BRAF mutation K-ras mutation BRAF/K-ras mutations Serrated polyps Goblet cell rich (n 66) 13 (20) 33 (50) 46 (70) Microvesicular (n 54) 38 (70) 6 (11) 45 (83) SSA (n 36) 28 (78) 3 (8) 31 (86) TSA (n 3) 2 (66) 0 (0) 2 (66) MP (n 7) 4 (57) 3 (43) 7 (100) Conventional adenomas Tubular adenoma (n 237) 1 (0.4) 12 (5) 13 (5.5) Tubulovillous adenoma (n 11) 0 (0) 6 (55) 6 (55) NOTE. All values are expressed as n (%). tion was detected in 248 conventional adenomas. This mutation occurred in a tubular adenoma from a 71-year-old female patient undergoing surveillance after previous polyps, who had on this occasion 9 tubular adenomas, 2 SSAs, and 2 hyperplastic polyps. Among adenomas, K-ras mutations were associated with tubulovillous histology (P.001). A much higher proportion of serrated polyps (hyperplastic polyps, SSAs, TSAs, and MPs) had demonstrable mutations affecting the mitogen-activated protein kinase pathway than did adenomas (79% [131/ 166] vs 8% [19/248]; P.0001). K-ras mutations were more common than BRAF mutations in goblet cell rich hyperplastic polyps (P.001). By contrast, BRAF mutations predominated in both microvesicular hyperplastic polyps (P.001) and SSAs (P.0001). BRAF/K-ras Mutation Relative to Anatomic Location and Polyp Size The relationship between BRAF/K-ras mutations and polyp location in the colorectum is shown in Table 5 and Figure 1. Among serrated polyps, BRAF mutations were more common in proximal polyps, with BRAF mutations detected in 62% of proximal polyps (42/68) compared with 44% of distal polyps (43/98) (P.05). Conversely, K-ras mutations were present in 15% of proximal polyps (10/68) and 34% of distal polyps (35/98) (P.01). BRAF mutation was correlated with polyp size in goblet cell rich hyperplastic polyps, where the mean size of the 13 polyps bearing a mutation was 4.9 mm and the mean size of the 53 wild-type polyps was 2.5 mm (P.001). In microvesicular hyperplastic polyps, the mean size of the 39 polyps bearing a mutation was 3.5 mm Figure 1. Percent of polyps with (A) BRAF and (B) K-ras mutation stratified for polyp size and location in the colorectum. versus 15 wild-type polyps with a mean size of 4.3 mm. In SSAs, 28 polyps with BRAF mutation had a mean size of 8.7 mm compared with 8 polyps with wild-type BRAF with a mean size of 7.8 mm. TSAs and MPs bearing BRAF mutations were also larger than those without BRAF mutations. Goblet cell rich hyperplastic polyps tended to be smaller if bearing ak-ras mutation (2.45 mm vs 3.57 mm; P.053), whereas K-ras mutation correlated with an increased size in tubular adenomas (8.33 mm vs 4.45 mm; P.001). Table 5. Polyp Location and BRAF and K-ras Mutation BRAF mutation K-ras mutation Polyp type Proximal Distal Proximal Distal Serrated polyps Goblet cell rich 7/21 (33) 6/45 (13) 7/21 (33) 26/45 (58) Microvesicular 7/14 (50) 31/40 (77) 2/14(14) 4/40 (10) SSA 23/27 (85) 5/9 (55) 0/27 (0) 3/9 (33) TSA 2/2 (100) 0/1 (0) 0/2 (0) 0/1 (0) MP 3/4 (75) 1/3 (33) 1/4 (25) 2/3 (66) Conventional adenomas Tubular adenoma 1/181 (0.6) 0/56 (0) 9/181 (5) 3/56 (5) Tubulovillous adenoma 0/6 (0) 0/5 (0) 4/6 (66) 2/5 (40) NOTE. All values are expressed as n (%).

6 November 2006 SERRATED POLYPS WITH BRAF MUTATIONS 1405 Characteristics of Patients Bearing BRAF/Kras Mutant Polyps To explore whether the occurrence of BRAF mutations was associated with particular patient characteristics, patients were divided into those found to have at least one polyp bearing the mutation of interest and those without any polyps mutant for that mutation. More patients with at least one polyp bearing a BRAF mutation had a family history of polyps (33% [17/52]) versus those with no BRAF mutant polyps (25% [21/ 84]), although this did not reach statistical significance. There was a trend toward an increased prevalence of family history in patients with polyps bearing K-ras mutations (35% [17/48]) versus those without (24% [21/88]). There was no correlation between the presence of BRAF or K-ras mutant polyps and a personal history of colorectal polyps. Presence of at least one polyp with a BRAF mutation was also associated with polyp multiplicity. In the 52 patients with at least one polyp with a mutant BRAF allele, the average number of polyps per patient was 3.75, compared with 2.61 polyps per patient in the remaining patients (P.05). K-ras mutation was also associated with a slightly increased average number of polyps per patient (3.58 vs 2.75 polyps per patient), although this was not statistically significant. Discussion The present study shows that SSAs constitute approximately 9% of all polyps and 22% of serrated polyps in a large series of patients undergoing colonoscopy for standard clinical indications. In the majority of earlier studies, SSAs had not been recognized as a distinct subgroup. However, in a retrospective series of sporadic serrated polyps in which the characteristics of SSAs were being defined, SSAs and TSAs together constituted 18% of all serrated polyps, but this study provided no information as to how representative this series was of polyps present at colonoscopy. 19 Before the present study, the clearest information regarding the proportion of polyps that are SSAs came from a study of all polyps obtained by colonoscopic polypectomy and submitted for pathologic examination to a single pathology department over a 2-year period. 10 Of these polyps, 2.2% were SSAs, 1.9% were TSAs, and 0.8% were MPs. Thus, the proportion of TSAs and MPs was similar to that in the present study, but SSAs were seen much less frequently. This difference is very unlikely to be due to differences in classification because the same pathologist (J.R.J.) classified polyps in both series. It is more likely that the Higuchi et al series did not fully represent the polyps present in patients because the series dates from the early 1990s, before the availability of the magnifying and dye-spray technique. This is supported by the fact that the mean number of polyps retrieved per patient was only 1.3, compared with the present series where the mean was 3.1. SSAs are probably less likely to be detected and removed by suboptimal colonoscopic techniques because they are sessile lesions in the proximal colon. Although it is possible that the populations of patients being screened were different, this seems less likely because both were from Western countries and only a decade apart in time. As in other series, the present study shows that SSAs are likely to be large and located in the proximal colon. 4,9,10,19 However, this study shows for the first time that there are distinct differences in demographic characteristics of patients with sporadic SSAs. They are more likely to have a family history of CRC in a first-degree relative, and there is a trend toward a female gender bias. CIMP-high cancers are associated with a positive family history 23 and are more common in women when the cancers are MSI-H 24 ; thus, these data supports SSAs being the precursors of these types of cancer. Furthermore, patients with SSAs are more likely to have multiple polyps, although not all of these additional polyps were SSAs. The percentage of polyps with a BRAF mutation was much higher in these patients. It has previously been shown that cancers bearing a BRAF mutation are associated with a positive family history. 24 These patients may have a genetic 22 and/or environmental predisposition to colorectal carcinogenesis with a tendency to DNA methylation and BRAF mutation. The association of SSAs with the occurrence of multiple polyps suggests that these patients may be at high risk for developing further polyps. Additional studies are thereby needed to determine if these patients would benefit from more frequent colonoscopic surveillance. Furthermore, lack of recognition of the importance of endoscopic therapy for SSAs may be contributing to the static incidence of proximal CRC despite the decreasing incidence of distal cancer. 25 Related to this is the interesting observation that adenomas were more common in the proximal colon than in the distal colon and rectum in this series of patients undergoing stateof-the-art colonoscopy. This contrasts with previous colonoscopic studies that reported the converse, 26,27 as have autopsy studies This could possibly reflect a true change in the epidemiology of adenomas, but it may also relate to more accurate detection of small lesions in the proximal colon using magnifying chromoendoscopy. Another study has shown that dye-spraying techniques detected more small and sessile lesions in the proximal colon compared with non dye-spray controls. 31 The analysis of BRAF and K-ras mutations revealed a high frequency of oncogenic BRAF mutation in microvesicular hyperplastic polyps as well as SSAs, TSAs, and MPs. The frequency was lower in goblet cell hyperplastic polyps, which instead seem to activate the mitogen-activated protein kinase pathway by K-ras mutation. As expected, no polyp had both BRAF and K-ras mutations. It is clear from our data that the use of highthroughput MassARRAY is a useful technique for screening a large series. However, this technique will miss a small fraction of mutations detected by AS-PCR, when mutations are present in less than 10% of the cell population. Another report on the use of AS-PCR for the detection of BRAF mutations determined the sensitivity of the assay to be 2% of cells or 1% of mutated DNA for monoallelic BRAF mutation. 32 Our results agree closely with 2 previous studies of selected retrospectively identified polyps 12,16 and thus show that this pattern of mutation is truly representative of sporadic polyps. In the study of O Brien et al, high levels of CIMP were present in SSAs but not in goblet cell rich hyperplastic polyps. 12 Microvesicular polyps had intermediate levels of methylation, but there is no information if this correlated with size and BRAF status. Further investigation of this intermediate group may give insight into whether BRAF mutation and heavy methylation are simultaneous processes or if one precedes the other. If microvesicular polyps do evolve directly into SSAs, TSAs, or MPs, they must do so quite rapidly because microvesicular polyps are uncommon in the proximal colon, where these polyps mostly occur, and the small number observed are not more likely to show BRAF mutations.

7 1406 SPRING ET AL GASTROENTEROLOGY Vol. 131, No. 5 In this study, BRAF mutations were almost never found in tubular or tubulovillous adenomas. Indeed, even K-ras mutations were uncommon except in the small number of adenomas, which have evolved a villous component. This finding is unlikely to be due to technical error because we were able to identify a high frequency of K-ras mutations even in small hyperplastic polyps. The BRAF findings are compatible with previous studies, 18,33 although these have not been as clear-cut because the selected retrospective series were likely contaminated by patients with hyperplastic polyposis. There is evidence that in hyperplastic polyposis 34 and in certain families with an inherited form of CRC, 22 polyps that appear to be conventional tubular adenomas can have BRAF mutations. The single tubular adenoma in which we identified a BRAF mutation was from a patient with multiple polyps who may fit the syndrome described by Young et al 22 despite the lack of a family history of CRC. The present study reinforces how unusual these polyps are, and indeed their presence may constitute a useful marker for these syndromes. It is unclear why activation of the mitogen-activated protein kinase pathway by BRAF mutation or even by K-ras mutation should be so rare in small adenomas while it is common in all hyperplastic polyps. In adenomas, the predominant abnormality is activation of the WNT pathway by mutation or deletion of APC, but this pathway is seldom activated in serrated polyps, where the main known genetic change is silencing of genes by promoter methylation associated with CIMP. 11,12,16 Our observations suggest that BRAF mutations rarely occur in colorectal cells in which the only other major abnormality is an activated WNT pathway but rather are strongly associated with CIMP positivity. To our knowledge, there is no information in the literature examining the effect of simultaneous BRAF mutation and WNT pathway activation. This combination of genetic alterations may not be advantageous at least until further alterations accumulate. An alternative explanation is that inactivation of important gene methylation targets particularly synergize with activation of the mitogen-activated protein kinase pathway. In summary, the present study has shown that the prevalence of SSAs is 9% in a prospective series of patients undergoing colonoscopy for standard clinical indications. These patients are more likely to have a family history of CRC and to have multiple polyps. BRAF mutations, which are strongly associated with CIMP-positive cancers, occurred frequently in SSAs, TSAs, MPs, and microvesicular hyperplastic polyps but not in adenomas, thus supporting the importance of serrated polyps as precursors of this subset of CRC. These findings have important implications for endoscopic CRC surveillance and reinforce the preliminary recommendations of Snover et al 9 that SSAs be completely removed endoscopically and that subsequent surveillance should follow the current guidelines for traditional adenomas. It is known that the presence of multiple polyps is a risk factor for missed polyps and further polyps, and this should be taken into account in planning surveillance. 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