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BRAF Mutations in Aberrant Crypt Foci and Hyperplastic Polyposis

      Patients with hyperplastic polyposis have multiple hyperplastic polyps (HPs) and increased risk of colorectal carcinomas. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesions in colorectal carcinogenesis. We evaluated BRAF mutations by DNA sequencing in 53 ACF from patients with sporadic colorectal carcinomas and familial adenomatous polyposis, in 18 sporadic HPs from patients with resected colorectal cancer, and in 70 HPs, 4 serrated adenomas, 3 admixed hyperplastic-adenomatous polyps, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status was compared with clinicopathological features and other genetic alterations by marginal logistic regression. BRAF mutation was present in only 2% of ACF and 6% of sporadic HPs. In contrast, BRAF mutation was present in 43% of HPs (P = 0.01 versus sporadic HPs), 75% of serrated adenomas, 33% of admixed hyperplastic-adenomatous polyps, 30% of tubular adenomas, and 33% of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. BRAF mutation status in patients with multiple/large HPs and/or hyperplastic polyposis correlated with HPs from the same patient (odds ratio, 5.8; P = 0.0002) but associated with younger age (odds ratio, 0.83; P = 0.006 compared to older age), with a large HP (odds ratio, 22.5; P = 0.01 compared with patients with multiple HPs), with location of HPs in the right colon (odds ratio, 3.0; P = 0.03), and with methylation of the p16 gene and the MINT31 locus [odds ratio, 12.2 (P = 0.0001) and 4.4 (P = 0.02), respectively]. Our study shows that BRAF mutation status is heterogeneous among patients with multiple/large HPs and/or hyperplastic polyposis, suggesting differences in pathogenesis of HPs that indicate subsets within this phenotype.
      Colorectal cancer is the second most common cause of cancer deaths in the United States. Most colorectal cancers develop from adenomatous polyps, and morphological and genetic progression in an adenoma-adenocarcinoma sequence and in hereditary colorectal cancer syndromes are well described.
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      and by the presence in some ACF of genetic and epigenetic alterations that are present in colorectal carcinomas, such as alterations in the adenomatous polyposis coli (APC) tumor suppressor gene, KRAS proto-oncogene mutations, microsatellite instability (MSI), and methylation of p16 gene and other CpG islands.
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      tubular adenomas, and carcinomas of patients with hyperplastic polyposis, these alterations include KRAS mutations, chromosome 1p loss, MSI, CpG island methylation of p16 gene and other loci, and CpG island methylator phenotype (CIMP) with concordant methylation of CpG islands.
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      The RAS-RAF-MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase)-ERK (extracellular signal-regulated kinase)-MAP (mitogen-activated protein) kinase pathway mediates cellular responses to growth signals. BRAF mutations have been found in a variety of human cancers including colorectal carcinomas and melanomas.
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      BRAF mutations have also been reported in sporadic HPs and in SAs, including a few from patients with hyperplastic polyposis.
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      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
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      BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum.
      In this study, we evaluated BRAF mutations in ACF from patients with FAP and sporadic colorectal cancers, in sporadic HPs, and in HPs, SAs, AHAPs, tubular adenomas, and colorectal carcinomas from the patients with multiple/large HPs and/or hyperplastic polyposis. We compared the BRAF mutation status with polyp and patient characteristics, including correlation among multiple HPs from the same patient.

      Materials and Methods

      Characteristics of Patients and Specimens

      All patients had given informed consent for the collection of specimens according to institutional guidelines. ACF were isolated from the grossly normal mucosa in 10 colectomy specimens from patients with sporadic colorectal cancers and from the nonpolypoid mucosa in two colectomy specimens from FAP patients with numerous polyps but no cancer. These ACF have been characterized previously.
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      Thirty ACF were from patients with sporadic colorectal cancers and 23 ACF from FAP patients. The ACF were classified as dysplastic, heteroplastic, or mixed (features of both dysplastic and heteroplastic ACF).
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      Eighteen sporadic HPs from 15 patients undergoing resection of colorectal cancer at The University of Texas MD Anderson Cancer Center, Houston, TX, and the patients and specimens from patients with multiple/large HPs and/or hyperplastic polyposis have been reported previously (Figure 1).
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      The patients were classified into three groups based on the number and size of HPs: large HPs (patients with HP greater than 1 cm), hyperplastic polyposis (patients with more than 20 HPs), and multiple HPs (patients with 5 to 10 HPs), as described previously.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      Predominance of HPs in the right colon and predominance of HPs in the left colorectum were defined by the location of the majority of HPs in the right colon or in the left colon and rectum, respectively.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      We evaluated 70 HPs, 4 SAs, 3 AHAPs, 10 tubular adenomas, and 6 carcinomas from 17 patients with multiple/large HPs and/or hyperplastic polyposis.
      Figure thumbnail gr1
      Figure 1Clinicopathological features of patients, BRAF mutation status, other genetic alterations, and CIMP status of HPs, SAs, AHAPs, tubular adenomas, and carcinomas from patients with large/multiple HPs, and/or hyperplastic polyposis.

      Sequencing of BRAF Gene

      Exons 11 and 15 of the BRAF gene were amplified and sequenced as previously described.
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      Exons 11 and 15 were amplified by genomic polymerase chain reaction using intronic primers and a commercial DNA sequencing kit according to the manufacturer's instructions (BigDye Terminator version 1.1 cycle sequencing kit; Applied Biosystems, Foster City, CA). The polymerase chain reaction products were analyzed with an Applied Biosystems 3730 automated sequencer using forward and reverse primers. All mutations were confirmed by an independent polymerase chain reaction amplification and sequencing. All BRAF mutations identified were a missense mutation at codon 599, exon 15 replacing GTG (valine) to GAG (glutamic acid). No mutations were identified in exon 11 or other codons of exon 15. Germline mutations were excluded by sequencing nonlesional DNA from these patients.

      KRAS Mutations, Loss of Heterozygosity of Chromosome 1p, MSI-High, CIMP Status

      KRAS mutation status of ACF, and KRAS mutations, loss of heterozygosity of chromosome 1p, MSI and CIMP status of sporadic HPs, and of HPs, SAs, AHAPs, tubular adenomas, and carcinomas from patients with multiple/large HPS and/or hyperplastic polyposis have been reported previously.
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      MSI-high was defined by presence of allelic shift in comparison with control DNA in at least 30% of evaluated markers. Methylation was assessed at the p16 gene and loci methylated in tumor (MINT): MINT1, MINT2, and MINT31. MINT1 is an island associated with a cDNA transcript of unknown function. MINT2 corresponds to a CpG island that is in the 5′ region of a cDNA with an open reading frame that has no protein homology. MINT31 is 2 kb upstream of the CACNAIG, a T-type calcium channel gene (J.P. Issa, unpublished data). HPs, SAs, adenomas, and carcinomas were classified as CIMP-high if two or more (50%) of the p16 gene or MINT loci were methylated, CIMP-low if one (25%) marker was methylated, and CIMP-negative if no marker was methylated.

      Statistical Analysis

      Patients with more than one HP were represented multiple times in this data set. To model correctly the correlation among polyps coming from the same patient as well as simultaneously partition out the effects of the various factors considered, marginal logistic regression models for correlated binary data
      • Carey V
      • Zeger SL
      • Diggle P
      Modelling multivariate binary data with alternating logistic regression.
      were used to assess associations between BRAF mutations and the various polyp and patient characteristics. These associations were tested for association with BRAF mutations and were represented as odds ratios, in which an odds ratio of greater than one suggests positive correlation of BRAF mutations with patients or polyp characteristics, respectively. We used three models. The first model with no factors was used to estimate the correlation among the BRAF mutation status in polyps from the same patient, without adjusting for other covariates. A second model included various patient- and polyp-level factors, including the methylation status of the p16 gene and MINT1, MINT2, and MINT31 loci as potential predictors of BRAF mutation status. A third model was used with CIMP status (CIMP-high versus CIMP-low and CIMP-negative) substituted in place of the methylation status of the p16 gene and MINT1, MINT2, MINT31 loci, individually. The statistical analysis was performed using PROC GENMOD in SAS (SAS Institute, Cary, NC), using an assumption that all polyps within a patient were equally correlated. In all models, factors with P values less than 0.05 were considered statistically significant.

      Results

      ACF

      Twenty-three ACF were from 2 FAP patients and 30 ACF from 10 patients with sporadic colorectal carcinomas. As previously reported,
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      91% (21 of 23) of ACF from FAP patients were dysplastic and 9% (2 of 23) were heteroplastic. In contrast, 87% (26 of 30) of ACF from patients with sporadic colorectal cancer were heteroplastic, only 10% (3 of 10) were dysplastic, and 3% (1 of 30) were mixed. BRAF mutation was present in 0% (0 of 23) of ACF from FAP patients and only 3% (1 of 30) of ACF from patients with sporadic colorectal cancers (Figure 2A). In contrast, KRAS mutation was present in 4% (1 of 23) of ACF from FAP patients and 40% (12 of 30) of ACF from patients with sporadic colorectal cancers. BRAF mutation was present in a heteroplastic ACF and was the missense point mutation at codon 599 as described in the Materials and Methods.
      Figure thumbnail gr2
      Figure 2A: Nucleotide sequencing of exon 15 of BRAF gene in ACF. B: Nucleotide sequencing of exon 15 of BRAF gene in HPs. The T to A missense point mutation at codon 599 with replacement of valine with glutamic acid is indicated by arrows. The wild-type and mutated nucleotide and amino acid sequences are shown on top.

      Sporadic HPs

      The sporadic HPs were from 12 men and 3 women, with a mean age of 64 ± 11 years (range, 48 to 80 years). The mean size of the polyp in this group was 0.3 cm (range, 0.1 to 0.7 cm). There were 3 HPs from the right colon and 15 from the left colorectum. BRAF mutation was present in 6% (1 of 18) of sporadic HPs. BRAF mutation was again the missense point mutation at codon 599 described above. No KRAS mutation or methylation of the p16 gene or MINT loci were present in sporadic HPs.

      Hyperplastic Polyposis

      There were 11 men and 6 women with multiple/large HPs and/or hyperplastic polyposis. The mean age was 64 ± 12 years (range, 46 to 84 years). The demographic data and characteristics of each individual patient and the number of HPs, adenomas, and carcinomas in each individual are summarized in Figure 1. BRAF mutations in patients are summarized in Figure 1, and representative examples of sequencing are shown in Figure 2B. BRAF mutations were present in 43% (30 of 70) of HPs (P = 0.01 versus sporadic HPs), 75% (3 of 4) of SAs, 33% (1 of 3) of AHAPs, 30% (3 of 10) of tubular adenomas, and 33% (2 of 6) of carcinomas from patients with multiple/large HPs and/or hyperplastic polyposis. All BRAF mutations in HPs and other lesions from patients with multiple/large HPs and/or hyperplastic polyposis were the missense point mutation at codon 599 as described in the Materials and Methods.
      We first examined if the BRAF mutation status was correlated within HPs from the same patient. We used a model with no factors except the correlation. We found that the correlation was statistically significant (odds ratio, 5.8; P = 0.0002; Figure 1 and Table 1). The odds ratio of 5.8 means that given the presence of a BRAF mutation in a polyp from a given patient, the probability of another polyp in the same patient having the BRAF mutation is 5.8 times greater than if the first polyp did not have the BRAF mutation.
      Table 1Patient and Polyp Characteristics in Relation to BRAF Mutation Status of Hyperplastic Polyps in Patients with Multiple/Large HPs and/or Hyperplastic Polyposis, Odds Ratio and 95% Confidence Intervals from GEE Marginal Regression Models
      BRAF mutations
      Model and factorsPresent % (fraction)Absent % (fraction)Odds ratio (95% confidence intervals)χ2P
      One
          Correlation5.8 (1.6, 2.3)14.50.0002
      Two
          Patient characteristics
              Correlation3.6 (1.3, 1.7)10.10.02
              Age (continuous, years)0.83 (0.7, 1.1)0.60.006
              Patients with large hyperplastic polyps50 (4/8)50 (4/8)22.5 (2.0, 3.5)259.90.01
              Patients with hyperplastic polyposis50 (20/40)50 (20/40)1.4 (0.2, 2.9)10.90.8
              Patients with multiple hyperplastic polyps27 (6/22)73 (18/22)1.0
              Patients with serrated adenoma or AHAP
      Admixed hyperplastic-adenomatous polyp, AHAP.
      56 (15/27)44 (12/27)4.1 (0.5, 2.9)34.60.2
              Patients without serrated adenoma or AHAP35 (15/43)65 (28/43)1.0
          Hyperplastic polyp characteristics
              Size (continuous, mean ± SD, mm)4.4 ± 4.23.2 ± 1.51.2 (0.7, 1.3)1.90.5
              Site
                  Right67 (20/30)33 (10/30)3.0 (1.1, 1.7)8.00.03
              Left25 (10/40)75 (30/40)1.00
          p16 methylation
                  Present80 (12/15)20 (3/15)12.2 (1.4, 6.1)24.30.0001
                  Absent32 (13/41)68 (28/41)1.00
                  Unassessed36 (5/14)64 (9/14)
              MINT1 methylation
                  Present75 (12/16)25 (4/16)0.8 (0.1, 3.2)7.40.8
                  Absent30 (15/50)70 (35/50)1.00
                  Unassessed75 (3/4)25 (1/4)
              MINT2 methylation
                  Present59 (22/37)41 (15/37)0.9 (0.1, 2.9)7.50.9
                  Absent18 (5/28)82 (23/28)1.00
                  Unassessed60 (3/5)40 (2/5)
              MIINT31 methylation
                  Present58 (22/38)42 (16/38)4.4 (1.2, 1.9)15.80.02
                  Absent23 (7/31)77 (24/31)1.00
                  Unassessed100 (1/1)0 (0/1)
      Three
      In this model CIMP status was substituted for methylation statuses of p16 gene, and MINT1, MINT2, MINT31.
              CIMP status
                  High64 (21/33)36 (12/33)1.7 (0.2, 2.8)12.70.6
                  Low or absent23 (8/35)77 (27/35)1.00
                  Unassessed50 (1/2)50 (1/2)
      * In this model CIMP status was substituted for methylation statuses of p16 gene, and MINT1, MINT2, MINT31.
      Admixed hyperplastic-adenomatous polyp, AHAP.
      We next examined associations of BRAF mutation status to various patient and polyp characteristics (summarized in Figure 1 and Table 1). We found that patient age, type of polyposis, polyp site, and methylation for p16 gene and MINT31 were significant predictors for BRAF mutation status. Specifically, we found that HPs from older patients were less likely to have the mutation (odds ratio, 0.83; P = 0.006). This odds ratio means that there is a 17% decrease in probability of BRAF mutation for every year older a given subject is. BRAF mutations were present in 50% (4 of 8) of HPs from patients with a large HP compared to 27% (6 of 22) of HPs from patients with multiple HPs (odds ratio, 22.5, compared with patients with multiple HPs; P = 0.01), in 67% (20 of 30) of HPs from the right colon compared to 25% (10 of 40) of HPs from the left colon and rectum (odds ratio, 3.0; P = 0.03), in 80% (12 of 15) of HPs with p16 gene methylation compared to 32% (13 of 41) without p16 methylation (odds ratio, 12.2; P = 0.0001), and in 58% (22 of 38) of HPs with methylation at MINT31 locus compared to 23% (7 of 31) without methylation at MINT31 (odds ratio, 4.4; P = 0.02). The correlation was statistically significant in this model (odds ratio, 3.6; P = 0.02), indicating that there was still evidence of correlation even after adjusting for the factors present in this model. The other factors were not independently associated with BRAF mutation status.
      In the third model, CIMP status was substituted for the methylation statuses of the p16 gene, and MINT1, MINT2, and MINT31 in model two (Table 1). HPs with CIMP-high were slightly more likely to have BRAF mutations, but this was not significant after adjusting for other factors. Other genetic alterations in HPs and other lesions have been previously reported.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      KRAS mutations were present in 9% (6 of 70) of HPs and 67% (2 of 3) of AHAPs but in none of 4 SAs, 10 tubular adenomas, or 6 carcinomas (Figure 1). Chromosome 1p loss was present in 4% (3 of 70) of HPs, 33% (1 of 3) of AHAPs, 10% (1 of 10) of tubular adenoma, and 17% (1 of 6) of carcinomas, but in none of four SAs. MSI was present in 33% (1 of 30) of AHAPs, 10% (1 of 10) of tubular adenomas, and 17% (1 of 6) of carcinomas, but in none of four SAs. Except for one carcinoma with BRAF mutation and MSI, all other genetic alterations were inversely related to BRAF mutations in all other lesions.

      Discussion

      We studied BRAF mutations in ACF from patients with FAP and sporadic colorectal cancers, sporadic HPs, and HPs and other colorectal lesions from patients with multiple/large HPs and/or hyperplastic polyposis. We found BRAF mutation status was correlated in HPs from patients with multiple/large HPs, or hyperplastic polyposis. BRAF mutations have been reported in sporadic colorectal carcinomas,
      • Davies H
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Clegg S
      • Teague J
      • Woffendin H
      • Garnett MJ
      • Bottomley W
      • Davis N
      • Dicks E
      • Ewing R
      • Floyd Y
      • Gray K
      • Hall S
      • Hawes R
      • Hughes J
      • Kosmidou V
      • Menzies A
      • Mould C
      • Parkar A
      • Stevens C
      • Watt S
      • Hooper S
      • Wilson R
      • Jayatilake H
      • Gusterson BA
      • Cooper C
      • Shipley J
      • Hargrave D
      • Pritchard-Jones K
      • Maitland N
      • Chenevix-Trench G
      • Riggins GJ
      • Bigner DD
      • Palmieri G
      • Cossu A
      • Flanagan A
      • Nicholson A
      • Ho JWC
      • Leung SY
      • Yuen ST
      • Yuen ST
      • Weber BL
      • Seigler HF
      • Darrow TL
      • Paterson H
      • Marais R
      • Marshall CJ
      • Wooster R
      • Stratton MR
      • Futreal PA
      Mutations of the BRAF gene in human cancer.
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      • Domingo E
      • Espin E
      • Armengol M
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Seruca R
      • Hamelin R
      • Yamamoto H
      • Schwartz Jr, S
      Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.
      • Fransen K
      • Klintenas M
      • Osterstrom A
      • Dimberg J
      • Monstein HJ
      • Soderkvist P
      Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Domingo E
      • Espin E
      • Armengol M
      • Yamamoto H
      • Hamelin R
      • Seruca R
      • Schwartz Jr, S
      BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.
      • Wang L
      • Cunningham JM
      • Winters JL
      • Guenther JC
      • French AJ
      • Boardman LA
      • Burgart LJ
      • McDonnell SK
      • Schaid DJ
      • Thibodeau SN
      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      • Wan PT
      • Garnett MJ
      • Roe SM
      • Lee S
      • Niculescu-Duvaz D
      • Good VM
      • Jones CM
      • Marshall CJ
      • Springer CJ
      • Barford D
      • Marais R
      Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      adenomas,
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      and HPs.
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      • Chan TL
      • Zhao W
      • Leung SY
      • Yuen ST
      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      • Yang S
      • Farraye FA
      • Mack C
      • Posnik O
      • O'Brien MJ
      BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum.
      However, BRAF mutations are uncommon in sporadic microsatellite-stable colorectal carcinomas but are more frequent in MSI-high carcinomas
      • Domingo E
      • Espin E
      • Armengol M
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Seruca R
      • Hamelin R
      • Yamamoto H
      • Schwartz Jr, S
      Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.
      • Fransen K
      • Klintenas M
      • Osterstrom A
      • Dimberg J
      • Monstein HJ
      • Soderkvist P
      Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Domingo E
      • Espin E
      • Armengol M
      • Yamamoto H
      • Hamelin R
      • Seruca R
      • Schwartz Jr, S
      BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.
      • Wang L
      • Cunningham JM
      • Winters JL
      • Guenther JC
      • French AJ
      • Boardman LA
      • Burgart LJ
      • McDonnell SK
      • Schaid DJ
      • Thibodeau SN
      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      because of methylation of hMLH1 gene.
      • Domingo E
      • Espin E
      • Armengol M
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Seruca R
      • Hamelin R
      • Yamamoto H
      • Schwartz Jr, S
      Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.
      • Wang L
      • Cunningham JM
      • Winters JL
      • Guenther JC
      • French AJ
      • Boardman LA
      • Burgart LJ
      • McDonnell SK
      • Schaid DJ
      • Thibodeau SN
      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      All BRAF mutations in our study were T to A missense point mutation at codon 599 with replacement of valine with glutamic acid. No mutation of exon 11 or other codons of exon 15 were found. In contrast, previous studies have reported BRAF mutations involving other codons, although infrequently, in colorectal cancers and other colorectal lesions including HPs.
      • Davies H
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Clegg S
      • Teague J
      • Woffendin H
      • Garnett MJ
      • Bottomley W
      • Davis N
      • Dicks E
      • Ewing R
      • Floyd Y
      • Gray K
      • Hall S
      • Hawes R
      • Hughes J
      • Kosmidou V
      • Menzies A
      • Mould C
      • Parkar A
      • Stevens C
      • Watt S
      • Hooper S
      • Wilson R
      • Jayatilake H
      • Gusterson BA
      • Cooper C
      • Shipley J
      • Hargrave D
      • Pritchard-Jones K
      • Maitland N
      • Chenevix-Trench G
      • Riggins GJ
      • Bigner DD
      • Palmieri G
      • Cossu A
      • Flanagan A
      • Nicholson A
      • Ho JWC
      • Leung SY
      • Yuen ST
      • Yuen ST
      • Weber BL
      • Seigler HF
      • Darrow TL
      • Paterson H
      • Marais R
      • Marshall CJ
      • Wooster R
      • Stratton MR
      • Futreal PA
      Mutations of the BRAF gene in human cancer.
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      • Fransen K
      • Klintenas M
      • Osterstrom A
      • Dimberg J
      • Monstein HJ
      • Soderkvist P
      Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.
      • Wang L
      • Cunningham JM
      • Winters JL
      • Guenther JC
      • French AJ
      • Boardman LA
      • Burgart LJ
      • McDonnell SK
      • Schaid DJ
      • Thibodeau SN
      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      • Chan TL
      • Zhao W
      • Leung SY
      • Yuen ST
      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
      The reported frequency of BRAF mutations in HPs and other serrated lesions is variable. In our study BRAF mutations were present in 43% of HPs, 75% of SAs, and 33% of AHAPs from patients with multiple/large HPs and/or hyperplastic polyposis, but were infrequent in sporadic HPs that were predominately from the left colon and rectum. These results are corroborated by another study that reported BRAF mutations in 13% of sporadic serrated polyps (including HPs, SAs, and AHAP) but in 88% of serrated polyps from four patients with hyperplastic polyposis.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      However, two previous studies reported higher frequencies of BRAF mutations in serrated polyps from sporadic patients.
      • Chan TL
      • Zhao W
      • Leung SY
      • Yuen ST
      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
      • Yang S
      • Farraye FA
      • Mack C
      • Posnik O
      • O'Brien MJ
      BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum.
      In one study BRAF mutations were present in 36% of HPs, 100% of SAs, and 20% of AHAP,
      • Chan TL
      • Zhao W
      • Leung SY
      • Yuen ST
      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
      and in the other 70% of HPs and 60% of SAs.
      • Yang S
      • Farraye FA
      • Mack C
      • Posnik O
      • O'Brien MJ
      BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum.
      These differences could be because of the methodology used for the detection of BRAF mutations, or to heterogeneity and selection bias of the study populations among our and previous studies.
      In our study, BRAF mutation status was correlated among multiple HPs from the same patient and was more frequent in younger patients, patients with a large HP and right-sided polyps. This is corroborated by reports of increased frequency of BRAF mutations in right-sided serrated polyps
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      and right-sided colonic carcinomas.
      • Domingo E
      • Espin E
      • Armengol M
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Seruca R
      • Hamelin R
      • Yamamoto H
      • Schwartz Jr, S
      Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.
      • Wang L
      • Cunningham JM
      • Winters JL
      • Guenther JC
      • French AJ
      • Boardman LA
      • Burgart LJ
      • McDonnell SK
      • Schaid DJ
      • Thibodeau SN
      BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair.
      We and others have previously reported more frequent CIMP-high in right-sided HPs,
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      • Wynter CVA
      • Walsh MD
      • Higuchi T
      • Leggett BA
      • Young J
      • Jass JR
      Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.
      and differences in topographic expression of p21Waf1/Cip1 cyclin-dependent kinase inhibitor and Ki-67 proliferation marker in right- and left-sided HPs from these patients.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      In addition, the HPs from the right colon are morphologically different from the HPs in the left colorectum.
      • Torlakovic E
      • Skovlund E
      • Snover DC
      • Torlakovic G
      • Nesland JM
      Morphologic reappraisal of serrated colorectal polyps.
      These data suggest that right-sided HPs are morphologically and genetically different from the left-sided HPs in patients with sporadic HPs and in those with multiple/large HPs and/or hyperplastic polyposis.
      The genetic alterations in sporadic HPs differ from the alterations in HPs from patients with multiple/large HPs, and/or hyperplastic polyposis. Sporadic HPs have more frequent KRAS mutations but less frequent BRAF mutations
      • Chan TL
      • Zhao W
      • Leung SY
      • Yuen ST
      BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      • Yang S
      • Farraye FA
      • Mack C
      • Posnik O
      • O'Brien MJ
      BRAF and KRAS mutations in hyperplastic polyps and serrated adenomas of the colorectum.
      or loss of chromosome 1p,
      • Jen J
      • Powell SM
      • Papadopoulos N
      • Smith KJ
      • Hamilton SR
      • Vogelstein B
      • Kinzler KW
      Molecular determinants of dysplasia in colorectal lesions.
      • Mukai K
      • Fujii T
      • Tajiri H
      • Yoshida S
      • Fukushima S
      • Esumi H
      High frequency of K-ras mutations in human colorectal hyperplastic polyps.
      • Lothe RA
      • Andersen SN
      • Hofstad B
      • Meling GI
      • Peltomaki P
      • Heim S
      • Brogger A
      • Vatn M
      • Rognum TO
      • Borresen AL
      Deletion of 1p loci and microsatellite instability in colorectal polyps.
      and lack CpG island methylation.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      • Wynter CVA
      • Walsh MD
      • Higuchi T
      • Leggett BA
      • Young J
      • Jass JR
      Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.
      In contrast, the present study and previous studies have reported that the HPs from patients with multiple/large HPs and/or hyperplastic polyposis have frequent BRAF mutations and CpG island methylation, but infrequent KRAS mutations or loss of chromosome 1p.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      • Wynter CVA
      • Walsh MD
      • Higuchi T
      • Leggett BA
      • Young J
      • Jass JR
      Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.
      Furthermore, KRAS mutation or loss of chromosome 1p was predominantly present in HPs from patients with predominance of HPs in left colorectum,
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
      • Giardiello FM
      • Hamilton SR
      Phenotypic and molecular characteristics of hyperplastic polyposis.
      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      a set of patients that lacked BRAF mutations or CIMP-high HPs.
      The data from our present study and previous studies
      • Kambara T
      • Simms LA
      • Whitehall VL
      • Spring KJ
      • Wynter CV
      • Walsh MD
      • Barker MA
      • Arnold S
      • McGivern A
      • Matsubara N
      • Tanaka N
      • Higuchi T
      • Young J
      • Jass JR
      • Leggett BA
      BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.
      • Wynter CVA
      • Walsh MD
      • Higuchi T
      • Leggett BA
      • Young J
      • Jass JR
      Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.
      suggest that HPs and other lesions from patients with multiple/large HPs and/or hyperplastic polyposis have BRAF mutations and CIMP-high but lack MSI. In contrast, sporadic colon carcinomas with BRAF mutations frequently have MSI.
      • Domingo E
      • Espin E
      • Armengol M
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Seruca R
      • Hamelin R
      • Yamamoto H
      • Schwartz Jr, S
      Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation.
      • Fransen K
      • Klintenas M
      • Osterstrom A
      • Dimberg J
      • Monstein HJ
      • Soderkvist P
      Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.
      • Oliveira C
      • Pinto M
      • Duval A
      • Brennetot C
      • Domingo E
      • Espin E
      • Armengol M
      • Yamamoto H
      • Hamelin R
      • Seruca R
      • Schwartz Jr, S
      BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.
      • Wang L
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      In our study, BRAF mutations were more frequent in tubular adenomas in patients with multiple/large HPs and/or hyperplastic polyposis compared to sporadic adenomas.
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      These data provide additional evidence that progression of colorectal carcinogenesis in patients with multiple/large HPs and/or hyperplastic polyposis is distinct from sporadic colorectal carcinomas. In some patients with multiple/large HPs and/or hyperplastic polyposis, HPs and other lesions have CIMP-high and BRAF mutations similar to sporadic CIMP-high colorectal carcinomas but lack MSI-high. Other patients have HPs and other lesions that lack BRAF mutation and CpG island methylation, as documented by four patients with loss of chromosome 1p or KRAS mutations in the majority of their HPs.
      • Rashid A
      • Houlihan PS
      • Booker S
      • Peterson GM
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      • Hamilton SR
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      • Chan AO
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      Concordant CpG island methylation in hyperplastic polyposis.
      In our study BRAF mutations were infrequent in ACF from patients with sporadic colorectal cancers and FAP. Dysplastic ACF are characterized by abnormal epithelial proliferation in the upper aspects of the crypts, lack of KRAS mutations and methylation, and presence of APC mutations in dysplastic ACF from FAP patients but not patients with sporadic colorectal cancers.
      • Otori K
      • Konishi M
      • Sugiyama K
      • Hasebe T
      • Shimoda T
      • Kikuchi-Yanoshita R
      • Mukai K
      • Fukushima S
      • Miyaki M
      • Esumi H
      Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.
      • Takayama T
      • Ohi M
      • Hayashi T
      • Miyanishi K
      • Nobuoka A
      • Nakajima T
      • Satoh T
      • Takimoto R
      • Kato J
      • Sakamaki S
      • Niitsu Y
      Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      The lack of BRAF mutations in dysplastic ACF or heteroplastic ACF from FAP patients is not surprising and is further corroborated by infrequent BRAF mutations in sporadic adenomas or adenomas from patients with FAP.
      • Yuen ST
      • Davies H
      • Chan TL
      • Ho JW
      • Bignell GR
      • Cox C
      • Stephens P
      • Edkins S
      • Tsui WW
      • Chan AS
      • Futreal PA
      • Stratton MR
      • Wooster R
      • Leung SY
      Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia.
      In contrast, heteroplastic ACF are characterized by lack of dysplasia, have proliferation mainly in the lower aspects of the crypts, have frequent KRAS mutations and methylation, and lack APC mutations.
      • Nucci MR
      • Robinson CR
      • Longo P
      • Campbell P
      • Hamilton SR
      Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.
      • Otori K
      • Konishi M
      • Sugiyama K
      • Hasebe T
      • Shimoda T
      • Kikuchi-Yanoshita R
      • Mukai K
      • Fukushima S
      • Miyaki M
      • Esumi H
      Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.
      • Takayama T
      • Ohi M
      • Hayashi T
      • Miyanishi K
      • Nobuoka A
      • Nakajima T
      • Satoh T
      • Takimoto R
      • Kato J
      • Sakamaki S
      • Niitsu Y
      Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.
      • Chan AO
      • Broaddus RB
      • Houlihan PS
      • Issa J-PJ
      • Morris JS
      • Hamilton SR
      • Rashid A
      CpG island methylation in aberrant crypt foci of the colorectum.
      Heteroplastic and mixed ACF resemble HPs and SAs histopathologically, respectively. These data suggest that either heteroplastic ACF are not precursors of HPs, SAs, and colorectal cancers with BRAF mutations, or BRAF mutation is a late event in a hyperplastic polyp-serrated adenoma-carcinoma sequence. Alternatively, these findings may be because of selection bias in our study with ACF from sporadic colorectal cancers and FAP, a patient-population that lacks BRAF mutations.
      Recent studies have suggested a hyperplastic polyp-serrated adenoma-carcinoma pathway in colorectal carcinogenesis.
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      Serrated route to colorectal cancer: back street or super highway?.
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      • Young J
      • Leggett BA
      Emerging concepts in colorectal neoplasia.
      Right-sided sporadic colon carcinomas often have CpG island methylation and BRAF mutations. In this study we show that some patients with multiple/large HPs and/or hyperplastic polyposis have these molecular characteristics in multiple colonic lesions including HPs. As a consequence, it appears that subsets of patients whose lesions have different pathogenesis have similar phenotypes. Molecular characteristics are needed to identify those subsets.

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