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BRAF mutation is associated with the CpG island methylator phenotype in colorectal cancer from young patients
Cancer Letters 273 (2009) 221–224
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
BRAF mutation is associated with the CpG island methylator phenotype in colorectal cancer from young patients Pei Woon Ang a,b, Wei Qi Li a,b, Richie Soong b, Barry Iacopetta a,* a b
School of Surgery M507, University of Western Australia, 35 Stirling Hwy, Nedlands 6009, Australia Oncology Research Institute, National University of Singapore, Centre for Life Sciences #02-14G, 28 Medical Drive, Singapore
a r t i c l e
i n f o
Article history: Received 12 May 2008 Received in revised form 12 May 2008 Accepted 4 August 2008
Keywords: BRAF CIMP Colorectal cancer APC
a b s t r a c t This study investigated the relationship between BRAF mutation, the CpG island methylator phenotype (CIMP+) and APC methylation in colorectal cancer (CRC) from young patients. The V600E BRAF mutation was found in 7% of cases and was strongly associated with the tumour features of proximal site, advanced stage and poor histological grade. More than half (53%) the tumours with BRAF mutation were also CIMP+ as evaluated by a standard panel of markers, compared to only 4% of tumours with wildtype BRAF (P < 0.0001). In contrast to CIMP+, APC methylation was inversely correlated with BRAF mutation (P = 0.02). BRAF mutation and CIMP+ are therefore likely to be involved in an alternate, albeit rare, pathway to APC inactivation during the development of CRC in younger patients. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is characterised by high levels of CpG methylation in multiple gene promoter regions. Distinctive clinicopathological features of CIMP+ CRC include origin in the proximal colon, more advanced age, female gender, mucinous histology and poor differentiation [1–5]. A panel of five markers was recently proposed to allow standardized assessment of CIMP+ [6]. This consists of CpG-rich regions within five genes: RUNX3, CACNA1G, IGF2, NEUROG1 and SOCS1. Using these markers, the frequency of CIMP+ in unselected series of CRC was estimated at approximately 15–18% [6–8]. Quantitative evaluation of methylation has also been recommended for the study of CIMP+ [7], with MethyLight being the most commonly used method. One of the distinguishing molecular features of CIMP+ tumours was a high incidence of mutation in the BRAF oncogene, particularly the V600E hotspot mutation [3,6–9]. Another feature recently reported for CIMP+ was an inverse correlation with APC methylation in an unselected CRC cohort [10].
* Corresponding author. Tel.: +61 8 9346 2085; fax: +61 8 9346 2416. E-mail address: [email protected] (B. Iacopetta). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.08.001
Although CIMP+ CRCs share many clinicopathological features with the microsatellite instability phenotype (MSI+), recent work has confirmed they comprise a distinct subgroup independently of MSI status [3,6–8]. Approximately half of all CIMP+ tumours are also MSI+ due to methylation-induced transcriptional silencing of the hMLH1 mismatch repair gene [6–8]. Compared to CIMP+/ MSI+ tumours, CIMP+/MSI tumours display higher stage at presentation, absence of lymphocytic infiltration and poor prognosis [5]. Recent reports also suggest the BRAF V600E mutation is associated with poor prognosis in CIMP+/MSI tumours [11–13]. These studies were carried out on CIMP+/MSI tumours originating predominantly in older patients and little is known of the frequency or the clinicopathological and molecular characteristics of such tumours in younger patients. The aim of the present study was therefore to investigate BRAF mutation and CIMP+ in MSI tumours from an exclusively younger cohort of sporadic CRC patients aged <60 years. 2. Materials and methods The 735 CRC samples investigated here were derived from a population-based screening study of hereditary non-polyposis colorectal cancer (HNPCC) in patients aged
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<60 years at diagnosis [14]. Only tumours judged to be MSI following analysis of the BAT-26 mononucleotide repeat were included in this study, thereby excluding HNPCC cases. Information on patient age and gender and on tumour characteristics including stage, site and grade were obtained from pathology reports. BRAF V600E mutations were identified by fluorescent-single strand conformation polymorphism as described previously [15]. A randomly selected subset of 32 tumours with BRAF mutation was matched for patient age and tumour site with 57 tumours having BRAF wildtype status. DNA was extracted from formalin-fixed, paraffin embedded tissues as described previously [16]. The minimum starting amount of genomic DNA to ensure reliable evaluation of DNA methylation following bisulfite modification was estimated by real-time quantification of amplifiable genomic DNA [17]. Sodium bisulfite modification was performed using the EZ DNA methylation kit (Zymo Research, Orange, CA) and eluted into 20 ll of 10 mmol/L Tris–HCl (pH 8). DNA methylation levels for RUNX3, CACNA1G, IGF2, NEUROG1, SOCS1 and APC were quantified using MethyLight as described previously [6,10]. The level of DNA methylation was calculated as a percentage of methylated reference (PMR) using b-actin to normalize for the amount of input bisulfite-treated DNA and SssI methylase-treated DNA as the methylated standard. A threshold PMR value of 4 was used to classify methylated loci [6,8]. Positive CIMP status (CIMP+) was defined as P3 of the RUNX3, CACNA1G, IGF2, NEUROG1 and SOCS1 markers being methylated. The v2 test was used to assess univariate relationships between the BRAF V600E mutation and categorical variables including clinicopathological characteristics and methylation at each locus. Fisher’s exact test was used when the sample size was less than 5. All P values were two-sided and the level of statistical significance was <0.05. Student’s t-test was used for comparisons of the mean number of methylated loci between the BRAF wildtype and BRAF mutant tumours. 3. Results The frequency of BRAF V600E mutation in 735 microsatellite stable (MSI ) CRCs from patients aged <60 years was 7% (Table 1). The presence of BRAF mutation was strongly associated with advanced stage, location in the proximal colon and high histological grade. A trend was also apparent for higher BRAF mutation frequency in tumours from very young patients (<40 years). The frequencies of gene hypermethylation and CIMP+ were investigated in 32 BRAF mutant and 57 BRAF wildtype tumours that were matched for patient age and tumour site. The median age of these patient groups was 52.0 and 51.5 years, respectively. A significantly higher frequency of methylation in each of the markers comprising the CIMP panel was observed in BRAF mutant compared to BRAF wildtype tumours (Table 2). Approximately half of the BRAF mutant tumours identified in this young cohort of MSI CRC were CIMP+, compared to just 4% for the BRAF wildtype tumours. Fig. 1 shows the distribution of the number of methylated CIMP markers for BRAF wildtype and BRAF mutant tumours. The mean number of methylated loci was higher in BRAF mutant (2.4 ± 1.54) compared to BRAF wildtype (0.3 ± 0.79) tumours (P < 0.0001, Student t-test). In contrast to the CIMP markers, a significant inverse correlation was observed between the presence of APC methylation and BRAF mutation (Table 2). All 8 tumours with APC hypermethylation were wildtype for BRAF and were amongst the 45 tumours that showed no methylation of any of the CIMP markers (Fig. 1).
Table 1 Associations between BRAF V600E mutation and clinicopathological features of microsatellite stable CRC from patients aged <60 years Feature (n)a
BRAF wildtype (%)
BRAF mutant (%)
Total (735)
685
50
Age (years) <30 (13) 30–39 (42) 40–49 (168) 50–59 (512)
10 (1) 38 (6) 159 (23) 478 (70)
3 (6) 4 (8) 9 (18) 34 (68)
0.07b
Sex Female (295) Male (440)
272 (40) 413 (60)
23 (46) 27 (54)
0.38
Stage (AJCC) In situ (38) I (116) II (115) III (223) IV (60)
36 (6) 113 (20) 151 (27) 205 (37) 50 (9)
2 (5) 3 (8) 4 (11) 18 (49) 10 (27)
0.0004c
Site Proximal (192) Distal (531)
163 (24) 511 (76)
29 (59) 20 (41)
<0.0001
Grade Low (561) High (89)
536 (88) 71 (12)
25 (58) 18 (42)
<0.0001
Mucinous histology Absent (599) 564 (82) Present (136) 121 (18)
35 (70) 15 (30)
0.03
P
a
Data for stage, site, grade was not available for 183, 30 and 103 cases, respectively. b Age <40 years vs. 40–59 years. c Stages III/IV vs. in situ/I/II.
4. Discussion Approximately 20–25% of CRC cases diagnosed in Western countries occur in patients aged <60 years [18]. The pathways by which these tumours develop are of special interest because the large majority of HNPCC cases arise in young patients. The MSI+ phenotype occurs in about 8% of CRC from patients aged <60 years [14] and many of these cases are associated with HNPCC. To investigate the pathways by which sporadic CRCs develop in younger patients, the present study investigated only MSI tumours from a large and consecutive series of patients aged <60 years. We were specifically interested in the associations between BRAF mutation, CIMP+ and APC methylation in these tumours. The frequency of BRAF mutation in this young patient cohort (7%) was within the range of previous reports of 3–9% in other population-based studies of unselected cases [11–13,15,19]. BRAF mutation was approximately 4-fold more frequent in tumours from the proximal colon (29/ 192, 15%) compared to distal colon and rectal tumours (20/531, 4%; P < 0.0001). In agreement with other studies, BRAF mutation was strongly associated with the poor prognosis features of advanced stage and high grade [11– 13,19]. Of the 37 BRAF mutant tumours in which stage could be ascertained from the pathology report, 28 (76%) were either stage III or IV. It is presently unclear why mutation of this oncogene is associated with such an aggressive tumour phenotype. The proportion of proximal
P.W. Ang et al. / Cancer Letters 273 (2009) 221–224 Table 2 Associations between gene hypermethylation and BRAF V600E mutation in microsatellite stable CRC from patients aged <60 years Gene methylation (n)a
BRAF wildtype (%)
BRAF mutant (%)
Total
57
32
RUNX3 Yes (22) No (67)
2 (4) 55 (96)
20 (62) 12 (38)
<0.0001
CACNA1G Yes (14) No (75)
3 (5) 54 (95)
11 (34) 21 (66)
0.0005
IGF2 Yes (23) No (66)
3 (5) 54 (95)
20 (61) 13 (39)
<0.0001
NEU Yes (29) No (60)
9 (16) 48 (84)
20 (62) 12 (38)
<0.0001
SOCS Yes (7) No (82)
1 (2) 56 (98)
6 (19) 26 (81)
0.007
CIMPb Positive (19) Negative (70)
2 (4) 55 (96)
17 (53) 15 (47)
<0.0001
APC Yes (8) No (81)
8 (14) 49 (86)
0 (0) 32 (100)
0.02
P
a
Yes, PMR P4; No, PMR <4. CIMP+ was defined as 3 or more of RUNX3, CACNA1G, IGF2, NEU and SOCS showing methylation. b
Fig. 1. Distribution of number of methylated CIMP markers for BRAF wildtype and BRAF mutant tumours.
BRAF mutant tumours with advanced stage was similar to that of distal BRAF mutant tumours, although this analysis was limited by the relatively small sample size. Similar to the findings in MSI tumours by Samowitz et al. [11], no association was observed between gender and CIMP+ in this cohort. Previous workers have demonstrated a strong association between BRAF mutation and CIMP+ [6–8,20,21] and indeed the former was used to help select markers to standardize the classification of CIMP+ [6]. The earlier studies linking BRAF mutation to promoter methylation of multiple cancer-related genes were carried out mostly on older subjects. The present study is the first to specifically investigate a younger patient cohort and in the absence of pos-
223
sible confounding effects from the MSI+ phenotype. A subset of 32 BRAF mutant and 57 BRAF wildtype tumours matched for patient age and anatomical site were selected for the study of CIMP+ using the quantitative MethyLight assay and a standardized panel of markers. All markers with the exception of NEU showed a low frequency of methylation (65%) in BRAF wildtype tumours, but significantly higher frequencies in BRAF mutant tumours (Table 2). Using the criterion of P3 methylated markers to define CIMP+, just over half (53%) of the BRAF mutant tumours were classified as CIMP+ compared to only 4% of the BRAF wildtype tumours. These results provide strong support for the notion that BRAF mutation and CIMP+ occur in the same pathway of CRC development, arising predominantly in the proximal colon. Although the proportion of stage III and IV tumours was higher in BRAF mutant (85%) compared to wildtype (58%) tumours, it is unlikely to account for the large difference observed in CIMP+ frequency between the two groups (Table 2). This is because previous studies have demonstrated only a weak association between methylation and tumour stage [1–3]. The overall frequency of CIMP+ tumours in young CRC patients (<60 years) was estimated for the first time in this study to be approximately 8%. This value comprises of 4% derived from patients with wildtype BRAF who were CIMP+, plus an additional 4% derived from patients with BRAF mutation (7% frequency) who were also CIMP+ (53%). This estimate is about half the frequency reported for CIMP+ in unselected CRC series [6,8,22]. The occurrence of CIMP+ CRC in young patients highlights the need to identify dietary and genetic risk factors that are specific for this tumour subgroup, thus allowing targeted screening. It was not possible to determine from the current results whether BRAF mutation precedes CIMP+ or vice versa. Co-segregation of BRAF mutations with extensive DNA methylation has been reported in serrated adenomas found mostly in the proximal colon and which have been proposed as precursor lesions for CIMP+ tumours [9,23,24]. The presence of BRAF mutation in serrated adenomas and CRC has been associated with a positive family history of this disease [11,24]. By extension, this may also imply genetic predisposition to the development of CIMP+, possibly via the level of DNA methylation in normal colonic mucosa [25]. In striking contrast to CIMP+ determined by the new panel of markers, methylation of APC was inversely correlated with BRAF mutation (Table 2). This confirms a previous observation in an unselected CRC series [10]. Moreover, Samowitz et al. have reported that mutations in APC are inversely correlated with the presence of BRAF mutation and CIMP+ in CRC [26]. Together, these results suggest that inactivation of the APC gene, either by methylation-induced silencing or by mutation, occur in a different pathway of CRC development to that which involves BRAF mutation and CIMP+ as defined by the new markers. The mechanism underlying the differential methylation of genes between these two tumour subtypes is unknown. Folate depletion has been shown to alter the level of APC expression in a normal human colon cell line [27], although it is unclear whether this is due to changes in
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the methylation status of APC. Hypermethylation of APC may be inversely associated with cellular folate status in comparison to methylation of the CIMP+ group of genes. In conclusion, the present results demonstrate that BRAF mutation is rare in MSI CRC from young patients, however when present it is associated with an aggressive tumour phenotype. Approximately half of all tumours with BRAF mutation are also CIMP+, compared to only a very low frequency of CIMP+ in tumours with wildtype BRAF. The inverse correlation observed here between APC methylation and BRAF mutation or CIMP+ provides further support for the existence of alternate pathways of CRC development. These findings demonstrate that the younger CRC population (<60 years) is also susceptible to the CIMP+ pathway of colorectal tumourigenesis, albeit at approximately half the frequency observed in older patients. Conflict of Interest All of the authors declare no conflict of interest with this work. Acknowledgements Pei Woon Ang was supported by an International Postgraduate Research Scholarship from the University of Western Australia. This work was funded by the Singapore Cancer Syndicate (SCS#BU51).
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
BRAF mutation is associated with the CpG island methylator phenotype in colorectal cancer from young patients Pei Woon Ang a,b, Wei Qi Li a,b, Richie Soong b, Barry Iacopetta a,* a b
School of Surgery M507, University of Western Australia, 35 Stirling Hwy, Nedlands 6009, Australia Oncology Research Institute, National University of Singapore, Centre for Life Sciences #02-14G, 28 Medical Drive, Singapore
a r t i c l e
i n f o
Article history: Received 12 May 2008 Received in revised form 12 May 2008 Accepted 4 August 2008
Keywords: BRAF CIMP Colorectal cancer APC
a b s t r a c t This study investigated the relationship between BRAF mutation, the CpG island methylator phenotype (CIMP+) and APC methylation in colorectal cancer (CRC) from young patients. The V600E BRAF mutation was found in 7% of cases and was strongly associated with the tumour features of proximal site, advanced stage and poor histological grade. More than half (53%) the tumours with BRAF mutation were also CIMP+ as evaluated by a standard panel of markers, compared to only 4% of tumours with wildtype BRAF (P < 0.0001). In contrast to CIMP+, APC methylation was inversely correlated with BRAF mutation (P = 0.02). BRAF mutation and CIMP+ are therefore likely to be involved in an alternate, albeit rare, pathway to APC inactivation during the development of CRC in younger patients. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is characterised by high levels of CpG methylation in multiple gene promoter regions. Distinctive clinicopathological features of CIMP+ CRC include origin in the proximal colon, more advanced age, female gender, mucinous histology and poor differentiation [1–5]. A panel of five markers was recently proposed to allow standardized assessment of CIMP+ [6]. This consists of CpG-rich regions within five genes: RUNX3, CACNA1G, IGF2, NEUROG1 and SOCS1. Using these markers, the frequency of CIMP+ in unselected series of CRC was estimated at approximately 15–18% [6–8]. Quantitative evaluation of methylation has also been recommended for the study of CIMP+ [7], with MethyLight being the most commonly used method. One of the distinguishing molecular features of CIMP+ tumours was a high incidence of mutation in the BRAF oncogene, particularly the V600E hotspot mutation [3,6–9]. Another feature recently reported for CIMP+ was an inverse correlation with APC methylation in an unselected CRC cohort [10].
* Corresponding author. Tel.: +61 8 9346 2085; fax: +61 8 9346 2416. E-mail address: [email protected] (B. Iacopetta). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.08.001
Although CIMP+ CRCs share many clinicopathological features with the microsatellite instability phenotype (MSI+), recent work has confirmed they comprise a distinct subgroup independently of MSI status [3,6–8]. Approximately half of all CIMP+ tumours are also MSI+ due to methylation-induced transcriptional silencing of the hMLH1 mismatch repair gene [6–8]. Compared to CIMP+/ MSI+ tumours, CIMP+/MSI tumours display higher stage at presentation, absence of lymphocytic infiltration and poor prognosis [5]. Recent reports also suggest the BRAF V600E mutation is associated with poor prognosis in CIMP+/MSI tumours [11–13]. These studies were carried out on CIMP+/MSI tumours originating predominantly in older patients and little is known of the frequency or the clinicopathological and molecular characteristics of such tumours in younger patients. The aim of the present study was therefore to investigate BRAF mutation and CIMP+ in MSI tumours from an exclusively younger cohort of sporadic CRC patients aged <60 years. 2. Materials and methods The 735 CRC samples investigated here were derived from a population-based screening study of hereditary non-polyposis colorectal cancer (HNPCC) in patients aged
222
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<60 years at diagnosis [14]. Only tumours judged to be MSI following analysis of the BAT-26 mononucleotide repeat were included in this study, thereby excluding HNPCC cases. Information on patient age and gender and on tumour characteristics including stage, site and grade were obtained from pathology reports. BRAF V600E mutations were identified by fluorescent-single strand conformation polymorphism as described previously [15]. A randomly selected subset of 32 tumours with BRAF mutation was matched for patient age and tumour site with 57 tumours having BRAF wildtype status. DNA was extracted from formalin-fixed, paraffin embedded tissues as described previously [16]. The minimum starting amount of genomic DNA to ensure reliable evaluation of DNA methylation following bisulfite modification was estimated by real-time quantification of amplifiable genomic DNA [17]. Sodium bisulfite modification was performed using the EZ DNA methylation kit (Zymo Research, Orange, CA) and eluted into 20 ll of 10 mmol/L Tris–HCl (pH 8). DNA methylation levels for RUNX3, CACNA1G, IGF2, NEUROG1, SOCS1 and APC were quantified using MethyLight as described previously [6,10]. The level of DNA methylation was calculated as a percentage of methylated reference (PMR) using b-actin to normalize for the amount of input bisulfite-treated DNA and SssI methylase-treated DNA as the methylated standard. A threshold PMR value of 4 was used to classify methylated loci [6,8]. Positive CIMP status (CIMP+) was defined as P3 of the RUNX3, CACNA1G, IGF2, NEUROG1 and SOCS1 markers being methylated. The v2 test was used to assess univariate relationships between the BRAF V600E mutation and categorical variables including clinicopathological characteristics and methylation at each locus. Fisher’s exact test was used when the sample size was less than 5. All P values were two-sided and the level of statistical significance was <0.05. Student’s t-test was used for comparisons of the mean number of methylated loci between the BRAF wildtype and BRAF mutant tumours. 3. Results The frequency of BRAF V600E mutation in 735 microsatellite stable (MSI ) CRCs from patients aged <60 years was 7% (Table 1). The presence of BRAF mutation was strongly associated with advanced stage, location in the proximal colon and high histological grade. A trend was also apparent for higher BRAF mutation frequency in tumours from very young patients (<40 years). The frequencies of gene hypermethylation and CIMP+ were investigated in 32 BRAF mutant and 57 BRAF wildtype tumours that were matched for patient age and tumour site. The median age of these patient groups was 52.0 and 51.5 years, respectively. A significantly higher frequency of methylation in each of the markers comprising the CIMP panel was observed in BRAF mutant compared to BRAF wildtype tumours (Table 2). Approximately half of the BRAF mutant tumours identified in this young cohort of MSI CRC were CIMP+, compared to just 4% for the BRAF wildtype tumours. Fig. 1 shows the distribution of the number of methylated CIMP markers for BRAF wildtype and BRAF mutant tumours. The mean number of methylated loci was higher in BRAF mutant (2.4 ± 1.54) compared to BRAF wildtype (0.3 ± 0.79) tumours (P < 0.0001, Student t-test). In contrast to the CIMP markers, a significant inverse correlation was observed between the presence of APC methylation and BRAF mutation (Table 2). All 8 tumours with APC hypermethylation were wildtype for BRAF and were amongst the 45 tumours that showed no methylation of any of the CIMP markers (Fig. 1).
Table 1 Associations between BRAF V600E mutation and clinicopathological features of microsatellite stable CRC from patients aged <60 years Feature (n)a
BRAF wildtype (%)
BRAF mutant (%)
Total (735)
685
50
Age (years) <30 (13) 30–39 (42) 40–49 (168) 50–59 (512)
10 (1) 38 (6) 159 (23) 478 (70)
3 (6) 4 (8) 9 (18) 34 (68)
0.07b
Sex Female (295) Male (440)
272 (40) 413 (60)
23 (46) 27 (54)
0.38
Stage (AJCC) In situ (38) I (116) II (115) III (223) IV (60)
36 (6) 113 (20) 151 (27) 205 (37) 50 (9)
2 (5) 3 (8) 4 (11) 18 (49) 10 (27)
0.0004c
Site Proximal (192) Distal (531)
163 (24) 511 (76)
29 (59) 20 (41)
<0.0001
Grade Low (561) High (89)
536 (88) 71 (12)
25 (58) 18 (42)
<0.0001
Mucinous histology Absent (599) 564 (82) Present (136) 121 (18)
35 (70) 15 (30)
0.03
P
a
Data for stage, site, grade was not available for 183, 30 and 103 cases, respectively. b Age <40 years vs. 40–59 years. c Stages III/IV vs. in situ/I/II.
4. Discussion Approximately 20–25% of CRC cases diagnosed in Western countries occur in patients aged <60 years [18]. The pathways by which these tumours develop are of special interest because the large majority of HNPCC cases arise in young patients. The MSI+ phenotype occurs in about 8% of CRC from patients aged <60 years [14] and many of these cases are associated with HNPCC. To investigate the pathways by which sporadic CRCs develop in younger patients, the present study investigated only MSI tumours from a large and consecutive series of patients aged <60 years. We were specifically interested in the associations between BRAF mutation, CIMP+ and APC methylation in these tumours. The frequency of BRAF mutation in this young patient cohort (7%) was within the range of previous reports of 3–9% in other population-based studies of unselected cases [11–13,15,19]. BRAF mutation was approximately 4-fold more frequent in tumours from the proximal colon (29/ 192, 15%) compared to distal colon and rectal tumours (20/531, 4%; P < 0.0001). In agreement with other studies, BRAF mutation was strongly associated with the poor prognosis features of advanced stage and high grade [11– 13,19]. Of the 37 BRAF mutant tumours in which stage could be ascertained from the pathology report, 28 (76%) were either stage III or IV. It is presently unclear why mutation of this oncogene is associated with such an aggressive tumour phenotype. The proportion of proximal
P.W. Ang et al. / Cancer Letters 273 (2009) 221–224 Table 2 Associations between gene hypermethylation and BRAF V600E mutation in microsatellite stable CRC from patients aged <60 years Gene methylation (n)a
BRAF wildtype (%)
BRAF mutant (%)
Total
57
32
RUNX3 Yes (22) No (67)
2 (4) 55 (96)
20 (62) 12 (38)
<0.0001
CACNA1G Yes (14) No (75)
3 (5) 54 (95)
11 (34) 21 (66)
0.0005
IGF2 Yes (23) No (66)
3 (5) 54 (95)
20 (61) 13 (39)
<0.0001
NEU Yes (29) No (60)
9 (16) 48 (84)
20 (62) 12 (38)
<0.0001
SOCS Yes (7) No (82)
1 (2) 56 (98)
6 (19) 26 (81)
0.007
CIMPb Positive (19) Negative (70)
2 (4) 55 (96)
17 (53) 15 (47)
<0.0001
APC Yes (8) No (81)
8 (14) 49 (86)
0 (0) 32 (100)
0.02
P
a
Yes, PMR P4; No, PMR <4. CIMP+ was defined as 3 or more of RUNX3, CACNA1G, IGF2, NEU and SOCS showing methylation. b
Fig. 1. Distribution of number of methylated CIMP markers for BRAF wildtype and BRAF mutant tumours.
BRAF mutant tumours with advanced stage was similar to that of distal BRAF mutant tumours, although this analysis was limited by the relatively small sample size. Similar to the findings in MSI tumours by Samowitz et al. [11], no association was observed between gender and CIMP+ in this cohort. Previous workers have demonstrated a strong association between BRAF mutation and CIMP+ [6–8,20,21] and indeed the former was used to help select markers to standardize the classification of CIMP+ [6]. The earlier studies linking BRAF mutation to promoter methylation of multiple cancer-related genes were carried out mostly on older subjects. The present study is the first to specifically investigate a younger patient cohort and in the absence of pos-
223
sible confounding effects from the MSI+ phenotype. A subset of 32 BRAF mutant and 57 BRAF wildtype tumours matched for patient age and anatomical site were selected for the study of CIMP+ using the quantitative MethyLight assay and a standardized panel of markers. All markers with the exception of NEU showed a low frequency of methylation (65%) in BRAF wildtype tumours, but significantly higher frequencies in BRAF mutant tumours (Table 2). Using the criterion of P3 methylated markers to define CIMP+, just over half (53%) of the BRAF mutant tumours were classified as CIMP+ compared to only 4% of the BRAF wildtype tumours. These results provide strong support for the notion that BRAF mutation and CIMP+ occur in the same pathway of CRC development, arising predominantly in the proximal colon. Although the proportion of stage III and IV tumours was higher in BRAF mutant (85%) compared to wildtype (58%) tumours, it is unlikely to account for the large difference observed in CIMP+ frequency between the two groups (Table 2). This is because previous studies have demonstrated only a weak association between methylation and tumour stage [1–3]. The overall frequency of CIMP+ tumours in young CRC patients (<60 years) was estimated for the first time in this study to be approximately 8%. This value comprises of 4% derived from patients with wildtype BRAF who were CIMP+, plus an additional 4% derived from patients with BRAF mutation (7% frequency) who were also CIMP+ (53%). This estimate is about half the frequency reported for CIMP+ in unselected CRC series [6,8,22]. The occurrence of CIMP+ CRC in young patients highlights the need to identify dietary and genetic risk factors that are specific for this tumour subgroup, thus allowing targeted screening. It was not possible to determine from the current results whether BRAF mutation precedes CIMP+ or vice versa. Co-segregation of BRAF mutations with extensive DNA methylation has been reported in serrated adenomas found mostly in the proximal colon and which have been proposed as precursor lesions for CIMP+ tumours [9,23,24]. The presence of BRAF mutation in serrated adenomas and CRC has been associated with a positive family history of this disease [11,24]. By extension, this may also imply genetic predisposition to the development of CIMP+, possibly via the level of DNA methylation in normal colonic mucosa [25]. In striking contrast to CIMP+ determined by the new panel of markers, methylation of APC was inversely correlated with BRAF mutation (Table 2). This confirms a previous observation in an unselected CRC series [10]. Moreover, Samowitz et al. have reported that mutations in APC are inversely correlated with the presence of BRAF mutation and CIMP+ in CRC [26]. Together, these results suggest that inactivation of the APC gene, either by methylation-induced silencing or by mutation, occur in a different pathway of CRC development to that which involves BRAF mutation and CIMP+ as defined by the new markers. The mechanism underlying the differential methylation of genes between these two tumour subtypes is unknown. Folate depletion has been shown to alter the level of APC expression in a normal human colon cell line [27], although it is unclear whether this is due to changes in
224
P.W. Ang et al. / Cancer Letters 273 (2009) 221–224
the methylation status of APC. Hypermethylation of APC may be inversely associated with cellular folate status in comparison to methylation of the CIMP+ group of genes. In conclusion, the present results demonstrate that BRAF mutation is rare in MSI CRC from young patients, however when present it is associated with an aggressive tumour phenotype. Approximately half of all tumours with BRAF mutation are also CIMP+, compared to only a very low frequency of CIMP+ in tumours with wildtype BRAF. The inverse correlation observed here between APC methylation and BRAF mutation or CIMP+ provides further support for the existence of alternate pathways of CRC development. These findings demonstrate that the younger CRC population (<60 years) is also susceptible to the CIMP+ pathway of colorectal tumourigenesis, albeit at approximately half the frequency observed in older patients. Conflict of Interest All of the authors declare no conflict of interest with this work. Acknowledgements Pei Woon Ang was supported by an International Postgraduate Research Scholarship from the University of Western Australia. This work was funded by the Singapore Cancer Syndicate (SCS#BU51).
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