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Phenotypic and genotypic differences in colorectal carcinoma among Caucasians, Asians, and Hispanics lack statistical significance
Pathology - Research and Practice 214 (2018) 720–726
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Phenotypic and genotypic differences in colorectal carcinoma among Caucasians, Asians, and Hispanics lack statistical significance
T
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Sara J. Hoffmana, , Mark Li-cheng Wub a b
University of California, Irvine School of Medicine, Division of Genetic and Genomic Medicine, Department of Pediatrics, Irvine, CA, USA University of California, Irvine School of Medicine, Department of Pathology and Laboratory Medicine, Irvine, CA, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Colorectal carcinoma Microsatellite instability Colon BRAF KRAS Ethnicity
Colorectal carcinoma (CRC) has been shown to have both genetic and environmental factors that can promote carcinoma development. Previous studies have found ethnic differences in the distribution of molecular phenotypes of CRC. Very little specific data exist regarding Hispanic CRC, and these data primarily focus on epidemiology or location of carcinoma. Our retrospective study analyzed 562 Caucasian, Asian, and Hispanic CRC patients at the UCI Medical Center from 2004 to 2012. The results showed that there were no statistically significant differences with respect to mean age, gender or site of carcinoma among the three ethnic groups. There were no statistically significant differences among the three ethnicities with respect to rates of MSI, mutated BRAF, and mutated KRAS. The Caucasian group had a non-significant higher rate of MSI (15%) and BRAF mutation (12%) than the Asian and Hispanic groups. Hispanics had a non-significant higher rate of KRAS mutation (59%) than Caucasians (38%) and Asians (37%). The results of this study demonstrated a higher rate of MSI and BRAF mutation in the Caucasian group and a higher rate of KRAS mutation in the Hispanic group, however differences were not statistically significant.
1. Introduction Colorectal carcinoma (CRC) is carcinoma which originates in the colon or the rectum, and it has been shown that both genetics and environment can promote carcinoma development [1]. Being the third most common carcinoma in both men and women, CRC is the second leading cause of carcinoma death in the United States. In 2010, there were 51,370 CRC deaths and 142,570 new cases of CRC [2]. The cumulative lifetime risk of developing CRC in the United States is 6% [3]. In developing (Western) societies, there are environmental factors that probably contribute to higher rates of CRC, such as diet, obesity, lack of physical activity, smoking, and alcohol consumption [4]. Because of the impact of this carcinoma on the general population, it is important to study colorectal carcinoma. Approximately 70% of all colorectal carcinoma is sporadic, which means that the carcinoma is caused by a series of genetic or epigenetic changes that accumulate, and are not caused by an inherited predisposition. About 20–30% of all CRC’s are familial, and genetic association and population studies have been discovering low penetrance susceptibility loci and polymorphisms. These loci and polymorphisms, along with shared environmental factors, are likely to increase risk of CRC in families that exhibit familial inheritance patterns of CRC. These
inheritance patterns do not fit well-established major gene patterns of inheritance. About 5% of CRC is due to an hereditary component, which is caused by highly penetrant mutations at major gene loci [5]. There are many different hereditary CRC syndromes. The most common are Lynch syndrome (previously known as hereditary nonpolyposis colorectal cancer) that accounts for roughly 1–3% of CRC cases and familial adenomatous polyposis (FAP) that accounts for less than 1% of CRC cases. Other rare hereditary CRC syndromes, which also account for less than 1% of CRC cases, are attenuated familial adenomatous polyposis (AFAP), MutY homolog-associated polyposis (MUTYH-associated polyposis, MAP), Peutz-Jeghers syndrome, juvenile polyposis syndrome, Cowden syndrome, hereditary mixed polyposis, and serrated polyposis (formerly hyperplastic polyposis) [6]. Lynch syndrome accounts for 1%-5% of all colorectal carcinomas, is the most common hereditary colorectal carcinoma, and is caused by germline mutations of hMLH1, hMSH2, hMSH6 and hPMS2, the genes that encode mismatch repair proteins, and the mutation of the related gene, EPCAM [7,8]. It is well known that the phenotypic and genotypic characteristics of colorectal carcinoma varies between individuals. Interestingly, there are many differences regarding the gross anatomy, microanatomy and
Abbreviations:MSI, microsatellite instability ⁎ Corresponding author at: Sutter Pacific Medical Foundation, 3883 Airway Dr, Ste 300, Santa Rosa, CA 95403, USA. E-mail address: [email protected] (S.J. Hoffman). https://doi.org/10.1016/j.prp.2018.03.008 Received 11 December 2017; Received in revised form 25 February 2018; Accepted 2 March 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
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of 15% found in the Caucasian population [21]. Because Hispanic and Asian cultures have long and contrasting histories with respect to geography, culture, diet, and environmental exposures, it seems reasonable to hypothesize that there may be significant differences in colorectal neoplasia between these ethnicities. Knowledge of the effect of ethnicity on CRC can potentially impact diagnosis and management, particularly in cases where histology or molecular phenotype is indeterminate or unavailable. For example, given that BRAF is mutated at a lower rate in the East, an indeterminate BRAF result might be more likely to be considered a wild-type (the typical form of the gene) result if the patient is of Asian ethnicity. Alternatively, it is known that neoadjuvant chemoradiation may alter molecular phenotype. If carcinoma is not tested for mutated BRAF prior to neoadjuvant chemoradiation, knowledge of Asian ethnicity may be the only way to infer the probable mutational status of BRAF. The University of California, Irvine Medical Center (UCI) is a large tertiary care hospital with an ethnically diverse population that includes large Hispanic and Asian populations, in addition to a Caucasian population. Furthermore, UCI also has surgeons, endoscopists, and a pathologist with special interest in colorectal pathology and ethnic diversity. Therefore, UCI provides an excellent opportunity to compare and contrast colorectal disease among Caucasian, Asian, and Hispanic ethnicities. The goal of our research study is to determine whether the prevalence of MSI, mutated BRAF, mutated KRAS, and hypermethylation of the MLH1 gene promoter in colorectal carcinoma differs among Caucasians, Asians, and Hispanics. Because Hispanics are generally closer to Caucasians than to East Asians with respect to geography, it is possible that the molecular phenotype of CRC in Hispanics more closely resembles that of Caucasians than that of East Asians. We hypothesize that the molecular and anatomic profiles of Hispanic carcinomas are expected to be closer to Caucasians than Asians.
physiology of the normal colorectum between individuals. In one study, it was shown that normal adult humans show much variation in the shape, measurement, and configuration of the sigmoid colon. There have been gender differences found in colorectal anatomy in the sigmoid part of the colon. In females, the mesocolon is broader rather than long, whereas in males, the mesocolon is more long than broad. The Eastern Indian patients in this study had a median vertical length of the sigmoid colon of 13 cm, which is longer than the median of 12 cm in Asian patients and the median of 11 cm in the Western patients. This is evidence that there are differences in colorectal anatomy among different ethnic populations. The sigmoid colon in males is longer than wide, and the sigmoid colon in females is wider than long; this may explain the finding that there is a higher incidence of sigmoid volvulus (twisting of the colon around its mesenteric attachment) in males [9]. Further evidence of the gender differences in colon anatomy were found in another study, in which the results showed that the female colon is longer than the male colon. This may explain why colonoscopy is more difficult to undertake in women than in men [10]. In a study comparing diverticula in individuals from Japan and people in the West, it was shown that diverticula are more often in the right colon in people from Japan and more often in the left colon in people from the West. It was concluded that this may represent a difference in the morphology of the colon between these two populations, rather than environmental differences [11]. Considering that ethnicity and gender are known to affect benign disease and anatomical differences, it seems reasonable to postulate that ethnicity and gender may also affect carcinoma. Ethnic differences may exist in the distribution of molecular phenotypes of colorectal carcinomas. For example, one study of colorectal carcinoma showed a lower rate of mutated BRAF in the East than in the West [12]. There appears to be conflicting data, potentially due to regional differences, with regards to the rate of mutated KRAS, microsatellite instability (MSI), and Lynch syndrome within East Asian populations. Some data suggest a lower rate of MSI than in the West. In one study of the A146 mutation of KRAS in a population in Hong Kong, the mutation rate was found to be 4.2% in those with CRC [13]. Another study of Japanese patients found a KRAS mutation rate of 42% in their population [14]. Additionally, a study of CRC among European, Asian, and Latin American populations found mutated KRAS in 36%, 22%, and 40% respectively [15]. Overall, there is variability in the frequency of mutated KRAS among different regional populations of Asia. In regards to MSI, a study done in the Philippines found MSI in about 20–22% of their study population with CRC [16]. In two different studies of the Japanese populations, MSI was found at a rate of 7% in the first population and 8% in the second for patients with CRC [17,18]. In a study comparing the Western and Asian populations, specifically Korea, it was found that the prevalence of MSI in CRC in the Western population is about 15% but is 7% in the Korean population. In this same study, it was stated that Lynch syndrome is present in 3% of the Asian (Chinese/Japanese) and Western populations [19]. A Taiwanese population study found a 2.3% incidence of Lynch syndrome in their patient cohort [20]. Therefore, there is a range in variability among ethnic groups, but most studies report a higher rate for MSI in Western populations than Asian populations. Rectal carcinoma occurs at a higher rate in Japanese individuals compared to those in the Western populations. Higher rates of rectal carcinoma, believed to be generally less affected by MSI, may play a role in the lower rates of MSI in the Asian population, and is another biological difference based on ethnicity [18]. The effect of Hispanic ethnicity on colorectal disease has yet to be fully studied. Very little specific data exist regarding Hispanic CRC, and these data primarily focus on epidemiology or location of carcinoma. To our knowledge, only one study regarding the molecular characteristics of CRC specifically in Hispanics has been published. This study showed a lower rate of MSI in a Puerto Rican population compared to Caucasians, as well as other differences. In particular, the rate of MSI in patients with CRC in this study was found to be 4.3% in Hispanics, which is lower than the rate
2. Materials and methods 2.1. Data sources 2.1.1. CoPath The primary patient search was performed in the CoPath database at the UCI Department of Pathology and Laboratory Medicine using the program InfoMaker Wizard. The title of the search used was “Natural Language Search by Pathologist”. The key words “adenocarcinoma; carcinoma; colon; rectum; colorectum; rectosigmoid; appendix; colonic; rectal; and colorectal” were used in the text search entry. The dates used to search for the cases were collected between 1/1/2004–8/1/2012. In order to obtain certain variables for the selected cases, their pathology reports were examined manually in CoPath. These variables were the diagnosis, immunostaining for hMLH1, hMSH2, hPMS2, and hMSH6, and the genetic testing for KRAS, BRAF, and hypermethylation of the MLH1 promoter. If any cases were benign or if they were carcinoma of another anatomical site, these cases were kept in the data list but were not used in data analysis. A mark was entered into the spreadsheet for these cases in order to keep them separate from cases that would be used in data analysis. Colorectal carcinoma cases with the following diagnoses concluded by the pathologist were considered appropriate for this study: adenocarcinoma, poorly differentiated carcinoma, suspicious for superficial invasive adenocarcinoma, signet ring carcinoma of the colon, intramucosal carcinoma, and any diagnosis which involved the word “adenocarcinoma”. Any other description of the diagnosis by the pathologist was excluded from the data set. There were 7 cases that were appendix carcinoma and were eliminated from this study, since this carcinoma is of a different and unique etiology from colorectal carcinoma. After cases that were not colorectal carcinoma were removed, 864 cases remained in the list of data to be analyzed in this study. 864 accessioned specimens, or cases, involving colorectal carcinoma were collected from CoPath. These cases included all incidences of 721
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PMS2 (EPR3947) was performed with Cell Marque Cat# 288R-18. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 64 min. It was prediluted at 37 ° Celsius incubated for 32 min.
pathologic examination, such as initial biopsy, resection, metastasectomy, and follow-up biopsy. Many patients had more than one case. Of these 864 cases, 562 unique patients were identified and were eligible for study. There were 23 cases that did not have a description of the specific site of the carcinoma in the pathology report, but instead had a description such as “30 cm from anus” or “colon, 30 cm, biopsy”. The site for these cases was recorded based on the division of the rectum and colon at the 15 cm mark, and approximated according to the estimated lengths of the different anatomical parts of the colon [10]. We defined MSI positive as an abnormal immunoprofile, namely, negative staining for any of the 4 mismatch repair proteins, hMLH1, hMSH2, hMSH6 or hPMS2. We defined MSI negative as a normal immunoprofile, namely, positive staining for all of the 4 mismatch repair proteins.
2.7. KRAS KRAS testing was performed at the Mayo Medical Laboratories (Rochester, Minnesota) with a PCR based assay employing allele specific amplification used to test for 7 mutations within codons 12 and 13 of the KRAS gene (Gly12Asp, Gly12Ala, Gly12Val, Gly12Ser, Gly12Arg. Gly12Cys, and Gly13Asp). 2.8. BRAF and MLH1 promoter hypermethylation BRAF and MLH1 hypermethylation testing was performed at the Mayo Medical Laboratories (Rochester, Minnesota) on tissue/tumor using a PCR based assay. Both tumor and normal DNA were tested for the presence of MLH1 promoter hypermethylation and tumor DNA was tested for the presence of the V600E (Val600Glu) mutation in the BRAF gene.
2.2. CDW (Clinical Data Warehouse) database Variables that were not possible to obtain from the CoPath database were collected from the CDW (Clinical Data Warehouse) which is the medical records database at the UCI Medical Center. A list of patients from the CoPath data search was sent to the Center for Bioinformatics. Gender, age, ethnicity, race, date of birth, date of diagnosis, and type of diagnosis were obtained from this department. The type of carcinoma and site of carcinoma were given in the form of ICD9 codes.
2.9. Statistical methods All statistical analyses were performed using the program Statistical Analysis Software (SAS) Version 4.3 (Cary, NC). For statistical analysis, Pearson Chi-square and Fisher’s exact tests were used for categorical variables that included MSI, KRAS, and BRAF. For the comparison of gender and ethnicity, and distal and proximal sites of carcinoma among the three ethnicities, the Pearson Chi-square test was used. The chisquare test was used for positive and negative MSI results and performed vs not performed by ethnicity. Finally, the Fisher’s exact test was used to compare mutant vs wild-type BRAF/KRAS among the three ethnicities and the chi-square test was used to compare performed vs not performed among the three ethnicities. For the continuous variables of age among subgroups, one- and two-way analysis of variance (ANOVA) tests were used. All tests were two-sided with a 0.05 significance level. Since there were no previous data (or very minute) to support using one-sided hypotheses, and despite the limited power for testing differences in genetic markers, the 2-tailed sided hypotheses were the most appropriate. With the available sample size, this study had 80% power to detect differences between ethnicities of an MSI rate of 15% using a chi-square test for equal proportions. With fewer subjects receiving BRAF and KRAS testing, there is 80% power to detect only large differences in mutation rates (> 25%). Thus power to detect small differences among ethnicities in this sample was limited.
2.3. Data review and correction For those patients who had more than one primary carcinoma, the case that was the primary carcinoma was chosen. This was due to the chance that using multiple carcinomas could possibly bias results in certain populations. For those patients who had more than one case per unique medical record number, the case that had testing performed was chosen. If there was no testing performed on any of the cases, then the first case, i.e. the case with the earliest date, was chosen. The CoPath and CDW databases were merged by medical record number and date of birth. 2.4. Ethnicity and race There were multiple categories for Ethnicity and Race in the CDW data. Only the categories Non-Hispanic/White, Hispanic/White, NonHispanic/Asian-Pacific, and Hispanic/Other were included in the data. 2.5. Site of carcinoma For the classification of proximal and distal carcinomas, the proximal locations were the cecum, ascending colon, hepatic flexure, transverse colon, and splenic flexure. The distal locations were the descending colon, sigmoid colon, and rectum.
3. Results Out of the total of 562 patients, 70% (n = 390) were Caucasian, 18% (n = 103) were Asian, and the 12% (n = 69) were Hispanic of the total patient population (Table 1). Overall, 58% (326/562) of the patients were male. Fifty-eight percent (228/390) of Caucasians, 52% (54/103) of Asians, and 64% (44/69) of Hispanics were male. There was no significant difference in gender distribution by ethnicity (p = .32). The mean age at diagnosis for all subjects was 61.8 (SD = 13.7). Mean age was 61.9 (SD = 13.8) in the Caucasian group, 61.4 (SD = 12.6) in the Asian group, and 61.3 (SD = 14.7) in the Hispanic group. The mean age among ethnic groups shown in Table 1. There was no significant difference in age distribution by ethnicity (p = .89). The largest group of patients with colorectal carcinoma was in the 60–69 year old range and the least number occurred in the 10–19 year old range. The age distribution was approximately normal for the patient population in this study (data not shown). The distribution of colorectal carcinomas by site and ethnicity is presented in Table 2. In all three ethnicities, the most frequent site was
2.6. Molecular testing protocol 2.6.1. Immunostaining All immunohistochemistry was performed at the UCI Medical Center Department of Pathology & Laboratory Medicine. Immunostaining tests were performed on the Ventana Benchmark Ultra (Tuscon, AZ) with Ultraview Universal DAB Detection, with Cell Marque (Rocklin, CA). MLH1 (G168-728) was performed with Cell Marque Cat# 285M-16. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 76 min. The dilution was one part antibody to 20 parts diluent at @ 37 ° Celsius incubated for 1 h. MSH2 (G219-1129) was performed with Cell Marque Cat# 286M-16. Antigen retrival was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 64 min. The dilution was one part antibody to 50 parts diluent at room temperature incubated for 32 min. MSH6 (44) was performed with Cell Marque Cat# 287M-16. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 36 min. The dilution was one part antibody to 25 parts diluent at room temperature for 44 min. 722
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Table 1 Total Numbers By Gender and Ethnicity. Gender
Male Female Mean age at diagnosis SD (age) Total
Table 3 Rate of MSI and Ethnicity.
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
228 (58) 162 (42) 61.9 13.8 390 (100)
54 (52) 49 (48) 61.4 12.6 103 (100)
44 (64) 25 (36) 61.3 14.7 69 (100)
MSI Result
Total Not performed Performed Positive Negative Unknown
For Gender and Ethnicity: Pearson chi-square (2 d.f) = 2.29, p = .32, n.s. For mean age by ethnicity: ANOVA: F(2,559 d.f) = 0.12, p = .89, n.s.
Cecum Ascending Hepatic Flexure Transverse Splenic Flexure Descending Sigmoid Rectum Colon, Unspecified Total Proximal Distal Total
Asian
Hispanic
Overall
N (%)
N (%)
N (%)
N (%)
20 (5) 54 (14) 6 (2) 15 (4) 4 (1) 14 (4) 79 (20) 190 (49) 5 (1) 387 (100) 99 (26) 283 (74) 382 (100)
2 (2) 13 (12) 3 (3) 6 (6) 2 (2) 6 (6) 30 (29) 40 (39) 1 (1) 103 (100) 26 (25) 76 (75) 102 (100)
3 (4) 10 (15) 1 (1) 4 (6) 0 (0) 2 (3) 16 (23) 32 (47) 1 (1) 69 (100) 18 (26) 50 (74) 68 (100)
25 (4) 77 (14) 10 (2) 25 (4) 6 (1) 22 (4) 125 (23) 262 (47) 7 (1) 559 (100) 143 (26) 409 (74) 552 (100)
Hispanic
N (%)
N (%)
N (%)
390 (100) 165 (42) 225 (58) 32 (15) 185 (85) 8
103 (100) 43 (42) 60 (58) 5 (8) 54 (92) 1
69 (100) 27 (39) 42 (61) 4 (10) 35 (90) 3
Table 4 BRAF Mutation/Wild-type Rate and Ethnicity. BRAF
Total Not performed Performed Mutant Wild type Insufficient material Indeterminate
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
390 (100) 312 (80) 78 (20) 9 (12) 66 (88) 2 1
103 (100) 84 (82) 19 (18) 1 (6) 17 (94) 0 1
69 (100) 52 (75) 17 (25) 1 (6) 16 (94) 0 0
Test for mutant vs wild type BRAF Result: Fisher’s Exact Test: p = .70, n.s. Test for Not Performed vs Performed: X2 (2) = 1.09, p = .58, n.s.
(10/27) in the Asian group, and 59% (10/17) in the Hispanic group (Table 5). Differences among ethnicities in KRAS mutation rates were not statistically significant (Fisher’s exact test, p = .08) (Table 5). Overall, the rate of KRAS mutation was 40%. The proportion of all patients who received KRAS testing did not differ significantly among ethnic groups (X2 (2) = 0.20, p = .58) (Table 5). The KRAS testing rate was 27% (106/390) in the Caucasian and Asian (28/103) populations, and 25% (17/69) in the Hispanic population. There is an 80% power to detect large (> 25%) differences in KRAS and BRAF mutation rates among the three ethnicities based on the number of patients receiving these two tests. In this study, there was limited power to detect small differences among the three ethnicities. Hypermethylation testing was ordered 9 times in the Caucasian group and 2 times in both the Asian and Hispanic groups. In the Caucasian group, 80% (8/9) were positive for hypermethylation. The Asian group had a 0% (0/2) positive rate for hypermethylation. Of those tested in the Hispanic group, 50% (1/2) were positive for hypermethylation. In two cases, results for hypermethylation testing were discrepant. The first case was a Caucasian patient who had no immunostaining performed but had hypermethylation testing which was positive and BRAF testing which was mutant. The second case was an Asian patient who had positive immunostaining results, negative
Table 2 Site of Carcinoma and Ethnicity. Caucasian
Asian
Test for Positive vs Negative MSI Result: X2 (2) = 1.91, p = .38, n.s. Test for Not Performed vs Performed: X2 (2) = 0.24, p = .89, n.s.
the rectum, and comprised 49% of Caucasians, 39% of Asians, and 47% of Hispanics. The sigmoid colon was the second most common site accounting for 20% in the Caucasian group, 29% in the Asian group, and 23% in the Hispanic group. In all three ethnicities, the majority of carcinomas were distal, being diagnosed in 74% of Caucasians, 75% of Asians, and 74% of Hispanics (Table 2). A total of 327 patients had MSI testing performed. The rate of positive test results for MSI was 15% (32/217) in the Caucasian group, 8% (5/59) in the Asian group, and 10% (4/39) in the Hispanic group (Table 3). There were no significant differences in the rate of positive tests for MSI among the three ethnic groups (X2 (2) = 1.91, p = .38, n.s). There were also no significant differences in the proportion of patients in each ethnic group who were tested for MSI (rate of testing), (X2 (2) = 0.24, p = .89) (Table 3). This study had 80% power to detect differences in MSI rates of 15% among the three ethnicities with the available sample size. Due to the small sample size, the power to detect small differences among the ethnicities is limited. Among patients with BRAF test results (n = 110), the Caucasian group had a BRAF mutation rate of 12% (9/75), and the Asian (1/18) and Hispanic (1/17) groups each had a 6% mutation rate (Table 4). A Fisher’s exact test for difference in BRAF mutation among the three ethnic groups resulted in a non-significant p value of 0.70 (Table 4). The overall rate of BRAF mutation was 10%. The proportion of all patients who had BRAF testing performed is shown in Table 4. The rate of BRAF testing was 20% (78/390) in Caucasians, 18% (19/103) in Asians, and 25% (17/69) in the Hispanic group. The number of patients who did and did not have BRAF testing did not differ significantly among the three ethnicities (X2 (2) = 1.09, p = .58) (Table 4). A total of 151 of 562 patients were tested for KRAS mutations. The rate of KRAS mutation was 38% (39/104) in the Caucasian group, 37%
SITE
Caucasian
Table 5 KRAS Mutation/Wild-type Rate and Ethnicity. KRAS
Total Not performed Performed Mutant Wild type Insufficient material
Pearson chi-square for proximal and distal carcinomas by ethnicity (2 d.f) = 0.02, p = .99, n.s. note: Three patients who had multiple primaries are excluded. Seven patients with carcinomas at unspecified sites excluded.
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
390 (100) 284 (73) 106 (27) 39 (38) 65 (62) 2
103 (100) 75 (73) 28 (27) 10 (37) 17 (63) 1
69 (100) 52 (75) 17 (25) 10 (59) 7 (41) 0
Test for mutant vs wild type KRAS Result: Fisher’s Exact Test: p = .08, n.s. Test for Not Performed vs Performed: X2 (2) = 0.20, p = .58, n.s. 723
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different Asian sub-group and 50% of their study population being under 40 years old. This younger population could be causing the higher rate of MSI given that the younger patients in this study showed a higher proportion of MSI [16]. A study by Gupta et al. [25] demonstrated a 10% rate of MSI in Hispanics. The study by Gonzalez-Keelan [21] showed a 4% rate of MSI in Hispanics. Furthermore, the MSI rate in the Hispanic population in our study (10%) agreed with the rate in the study by Gupta et al. (10%) [25], however it was higher than the rate found in the study by Gonzalez-Keelan (4%) [21]. There was agreement in our study and the majority of previous studies of a higher rate of MSI in Caucasians compared to Asians and Hispanics. However, the previous studies did not conduct statistical testing and the majority of these studies did not include the Caucasian population for comparison. For example, the study by Kang [19] stated a 7% rate of MSI compared to a 15% rate in Caucasians, but these were descriptive findings and were not tested. The findings in the article by Vasovcak et al. [24] were also descriptive and were not statistically tested or compared with other ethnic groups. Similarly, the study by Gonzalez-Keelan [21] showed a 4% rate of MSI in Hispanics and cited a 20% rate of MSI from an American study which was comprised mostly (90%) of Caucasians, but there were no statistical tests done to validate these results. The difference between the results of our study and these other studies could be due to small numbers or could be actual differences. Although our results were not statistically significant, they did agree with the previous studies showing a higher rate of MSI in Caucasians than in Asians and Hispanics. If the non-significant differences among ethnicities in rates of MSI mutation reflect real differences, the implications for these differences need to be addressed. Colorectal carcinoma is known to be caused by both genetic and environmental factors. The Caucasian group in our study had the highest rate of MSI, and this means there is a possibility that MSI may be a more important contributing factor in causing CRC in this group than in the Asian and Hispanic groups. BRAF mutation is associated with MSI-H due to hypermethylation of the promoter of hMLH1. The Caucasian population shows a higher rate of BRAF mutation than the Asian and Hispanic population, thus we would expect the rate of MSI to be higher in the Caucasian group. Furthermore, it has been shown that patients with CRC who have MSI have an overall better prognosis [26]. Given that Caucasians with CRC have an overall higher survival rate than Hispanics, and that in our study Caucasians were found to have a higher rate of MSI, it is possible that MSI could be a component in better CRC survival [27]. Larger population studies in the future will be needed to see if the difference in the rate of MSI between the three ethnicities represents a true difference. The Caucasian group had a non-statistically significant higher rate of BRAF mutation (12%) compared to the Asian (6%) and Hispanic (6%) populations (p = .70). In a previous study, it was stated that a literature review found a 4.2–5.1% rate of BRAF mutation in Asians and a 9.5–11.5% in Caucasians [12]. There were no statistical tests done in this study because these BRAF mutation rates were taken from four independent studies. These four studies did not compare BRAF mutation rates between Caucasians and Asians, and thus it isn’t certain whether rates represent true differences. Our results of BRAF mutation in the Caucasian group were similar to those reported by a literature review referenced by Lee et al. The Caucasians in our study had a 12% rate and the previous studies showed a 9.5–11.5% rate. In addition, our results for BRAF mutation in the Asian population (6%) were similar to the studies in the literature review by Lee et al. (4.2–5.1%) [12]. This study showed a non-significant lower rate of BRAF mutation in the Asian and Hispanic groups compared to the Caucasian group, which agrees with the results by Lee et al. If these non-significant higher rates for BRAF mutations in Caucasians do represent a true difference, there is a possibility that BRAF mutations could be a more significant, genetic contributing factor for CRC in the Caucasian group than in Asians and Hispanics. A previous study noted that CRC patients whose tumors exhibit a BRAF
hypermethylation test, and a BRAF mutation (data not shown). 4. Discussion This study tested the alternative hypothesis that molecular profiles for CRC differ by ethnicity against the null hypothesis of no differences between ethnic groups. The results showed that there were no statistically significant differences among the three groups with respect to mean age and gender distribution. There were more males than females with CRC in this study in all three ethnicities. Males comprised 58%, 52%, and 64% of the Caucasian, Asian and Hispanic groups, respectively. The difference in the percentage of males between the three ethnicities was not statistically significant. There was also no statistically significant difference in sites of carcinoma among the three ethnic groups. There were no statistically significant differences for the rates of MSI, KRAS mutation, or BRAF mutation for the 3 ethnic groups. The gender and age distribution differed from those found in the data from the Surveillance Epidemiology and End Results database [22]. In the SEER study, 51% of the CRC patients were male. The mean age at diagnosis of the population in this study was 61.8 and the mean age in the SEER study was 69 years of age. Compared to the SEER study, there were 7% more males and a lower age at diagnosis in the population in this study. It is possible that the UCI Medical Center population is being screened for CRC at a younger age and thus getting treatment earlier compared to the SEER study population. In addition, the SEER study includes all CRC cases including those diagnosed on death certificate only whereas our study population includes no cases diagnosed only on the death certificate. This could be the reason for the older mean age at diagnosis. It is unclear why there are more males than females, and why this was consistent between ethnicities. There are wide differences in diet between these ethnicities, so that dietary differences are not likely to result in the gender difference. It is possible that exposure to carcinogenic toxins, that might be more common in men than women regardless of ethnicity, such as alcohol or tobacco, could result in carcinoma being more common in men than in women across ethnicities. The most common site for carcinoma in our study population was the rectum in all three ethnicities, with the sigmoid colon being the second most common site. Thus, the majority of colorectal carcinomas in our study were distal. Difference in the site distribution (proximal or distal) of carcinoma among the three ethnic groups did not reach statistical significance. A previous larger study, by the Center for Disease Control and Prevention (CDC) [23], which included data from the National Program of Cancer Registries and SEER study, showed that 52.7% of colorectal carcinomas occurred in the rectum and distal colon. Our study revealed that 74% of CRC were distal. Both our study and the CDC study show that CRC is more commonly distal than proximal. The Caucasian population had the highest rate (15%) of MSI of all three groups. The Asian population had a rate of 8% and the Hispanic population had a rate of 10%. However, there was no statistically significant difference in rate of MSI among the three groups. The rate of MSI in Caucasians was cited to be 15% in a study of the molecular pathways of CRC in Koreans and Caucasians [19]. Another study by Vasovcak et al. [24] that studied 103 sporadic colorectal carcinomas in Czech Patients found a MSI rate of 24.3%. The rate of MSI in the Caucasian group in our study was the same as that found in the study by Kang (15%) [19], and lower than the rate found in the study by Vasovcak et al. (24.3%) [24]. In two studies that examined the rate of MSI in the Japanese population, Hayashi et al. [17] found an 8% rate and a study by Ishikubo et al. [18] found a 7% rate. The study by Kang [19] stated a 7% rate in the Korean population. Interestingly, another study in the Philippines showed a 20–22% rate of MSI in their population [16]. The rate of MSI in the Asian group in this study (8%) agreed with previous studies, except for the 20–22% rate of MSI in the study from the Philippines. Possible reasons for this large difference in the rate of MSI in the study from the Philippines include this representing a 724
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generational differences, and it is possible that data generated from the first generation of an ethnicity might be discordant with that from second or subsequent generations. We did not account for patients that lacked medical insurance. It is possible that indigent patients without insurance would be unable to undergo the complete surgical, pathological, and molecular workup simply due to financial inability to pay. All of these issues potentially affect our data and without very detailed knowledge of the precise methodology of other studies, comparisons between our study and other studies is difficult at best. We studied a subset of the total UCI population. At the UCI Medical Center, East Indians are included in the Asian/Pacific Islander race category, and it was not possible to separate East Indians from the rest of the Asian/Pacific Islanders. Ethnicity and race are self-reported voluntarily by the patients. Since identification of Asian subgroups was not possible, there may be differences between the results of our Asian group compared to other studies which were able to separate East Asians and East Indians. There may be differences in the molecular profile of CRC in some Asian subgroups. This study does not focus on the East Indian population, which is small at UCI and thus most likely does not significantly affect the results for the Asian/Pacific Islander category. Furthermore, in the CoPath/CDW data, there were a set of patients with unknown ethnicity. Had information been available for this set of patients, it may have contributed to the overall size of the study population and the data for the tests that were analyzed. Although the total number of CRC patients in this study was large, the number of patients for whom molecular testing was performed was much smaller. Thus, differences in MSI, BRAF and KRAS mutation rates are small and it is difficult to detect differences in this study due to limited power. We estimate that if we had an approximately 90% increase in the number of patients, or double the number of patients, there would be a higher likelihood for the results to reach statistical significance.
mutation have a poorer prognosis than patients without BRAF mutations [28]. However, Caucasian patients with CRC have higher survival rates than do Hispanics with CRC. Survival rates in Asians, however, are higher than in Caucasians [27]. Because there is a contradiction between the higher survival rate of Caucasians with CRC and prevalence of BRAF mutations indicating higher mortality in those with CRC, there are likely other factors that are contributing to the differences in the survival rates among the three ethnic groups. The Hispanic group in our study had a non-significantly higher rate of KRAS mutation (59%) compared to Asians (37%) and Caucasians (38%) (p = .08). In a previous study, it was shown that Caucasians had a KRAS mutation rate of 36% [15]. The rate of KRAS mutation in our study in the Caucasian group (38%) was similar to the rate found by Ciardiello et al. [15]. This same study showed that Asians had a KRAS mutation rate of 22% [15]. In one study, a specific KRAS mutation in codon 146 (A146) was tested in populations from Hong Kong and the US. The rate of this specific KRAS mutation was 7/126 in the Hong Kong population, approximately 5.5% [13]. Another study in the Japanese population showed a 42% rate of KRAS mutation [14]. A possible reason for the higher rate observed by Doi et al. [14] is the small study population of 24 patients. Rates for KRAS mutations vary widely from one study to another, reflecting small numbers and limited power to detect differences between ethnic groups. The rate of KRAS mutation in the Asian group in our study (37%) were similar to the results in the study by Doi et al. (42%) [14] but not to those found in the study by Edkins et al. (4.2%) [13]. However, there was large variability in KRAS mutation rates in Asians from difference studies. The study by Ciardiello et al. [15] showed a KRAS mutation rate in Hispanics to be 40%, which was lower than the rate in our study (59%). In our study, the Caucasian group had a higher rate of KRAS mutation than in the Asian group. This agrees with previous study by Ciardiello et al. [15]. Our results also agree with Ciardiello’s study showing that Hispanics had a higher rate of KRAS mutation than in the Caucasians. It is important to note that the differences found in the previous studies did not have statistical testing, and they may not reflect true differences because there was no statistical analysis comparing Asians and Hispanics to Caucasians. The higher rate of KRAS mutation in Hispanics was close to significance in our study (p = .08) and thus did give some support for a higher rate in Hispanics. The interpretation of the data regarding KRAS mutations is limited by small sample size. The frequency of KRAS mutations in the Hispanic population was approximately 50% higher than that of the Caucasians and Asian groups. Although this did not quite reach statistical significance, if this represents a true difference, this implies a much higher rate of mutation in Hispanics and therefore a need for the population to be tested more carefully. Furthermore, KRAS mutations could be a larger contributing factor in the cause of CRC in the Hispanic population than in the Caucasian and Asian populations. In a study by Tan et al. [29], it was found that there are contradicting conclusions as to whether KRAS mutations improve or worsen prognosis in patients with CRC. Because patients with KRAS mutations respond differently to anti-EGFR therapy, it is important to test for KRAS and especially important to test if one ethnic group is at higher risk for KRAS mutations than others in order to give patients the best treatment possible. This cursory meta-analysis of the literature demonstrates that our results differ in certain respects and are similar in other respects with prior studies. Although a formal meta-analysis is beyond the scope of our project, the differences and similarities seemingly lack a recognizable pattern when viewed as a whole. There are a number of possible factors that might contribute to these differences and similarities. Our study was based on only one institution, whereas other studies may have pooled data from multiple institutions. We were not able to control for important dietary variables. It is quite possible that the three ethnicities had nearly identical diets, given that the patients were all living within the same county. We were not able to control for
5. Conclusions The results of this study are consistent with the null hypothesis that there are no statistically significant differences in the molecular profiles of colorectal carcinoma among the Caucasian, Asian, and Hispanic populations at the UCI Medical Center. This study did show non-significantly higher rates in the Caucasian group for MSI (15% vs. 8% in Asians and 10% in Hispanics) and mutated BRAF (12% vs. 6% in Asians and 6% in Hispanics) and a non-significantly higher rate of KRAS mutation in the Hispanic population (59% vs. 37% in Asians and 38% in Caucasians). However, our study population was not large enough to detect statistically significant differences among ethnic groups. The characteristics of colorectal carcinoma are similar in some respects and different in other respects among the Hispanic and other groups. The progress in our knowledge of genetics and our understanding of the causes of carcinoma and the implications for treatment has increased and will lead to better prevention, earlier diagnosis, and better treatments for colorectal carcinoma. As more tests are ordered, the differences among the ethnicities may become more evident. Larger population studies will be needed to reveal whether our non-significant findings of ethnic differences in molecular profiles which were too small to achieve statistical significance represent real differences. Colorectal carcinoma is a significant public health problem and affects people of both genders and from all ethnic groups and ages. It is important for genetic research to continue in order to gather more data about the molecular characteristics of colorectal carcinoma among different ethnic groups, particularly in the Hispanic population. Evaluation and interpretation of these data are essential for improved prevention, diagnosis, and treatment for colorectal carcinoma. Conflicts of interest The authors have no conflicts of interest to declare. 725
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Funding
[15] F. Ciardiello, S. Tejpar, N. Normanno, D. Mercadante, T. Teague, B. Wohlschlegel, E. Van Cutsem, Uptake of KRAS mutation testing in patients with metastatic colorectal cancer in Europe, Latin America and Asia, Targeted Oncol. 6 (3) (2011) 133–145. [16] G.B. Uy, L.L. Kaw, C.K. Punzalan, R.I.L.C. Querol, E.V. Koustova, M.W. Bowyer, C.M. Hobbs, et al., Clinical and molecular biologic characteristics of early-onset versus late-onset colorectal carcinoma in Filipinos, World J. Surg. 28 (2) (2004) 117–123. [17] T. Hayashi, M. Arai, M. Ueno, H. Kinoshita, Y. Tada, K. Koizumi, Y. Miki, et al., Frequency of immunohistochemical loss of mismatch repair protein in double primary cancers of the colorectum and stomach in Japan, Dis. Colon Rectum 49 (10 Suppl) (2006) S23–9. [18] T. Ishikubo, Y. Nishimura, K. Yamaguchi, U. Khansuwan, Y. Arai, T. Kobayashi, Y. Ohkura, et al., The clinical features of rectal cancers with high-frequency microsatellite instability (MSI-H) in Japanese males, Cancer Lett. 216 (1) (2004) 55–62. [19] G.H. Kang, Four molecular subtypes of colorectal cancer and their precursor lesions, Arch. Pathol. Lab. Med. 135 (2011) 698–703. [20] S.-C. Chang, P.-C. Lin, S.-H. Yang, H.-S. Wang, W.-Y. Liang, J.-K. Lin, Taiwan hospital-based detection of Lynch syndrome distinguishes 2 types of microsatellite instabilities in colorectal cancers, Surgery 147 (5) (2010) 720–728. [21] C. Gonzalez-Keelan, S.R. Hamilton, M. Cruz-Correa, Mismatch repair protein expression and colorectal cancer in Hispanics from Puerto Rico, Fam. Cancer 9 (2) (2010) 155–166. [22] SEER Stat Fact Sheets: Colon and Rectum. (n.d.). Surveillance Epidemiology and End Results. Retrieved May 13, 2013, from http://seer.cancer.gov/statfacts/html/ colorect.html. [23] S.H. Rim, L. Seeff, F. Ahmed, J.B. King, S.S. Coughlin, Colorectal cancer incidence in the United States, 1999–2004: an updated analysis of data from the National Program of Cancer Registries and the Surveillance, Epidemiology, and End Results Program, Cancer 115 (9) (2009) 1967–1976. [24] P. Vasovcak, K. Pavlikova, Z. Sedlacek, P. Skapa, M. Kouda, J. Hoch, A. Krepelova, Molecular genetic analysis of 103 sporadic colorectal tumours in Czech patients, PLoS One 6 (8) (2011) e24114. [25] S. Gupta, R. Ashfaq, P. Kapur, B.B. Afonso, T.-P.T. Nguyen, F. Ansari, C.R. Boland, et al., Microsatellite instability among individuals of Hispanic origin with colorectal cancer, Cancer 116 (21) (2010) 4965–4972. [26] L. Laghi, A. Malesci, Microsatellite instability and therapeutic consequences in colorectal cancer, Dig. Dis. (Basel, Switzerland) 30 (3) (2012) 304–309. [27] C. Chien, L.M. Morimoto, J. Tom, C.I. Li, Differences in colorectal carcinoma stage and survival by race and ethnicity, Cancer 104 (3) (2005) 629–639. [28] A.I. Phipps, D.D. Buchanan, K.W. Makar, A.N. Burnett-Hartman, A.E. Coghill, M.N. Passarelli, J.A. Baron, et al., BRAF mutation status and survival after colorectal cancer diagnosis according to patient and tumor characteristics, Cancer Epidemiol. 21 (10) (2012) 1792–1798. [29] C. Tan, X. Du, KRAS mutation testing in metastatic colorectal cancer, World J. Gastroenterol.: WJG 18 (37) (2012) 5171–5180.
This study did not require funding. Acknowledgments The authors thank Professor Emerita M. Anne Spence, PhD, Pamela Flodman, MSc, MS, LCGC, and Adjunct Professor Kathryn E. Osann, PhD for their help with this project. References [1] J. Zeller, Colon cancer screening, JAMA 300 (23) (2008) 2816. [2] A. Jemal, R. Siegel, J. Xu, E. Ward, Cancer statistics, 2010 Both sexes female both sexes estimated deaths, CA: Cancer J. Clin. 60 (5) (2010) 277–300. [3] E.T. Hawk, B. Levin, Colorectal cancer prevention, J. Clin. Oncol. 23 (2) (2005) 378–391. [4] A.J.M. Watson, P.D. Collins, Colon cancer: a civilization disorder, Dig. Dis. (Basel, Switzerland) 29 (2) (2011) 222–228. [5] M. Goodenberger, N.M. Lindor, Lynch syndrome and MYH-associated polyposis: review and testing strategy, J. Clin. Gastroenterol. 45 (6) (2011) 488–500. [6] R. Burt, Inheritance of colorectal cancer, Drug Discov. Today: Dis. Mech. 4 (4) (2007) 293–300. [7] F.J. Backes, D.E. Cohn, Lynch syndrome, Clin. Obstet. Gynecol. 54 (2) (2011) 199–214. [8] A.N. Bellizzi, W.L. Frankel, Colorectal cancer due to deficiency in DNA mismatch repair function: a review, Adv. Anat. Pathol. 16 (6) (2009) 405–417. [9] B.N.S. Bhatnagar, C.L.N. Sharma, S.N. Gupta, M.M. Mathur, D.C.S. Reddy, Study on the anatomical dimensions of the human sigmoid colon, Clin. Anat. 17 (3) (2004) 236–243. [10] B.P. Saunders, M. Fukumoto, S. Halligan, C. Jobling, M.E. Moussa, C.I. Bartram, C.B. Williams, Why is colonoscopy more difficult in women? Gastrointest. Endosc. 43 (2) (1996) 124–126. [11] S. Nakaji, K. Danjo, A. Munakata, K. Sugawara, D. MacAuley, G. Kernohan, D. Baxter, Comparison of etiology of right-sided diverticula in Japan with that of left-sided diverticula in the West, Int. J. Colorectal Dis. 17 (6) (2002) 365–373. [12] S. Lee, N.-Y. Cho, M. Choi, E.J. Yoo, J.-H. Kim, G.H. Kang, Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation, Pathol. Int. 58 (2) (2008) 104–113. [13] S. Edkins, S.O. Meara, A. Parker, C. Stevens, M. Reis, C. Greenman, H. Davies, et al., Recurrent KRAS codon 146 mutations in human colorectal cancer, Cancer Biol. Ther. 5 (8) (2006) 928–932. [14] T. Doi, M. Tahara, T. Yoshino, K. Yamazaki, T. Tamura, Y. Yamada, B.-B. Yang, et al., Tumor KRAS status predicts responsiveness to panitumumab in Japanese patients with metastatic colorectal cancer, Jpn. J. Clin. Oncol. 41 (2) (2011) 210–216.
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Phenotypic and genotypic differences in colorectal carcinoma among Caucasians, Asians, and Hispanics lack statistical significance
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Sara J. Hoffmana, , Mark Li-cheng Wub a b
University of California, Irvine School of Medicine, Division of Genetic and Genomic Medicine, Department of Pediatrics, Irvine, CA, USA University of California, Irvine School of Medicine, Department of Pathology and Laboratory Medicine, Irvine, CA, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Colorectal carcinoma Microsatellite instability Colon BRAF KRAS Ethnicity
Colorectal carcinoma (CRC) has been shown to have both genetic and environmental factors that can promote carcinoma development. Previous studies have found ethnic differences in the distribution of molecular phenotypes of CRC. Very little specific data exist regarding Hispanic CRC, and these data primarily focus on epidemiology or location of carcinoma. Our retrospective study analyzed 562 Caucasian, Asian, and Hispanic CRC patients at the UCI Medical Center from 2004 to 2012. The results showed that there were no statistically significant differences with respect to mean age, gender or site of carcinoma among the three ethnic groups. There were no statistically significant differences among the three ethnicities with respect to rates of MSI, mutated BRAF, and mutated KRAS. The Caucasian group had a non-significant higher rate of MSI (15%) and BRAF mutation (12%) than the Asian and Hispanic groups. Hispanics had a non-significant higher rate of KRAS mutation (59%) than Caucasians (38%) and Asians (37%). The results of this study demonstrated a higher rate of MSI and BRAF mutation in the Caucasian group and a higher rate of KRAS mutation in the Hispanic group, however differences were not statistically significant.
1. Introduction Colorectal carcinoma (CRC) is carcinoma which originates in the colon or the rectum, and it has been shown that both genetics and environment can promote carcinoma development [1]. Being the third most common carcinoma in both men and women, CRC is the second leading cause of carcinoma death in the United States. In 2010, there were 51,370 CRC deaths and 142,570 new cases of CRC [2]. The cumulative lifetime risk of developing CRC in the United States is 6% [3]. In developing (Western) societies, there are environmental factors that probably contribute to higher rates of CRC, such as diet, obesity, lack of physical activity, smoking, and alcohol consumption [4]. Because of the impact of this carcinoma on the general population, it is important to study colorectal carcinoma. Approximately 70% of all colorectal carcinoma is sporadic, which means that the carcinoma is caused by a series of genetic or epigenetic changes that accumulate, and are not caused by an inherited predisposition. About 20–30% of all CRC’s are familial, and genetic association and population studies have been discovering low penetrance susceptibility loci and polymorphisms. These loci and polymorphisms, along with shared environmental factors, are likely to increase risk of CRC in families that exhibit familial inheritance patterns of CRC. These
inheritance patterns do not fit well-established major gene patterns of inheritance. About 5% of CRC is due to an hereditary component, which is caused by highly penetrant mutations at major gene loci [5]. There are many different hereditary CRC syndromes. The most common are Lynch syndrome (previously known as hereditary nonpolyposis colorectal cancer) that accounts for roughly 1–3% of CRC cases and familial adenomatous polyposis (FAP) that accounts for less than 1% of CRC cases. Other rare hereditary CRC syndromes, which also account for less than 1% of CRC cases, are attenuated familial adenomatous polyposis (AFAP), MutY homolog-associated polyposis (MUTYH-associated polyposis, MAP), Peutz-Jeghers syndrome, juvenile polyposis syndrome, Cowden syndrome, hereditary mixed polyposis, and serrated polyposis (formerly hyperplastic polyposis) [6]. Lynch syndrome accounts for 1%-5% of all colorectal carcinomas, is the most common hereditary colorectal carcinoma, and is caused by germline mutations of hMLH1, hMSH2, hMSH6 and hPMS2, the genes that encode mismatch repair proteins, and the mutation of the related gene, EPCAM [7,8]. It is well known that the phenotypic and genotypic characteristics of colorectal carcinoma varies between individuals. Interestingly, there are many differences regarding the gross anatomy, microanatomy and
Abbreviations:MSI, microsatellite instability ⁎ Corresponding author at: Sutter Pacific Medical Foundation, 3883 Airway Dr, Ste 300, Santa Rosa, CA 95403, USA. E-mail address: [email protected] (S.J. Hoffman). https://doi.org/10.1016/j.prp.2018.03.008 Received 11 December 2017; Received in revised form 25 February 2018; Accepted 2 March 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
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of 15% found in the Caucasian population [21]. Because Hispanic and Asian cultures have long and contrasting histories with respect to geography, culture, diet, and environmental exposures, it seems reasonable to hypothesize that there may be significant differences in colorectal neoplasia between these ethnicities. Knowledge of the effect of ethnicity on CRC can potentially impact diagnosis and management, particularly in cases where histology or molecular phenotype is indeterminate or unavailable. For example, given that BRAF is mutated at a lower rate in the East, an indeterminate BRAF result might be more likely to be considered a wild-type (the typical form of the gene) result if the patient is of Asian ethnicity. Alternatively, it is known that neoadjuvant chemoradiation may alter molecular phenotype. If carcinoma is not tested for mutated BRAF prior to neoadjuvant chemoradiation, knowledge of Asian ethnicity may be the only way to infer the probable mutational status of BRAF. The University of California, Irvine Medical Center (UCI) is a large tertiary care hospital with an ethnically diverse population that includes large Hispanic and Asian populations, in addition to a Caucasian population. Furthermore, UCI also has surgeons, endoscopists, and a pathologist with special interest in colorectal pathology and ethnic diversity. Therefore, UCI provides an excellent opportunity to compare and contrast colorectal disease among Caucasian, Asian, and Hispanic ethnicities. The goal of our research study is to determine whether the prevalence of MSI, mutated BRAF, mutated KRAS, and hypermethylation of the MLH1 gene promoter in colorectal carcinoma differs among Caucasians, Asians, and Hispanics. Because Hispanics are generally closer to Caucasians than to East Asians with respect to geography, it is possible that the molecular phenotype of CRC in Hispanics more closely resembles that of Caucasians than that of East Asians. We hypothesize that the molecular and anatomic profiles of Hispanic carcinomas are expected to be closer to Caucasians than Asians.
physiology of the normal colorectum between individuals. In one study, it was shown that normal adult humans show much variation in the shape, measurement, and configuration of the sigmoid colon. There have been gender differences found in colorectal anatomy in the sigmoid part of the colon. In females, the mesocolon is broader rather than long, whereas in males, the mesocolon is more long than broad. The Eastern Indian patients in this study had a median vertical length of the sigmoid colon of 13 cm, which is longer than the median of 12 cm in Asian patients and the median of 11 cm in the Western patients. This is evidence that there are differences in colorectal anatomy among different ethnic populations. The sigmoid colon in males is longer than wide, and the sigmoid colon in females is wider than long; this may explain the finding that there is a higher incidence of sigmoid volvulus (twisting of the colon around its mesenteric attachment) in males [9]. Further evidence of the gender differences in colon anatomy were found in another study, in which the results showed that the female colon is longer than the male colon. This may explain why colonoscopy is more difficult to undertake in women than in men [10]. In a study comparing diverticula in individuals from Japan and people in the West, it was shown that diverticula are more often in the right colon in people from Japan and more often in the left colon in people from the West. It was concluded that this may represent a difference in the morphology of the colon between these two populations, rather than environmental differences [11]. Considering that ethnicity and gender are known to affect benign disease and anatomical differences, it seems reasonable to postulate that ethnicity and gender may also affect carcinoma. Ethnic differences may exist in the distribution of molecular phenotypes of colorectal carcinomas. For example, one study of colorectal carcinoma showed a lower rate of mutated BRAF in the East than in the West [12]. There appears to be conflicting data, potentially due to regional differences, with regards to the rate of mutated KRAS, microsatellite instability (MSI), and Lynch syndrome within East Asian populations. Some data suggest a lower rate of MSI than in the West. In one study of the A146 mutation of KRAS in a population in Hong Kong, the mutation rate was found to be 4.2% in those with CRC [13]. Another study of Japanese patients found a KRAS mutation rate of 42% in their population [14]. Additionally, a study of CRC among European, Asian, and Latin American populations found mutated KRAS in 36%, 22%, and 40% respectively [15]. Overall, there is variability in the frequency of mutated KRAS among different regional populations of Asia. In regards to MSI, a study done in the Philippines found MSI in about 20–22% of their study population with CRC [16]. In two different studies of the Japanese populations, MSI was found at a rate of 7% in the first population and 8% in the second for patients with CRC [17,18]. In a study comparing the Western and Asian populations, specifically Korea, it was found that the prevalence of MSI in CRC in the Western population is about 15% but is 7% in the Korean population. In this same study, it was stated that Lynch syndrome is present in 3% of the Asian (Chinese/Japanese) and Western populations [19]. A Taiwanese population study found a 2.3% incidence of Lynch syndrome in their patient cohort [20]. Therefore, there is a range in variability among ethnic groups, but most studies report a higher rate for MSI in Western populations than Asian populations. Rectal carcinoma occurs at a higher rate in Japanese individuals compared to those in the Western populations. Higher rates of rectal carcinoma, believed to be generally less affected by MSI, may play a role in the lower rates of MSI in the Asian population, and is another biological difference based on ethnicity [18]. The effect of Hispanic ethnicity on colorectal disease has yet to be fully studied. Very little specific data exist regarding Hispanic CRC, and these data primarily focus on epidemiology or location of carcinoma. To our knowledge, only one study regarding the molecular characteristics of CRC specifically in Hispanics has been published. This study showed a lower rate of MSI in a Puerto Rican population compared to Caucasians, as well as other differences. In particular, the rate of MSI in patients with CRC in this study was found to be 4.3% in Hispanics, which is lower than the rate
2. Materials and methods 2.1. Data sources 2.1.1. CoPath The primary patient search was performed in the CoPath database at the UCI Department of Pathology and Laboratory Medicine using the program InfoMaker Wizard. The title of the search used was “Natural Language Search by Pathologist”. The key words “adenocarcinoma; carcinoma; colon; rectum; colorectum; rectosigmoid; appendix; colonic; rectal; and colorectal” were used in the text search entry. The dates used to search for the cases were collected between 1/1/2004–8/1/2012. In order to obtain certain variables for the selected cases, their pathology reports were examined manually in CoPath. These variables were the diagnosis, immunostaining for hMLH1, hMSH2, hPMS2, and hMSH6, and the genetic testing for KRAS, BRAF, and hypermethylation of the MLH1 promoter. If any cases were benign or if they were carcinoma of another anatomical site, these cases were kept in the data list but were not used in data analysis. A mark was entered into the spreadsheet for these cases in order to keep them separate from cases that would be used in data analysis. Colorectal carcinoma cases with the following diagnoses concluded by the pathologist were considered appropriate for this study: adenocarcinoma, poorly differentiated carcinoma, suspicious for superficial invasive adenocarcinoma, signet ring carcinoma of the colon, intramucosal carcinoma, and any diagnosis which involved the word “adenocarcinoma”. Any other description of the diagnosis by the pathologist was excluded from the data set. There were 7 cases that were appendix carcinoma and were eliminated from this study, since this carcinoma is of a different and unique etiology from colorectal carcinoma. After cases that were not colorectal carcinoma were removed, 864 cases remained in the list of data to be analyzed in this study. 864 accessioned specimens, or cases, involving colorectal carcinoma were collected from CoPath. These cases included all incidences of 721
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PMS2 (EPR3947) was performed with Cell Marque Cat# 288R-18. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 64 min. It was prediluted at 37 ° Celsius incubated for 32 min.
pathologic examination, such as initial biopsy, resection, metastasectomy, and follow-up biopsy. Many patients had more than one case. Of these 864 cases, 562 unique patients were identified and were eligible for study. There were 23 cases that did not have a description of the specific site of the carcinoma in the pathology report, but instead had a description such as “30 cm from anus” or “colon, 30 cm, biopsy”. The site for these cases was recorded based on the division of the rectum and colon at the 15 cm mark, and approximated according to the estimated lengths of the different anatomical parts of the colon [10]. We defined MSI positive as an abnormal immunoprofile, namely, negative staining for any of the 4 mismatch repair proteins, hMLH1, hMSH2, hMSH6 or hPMS2. We defined MSI negative as a normal immunoprofile, namely, positive staining for all of the 4 mismatch repair proteins.
2.7. KRAS KRAS testing was performed at the Mayo Medical Laboratories (Rochester, Minnesota) with a PCR based assay employing allele specific amplification used to test for 7 mutations within codons 12 and 13 of the KRAS gene (Gly12Asp, Gly12Ala, Gly12Val, Gly12Ser, Gly12Arg. Gly12Cys, and Gly13Asp). 2.8. BRAF and MLH1 promoter hypermethylation BRAF and MLH1 hypermethylation testing was performed at the Mayo Medical Laboratories (Rochester, Minnesota) on tissue/tumor using a PCR based assay. Both tumor and normal DNA were tested for the presence of MLH1 promoter hypermethylation and tumor DNA was tested for the presence of the V600E (Val600Glu) mutation in the BRAF gene.
2.2. CDW (Clinical Data Warehouse) database Variables that were not possible to obtain from the CoPath database were collected from the CDW (Clinical Data Warehouse) which is the medical records database at the UCI Medical Center. A list of patients from the CoPath data search was sent to the Center for Bioinformatics. Gender, age, ethnicity, race, date of birth, date of diagnosis, and type of diagnosis were obtained from this department. The type of carcinoma and site of carcinoma were given in the form of ICD9 codes.
2.9. Statistical methods All statistical analyses were performed using the program Statistical Analysis Software (SAS) Version 4.3 (Cary, NC). For statistical analysis, Pearson Chi-square and Fisher’s exact tests were used for categorical variables that included MSI, KRAS, and BRAF. For the comparison of gender and ethnicity, and distal and proximal sites of carcinoma among the three ethnicities, the Pearson Chi-square test was used. The chisquare test was used for positive and negative MSI results and performed vs not performed by ethnicity. Finally, the Fisher’s exact test was used to compare mutant vs wild-type BRAF/KRAS among the three ethnicities and the chi-square test was used to compare performed vs not performed among the three ethnicities. For the continuous variables of age among subgroups, one- and two-way analysis of variance (ANOVA) tests were used. All tests were two-sided with a 0.05 significance level. Since there were no previous data (or very minute) to support using one-sided hypotheses, and despite the limited power for testing differences in genetic markers, the 2-tailed sided hypotheses were the most appropriate. With the available sample size, this study had 80% power to detect differences between ethnicities of an MSI rate of 15% using a chi-square test for equal proportions. With fewer subjects receiving BRAF and KRAS testing, there is 80% power to detect only large differences in mutation rates (> 25%). Thus power to detect small differences among ethnicities in this sample was limited.
2.3. Data review and correction For those patients who had more than one primary carcinoma, the case that was the primary carcinoma was chosen. This was due to the chance that using multiple carcinomas could possibly bias results in certain populations. For those patients who had more than one case per unique medical record number, the case that had testing performed was chosen. If there was no testing performed on any of the cases, then the first case, i.e. the case with the earliest date, was chosen. The CoPath and CDW databases were merged by medical record number and date of birth. 2.4. Ethnicity and race There were multiple categories for Ethnicity and Race in the CDW data. Only the categories Non-Hispanic/White, Hispanic/White, NonHispanic/Asian-Pacific, and Hispanic/Other were included in the data. 2.5. Site of carcinoma For the classification of proximal and distal carcinomas, the proximal locations were the cecum, ascending colon, hepatic flexure, transverse colon, and splenic flexure. The distal locations were the descending colon, sigmoid colon, and rectum.
3. Results Out of the total of 562 patients, 70% (n = 390) were Caucasian, 18% (n = 103) were Asian, and the 12% (n = 69) were Hispanic of the total patient population (Table 1). Overall, 58% (326/562) of the patients were male. Fifty-eight percent (228/390) of Caucasians, 52% (54/103) of Asians, and 64% (44/69) of Hispanics were male. There was no significant difference in gender distribution by ethnicity (p = .32). The mean age at diagnosis for all subjects was 61.8 (SD = 13.7). Mean age was 61.9 (SD = 13.8) in the Caucasian group, 61.4 (SD = 12.6) in the Asian group, and 61.3 (SD = 14.7) in the Hispanic group. The mean age among ethnic groups shown in Table 1. There was no significant difference in age distribution by ethnicity (p = .89). The largest group of patients with colorectal carcinoma was in the 60–69 year old range and the least number occurred in the 10–19 year old range. The age distribution was approximately normal for the patient population in this study (data not shown). The distribution of colorectal carcinomas by site and ethnicity is presented in Table 2. In all three ethnicities, the most frequent site was
2.6. Molecular testing protocol 2.6.1. Immunostaining All immunohistochemistry was performed at the UCI Medical Center Department of Pathology & Laboratory Medicine. Immunostaining tests were performed on the Ventana Benchmark Ultra (Tuscon, AZ) with Ultraview Universal DAB Detection, with Cell Marque (Rocklin, CA). MLH1 (G168-728) was performed with Cell Marque Cat# 285M-16. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 76 min. The dilution was one part antibody to 20 parts diluent at @ 37 ° Celsius incubated for 1 h. MSH2 (G219-1129) was performed with Cell Marque Cat# 286M-16. Antigen retrival was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 64 min. The dilution was one part antibody to 50 parts diluent at room temperature incubated for 32 min. MSH6 (44) was performed with Cell Marque Cat# 287M-16. Antigen retrieval was done at 95 ° Celsius with Ultra CC1 at a pH of 8.0 for 36 min. The dilution was one part antibody to 25 parts diluent at room temperature for 44 min. 722
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Table 1 Total Numbers By Gender and Ethnicity. Gender
Male Female Mean age at diagnosis SD (age) Total
Table 3 Rate of MSI and Ethnicity.
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
228 (58) 162 (42) 61.9 13.8 390 (100)
54 (52) 49 (48) 61.4 12.6 103 (100)
44 (64) 25 (36) 61.3 14.7 69 (100)
MSI Result
Total Not performed Performed Positive Negative Unknown
For Gender and Ethnicity: Pearson chi-square (2 d.f) = 2.29, p = .32, n.s. For mean age by ethnicity: ANOVA: F(2,559 d.f) = 0.12, p = .89, n.s.
Cecum Ascending Hepatic Flexure Transverse Splenic Flexure Descending Sigmoid Rectum Colon, Unspecified Total Proximal Distal Total
Asian
Hispanic
Overall
N (%)
N (%)
N (%)
N (%)
20 (5) 54 (14) 6 (2) 15 (4) 4 (1) 14 (4) 79 (20) 190 (49) 5 (1) 387 (100) 99 (26) 283 (74) 382 (100)
2 (2) 13 (12) 3 (3) 6 (6) 2 (2) 6 (6) 30 (29) 40 (39) 1 (1) 103 (100) 26 (25) 76 (75) 102 (100)
3 (4) 10 (15) 1 (1) 4 (6) 0 (0) 2 (3) 16 (23) 32 (47) 1 (1) 69 (100) 18 (26) 50 (74) 68 (100)
25 (4) 77 (14) 10 (2) 25 (4) 6 (1) 22 (4) 125 (23) 262 (47) 7 (1) 559 (100) 143 (26) 409 (74) 552 (100)
Hispanic
N (%)
N (%)
N (%)
390 (100) 165 (42) 225 (58) 32 (15) 185 (85) 8
103 (100) 43 (42) 60 (58) 5 (8) 54 (92) 1
69 (100) 27 (39) 42 (61) 4 (10) 35 (90) 3
Table 4 BRAF Mutation/Wild-type Rate and Ethnicity. BRAF
Total Not performed Performed Mutant Wild type Insufficient material Indeterminate
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
390 (100) 312 (80) 78 (20) 9 (12) 66 (88) 2 1
103 (100) 84 (82) 19 (18) 1 (6) 17 (94) 0 1
69 (100) 52 (75) 17 (25) 1 (6) 16 (94) 0 0
Test for mutant vs wild type BRAF Result: Fisher’s Exact Test: p = .70, n.s. Test for Not Performed vs Performed: X2 (2) = 1.09, p = .58, n.s.
(10/27) in the Asian group, and 59% (10/17) in the Hispanic group (Table 5). Differences among ethnicities in KRAS mutation rates were not statistically significant (Fisher’s exact test, p = .08) (Table 5). Overall, the rate of KRAS mutation was 40%. The proportion of all patients who received KRAS testing did not differ significantly among ethnic groups (X2 (2) = 0.20, p = .58) (Table 5). The KRAS testing rate was 27% (106/390) in the Caucasian and Asian (28/103) populations, and 25% (17/69) in the Hispanic population. There is an 80% power to detect large (> 25%) differences in KRAS and BRAF mutation rates among the three ethnicities based on the number of patients receiving these two tests. In this study, there was limited power to detect small differences among the three ethnicities. Hypermethylation testing was ordered 9 times in the Caucasian group and 2 times in both the Asian and Hispanic groups. In the Caucasian group, 80% (8/9) were positive for hypermethylation. The Asian group had a 0% (0/2) positive rate for hypermethylation. Of those tested in the Hispanic group, 50% (1/2) were positive for hypermethylation. In two cases, results for hypermethylation testing were discrepant. The first case was a Caucasian patient who had no immunostaining performed but had hypermethylation testing which was positive and BRAF testing which was mutant. The second case was an Asian patient who had positive immunostaining results, negative
Table 2 Site of Carcinoma and Ethnicity. Caucasian
Asian
Test for Positive vs Negative MSI Result: X2 (2) = 1.91, p = .38, n.s. Test for Not Performed vs Performed: X2 (2) = 0.24, p = .89, n.s.
the rectum, and comprised 49% of Caucasians, 39% of Asians, and 47% of Hispanics. The sigmoid colon was the second most common site accounting for 20% in the Caucasian group, 29% in the Asian group, and 23% in the Hispanic group. In all three ethnicities, the majority of carcinomas were distal, being diagnosed in 74% of Caucasians, 75% of Asians, and 74% of Hispanics (Table 2). A total of 327 patients had MSI testing performed. The rate of positive test results for MSI was 15% (32/217) in the Caucasian group, 8% (5/59) in the Asian group, and 10% (4/39) in the Hispanic group (Table 3). There were no significant differences in the rate of positive tests for MSI among the three ethnic groups (X2 (2) = 1.91, p = .38, n.s). There were also no significant differences in the proportion of patients in each ethnic group who were tested for MSI (rate of testing), (X2 (2) = 0.24, p = .89) (Table 3). This study had 80% power to detect differences in MSI rates of 15% among the three ethnicities with the available sample size. Due to the small sample size, the power to detect small differences among the ethnicities is limited. Among patients with BRAF test results (n = 110), the Caucasian group had a BRAF mutation rate of 12% (9/75), and the Asian (1/18) and Hispanic (1/17) groups each had a 6% mutation rate (Table 4). A Fisher’s exact test for difference in BRAF mutation among the three ethnic groups resulted in a non-significant p value of 0.70 (Table 4). The overall rate of BRAF mutation was 10%. The proportion of all patients who had BRAF testing performed is shown in Table 4. The rate of BRAF testing was 20% (78/390) in Caucasians, 18% (19/103) in Asians, and 25% (17/69) in the Hispanic group. The number of patients who did and did not have BRAF testing did not differ significantly among the three ethnicities (X2 (2) = 1.09, p = .58) (Table 4). A total of 151 of 562 patients were tested for KRAS mutations. The rate of KRAS mutation was 38% (39/104) in the Caucasian group, 37%
SITE
Caucasian
Table 5 KRAS Mutation/Wild-type Rate and Ethnicity. KRAS
Total Not performed Performed Mutant Wild type Insufficient material
Pearson chi-square for proximal and distal carcinomas by ethnicity (2 d.f) = 0.02, p = .99, n.s. note: Three patients who had multiple primaries are excluded. Seven patients with carcinomas at unspecified sites excluded.
Caucasian
Asian
Hispanic
N (%)
N (%)
N (%)
390 (100) 284 (73) 106 (27) 39 (38) 65 (62) 2
103 (100) 75 (73) 28 (27) 10 (37) 17 (63) 1
69 (100) 52 (75) 17 (25) 10 (59) 7 (41) 0
Test for mutant vs wild type KRAS Result: Fisher’s Exact Test: p = .08, n.s. Test for Not Performed vs Performed: X2 (2) = 0.20, p = .58, n.s. 723
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different Asian sub-group and 50% of their study population being under 40 years old. This younger population could be causing the higher rate of MSI given that the younger patients in this study showed a higher proportion of MSI [16]. A study by Gupta et al. [25] demonstrated a 10% rate of MSI in Hispanics. The study by Gonzalez-Keelan [21] showed a 4% rate of MSI in Hispanics. Furthermore, the MSI rate in the Hispanic population in our study (10%) agreed with the rate in the study by Gupta et al. (10%) [25], however it was higher than the rate found in the study by Gonzalez-Keelan (4%) [21]. There was agreement in our study and the majority of previous studies of a higher rate of MSI in Caucasians compared to Asians and Hispanics. However, the previous studies did not conduct statistical testing and the majority of these studies did not include the Caucasian population for comparison. For example, the study by Kang [19] stated a 7% rate of MSI compared to a 15% rate in Caucasians, but these were descriptive findings and were not tested. The findings in the article by Vasovcak et al. [24] were also descriptive and were not statistically tested or compared with other ethnic groups. Similarly, the study by Gonzalez-Keelan [21] showed a 4% rate of MSI in Hispanics and cited a 20% rate of MSI from an American study which was comprised mostly (90%) of Caucasians, but there were no statistical tests done to validate these results. The difference between the results of our study and these other studies could be due to small numbers or could be actual differences. Although our results were not statistically significant, they did agree with the previous studies showing a higher rate of MSI in Caucasians than in Asians and Hispanics. If the non-significant differences among ethnicities in rates of MSI mutation reflect real differences, the implications for these differences need to be addressed. Colorectal carcinoma is known to be caused by both genetic and environmental factors. The Caucasian group in our study had the highest rate of MSI, and this means there is a possibility that MSI may be a more important contributing factor in causing CRC in this group than in the Asian and Hispanic groups. BRAF mutation is associated with MSI-H due to hypermethylation of the promoter of hMLH1. The Caucasian population shows a higher rate of BRAF mutation than the Asian and Hispanic population, thus we would expect the rate of MSI to be higher in the Caucasian group. Furthermore, it has been shown that patients with CRC who have MSI have an overall better prognosis [26]. Given that Caucasians with CRC have an overall higher survival rate than Hispanics, and that in our study Caucasians were found to have a higher rate of MSI, it is possible that MSI could be a component in better CRC survival [27]. Larger population studies in the future will be needed to see if the difference in the rate of MSI between the three ethnicities represents a true difference. The Caucasian group had a non-statistically significant higher rate of BRAF mutation (12%) compared to the Asian (6%) and Hispanic (6%) populations (p = .70). In a previous study, it was stated that a literature review found a 4.2–5.1% rate of BRAF mutation in Asians and a 9.5–11.5% in Caucasians [12]. There were no statistical tests done in this study because these BRAF mutation rates were taken from four independent studies. These four studies did not compare BRAF mutation rates between Caucasians and Asians, and thus it isn’t certain whether rates represent true differences. Our results of BRAF mutation in the Caucasian group were similar to those reported by a literature review referenced by Lee et al. The Caucasians in our study had a 12% rate and the previous studies showed a 9.5–11.5% rate. In addition, our results for BRAF mutation in the Asian population (6%) were similar to the studies in the literature review by Lee et al. (4.2–5.1%) [12]. This study showed a non-significant lower rate of BRAF mutation in the Asian and Hispanic groups compared to the Caucasian group, which agrees with the results by Lee et al. If these non-significant higher rates for BRAF mutations in Caucasians do represent a true difference, there is a possibility that BRAF mutations could be a more significant, genetic contributing factor for CRC in the Caucasian group than in Asians and Hispanics. A previous study noted that CRC patients whose tumors exhibit a BRAF
hypermethylation test, and a BRAF mutation (data not shown). 4. Discussion This study tested the alternative hypothesis that molecular profiles for CRC differ by ethnicity against the null hypothesis of no differences between ethnic groups. The results showed that there were no statistically significant differences among the three groups with respect to mean age and gender distribution. There were more males than females with CRC in this study in all three ethnicities. Males comprised 58%, 52%, and 64% of the Caucasian, Asian and Hispanic groups, respectively. The difference in the percentage of males between the three ethnicities was not statistically significant. There was also no statistically significant difference in sites of carcinoma among the three ethnic groups. There were no statistically significant differences for the rates of MSI, KRAS mutation, or BRAF mutation for the 3 ethnic groups. The gender and age distribution differed from those found in the data from the Surveillance Epidemiology and End Results database [22]. In the SEER study, 51% of the CRC patients were male. The mean age at diagnosis of the population in this study was 61.8 and the mean age in the SEER study was 69 years of age. Compared to the SEER study, there were 7% more males and a lower age at diagnosis in the population in this study. It is possible that the UCI Medical Center population is being screened for CRC at a younger age and thus getting treatment earlier compared to the SEER study population. In addition, the SEER study includes all CRC cases including those diagnosed on death certificate only whereas our study population includes no cases diagnosed only on the death certificate. This could be the reason for the older mean age at diagnosis. It is unclear why there are more males than females, and why this was consistent between ethnicities. There are wide differences in diet between these ethnicities, so that dietary differences are not likely to result in the gender difference. It is possible that exposure to carcinogenic toxins, that might be more common in men than women regardless of ethnicity, such as alcohol or tobacco, could result in carcinoma being more common in men than in women across ethnicities. The most common site for carcinoma in our study population was the rectum in all three ethnicities, with the sigmoid colon being the second most common site. Thus, the majority of colorectal carcinomas in our study were distal. Difference in the site distribution (proximal or distal) of carcinoma among the three ethnic groups did not reach statistical significance. A previous larger study, by the Center for Disease Control and Prevention (CDC) [23], which included data from the National Program of Cancer Registries and SEER study, showed that 52.7% of colorectal carcinomas occurred in the rectum and distal colon. Our study revealed that 74% of CRC were distal. Both our study and the CDC study show that CRC is more commonly distal than proximal. The Caucasian population had the highest rate (15%) of MSI of all three groups. The Asian population had a rate of 8% and the Hispanic population had a rate of 10%. However, there was no statistically significant difference in rate of MSI among the three groups. The rate of MSI in Caucasians was cited to be 15% in a study of the molecular pathways of CRC in Koreans and Caucasians [19]. Another study by Vasovcak et al. [24] that studied 103 sporadic colorectal carcinomas in Czech Patients found a MSI rate of 24.3%. The rate of MSI in the Caucasian group in our study was the same as that found in the study by Kang (15%) [19], and lower than the rate found in the study by Vasovcak et al. (24.3%) [24]. In two studies that examined the rate of MSI in the Japanese population, Hayashi et al. [17] found an 8% rate and a study by Ishikubo et al. [18] found a 7% rate. The study by Kang [19] stated a 7% rate in the Korean population. Interestingly, another study in the Philippines showed a 20–22% rate of MSI in their population [16]. The rate of MSI in the Asian group in this study (8%) agreed with previous studies, except for the 20–22% rate of MSI in the study from the Philippines. Possible reasons for this large difference in the rate of MSI in the study from the Philippines include this representing a 724
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generational differences, and it is possible that data generated from the first generation of an ethnicity might be discordant with that from second or subsequent generations. We did not account for patients that lacked medical insurance. It is possible that indigent patients without insurance would be unable to undergo the complete surgical, pathological, and molecular workup simply due to financial inability to pay. All of these issues potentially affect our data and without very detailed knowledge of the precise methodology of other studies, comparisons between our study and other studies is difficult at best. We studied a subset of the total UCI population. At the UCI Medical Center, East Indians are included in the Asian/Pacific Islander race category, and it was not possible to separate East Indians from the rest of the Asian/Pacific Islanders. Ethnicity and race are self-reported voluntarily by the patients. Since identification of Asian subgroups was not possible, there may be differences between the results of our Asian group compared to other studies which were able to separate East Asians and East Indians. There may be differences in the molecular profile of CRC in some Asian subgroups. This study does not focus on the East Indian population, which is small at UCI and thus most likely does not significantly affect the results for the Asian/Pacific Islander category. Furthermore, in the CoPath/CDW data, there were a set of patients with unknown ethnicity. Had information been available for this set of patients, it may have contributed to the overall size of the study population and the data for the tests that were analyzed. Although the total number of CRC patients in this study was large, the number of patients for whom molecular testing was performed was much smaller. Thus, differences in MSI, BRAF and KRAS mutation rates are small and it is difficult to detect differences in this study due to limited power. We estimate that if we had an approximately 90% increase in the number of patients, or double the number of patients, there would be a higher likelihood for the results to reach statistical significance.
mutation have a poorer prognosis than patients without BRAF mutations [28]. However, Caucasian patients with CRC have higher survival rates than do Hispanics with CRC. Survival rates in Asians, however, are higher than in Caucasians [27]. Because there is a contradiction between the higher survival rate of Caucasians with CRC and prevalence of BRAF mutations indicating higher mortality in those with CRC, there are likely other factors that are contributing to the differences in the survival rates among the three ethnic groups. The Hispanic group in our study had a non-significantly higher rate of KRAS mutation (59%) compared to Asians (37%) and Caucasians (38%) (p = .08). In a previous study, it was shown that Caucasians had a KRAS mutation rate of 36% [15]. The rate of KRAS mutation in our study in the Caucasian group (38%) was similar to the rate found by Ciardiello et al. [15]. This same study showed that Asians had a KRAS mutation rate of 22% [15]. In one study, a specific KRAS mutation in codon 146 (A146) was tested in populations from Hong Kong and the US. The rate of this specific KRAS mutation was 7/126 in the Hong Kong population, approximately 5.5% [13]. Another study in the Japanese population showed a 42% rate of KRAS mutation [14]. A possible reason for the higher rate observed by Doi et al. [14] is the small study population of 24 patients. Rates for KRAS mutations vary widely from one study to another, reflecting small numbers and limited power to detect differences between ethnic groups. The rate of KRAS mutation in the Asian group in our study (37%) were similar to the results in the study by Doi et al. (42%) [14] but not to those found in the study by Edkins et al. (4.2%) [13]. However, there was large variability in KRAS mutation rates in Asians from difference studies. The study by Ciardiello et al. [15] showed a KRAS mutation rate in Hispanics to be 40%, which was lower than the rate in our study (59%). In our study, the Caucasian group had a higher rate of KRAS mutation than in the Asian group. This agrees with previous study by Ciardiello et al. [15]. Our results also agree with Ciardiello’s study showing that Hispanics had a higher rate of KRAS mutation than in the Caucasians. It is important to note that the differences found in the previous studies did not have statistical testing, and they may not reflect true differences because there was no statistical analysis comparing Asians and Hispanics to Caucasians. The higher rate of KRAS mutation in Hispanics was close to significance in our study (p = .08) and thus did give some support for a higher rate in Hispanics. The interpretation of the data regarding KRAS mutations is limited by small sample size. The frequency of KRAS mutations in the Hispanic population was approximately 50% higher than that of the Caucasians and Asian groups. Although this did not quite reach statistical significance, if this represents a true difference, this implies a much higher rate of mutation in Hispanics and therefore a need for the population to be tested more carefully. Furthermore, KRAS mutations could be a larger contributing factor in the cause of CRC in the Hispanic population than in the Caucasian and Asian populations. In a study by Tan et al. [29], it was found that there are contradicting conclusions as to whether KRAS mutations improve or worsen prognosis in patients with CRC. Because patients with KRAS mutations respond differently to anti-EGFR therapy, it is important to test for KRAS and especially important to test if one ethnic group is at higher risk for KRAS mutations than others in order to give patients the best treatment possible. This cursory meta-analysis of the literature demonstrates that our results differ in certain respects and are similar in other respects with prior studies. Although a formal meta-analysis is beyond the scope of our project, the differences and similarities seemingly lack a recognizable pattern when viewed as a whole. There are a number of possible factors that might contribute to these differences and similarities. Our study was based on only one institution, whereas other studies may have pooled data from multiple institutions. We were not able to control for important dietary variables. It is quite possible that the three ethnicities had nearly identical diets, given that the patients were all living within the same county. We were not able to control for
5. Conclusions The results of this study are consistent with the null hypothesis that there are no statistically significant differences in the molecular profiles of colorectal carcinoma among the Caucasian, Asian, and Hispanic populations at the UCI Medical Center. This study did show non-significantly higher rates in the Caucasian group for MSI (15% vs. 8% in Asians and 10% in Hispanics) and mutated BRAF (12% vs. 6% in Asians and 6% in Hispanics) and a non-significantly higher rate of KRAS mutation in the Hispanic population (59% vs. 37% in Asians and 38% in Caucasians). However, our study population was not large enough to detect statistically significant differences among ethnic groups. The characteristics of colorectal carcinoma are similar in some respects and different in other respects among the Hispanic and other groups. The progress in our knowledge of genetics and our understanding of the causes of carcinoma and the implications for treatment has increased and will lead to better prevention, earlier diagnosis, and better treatments for colorectal carcinoma. As more tests are ordered, the differences among the ethnicities may become more evident. Larger population studies will be needed to reveal whether our non-significant findings of ethnic differences in molecular profiles which were too small to achieve statistical significance represent real differences. Colorectal carcinoma is a significant public health problem and affects people of both genders and from all ethnic groups and ages. It is important for genetic research to continue in order to gather more data about the molecular characteristics of colorectal carcinoma among different ethnic groups, particularly in the Hispanic population. Evaluation and interpretation of these data are essential for improved prevention, diagnosis, and treatment for colorectal carcinoma. Conflicts of interest The authors have no conflicts of interest to declare. 725
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Funding
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