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Microsatellite instability and mutations in BRAF and KRAS are significant predictors of disseminated disease in colon cancer

Abstract

Background

Molecular alterations are well studied in colon cancer, however there is still need for an improved understanding of their prognostic impact. This study aims to characterize colon cancer with regard to KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), and average DNA copy number, in connection with tumour dissemination and recurrence in patients with colon cancer.

Methods

Disease stage II-IV colon cancer patients (n = 121) were selected. KRAS, BRAF, and PIK3CA mutation status was assessed by pyrosequencing and MSI was determined by analysis of mononucleotide repeat markers. Genome-wide average DNA copy number and allelic imbalance was evaluated by SNP array analysis.

Results

Patients with mutated KRAS were more likely to experience disease dissemination (OR 2.75; 95% CI 1.28-6.04), whereas the opposite was observed for patients with BRAF mutation (OR 0.34; 95% 0.14-0.81) or MSI (OR 0.24; 95% 0.09-0.64). Also in the subset of patients with stage II-III disease, both MSI (OR 0.29; 95% 0.10-0.86) and BRAF mutation (OR 0.32; 95% 0.16-0.91) were related to lower risk of distant recurrence. However, average DNA copy number and PIK3CA mutations were not associated with disease dissemination.

Conclusions

The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with MSI and BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.

Peer Review reports

Background

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death in Sweden [1]. Metastatic disease is present at diagnosis in 20-25% of patients and another 20-25% develops metastases in the course of the follow-up time. As local disease nowadays rarely is a cause of death in cancer of the colon and rectum [2], tumour cell dissemination may be considered a prerequisite for tumour death. To be able to improve survival by more appropriate treatment selection in primary disease, focus must therefore be on the identification of tumours with the capability to disseminate, whether clinically apparent at diagnosis (stage IV) or detected during follow-up after curative surgery (stages II and III).

The TNM (tumour-node-metastasis) classification based on radiologic and histopathological evaluation is currently the most reliable method for treatment selection and prognostic prediction in patients with CRC [3]. Patients curatively operated for stage II disease have around 15% risk of developing disease recurrence [4] if staged appropriately, operated according to modern principles and assessed with high quality pathology. Due to low risk of recurrence, these patients are regularly not given adjuvant chemotherapy, unless they are considered to be at “high risk” due to poor prognostic features such as T4, emergency operation or vascular invasion [5,6]. Patients with stage III disease have approximately a 40% risk to develop recurrent disease. Adjuvant therapy with 5-fluorouracil (5-FU)/leucovorin in patients with stage III disease reduces this risk by approximately 30%. If 5-FU/leucovorin is combined with oxaliplatin, the recurrence rate is further decreased with 15-20% [7]. Obviously, a subgroup of patients with stage III disease is given adjuvant chemotherapy with limited survival benefits. At the same time, there is an under-treatment of the subset of stage II patients that eventually develop recurrent disease.

CRC is heterogeneous with regard to molecular alterations and characterization of the molecular aetiology of sporadic CRC has identified different oncogenic pathways. The two major genomic instability pathways are the “traditional” chromosomal instability (CIN), or aneuploidy pathway, and the microsatellite instability (MSI) pathway [8-11]. These two pathways have been described as mutually exclusive, as the CIN tumours are microsatellite stable (MSS) [12]. CIN positive tumours constitute 65-70% of CRCs and have been associated with an aggressive clinical behaviour and distal location [10,13]. Tumours with CIN usually have large genomic aberrations that lead to higher average DNA copy number compared with MSI tumours [14]. Absolute DNA copy numbers can be assayed by SNP arrays and subsequent allele-specific analysis [15]. The MSI phenotype is the result of gene silencing of DNA mismatch repair (MMR) genes that cause accumulation of mutations in tumour suppressor genes and oncogenes. The MSI phenotype is therefore also referred to as the MMR deficient or mutator phenotype. CRC with MSI accounts for approximately 15% of sporadic CRCs and is characterized by a more proximal location, mucinous differentiation, near-diploid chromosome set and better prognosis compared to MMR proficient, frequently CIN positive, CRC [16-19]. Some CRC tumours also display epigenetic instability manifested as CpG island methylator phenotype (CIMP) or global DNA hypomethylation. CIMP-positive tumours are strongly associated with the MSI phenotype and the presence of BRAF mutations [20,21]. An additional CRC subtype comprises MSS CIN negative (diploid) tumours that also frequently are CIMP positive and BRAF mutated [12].

CRC tumourigenesis is also dependent on mutations in genes that deregulate intracellular signaling pathways, e.g. the EGFR mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Frequently mutated genes in these pathways are KRAS, BRAF and PIK3CA. Similar to CIN and MSI, these genes have been suggested as prognostic biomarkers, but although examined in many previous studies, the precise prognostic role of mutations in these genes remains unclear [22,23]. Based on the increased molecular knowledge of CRC, a classification of sporadic CRC into five different entities has been proposed [12]. However, the clinical value of these entities is still unclear and conflicting data exists among studies, probably a result of the heterogeneity of CRC resulting in overlap between the different pathways involved in CRC tumourigenesis.

In order to better understand tumour cell characteristics in primary colon cancers associated with tumour cell dissemination, and disease recurrence, the aim of this study was to characterize colon tumours, stratified by tumour stage and presence or development of metastatic disease, with regard to KRAS, BRAF, and PIK3CA mutations, MSI, and average DNA copy number.

Methods

Patient material and study design

Fresh frozen tumour material was available for molecular analysis from over 600 patients with primary colon and rectal cancer operated at the Uppsala University Hospital, Sweden, between 1987 and 2006, or at the Central District Hospital in Västerås, Sweden, between 2000 and 2003. From this population patients with stage II and III tumours, with and without recurrent disease, and patients with stage IV disease at diagnosis, were identified. To enable comparisons of tumours with and without metastatic capability, patients with synchronous metastases at diagnosis were considered equivalent to those with metastases appearing during the follow-up period, as both synchronous and metachronous metastases develop from the primary tumour and may indicate the presence of certain traits. The terms “non-disseminated” was used for patients with stage II and III tumours without recurrence and “disseminated” for stages II and III with recurrence together with stage IV.

Only colon cancers were selected as rectal cancers are often treated preoperatively with radiation and/or chemotherapy and rectal cancer can differ from colon cancer in the mutation profile. To ensure the high quality of the study population, only radiologically adequately staged patients and those operated abdominally according to either right-sided or left-sided hemicolectomy or sigmoidectomy were included. No preoperative therapy was allowed and the surgery was required to be radical (R0). Patients with stage II disease were only included if at least 10 lymph nodes were analyzed. Moreover, patients with stages II-III, with no disease recurrence were only included if the follow-up time was longer than 5 years.

Haematoxylin-eosin stained tissue sections were prepared from OCT-embedded fresh-frozen specimens using a cryostat and the CryoJane tape-transfer system (Instrumedics, Richmond, IL). The tumour tissue sections were examined by a trained pathologist to ensure that only representative samples containing more than 40% tumour cells were included.

Based on the above-mentioned criteria, tumour tissue from 121 patients was selected for analysis; 25 with disease stage II and 28 with stage III without disease recurrence; 15 with stage II and 27 with stage III with distant recurrence and 26 with stage IV disease. Totally 68 patients were therefore regarded as disseminated and 53 as non-disseminated. The stage II group with disease recurrence had to be limited to 15 cases as no more eligible patients could be identified; otherwise the aim was to include at least 25 patients in each group. Basic clinical and histopathological information of the selected cohort is given in Additional file 1: Table S1.

DNA extraction

Genomic DNA was extracted from 5-10 frozen tissue sections (10 μm) using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s recommendations. The purityand concentration of the extracted DNA was assessed using a NanoDrop instrument (Thermo Scientific, Wilmington, DE).

Pyrosequencing

The PyroMark Q24 BRAF and KRAS v2.0 assays (Qiagen) were used to detect mutations in BRAF (codon 600) and KRAS (codons 12, 13 and 61 in exons 2 and 3) according to the manufacturer’s recommendations. Novel pyrosequencing assays were developed for the analysis of known PIK3CA mutation hotspots in exon 9 (codons 542, 545, and 546) and exon 20 (codons 1043 and 1047). PCR primers and sequencing primers were designed using the PyroMark Assay Design 2.0 software (Qiagen). Forward (F) and reverse (R) PCR primers and sequencing primers (S) for PIK3CA were as follows (5’-3’): 9-F CAGCTCAAAGCAATTTCTACACG (biotin); 9-R CTCCATTTTAGCACTTACCTGTGAC; 9-S TG ACTCCATAGAAAATCTTT; 20-F GCAAGAGGCTTTGGAGTATTTC (biotin); 20-R AG ATCCAATCATTTTTGTTGTC; 20-S TTTTGTTGTCCAGCC. Briefly, ten nanogram of genomic DNA was used in 25 μl PCR reactions. Eight (PIK3CA) or 20 μl (BRAF and KRAS) of the PCR product was subsequently subjected to pyrosequencing using Streptavidin Sepharose High Performance (GE Healthcare, Uppsala, Sweden), PyroMark Gold Q96 reagents, PyroMark Q24 1.0.9 software, and a Q24 instrument (QIAGEN). All identified mutations were confirmed in a second analysis.

MSI analysis

Determination of MSI status was performed using MSI Analysis System, version 1.2 (Promega, Madison, WI) with 6 ng genomic DNA and analysis of five mononucleotide repeat markers (BAT25, BAT26, NR-21, NR-24 and MONO-27). Analyses were performed on a 3130xl genetic analyzer (Applied Biosystems, Foster City, CA). According to guidelines from a National Cancer Institute workshop in 1997, samples were denoted MSI-High (MSI-H) if two or more of the five markers show instability, MSI-Low (MSI-L) if only one marker shows instability and microsatellite stable (MSS) if no markers display instability. In this study, MSI-L and MSS was grouped together in the interpretation of MSI data, therefore MSI refers to MSI-H and MSS refers to both MSS and MSI-L.

SNP array analysis

Array experiments were performed according to the standard protocols for AffymetrixGeneChip® Mapping SNP 6.0 arrays (AffymetrixCytogenetics Copy Number Assay User Guide (P/N 702607 Rev2.), Affymetrix Inc., Santa Clara, CA). Briefly, 500 ng total genomicDNA was digested with a restriction enzyme (Nsp, Sty), ligated to an appropriate adapter for the enzyme, and subjected to PCR amplification using a single primer. After digestionwith DNase I, the PCR products were labeled with a biotinylatednucleotide analogue using terminal deoxynucleotidyltransferaseand hybridized to the microarray. Hybridized probes were captured by streptavidin-phycoerythrin conjugates using the Fluidics Station 450 and the arrays were finally scanned using the GeneChip® Scanner 3000 7G. Normalization and segmentation of genomic data was performed using BioDiscovery Nexus Copy Number 6.0 and the SNP Rank Segmentation algorithm [24,25] with default settings. Genome-wide average DNA copy number (ploidy) and the proportion of the genome with allelic imbalance were determined using Tumour Aberration Prediction Suite (TAPS) [15]. Average DNA copy number was calculated as the mean copy number of all genomic segments, weighted on segment length. Near diploid tumours were defined to have average copy number <2.5 and aneuploid tumours to have average copy number ≥2.5. SNP array data is available at GEO with accession number: (GSE62875).

Statistical analyses

The Mann-Whitney U test was used in comparisons of non-parametric two group parameters, the Kruskal-Wallis test for multiple groups and the Chi-square test for dichotomous response parameters and to test differences in proportions between groups. A two-sided Fisher’s exact test was used instead of the Chi-square test when fewer than 30 cases where analysed in total or less than 10 cases in each group. Spearman’s rho was used to calculate the correlation coefficient (r). The odds ratio (OR) and the 95% confidence intervals (CI) were calculated according to Ahlbom et al. [26]. Differences were considered statistically significant if p < 0.05.

Ethics

Ethical approval was obtained from the Ethics committee at Uppsala University, Uppsala, Sweden.

Results

Of the 121 tumours analysed, 48 (40%) had KRAS mutations, the mutations where located in codon 12 (65%), codon 13 (31%) and codon 61 (4%). BRAF mutations were detected in 28 (23%) of the tumours and PIK3CA mutations were seen in 22 (18%) tumours mainly in exon 9 (n = 18; 82%) with 4 mutations in exon 20 (18%). MSI-H was detected in 24 (20%) tumours and MSI-L in 7 (6%). DNA copy number <2.5 were seen in 66 out of 116 (57%) tumours analysed. In Table 1 the main clinical and histopathological characteristics of the cohort are shown in relations to KRAS, BRAF and PIK3CA mutations and MSI and DNA copy number. The main findings were that KRAS mutation was associated with advanced disease stage, BRAF mutations were mainly found in right colon, PIK3CA was associated with poor tumour differentiation, MSI was more commonly seen in lower disease stage, larger and more poorly differentiated tumours. However, DNA copy number did not reveal any associations to the variables analysed (Table 1). The well-known mutual exclusiveness of KRAS and BRAF mutations was observed (Table 2 and Figure 1), and MSI was more prevalent in KRAS wild-type and BRAF mutated tumours (Table 2). PIK3CA mutations were in this cohort significantly associated with the presence of BRAF mutations and MSI (Table 2) and, in contrast to the mutual exclusive pattern of KRAS and BRAF mutations, PIK3CA mutations coexisted with mutations in the two other genes.

Table 1 Clinical and histopathological relations of KRAS, BRAF and PIK3CA mutations and MSI (n = 121) and DNA copy number (n = 116) in primary tumours of patients with colon cancer
Table 2 Correlations between KRAS, BRAF and PIK3CA mutations, MSI (n = 121) and DNA copy number (n = 115) in primary tumours from patients with colon cancer
Figure 1
figure1

Venn diagrams representing the interrelations of KRAS, BRAF, PIK3CA mutations and MSI in primary tumours from patients with colon cancer; a) the entire cohort (n = 121); b) non-disseminated disease (n = 53) and c) disseminated disease (n = 68).

Tumours with average DNA copy number <2.5 frequently exhibited MSI and mutated BRAF. None of the tumours with MSI demonstrated an average DNA copy number ≥2.5 (Table 2 and Figure 2). On the contrary, 51 percent of the MSS tumours demonstrated an average DNA copy number ≥2.5, and were in all cases accompanied by a high proportion of the genome affected by allelic imbalance (Figure 2). However, average DNA copy number was neither associated with KRAS, nor PIK3CA mutation status (Table 2).

Figure 2
figure2

MSS/MSI-L and MSI-H samples were plotted according to average DNA copy number and proportion of the genome with allelic imbalance (%).

DNA copy number or PIK3CA mutations revealed no associations with disseminated disease or recurrence in the whole study cohort, or in various subgroup combinations of the cohort, and were therefore excluded from further analysis.

KRAS mutated tumours were more commonly seen in patients with disseminated disease. In contrast, BRAF mutations or MSI were less common in tumours from patients with disseminated disease or in those developing recurrence in disease stages II and III (Table 3). No statistically significant associations were seen when disease stages II and III were analysed separately (data not shown).

Table 3 The associations of KRAS and BRAF mutations and MSI to the risk of recurrence and dissemination in patients with colon cancer

Higher frequency of KRAS mutations was observed in tumours from patients with higher disease stages; 28% in stage II; 38% in stage III and 62% in stage IV. Whereas mutated BRAF, as well as MSI, were more frequent in lower disease stages; BRAF mutation frequency was 30% in stage II; 22% in stage III and 15% in stage IV and the frequency of MSI was: 33% in stage II; 16% in stage III and 8% in stage IV. When these genotypes were analysed separately in left and right colon, MSI and BRAF mutations were observed more frequently in the right colon and these molecular changes were present in both tumours from patients with, or without, recurrence in disease stages II and III and in disseminated disease (Table 4). For left colon, MSI and BRAF mutations could not be found in tumours from patients developing disease recurrence in stages II or III and were rare in those with disseminated disease (Table 4). On the contrary, KRAS mutations had a stronger association with disseminated disease in left compared with right colon (Table 4). Overall KRAS was the most frequently mutated gene in patients with disseminated disease (Figure 1c) and KRAS codon 12 glycine to valine mutations was seen in 10 of 34 KRAS mutated tumours in patients with disseminated disease compared to 2 of 14 KRAS mutated tumours in patients with non-disseminated disease (data not shown).

Table 4 The prognostic associations of KRAS mutation, BRAF mutation and MSI in right versus left colon in 121 patients with colon cancer

In Table 3, patients with MSS tumours only, KRAS wild type only and BRAF wild type only are also presented according to molecular status, dissemination and recurrence. Among these subgroups, patients with KRAS wild type tumours that are MSI are less likely (p = 0.041) to have disseminated disease. Patients with KRAS mutated MSS tumours appear more likely to have disseminated disease, but recurrences in stages II and III disease were not more frequent when MSS tumours were KRAS mutated. The same trend for dissemination can be seen for BRAF wild type tumours with a KRAS mutation (Table 3). The OR for dissemination for BRAF mutated tumours is low both in MSS tumours and in KRAS wild type tumours; however these results are statistically non-significant.

In an attempt to identify specific subgroups of molecular markers that could help to detect patients with high or low risk of disease dissemination, or recurrence in stages II and III, several combinations of markers were of interest. Patients with tumours presenting both KRAS wild type and MSI had a reduced risk of dissemination (OR 0.22; 95% CI 0.08-0.62) and recurrence in disease stages II and III (OR 0.31; 95% CI 0.10-0.94) compared with all other groups. On the other hand, patients with tumours harbouring both BRAF wild type and MSS presented a higher risk of disseminated disease, and disease recurrence in stages II and III compared with all other groups (Table 3). Tumours with both BRAF mutation and MSI had the lowest risk for dissemination also marginally significant for lower risk for disease recurrence in stages II and III (Table 3). No statistically significant differences were seen when stages II and III were analysed separately with aforementioned subgroups (data not shown).

Discussion

The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with microsatellite instable (MSI) disease or mutated BRAF, as compared to patients with MSS or BRAF wild-type tumours. On the contrary, disseminated disease was more commonly observed in patients with mutated KRAS, as compared to their KRAS wild-type counterparts.

This study is among the first that describes frequencies of mutations and microsatellite instability in association with disease dissemination (metastatic disease either present at the time of diagnosis or developed during follow-up time) in a selected subset of colon cancer patients. The rationale behind including patients with stage II and III colon cancer, with and without recurrent metastatic disease, together with stage IV patients (metastatic disease at diagnosis), was to facilitate the detection of predictive genotypes in a cost-effective way. The applied unmatched case-control design enabled a smaller number of samples to be analysed, while the number of critical events was maintained. However, it should be noted that the reduced sample size of each subgroup, as a result of the applied selection criteria, also might limit the power to detect statistically significant differences between the subgroups. Furthermore, even based on a large material of over 600 frozen tissue samples, we were unable to include the planned number of stage II patients with metastatic recurrence. The strict quality requirements with regard to staging, surgery, and pathology contributed to this inability, but at the same time likely increased the validity of the results, as the influence of unrelated factors was minimised.

The observed mutation frequencies in the present investigation should be interpreted with caution, as this cohort is not population-based. Even so, the KRAS mutation frequency of 40% in this cohort was in good agreement with other published studies [27-29]. Moreover, we observed that the proportion of KRAS mutated patients increased with higher disease stage, a finding supported by Eklöf et al. [30], but not uniformly seen in other cohorts [31,32].Today KRAS mutation status is routinely analysed because of its predictive nature in patients receiving therapeutic antibodies against EGFR, with treatment restricted to patients with KRAS wild type tumours [33,34]. In addition to predictive power with regard to treatment response, the prognostic impact of mutated KRAS has been thoroughly studied in CRC. In the RASCAL II study, KRAS mutations were associated with worse prognosis compared to KRAS wild type in over 3000 patients with CRC, an association that was stronger in stage III than in stage II [31]. The association to worse prognosis was however restricted to KRAS 12Gly > Val in stage III disease [31,35]. In the present study, a similar trend of worse prognosis for KRAS 12Gly > Val mutated patients was observed. Additional studies have confirmed the association of KRAS mutations and poor prognosis [30,32,36-38]. Contrary to these results, two other prospective studies, including 1,404 and 315 patients respectively, did not demonstrate any major impact of KRAS mutations on prognosis [39,40].

In the present study, the BRAF mutation frequency (23%) was higher compared to the 5-17% previously reported in colorectal cancer [30,32,41], possibly explained by the fact that right-sided tumours were predominant in our cohort and BRAF mutations have been reported to mainly occur in tumours of the right colon [30,37,39-41]. BRAF mutations were associated with lower likelihood of tumour dissemination in the whole cohort, as well as lower likelihood of metastatic recurrence in a separate analysis of stage II and III tumours. This is in contrast to a majority of published studies, where BRAF mutations were mostly associated with worse prognosis [28,30,37,39,40,42,43] or did not exhibit a prognostic impact [30,38]. Of interest is that two recent studies showed that BRAF mutations were related to worse overall survival, but not to relapse-free survival [44,45], which may be explained by higher frequencies of BRAF mutations in older individuals [30,45].

BRAF and KRAS mutations were confirmed to be mutually exclusive in this study, as previously reported [46]. BRAF mutations were moreover significantly associated with MSI, also this in agreement with previous findings [37,47]. The good prognostic feature of patients with the MSI tumour type, also seen here, is well-established [38,48-50] and MSI has been reported to be prognostic in both stages II and III [48], stage II only [48,50] and stage III only [19]. As observed by others and similarly to BRAF mutations, MSI tumours were found to have larger tumour size, association with lower disease stage and poor differentiation. However, the frequently seen associations of MSI with right colon, mucinous tumour type and female gender was not seen in the present cohort possibly reflecting the differences in selection of patients compared with consecutive cohorts. Interestingly, of the patients with left-sided MSI tumours in the present cohort none developed recurrence. It is tempting to omit MSI analysis in left-sided colon cancers, as only about 5% of left-sided tumours are expected to be MSI, however this study indicates that MSI analysis can assist when selecting patients for adjuvant treatment even for left sided tumours. We were unable to find any publications that analysed the prognostic impact of MSI in left-sided colon cancers, as most studies state that the case number is too low for meaningful investigations of this subset [38].

MSI tumours are characterised by a defective DNA mismatch repairsystem and the consequential accumulation of mutations in tumour suppressor genes and oncogenes. Tumours that are MSS commonly exhibit another type of instability, CIN, with abundant large-scale genomic alterations that often lead to a higher average DNA copy number. In contrast to MSI, average DNA copy number is not routinely assessed. Therefore, in the present study, average DNA copy number was determined based on genome-wide SNP array analysis. A low average DNA copy number was associated with the presence of BRAF mutation and MSI, but no association with tumour dissemination nor disease recurrence was found, suggesting that the analysis of average DNA copy number would not improve routine diagnostics.

In addition to KRAS and BRAF mutations, it has been put forward that mutations in PIK3CA, the p110α catalytic subunit of phosphatidylinositol-4,5-bisphosphonate 3-kinase (PI3K) and a main player in the PI3K/AKT/mTOR pathway, might be of clinical relevance. Coexistence of PIK3CA exon 9 and 20 mutations has, mainly by one group, revealed worse prognosis in CRC [22,51]. The present study revealed that PIK3CA mutations were more common in MSI and BRAF mutated tumours. However, no significant association with tumour dissemination was observed, an observation supported by others [30].

Molecular analysis methods to detect the presence of mutations and chromosomal or microsatellite instability are unlikely to replace conventional pathological analysis, but can potentially help oncologists decide whether or not colon cancer patients should receive chemotherapy as an adjuvant treatment to reduce the risk of metastatic recurrence.

Conclusions

The present study revealed that tumour dissemination is less likely to occur in colon cancer patients displaying MSI or BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.

References

  1. 1.

    NORDCAN database. [Available from http://www.ancr.nu, accessed on 16/04/2013.]

  2. 2.

    Glimelius B. Multidisciplinary treatment of patients with rectal cancer: Development during the past decades and plans for the future. Ups J Med Sci. 2012;117(2):225–36.

    Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Sobin LH, Gospodarowicz MK, Witteking C. TNM classification of malignant tumors. 7th ed. Oxford: Wiley-Blackwell; 2009.

    Google Scholar 

  4. 4.

    Birgisson H, Wallin U, Holmberg L, Glimelius B. Survival endpoints in colorectal cancer and the effect of second primary other cancer on disease free survival. BMC Cancer. 2011;11:438.

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Labianca R, Nordlinger B, Beretta GD, Mosconi S, Mandala M, Cervantes A, et al. Early colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013;24 Suppl 6:vi64–72.

    Article  PubMed  Google Scholar 

  6. 6.

    Schmoll HJ, Van Cutsem E, Stein A, Valentini V, Glimelius B, Haustermans K, et al. ESMO Consensus Guidelines for management of patients with colon and rectal cancer: a personalized approach to clinical decision making. Ann Oncol. 2012;23(10):2479–516.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Andre T, Boni C, Navarro M, Tabernero J, Hickish T, Topham C, et al. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J Clin Oncol. 2009;27(19):3109–16.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Cappell MS. From colonic polyps to colon cancer: pathophysiology, clinical presentation, and diagnosis. Clin Lab Med. 2005;25(1):135–77.

    Article  PubMed  Google Scholar 

  9. 9.

    Higuchi T, Jass JR. My approach to serrated polyps of the colorectum. J Clin Pathol. 2004;57(7):682–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Walther A, Johnstone E, Swanton C, Midgley R, Tomlinson I, Kerr D. Genetic prognostic and predictive markers in colorectal cancer. Nat Rev Cancer. 2009;9(7):489–99.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319(9):525–32.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology. 2007;50(1):113–30.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Walther A, Houlston R, Tomlinson I. Association between chromosomal instability and prognosis in colorectal cancer: a meta-analysis. Gut. 2008;57(7):941–50.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330–7.

    Article  Google Scholar 

  15. 15.

    Rasmussen M, Sundstrom M, Goransson Kultima H, Botling J, Micke P, Birgisson H, et al. Allele-specific copy number analysis of tumor samples with aneuploidy and tumor heterogeneity. Genome Biol. 2011;12(10):R108.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23(3):609–18.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28(20):3219–26.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Venook AP, Niedzwiecki D, Lopatin M, Ye X, Lee M, Friedman PN, et al. Biologic determinants of tumor recurrence in stage II colon cancer: validation study of the 12-gene recurrence score in cancer and leukemia group B (CALGB) 9581. J Clin Oncol. 2013;31(14):1775–81.

    Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Sinicrope FA, Foster NR, Thibodeau SN, Marsoni S, Monges G, Labianca R, et al. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst. 2011;103(11):863–75.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hinoue T, Weisenberger DJ, Pan F, Campan M, Kim M, Young J, et al. Analysis of the association between CIMP and BRAF in colorectal cancer by DNA methylation profiling. PLoS One. 2009;4(12):e8357.

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38(7):787–93.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Liao X, Morikawa T, Lochhead P, Imamura Y, Kuchiba A, Yamauchi M, et al. Prognostic role of PIK3CA mutation in colorectal cancer: cohort study and literature review. Clin Cancer Res. 2012;18(8):2257–68.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Pritchard CC, Grady WM. Colorectal cancer molecular biology moves into clinical practice. Gut. 2011;60(1):116–29.

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Olshen AB, Venkatraman ES, Lucito R, Wigler M. Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics. 2004;5(4):557–72.

    Article  PubMed  Google Scholar 

  25. 25.

    Rabbee N, Speed TP. A genotype calling algorithm for affymetrix SNP arrays. Bioinformatics. 2006;22(1):7–12.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Ahlbom A, Norell S. Introduction to modern epidemiology. Chestnut Hill, MA: Epidemiology Resources Inc.; 1990.

    Google Scholar 

  27. 27.

    Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58(1):90–6.

    Article  PubMed  Google Scholar 

  28. 28.

    French AJ, Sargent DJ, Burgart LJ, Foster NR, Kabat BF, Goldberg R, et al. Prognostic significance of defective mismatch repair and BRAF V600E in patients with colon cancer. Clin Cancer Res. 2008;14(11):3408–15.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 2010;11(8):753–62.

    Article  PubMed  Google Scholar 

  30. 30.

    Eklof V, Wikberg ML, Edin S, Dahlin AM, Jonsson BA, Oberg A, et al. The prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer. Br J Cancer. 2013;108(10):2153–63.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Andreyev HJ, Norman AR, Cunningham D, Oates J, Dix BR, Iacopetta BJ, et al. Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study. Br J Cancer. 2001;85(5):692–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Phipps AI, Buchanan DD, Makar KW, Win AK, Baron JA, Lindor NM, et al. KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers. Br J Cancer. 2013;108(8):1757–64.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26(10):1626–34.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 2008;26(3):374–9.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Imamura Y, Morikawa T, Liao X, Lochhead P, Kuchiba A, Yamauchi M, et al. Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075 BRAF wild-type colorectal cancers. Clin Cncer Res. 2012;18(17):4753–63.

    CAS  Article  Google Scholar 

  36. 36.

    Conlin A, Smith G, Carey FA, Wolf CR, Steele RJ. The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma. Gut. 2005;54(9):1283–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Farina-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, Rutten HJ, et al. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol. 2010;21(12):2396–402.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Hutchins G, Southward K, Handley K, Magill L, Beaumont C, Stahlschmidt J, et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol. 2011;29(10):1261–70.

    Article  PubMed  Google Scholar 

  39. 39.

    Price TJ, Hardingham JE, Lee CK, Weickhardt A, Townsend AR, Wrin JW, et al. Impact of KRAS and BRAF Gene Mutation Status on Outcomes From the Phase III AGITG MAX Trial of Capecitabine Alone or in Combination With Bevacizumab and Mitomycin in Advanced Colorectal Cancer. J Clin Oncol. 2011;29(19):2675–82.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D, et al. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol. 2010;28(3):466–74.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, et al. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 2005;65(14):6063–9.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Lochhead P, Kuchiba A, Imamura Y, Liao X, Yamauchi M, Nishihara R, et al. Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication. J Natl Cancer Inst. 2013;105(15):1151–6.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Rosty C, Williamson EJ, Clendenning M, Walters RJ, Walsh MD, Win AK et al: Re: Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication. J Natl Cancer Inst 2014, 106(8)

  44. 44.

    Roth AD, Delorenzi M, Tejpar S, Yan P, Klingbiel D, Fiocca R, et al. Integrated analysis of molecular and clinical prognostic factors in stage II/III colon cancer. J Natl Cancer Inst. 2012;104(21):1635–46.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Ogino S, Shima K, Meyerhardt JA, McCleary NJ, Ng K, Hollis D, et al. Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803. Clin Cancer Res. 2012;18(3):890–900.

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Rajagopalan H, Bardelli A, Lengauer C, Kinzler K, Vogelstein B, Velculescu V. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature. 2002;418(6901):934.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Gavin PG, Colangelo LH, Fumagalli D, Tanaka N, Remillard MY, Yothers G, et al. Mutation profiling and microsatellite instability in stage II and III colon cancer: an assessment of their prognostic and oxaliplatin predictive value. Clin Cancer Res. 2012;18(23):6531–41.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Lanza G, Gafa R, Santini A, Maestri I, Guerzoni L, Cavazzini L. Immunohistochemical test for MLH1 and MSH2 expression predicts clinical outcome in stage II and III colorectal cancer patients. J Clin Oncol. 2006;24(15):2359–67.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med. 2000;342(2):69–77.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Merok MA, Ahlquist T, Royrvik EC, Tufteland KF, Hektoen M, Sjo OH, et al. Microsatellite instability has a positive prognostic impact on stage II colorectal cancer after complete resection: results from a large, consecutive Norwegian series. Ann Oncol. 2013;24(5):1274–82.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Ogino S, Nosho K, Kirkner GJ, Shima K, Irahara N, Kure S, et al. PIK3CA mutation is associated with poor prognosis among patients with curatively resected colon cancer. J Clin Oncol. 2009;27(9):1477–84.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

To Lions cancer foundation and Erik, Karin and Gösta Selanders foundation who supported the study. The authors would like to express our gratitude to SiminTahmasebpoor for expert fresh frozen tissue management and sectioning.

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Correspondence to Helgi Birgisson.

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Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HB, BG, LP, JB, PM, AI and MS were involved in the study design. HB, MS, KE and UW: Gathered tumour samples and clinical information; JB and PM; Carried out histopathological examination; MS and KE carried out the DNA extraction, pyrosequensing and MSI analysis; HGK, MM and AI: Carried out SNP array analysis; HB and UW: made statistical analysis; HB, UW, MS and BG; were responsible for the drafting of the manuscript. All authors were involved in the revision of the manuscript and gave the final approval of the manuscript.

Additional file

Additional file 1: Table S1.

Clinical and histopathological data of the study cohort including 121 cases with primary colon cancer.

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Birgisson, H., Edlund, K., Wallin, U. et al. Microsatellite instability and mutations in BRAF and KRAS are significant predictors of disseminated disease in colon cancer. BMC Cancer 15, 125 (2015). https://doi.org/10.1186/s12885-015-1144-x

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Keywords

  • Colon cancer
  • MSI
  • BRAF
  • KRAS
  • PIK3CA
  • DNA copy number
  • Prognosis