Colorectal cancer without high microsatellite instability and chromosomal instability—an alternative genetic pathway to human colorectal cancer

Reiping Tang1, Chung Rong Changchien1, Meng-Chih Wu1, Chung-Wei Fan1, Kwang-Wen Liu1, Jinn-Shiun Chen1, Huei-Tzu Chien2 and Ling-Ling Hsieh2,3

1 Colorectal Section, Department of Surgery, Chang Gung Memorial Hospital, Lin-ko and 2 Department of Public Health, Chang Gung University, Tao-Yuan, Taiwan

3 To whom correspondence should be addressed Email: llhsieh{at}mail.cgu.edu.tw


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At least two forms of genomic instability have been described in colorectal cancers (CRCs): microsatellite instability (MIN), which is characterized by a high frequency of microsatellite instability (MSI-H) and chromosomal instability (CIN), which is characterized by losses and gains of chromosomes (aneuploidy), as well as chromosome rearrangements. Morphological and molecular heterogeneity within MIN(–) CRCs have been described, but the distinctions between MIN(–) tumors with CIN and those without CIN remain largely unknown. We studied 179 colorectal cancers to elucidate the clinicopathological characteristics and molecular events in CRCs arising along these pathways. Loss of heterozygosity, MIN, DNA content, mutation of p53 and K-ras, and expression of p53, hMLH1 and hMSH2 were examined. We found that a subtype of tumors (17%) with MIN(–) and CIN(–), differed from MIN(–)CIN(+) tumors with respect to clinicopathological and genetic characteristics. This subtype was associated with a greater frequency of poorly differentiated and/or mucinous tumors (26%). This subtype of tumors had an extremely low p53 gene mutation rate (11%) and a relatively high p53 protein accumulation rate (55%). The dissociation between the p53 gene mutation and protein accumulation suggests that stabilization of p53 protein in the absence of p53 gene mutation may be an important event on a distinct pathway.

Abbreviations: CIN, chromosomal instability; CRC, colorectal cancer; IHC, immunohistochemistry; LOH, losses of heterozygosity; MIN, microsatellite instability; MSI-H, high frequency of microsatellite instability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most aggressive human cancers are characterized by genomic instability (1). Although it remains subject to debate whether genomic instability is strictly required for human colorectal tumorigenesis (2), genomic instability is proposed to be a driving force in the initiation of human colorectal cancer (CRC) development (3). At least two forms of genomic instability have been described in CRCs: chromosomal instability (CIN) and microsatellite instability (MIN) (4). About 60% of CRCs develop through the CIN pathway, which is characterized by losses and gains of chromosomes (aneuploidy), as well as losses of heterozygosity (LOH), i.e. loss of one of the parental alleles present in the cells (5). The consequence of CIN is an imbalance in the number of chromosomes per cell (aneuploidy) and an enhanced rate of LOH (6). Up to 15% of sporadic CRCs show a high frequency of microsatellite instability (MSI-H or MIN) (7), which is a diagnostic of defects in the DNA mismatch repair system (8). MIN in sporadic CRC is commonly the result of silencing of the human mut-L homolog 1 (hMLH1) gene, secondary to promoter methylation (9). These tumors acquire somatic frameshift mutations of coding repeats within several cancer-associated genes, which constitute the distinct tumorigenesis pathway (10).

Recently, existence of multiple alternative genetic pathways has been suggested (11). Stratification of human CRCs based on microsatellite and chromosomal instability results in four subsets: MIN(+)CIN(+), MIN(+)CIN(–), MIN(–)CIN(+) and MIN(–)CIN(–). Goel et al. (12) observed a significant degree of overlap between MIN and CIN pathways. These MIN(+) tumors have well-established clinical and pathological correlates (13). The distinction between MIN(–) tumors with CIN and those without CIN is less characterized. It has been proposed that the MIN(–)CIN(–) phenotype of CRCs is distinct from MIN(–)CIN(+) phenotype of CRCs, according to clinical, pathological and molecular features (1417). To further explore this hypothesis we examined the distribution of multiple clinicopathological and molecular variables across sporadic CRCs stratified by both MSI and DNA ploidy status/ LOH. Our study confirmed that the MIN(–)CIN(–) phenotype has distinct clinicopathological and genetic features that differ from those of MIN(–)CIN(+) phenotype.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The material was derived from a prospectively collected, hospital-based frozen bank comprising 181 CRC specimens, in which 57 p53 gene mutations were found (18). Colorectal tissue specimens were collected from these patients and frozen at –80°C. For each case a pair of tumor tissue and normal mucosa, at least 10 cm away from the tumor, were obtained. A total of 179 tumors had sufficient DNA samples for the following analyses.

DNA flow cytometry
Tumor cells were extracted from fresh-frozen tissue. DNA content was analyzed using a Coulter Epics Analyzer (Beckman Coulter, Brea, CA), using the Multicycle AV DNA Analysis Package (Phoenix Flow Systems, San Diego, CA). 20 000 stained nuclei were analyzed from each sample. A diploid tumor was defined as having single G0/G1, S and G2M peaks with a ratio of the mode channel number of the G2M and G0/G1 peaks of 1.9–2.1 and with 10% or fewer of the nuclei in the G2M peak. Non-diploid histograms included aneuploid (two or more separable G0/G1 peaks) and tetraploid (>10% of nuclei in the G2/M peak) histograms (19).

Microsatellite analysis
For MSI analysis, normal and tumor DNA was extracted from fresh-frozen tissue. Polymerase chain reaction (PCR) was performed for 30 cycles in 10 µl of reaction mixture comprising 1 µl of the 100 ng DNA sample and 9 µl of reaction mixture containing oligonucleotide primer pair (one end-labeled with 32P), 25 µM of each deoxynucleotide triphosphate (dNTP), PCR reaction buffer, and 0.5 U Taq polymerase. After denaturation with an equal volume of formamide at 95°C for 5 min, the amplified PCR products were electrophoresed on 6% denaturing polyacrylamide gels at 35 W for 3–4 h. The gel was dried on filter paper and exposed to X-ray film. The primer panel included the reference panel of markers (BAT25, BAT26, D2S123 on 2p16, D5S346 on 5q21-22 and D17S250 on 17q11-12) (9), and eight markers (all dinucleotide) at alternative foci (D4S402 on 4pter–4qter, D5S406 on 5pter–5qter, D10S197 on 10qter, D11S904 on 11p114/13, D11S925 on 11q23.3, D13S175 on 13q11, D18S58 on 18q22.3 and D18S69 on 18q21). MSI was defined by the presence of novel bands following PCR amplification of tumor DNA that were not present in the PCR products of the corresponding normal DNA. International criteria for the determination of MSI in CRC were used to differentiate MSI-H from MSI-L/MSS tumors (7). MSI-H tumors were defined as having instability in two or more markers using the reference panel or as having instability in four or more markers (>=30%) of the 13 markers tested (7). For LOH assessment, 11 dinucleotide markers were used. LOH was defined as at least a 50% reduction in the relative intensity of the allele from the tumor DNA compared with that of the normal mucosa DNA. Since karyotypic studies have shown that the majority of cancers have lost or gained chromosomes, and molecular studies indicated that karyotypic analysis might actually underestimate the true extent of such changes (see review in ref. 6), we defined CIN(+) as tumors showing either aneuploidy or LOH at one or more dinucleotide microsatellite markers.

K-ras and p53 gene mutation
p53 mutational status in exons 5–9 were known from a previous study (18). The methods described by Jiang et al. (20) were used for detection of the K-ras mutation at codon 12, and the methods described by Nollau et al. (21) were used for detection of K-ras mutation at codon 13. Briefly, PCR was performed in a 25-µl solution containing 500 ng of genomic DNA, 200 mM dNTP, 1 U Taq polymerase, 1x reaction buffer and 10 pmol of each primer for 30 cycles. The resulting PCR product was digested, electrophoresed on a 6% polyacrylamide gel, and stained with ethidium bromide to identify specific bands resulting from mutations. Direct DNA sequencing was then performed to determine the position and type of the mutation at codons 12 and 13.

Immunohistochemistry of p53, hMLH1 and hMSH2
Of the 179 tumors, 165 paraffin sections of tumor tissue were available and subjected to immunohistochemistry (IHC) analyses. For each of these tumors, 5-mm sections from representative blocks of the tumors were deparaffinized in xylene and absolute alcohol, retrieved with heat, and treated with 3% hydrogen peroxide to remove endogenous peroxidase activity. Immunohistochemical staining for p53 protein was processed using anti-p53 monoclonal antibody DO7 (1:100) (Dako, Glostrup, Denmark), which recognizes an epitope in the N-terminal part of the human p53 protein between amino acids 1 and 45, as the primary antibody and the DAKO LSAB2 System with DAKO DAB chromogen solution (Dako). The primary antibodies for MLH1 and MSH2 were G168-15 (1:100) (Becton-Dickinson Biosciences, Franklin Lakes, NJ) and NA27 (1:100) (Oncogene, Darmstadt, Germany), respectively. The slides were then counterstained with hematoxylin, overslipped with Permount and examined for the extent and intensity of nuclear and cytoplasmic staining in tumor cells and for background staining in the stroma. At least five high-power fields at 40x magnification were scored for the extent of nuclear staining (1, 1–20%; 2, 20–40%; 4, 40–60%; 6, 60–80%; 8, 80–100%) and intensity of staining (0, no nuclear stain; 1, weak nuclear staining; 2, intermediate staining; 3, strong staining) by an investigator (M.C.W.). The score for intensity was always that of the most strongly stained nucleus. In occasional cases of doubt as to the interpretation of staining results, a joint decision was made with a second investigator (R.T.). We defined the staining score as the extent of nuclear staining times the intensity grade. p53 staining was designated as none/low if the p53 staining score was 0–3, moderate if score was 4–12 and high if score was 16–32. For purposes of statistical analyses, p53 staining was treated as positive if any tumor had moderate to high nuclear staining for p53 and as negative if it had no or low p53 staining.

Statistical analyses
Comparisons between groups were performed using {chi}2 analysis or Fisher's exact test whenever appropriate. Statistical analyses were conducted using either SPSS (SPSS 10.0, Chicago, IL) or SAS (version 8.01) software. Statistical significance was set at P < 0.05. All P were estimated from two-sided tests.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Molecular characteristics of tumors
Among the 13 microsatellite markers analyzed (Figure 1A), the frequency of MSI ranged from 5.6 (D11S904) to 11% (BAT25 and D17S250), whereas the frequency of LOH ranged from 1.4 (D2S123) to 26.7% (D18S69). Of the 19 tumors with MSI-H (instability in four or more markers), 16 tumors (84%) showed instability of eight or more markers out of 13; while among 32 tumors with MSI-L (instability in three or less markers), 27 tumors (84%) showed instability in only one marker (Figure 1B). Two of the three MSI-H cases showing instability in four or five markers had mutations in both mononucleotide markers (BAT25 and BAT26). The third case had instability in BAT25. Figure 1C shows that LOH in two or more markers was more frequently seen in the aneuploid tumors than in the diploid tumors (30.5 versus 5.2%, P = 0.003). K-ras mutation was found in 27% of the samples studied. The majority of K-ras mutations (82.6%) were found in codon 12, and the most common type of mutation was G to A transition both in codons 12 and 13. Of 165 tumors with p53 IHC staining, 71 (43.0%) showed no or low nuclear staining, 35 (21.2%) showed moderate staining, and 59 (35.8%) showed high p53 nuclear staining. Tumors with moderate to high nuclear staining were designated as positive. Among the 55 tumors with p53 mutations in exons 5–9, 42 (76%) were positive on IHC staining.



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Fig. 1. (A) The frequency of LOH (white) and MSI (black) for each of the markers listed in 179 sporadic CRCs. The frequency of tumors with heterozygosity (informative data) for each marker ranged from 61 (for D5S406) to 78% (for D2S123). (B) Distribution of MIN in 179 sporadic CRCs. Graph shows percentage of colorectal tumors with the number of loci showing MIN. Those tumors (n = 32) with one to three markers demonstrating instability was defined as MSI-L (gray) and those (n = 19) with four or more markers demonstrating instability was MIS-H (black). (C) The frequency of loss of heterozygosity (LOH) on one (white), two to three (gray) and four or more markers (black) in diploid versus aneuploid tumors. The frequency of LOH on two or more markers in aneuploid tumors (30.5%) was significantly higher than diploid tumors (5.2%, P = 0.0003).

 
Patterns of genomic instability
Of the 179 tumors, there were eight (5%) tumors displaying MIN(+)CIN(–), 11(6%) MIN(+)CIN(+), 129 (72%) MIN(–) CIN(+) and 31 (17%) MIN(–)CIN(–). No statistical differences were noted between MIN(+) tumors with CIN(+) and those with CIN(–) in regards to clinicopathological and molecular profile (data not shown). Therefore, we pooled these two groups together and designated it as MIN(+). Compared with MIN(–) (including tumors with or without CIN), MIN(+) tumors tended to be bigger (89 versus 50% with a diameter of 5 cm or more, P = 0.001), more often located in the right colon (47 versus 16%, P = 0.002), more likely to show mucinous or poorly differentiated histology (53 versus 14%, P = 0.0002), more likely to be diploid (68 versus 28%, P = 0.001), less often harbored a p53 alteration (mutation or protein over-expression, 37 versus 69%, P = 0.007) and associated with only minimal chromosomal rearrangement (0 versus 25% with LOH in at least two out of 11 markers, P < 0.0001). K-ras mutations occurred as frequently in MIN(+) as in MIN(–) CRCs (32 versus 25%, P = 0.535). Eight of 11 (73%) MIN(+) tumors were IHC-deficient for expression of either MLH1 (n = 4) or MSH2 protein (n = 4), whereas only one of 95 (1%) MIN(–) tumors was IHC-deficient for expression of MSH2 (P < 0.0001). The results were not different after excluding three cases with instability in four or five markers for statistical analyses.

The MIN(–) tumors without chromosomal instabiltiy were distinct from those with chromosomal instability in several ways. At the clinicopathological level (Table I), they tended to be more frequently associated with poorly differentiated or mucinous tumors (26 versus 12%, P = 0.043). At the genetic level, (Table II), the differences between these two groups were seen mainly with respect to p53 alterations. No significant differences in the frequency of p53 protein over-expression were observed between these two groups. However, a strikingly lower p53 mutation rate was noted in the MIN(–)CIN(–) tumors than in MIN(–)CIN(+) tumors (10 versus 36%, P = 0.005). We further explored the association between p53 mutations and LOH in diploid MIN(–) tumors and found that 36% (five out of 14) of diploid MIN(–) with LOH tumors harbored p53 mutations while <10% (3/31) in those without LOH (P = 0.085 by Fisher's exact test). No frameshift mutation was noted in the MIN(–)CIN(–) tumors (Table II). Figure 2 further demonstrates the dissociation of frequency of p53 gene mutation and p53 protein over-expression observed the MIN(–) CIN(–) tumors. Only a 20% p53 mutation rate was observed in the MIN(–)CIN(–) tumors with nuclear p53 accumulation, compared with 47% in MIN(–)CIN(+) tumors with nuclear p53 accumulation (P = 0.022). Only one MIN(–) tumor had deficient MSH2 stain and none had deficient MLH1 stain.


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Table I. Clinicopathological differences in MIN(–) CRC with and without chromosomal instability

 

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Table II. Genetic changes in MIN(–) CRC with and without chromosomal instability

 


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Fig. 2. Frequency of p53 mutation (white) and p53 staining (black or gray) in CRCs without high MIN. The frequency of p53 gene mutation (white) in tumors with MIN(–)CIN(–) phenotype (10%) was significantly lower than in those with the CIN(+) counterpart (36%). The frequency of moderate or high p53 staining in tumors with MIN(–)CIN(–) phenotype was not different from that in the CIN(+) counterpart.

 

    Discussion
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 Materials and methods
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The current data confirm that a small portion of (17%) sporadic colorectal tumors without MIN or CIN exhibit a distinct clinicopathological and genetic presentation, suggesting that these tumors might arise from another distinct pathway (1417). Hawkins et al. (15), found 14 (30%) tumors out of 46 consecutive sporadic CRC were diploid and microsatellite-stable and were associated with the early development of metastasis. Yao et al. (16) reported one-fifth of 65 sporadic CRC tumors (from 35 patients aged 40 years or less and 30 patients aged 60 or more) were diploid and microsatellite-stable. They did not find an association of young age with the particular type of tumors. Whereas, Chan et al. (17), in a study including 22 early-onset and 16 late-onset tumors, reported a much greater rate of diploid and microsatellite-stable tumors in early-onset as compared with late-onset tumors (64 versus 13%). All four papers found a significant portion of sporadic CRC showing no evidence of either the CIN or MIN pathways, although the patient studied was highly selective except in one paper, their sample sizes were relatively small, and the criteria and methods to define MIN or CIN were varied. No genetic association was demonstrated in these four published papers.

In the current study, we analyzed 179 consecutive CRCs and found that the MIN(–)CIN(–) tumors were associated with a greater frequency of poorly differentiated and/or mucinous tumors compared with that of MIN(–)CIN(+) tumors (26 versus 12%). This form of CRC has a reduced frequency of p53 mutation (10%), but with a much greater frequency of p53 protein accumulation (56%), reflecting that stabilization of p53 protein in the absence of p53 gene mutation is an important event in this particular pathway (Figure 2). Because the distribution of TNM stages is similar between these two types of tumors, it is unlikely that the MIN(–)CIN(–) tumor represents an earlier stage of progression of the MIN (–)CIN(+) counterpart. These tumors, for which no mutation was detected, have increased p53 protein expression, reminiscent of the situation in adenomas reported by Tominaga et al. (22), who observed elevation of p53 protein in the absence of p53 mutation in 26 of 28 adenomas.

The p53 gene mutation rates (range, 30–63%) as well as the spectrum of p53 mutations vary in different geographic areas (18). The frequency of positive p53 staining was consistent with the reported data, ranging from 44.8 to 60.8% (2325). The frequency of p53 reactivity depended on the IHC procedures used for detection, IHC antibodies used, types of p53 mutation and cut-off values used for defining positivity (26). One tumor displayed only cytoplasmic reactivity. An association between IHC p53 protein reactivity and p53 gene mutations in ~70% of CRCs has been reported in the literature (27).

Of the 179 tumors, the frequency of tumors displaying MIN was in line with other reports (9,28,29). Compared with MIN(–), MIN(+) tumors tended to be bigger, more often located in the right colon, more likely to show mucinous or poorly differentiated histology, more likely to be diploid and associated with only minimal chromosomal rearrangement (29,30). K-ras mutations occurred as frequently in MIN(+) as in MIN(–) CRCs, which is in agreement with the findings of some investigators (30,31), but in contrast to those of others, who showed that MIN tumors were less likely to have K-ras mutations (32). Consistent with previous reports (26,31,32), eight of 11 (73%) MIN(+) tumors were IHC-deficient for expression of either MLH1 or MSH2 protein, whereas only one of 95 (1%) MIN(–) tumors was IHC-deficient.

The spectrum of p53 mutations in human tumors provides clues to the etiology and molecular pathogenesis (33). p53 expression is normally maintained at a low level (thus, it cannot be detected using IHC) through a negative feedback loop in which the p53-induced Mdm2 oncogene product interacts with p53 and targets it for proteasomal degradation (34,35). Yin et al. (36) showed that post-translational modifications that stabilize Mdm2 and inhibit Mdm2-mediated degradation of p53, such as p14ARF binding to Mdm2, or changes in growth conditions could block the degradation of p53 protein. It is well established that p53 protein accumulation detected by IHC may be found in p53 wild-type tumors as a result of alternative mechanisms leading to p53 protein stabilization (26,27). Thus, functional inactivation of p53 protein could circumvent the need for mutation (37,38). One of the major factors contributing to p53 stabilization in p53 wild-type tumors is stabilization of p53 via expression of p14ARF (39,40).

Several potential misclassifications should be considered. First, the three cases with instability in four or five markers among MIN(+) group might represent the high end of the MSI-L range. The possibility is low since at least one of two mononucleotide markers was mutated in these cases. Secondly, there was one MIN(–)CIN(–) tumor in the current study that might have been an MIN(+) tumor, as evidenced by the fact that this tumor was MSI-H (as classified by a panel of five markers) and also lacked hMSH2 expression. This misclassification played a trivial role in this study. Finally, since p53 mutations are generally screened on exons 5–9 (as with our data), it could be that some mutations have been overlooked. Nonetheless, bias caused by differences in mutations in the rarely studied exons is likely to be small.

In conclusion, we confirmed that CRC without high MIN and chromosomal instability is a biologically distinct form of CRC. A different type of chromosomal instability or a lesser degree of genomic instability is proposed to be involved in this particular type of tumor. Future work will focus on the identification of the genes involved in this fundamental cellular process. Further characterization and elucidation of this process may be expected to yield important new insights in the understanding and management of CRC.


    Acknowledgments
 
This study was supported by grant NSC 85-2331-B182-108 from the National Science Council and grants DOH85-HR-516, DOH86-HR-516, and DOH87-HR-516 from the National Health Research Institute, Department of Health, Executive Yuan, ROC.


    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Received September 5, 2003; revised December 18, 2003; accepted December 19, 2003.