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Loss of heterozygosity: An independent prognostic factor of colorectal cancer

AIM: Colorectal cancers result from the accumulation of several distinct genetic alterations. This study was to investigate the frequency and prognostic value of loss of heterozygosity (LOH) and microsatellite instability (MSI) at 14 genetic loci located near or within regions containing important genes implicated in colorectal tumorigenesis. METHODS: We studied colorectal cancers with corresponding normal mucosae in 207 patients (139 males and 68 females, mean age at the time of tumor resection 66.2±12.4 years, range 22-88 years). There were 37 right-sided colonic tumors, 85 left-sided colonic tumors and 85 rectal tumors. The distribution of tumor staging was stage I in 25, stage II in 73, stage III in 68, and stage IV in 41. We analyzed the LOH and MSI of HPC1, hMSH2, hMLH1, APC, MET, P53, NH23-H1, DCC, BAT25, BAT26, D17S250, MYCL1 and D8S254 with fluorescent polymerase chain reaction and denatured gel electrophoresis. High-frequency LOH was determined to be greater than three, or more than 50% of the informative marker with LOH. High-frequency MSI (MSI-H) was determined as more than four markers with instability (>30%). Correlations of LOH and MSI with clinical outcomes and pathological features were analyzed and compared. RESULTS: The occurrence of MSI-H was 7.25%, located predominantly in the right colons (7/15) and had a higher frequency of poor differentiation (6/15) and mucin production (7/15). LOH in at least one genetic locus occurred in 78.7% of the tumors and was significantly associated with disease progression. Of the 166 potentially cured patients, 45 developed tumor recurrence within 36 mo of follow-up. Clinicopathological factors affecting 3-year disease-free survival (DFS) were TNM staging, grade of differentiation, preoperative CEA level, and high LOH status. Patients with high LOH tumors had a significantly lower DFS (50%) compared with patients with low LOH tumors (84%). Of the patients developing subsequent tumor recurrence, the number and percentage of LOH were 2.97 and 46.8% respectively, similar to the stage IV disease patients. TNM staging had the most significant impact on DFS, followed by high LOH status. CONCLUSION: Clinical manifestations of LOH and MSI are different in colorectal cancer patients. High-frequency LOH is associated with high metastatic potential of colorectal cancers.


INTRODUCTION
Cumulated genetic alterations could affect proto-oncogenes such as Ki-ras and tumor suppressor genes, including APC, DCC and TP53 [1,2] . The common event that inactivates tumor suppressor genes is the loss of one allele frequently associated with an inactivating point mutation in the remaining wildtype allele. This process is referred to as loss of heterozygosity (LOH) [3] .
In another mutational pathway, colorectal cancers display increased rates of intragenic mutation characterized by generalized instability of short, tandemly repeated DNA sequences known as microsatellites [4] . High frequency of microsatellite instability (MSI-H) has been found in more than 90% of tumors in patients with hereditary nonpolyposis colorectal cancer [5,6] . The presence of MSI could be the result of defective DNA mismatch repair genes that were misaligned in repetitive sequences (caused by slippage of polymerases) and left unrepaired. In sporadic cases of colorectal cancer, MSI-H occurred in approximately 15% of tumors [4,5,7] .
Previous studies have demonstrated that tumors with more alleles lost have a considerably worse prognosis [8,9] . In contrast, the presence of MSI-H carries significant prognostic implications for colorectal cancer patients [10,11] . In this study, we evaluated 207 colorectal cancer patients. LOH and MSI at 14 markers were detected by microsatellite analysis to evaluate the clinical manifestations of LOH and MSI in sporadic colorectal cancers.

MATERIALS AND METHODS Patients and clinicopathological data
A total of 207 patients with colorectal adenocarcinoma who underwent resection in Taipei Veterans General Hospital from January 1999 to December 1999 were enrolled in this study. Patients were excluded if they received preoperative chemotherapy and/or radiotherapy, and had evidence of hereditary non-polyposis colorectal cancer according to the criteria of Amsterdam or familial adenomatous polyposis, a malignant tumor outside the colon within the previous five years, or died from surgical complications. Clinical data were recorded prospectively and stored in computerized files. The database included (1) patients' names, gender, age, family history, and major medical problems, (2) location, size, gross appearance, tumor-node-metastasis (TNM) stage, differentiation, and relevant pathologic prognostic features, and (3) types of operations, complications, recurrence of disease, and follow-up conditions. After operation, patients were followed up every 3 mo in the first 2 years, every 6 mo between 2 and 5 years, and annually thereafter. All patients were followed up for at least 3 years after operation or until death.

Tumor tissues
The Institutional Review Board of the Taipei Veterans General Hospital approved the study program. Consents to tissue collection were obtained from all patients. Following removal, specimens were cleaned thoroughly. The tumors were dissected meticulously and samples were collected from 4 different quadrants of the tumor for consideration of intratumoral heterogeneity. The corresponding normal mucosae taken at least 10 cm away from the primary tumor were collected. The fragments of samples were immediately frozen in liquid nitrogen and stored at -70 ℃. Sections of cancer tissues and the corresponding normal mucosae were identified, reviewed, and analyzed by a senior gastrointestinal pathologist who did not know the clinical outcome of the patients. The stage of the disease was classified according to the TNM classification of the International Union Against Cancer [12] . There were 139 (67.1%) men and 68 (32.9%) women in the study group.

LOH and MSI analysis
High-molecular-weight genomic DNA was prepared from each tumor and the corresponding normal tissue using the QIAamp tissue kit (QIAGEN GmbH, Hilden, Germany). Normal and tumor genomic DNA sample pairs were amplified using the microsatellite instability MSI/LOH assay starter kit (Applied Biosystems, Foster City, CA). According to the international criteria for the determination of MSI [13] , five reference markers including D2S123, D5S345, Bat25, Bat26 and D17S250 were enrolled in our microsatellite panel. The chromosomal location of the microsatellite markers and genes surrounding the markers was described in a previous report [14] . In brief, 25 ng template DNA was amplified with fluorescent-labeled primer. PCR was carried out in a GeneAmp PCR System 9, 600 thermal cycler (Applied Biosystems) as follows: a 10-min pre-PCR incubation step at 95 ℃, 30 cycles of amplification, each at 96 ℃ for 10 s, at 55 ℃ for 30 s, at 70 ℃ for 3 min, and a final extension at 70 ℃ for 30 min.
The amplification reactions were pooled and loaded onto a denaturing polyacrylamide gel, and the data were collected with an ABI 377 automated sequencer (Applied Biosystems). At the end of the run, each fluorescent peak was quantitated in terms of size (in base pairs), peak height, and area. Normal and tumor DNA pairs were compared for changes in peak height of each microsatellite marker via GeneScan analysis software (Applied Biosystems).
The LOH index was calculated using the modified method described by Cawkwell [15] for each paired normal and tumor samples. The peak height of two alleles in each tumor was divided by the peak height in normal samples: T1:T2/N1:N2 where T1 and N1 are the peak heights of the tumor and normal samples, respectively for the corresponding allele one, and T2 and N2 for the corresponding allele two. Allele loss, according to the manufacturer's instructions, occurred with an LOH index of ≤0.67 or ≥1.5. This allele loss also translated to a 33% decrease in peak height of one of the tumor alleles as compared with the normal alleles. The result of LOH analysis of a representative sample is shown in Figure 1. The tumors that exhibited LOH at more than 3 markers, or showed more than 50% of informative markers, were classified as the highfrequency LOH group (LOH-high). Others were classified as the low-frequency LOH group (LOH-low).
Tumor samples that exhibited novel allele peaks compared with the corresponding normal samples were classified as MSI at that marker. Such markers were considered uninformative for the LOH study. The pattern of MSI of a representative sample is shown in Figure 2. The analysis was performed twice if the data were controversial. According to the international criteria for the determination of MSI [13] , MSI in at least 4 or more loci was defined as high microsatellite instability (MSI-H); otherwise it was considered microsatellite stability (MSS).

Statistical analysis
The statistical end point in our analysis was the occurrence of metastasis or death. The group distribution of each clinicopathological characteristic according to the presence or absence of LOH or MSI was compared using the  2 test. The numerical value was compared with the Student's t test. Statistical significance was defined as P<0.05 (SPSS for Windows version 10.0). The variables that resulted in P values <0.1 were entered into the Cox proportional hazards model for the determination of the independent prognostic factor for colorectal cancer.

RESULTS
Markers with detectable heterozygous alleles were defined as informative. One hundred and sixty-three tumors (78.8%) exhibited LOH in at least one microsatellite marker. LOH in more than 3 markers (LOH-high) was noted in 93 tumors (44.9%). The highest frequency of LOH in the tumor suppressor gene was TP53.alu (65%), followed by DCC (64.3%), D8S254 (51.7%) and APC (47.8%) (Table 1, Figure 1A). There was no difference in the distribution of gender, age, preoperative CEA level and location of tumors between the LOH-high and LOH-low groups. Considering the pathological variables, there was a linear trend between TNM staging and the LOH-high tumors (P<0.001). The mean number and percentage of LOH in stage I tumors were 1.52% and 22.8%, respectively, with an increase to 3.02% and 48%, respectively, in stage IV tumors ( Figure 2). The other pathological variables such as lymph-vascular invasion, mucin component, invasive pattern of tumors, and grade of differentiation showed no significant association with the LOH-high tumors ( Table 2). Reference markers according to the international criteria for the determination of MSI [13] .

DISCUSSION
Our approach consisted of examining 14 microsatellite markers located near or within regions containing important genes implicated in the complex process of colorectal tumorigenesis. The use of automated assessment of loss of heterozygosity and microsatellite instability with fluorescent-PCR was described by Cawkwell [15] and could be adapted to the analysis of both LOH and MSI, two important mechanisms of colorectal carcinogenesis. In our results, the frequency of MSI exhibited a trend to cluster in hMSH2, BAT25 and BAT26 loci, but not in markers in regions displaying high levels of allelic loss (e.g., p53 and DCC). The higher susceptibility of mononucleotide repeats to instability in MSI tumors has been reported by Ionov [4] and Dietmaier [16] . The microsatellite markers with a low frequency MSI might provide prognostic information about LOH. The present study demonstrated a significant inverse correlation between the two molecular events (LOH vs MSI).
The tumors with MSI-H phenotypes had a lower frequency of LOH than those with MSS phenotype. In the present series of patients with resected primary colorectal carcinomas, the frequency of MSI-H phenotypes was 7.25%, lower than in previous reports [7,17] . This may be attributed to several markers having a low-frequency MSI. If only considering five reference markers [13] as the current definition of MSI-H, the frequency of MSI-H could be as high as 11.1% of tumors. Previous reports indicated that MSI-H colorectal cancers were associated with distinct clinicopathological features, including poor differentiation, extracellular mucin accumulation, younger age at onset, and proximal tumor location [18][19][20] . Our MSI-H tumors displayed similar clinicopathological findings.
Loss of heterozygosity, i.e., loss of one allele at a constitutional heterozygous locus indicates the probability of loss of a tumor suppressor gene, which might promote neoplastic progression [21,22] . The frequency of an informative tumor for each microsatellite marker varied in the present study (range 14.5-73.9%). Analysis of a single marker may be biased by non-informative tumors, especially in the marker in which the frequency of an informative tumor was less than 50%. Therefore, we defined the LOH-high group as LOH at more than 3 markers, or more than 50% of informative markers for considering both the number and percentage of LOH. In our results, high-frequency LOH could be an independent prognostic factor affecting tumor recurrence. Of the 166 potentially cured patients, those with a high LOH tumor showed a DFS of 50 %, which was poorer than those with low LOH tumors (84%). Of the 45 patients who developed tumor recurrence, the mean number of LOH was 2.97 and the percentage of LOH in the primary tumor was 46.8%, which were similar to those tumors that already had distant metastasis at operation. These findings imply that high LOH tumors behave comparably to those stage IV diseases.
Metastasis is a complicated multistep process. The cells must detach and acquire motility, invade through the basement membrane and extracellular matrix, gain access to and survive in the vascular system, adhere to and immigrate into the target organ, and then proliferate in the new site [23] . It is reasonable to believe that multiple genes control the complicated process. Previous reports have hypothesized that accumulation of various genetic abnormalities is more important in tumorigenesis than the sequence of genetic alterations [2,24] .
Additionally, a previous report demonstrated that p53 and DCC were the most common mutated tumor suppressor genes in colorectal cancer, and occurred mainly at the late phase of carcinogenesis [1] . In our study, the frequency of LOH was the highest at p53 (65.0%) followed by DCC (64.3%), D8S254 (51.7%) and APC (47.8%). There were four markers associated with metastatic potential, including p53, DCC, and a tumor suppressor gene located in the 8p22. In our series, the incidence of LOH of p53 was similar to a previous report [24] . The p53 gene has been thought to modulate two discrete functions, both in response to stress (particularly DNA damage), cell cycle arrest, and apoptosis [25,26] . The loss of p53 in the final stages of colon cancer not only allows the cells to divide at an unrestrained rate, but this rapid replication further promotes the development of even more genetic mutations because the cell cycle is not interrupted when errors occur. Several authors have found that mutations of p53 or allelic loss of the p53 was correlated with poor outcomes [27][28][29] . Our study yielded a similar finding. The incidence of allele loss of DCC in colorectal cancer has been reported to be from 33% [15] to 75% [29][30][31] . According to previous reports, LOH or decreased DCC expression was associated with liver and lymph node metastasis [29,31] . In our study, LOH of DCC was significantly associated with lymph node metastasis (data not shown). The DCC protein was a transmembrane glycoprotein similar to the neural cell adhesion molecules [32] . LOH of DCC could thus lead to impaired contacts between cells, thereby contributing to tumor growth and invasion. The prognostic value of LOH of DCC has been confirmed in several previous studies [31,33,34] . Our observations also showed that LOH of DCC was significantly associated with metastasis potential.
Another putative tumor suppressor gene located in the short arm of chromosome 8 had a high frequency of LOH in our study, as shown in a previous report [35] . Several studies showed that this novel tumor suppressor gene locus was related to the stage progression of colorectal cancer [36,37] , similar to our findings.
In the multivariate analysis, TNM stage was the most significantly independent prognostic factor for colorectal cancer, followed by high-frequency LOH and preoperative CEA level. The outcome of patients with colorectal cancer was determined by the presence or absence of metastasis at the time of operation. We further analyzed the prognostic impact of high LOH status on early state tumors (stage I or II). Of the 29 stage I or II tumor patients with high-frequency LOH, 9 patients (32.1%), developed distant metastasis within three years after surgery, which was significantly higher than those with low-frequency LOH (8.7%, P = 0.037). This finding implies that early stage tumors with LOH-high have a stronger metastatic potential.
In conclusion, genetic change in both MSI and LOH phenotypes may occur in sporadic colorectal tumors, as established by microsatellite analysis. Variation of genetic phenotypes is related to different clinical manifestations, an important finding that may be useful in identifying subgroups of patients with different prognostic risks.