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10.1245/ASO.2003.12.014
Annals of Surgical Oncology 10:1086-1094 (2003)
© 2003 Society of Surgical Oncology
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ORIGINAL ARTICLES

Loss of Heterozygosity and DNA Aneuploidy in Colorectal Adenocarcinoma

Jen-Kou Lin, MD, PhD, Shih-Ching Chang, MD, Ya-Chien Yang, PhD and Anna Fen-Yau Li, MD

From the Department of Surgery, Division of Colon & Rectal Surgery (J-KL, S-CC), and Department of Pathology (AF-YL), Veterans General Hospital, Taipei, Taiwan; Institute of Clinical Medicine (S-CC), National Yang-Ming University, Taipei, Taiwan; School of Medical Technology (Y-CY), College of Medicine, Institute of Epidemiology, College of Public Health; and Department of Laboratory Medicine (Y-CY), College of Medicine, National Taiwan University, Taipei, Taiwan.

Correspondence: Address correspondence and reprint requests to: Jen-Kou Lin, MD, PhD, Division of Colon & Rectal Surgery, Department of Surgery, Veterans General Hospital Taipei, No. 201, Sec. 2, Shih-Pai Rd., Taipei 11217, Taiwan, ROC; Fax: 886-2-287-57639; E-mail: jklin{at}vghtpe.gov.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: This study evaluated the relationship between DNA aneuploidy and loss of heterozygosity (LOH) at different genetic loci in colorectal adenocarcinoma.

Methods: A total of 112 patients with surgically removed colorectal adenocarcinoma in Taipei Veterans General Hospital from January 1999 to July 2001 were included in this study. The pattern of DNA ploidy was determined with DNA flow cytometry, and the LOH of various genetic loci was determined with fluorescence polymerase chain reaction and denaturing gradient gel electrophoresis. The relationship between DNA ploidy, LOH of various genetic loci, and clinicopathologic variables was analyzed with the {chi}2 test with Yates’ correction as well as by multivariate binary logistic regression analysis.

Results: Seventy-one (63.4%) of the 112 carcinomas had DNA aneuploidy. The DNA aneuploidy was not associated with any clinicopathologic variable. Ninety-one tumors (81.3%) exhibited LOH in at least one genetic locus. In the univariate analysis, the DNA aneuploidy was associated with LOH of Tp53-penta, D8S254, D5S346, and high-frequency LOH (P = .001, P = .016, P = .041, and P < .001, respectively). In the multivariate analysis, the most significant factor influencing DNA aneuploidy was D8S254, followed by Tp53-penta, high-frequency LOH, and D5S346.

Conclusions: DNA aneuploidy is strongly associated with LOH at specific genetic loci.

Key Words: Loss of heterozygosity • Aneuploidy • Microsatellite instability • Colorectal neoplasms • p53 • APC


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal cancers result from the accumulation of several distinct genetic alterations that occur at the chromosomal and DNA levels. It has been shown that the development of most colorectal carcinomas is associated with allelic losses of the tumor-suppressor 17p (p53) gene, the 5q (adenomatous polyposis coli; APC) gene, and the 18q (deleted in colorectal cancer; DCC) gene.1

At the chromosomal level, the abnormal nuclear content of DNA (aneuploidy), as determined by flow cytometry, is a well-known finding in colorectal cancer.2–4 However, reports of the prognostic value of flow cytometric DNA ploidy in patients with colorectal carcinoma are contradictory.5,6

The relationship between DNA ploidy and molecular genetic alterations is of interest. Numerous in vitro and in vivo studies have shown that tumors with a mutated p53 gene frequently exhibit abnormalities in chromosome numbers7,8 and suggest that loss of p53 function may lead to abnormal regulation of mitosis and segregation of chromosomes.9 However, several other lines of evidence do not support a direct role for p53 mutation in the genesis of this form of chromosomal instability.10,11 Some diploid tumor cell lines that exhibit a stable karyotype also contain mutant p53.12

The purpose of this study was to correlate the aneuploidy of the tumors determined by flow cytometry with loss of heterozygosity (LOH) of specific genetic loci in primary sporadic colorectal carcinomas. In addition, the association of DNA ploidy and the LOH of specific loci with various tumor characteristics was evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient and Clinicopathologic Data
A total of 112 patients with sporadic colorectal adenocarcinoma who underwent resection in the Taipei Veterans General Hospital between January 1999 and July 2001 were included in this study. Patients who received preoperative chemotherapy, radiotherapy, or both; died within 30 days after surgery; or had evidence of hereditary nonpolyposis colorectal cancer (according to the criteria of Amsterdam) or familial adenomatous polyposis were excluded. Clinical data were recorded prospectively and stored in computer files. The database included (1) name, sex, age, family history, and major medical problems of the patients and (2) location, size, gross appearance, tumor-node-metastasis stage, differentiation, and important pathologic prognostic features of the tumor.

There were 80 men and 32 women in the study group. The mean age at the time of tumor resection was 66.7 ± 10.7 years (range, 23–86 years). There were 25 right-sided tumors (cecum to distal transverse colon) and 87 left-sided tumors (splenic flexure to rectum). The distribution of tumor staging was 14 stage I (12.5%), 44 stage II (39.3%), 33 stage III (29.5%), and 21 stage IV (18.8%).

Tumor Tissue
Tumors were dissected meticulously, and samples were collected from four different quadrants of the tumor for consideration of intratumoral heterogeneity. The corresponding normal mucosa at least 10 cm away from the primary tumor was also collected. The fragments of the tissue were immediately frozen in liquid nitrogen and stored at -70°C. Sections of cancerous tissue and its corresponding normal tissue were 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 tumor-node-metastasis classification of the International Union Against Cancer.13 The study was performed in accordance with the Institutional Review Board of the Taipei Veterans General Hospital, and written, informed consent for tissue collection was obtained from all patients.

Flow Cytometry for DNA Ploidy
The DNA ploidy in 112 tumors was analyzed with flow cytometry by following the method of Dressler et al.,14 with some modifications. DNA ploidy was quantitated by the DNA index (DI), that is, the ratio of the mean fluorescence intensity of the G0G1 peak of the tumor cell population to that of the normal diploid population. Specimens were considered diploid (DI = 1) if they had a single G0G1 peak and aneuploid (DI != 1) if they exhibited two or more discrete peaks, including abnormal G0G1 peaks (each peak equivalent to the fluorescence of at least 20% of the total sample nuclei) and a corresponding G2M peak. Samples with coefficients of variation >8% were excluded from further analysis. Tumors with both diploid and aneuploid subpopulations were classified as having DNA aneuploidy. The mean coefficients of variation were 6.4% (tumor) and 2.4% (normal colon mucosa). A representative aneuploid sample is shown in Fig. 1.



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  		FIG. 1. Representative flow cytometry results show DNA diploidy and aneuploidy. (A) The tumor of a patient with stage II disease had DNA diploidy. Flow cytometry shows a single G0G1 peak (4.6% coefficient of variation [CV]). (B) The tumor of another patient with stage III disease had DNA aneuploidy. The sample exhibited two discrete G0G1 peaks, with an abnormal G0G1 peak containing 66.6% of the total sample nuclei. The DNA index of the abnormal G0G1 peak was 1.597, and the CV was 5.2%.

 
LOH Analysis
High-molecular-weight genomic DNA was prepared from each tumor and corresponding normal tissue by using the QIAamp Tissue Kit (Qiagen GmbH, Hilden, Germany) and proteinase K. Normal and tumor genomic DNA sample pairs were amplified by using the Microsatellite Instability RER/LOH Assay Starter Kit (Applied Biosystems, Foster City, CA). The microsatellite markers included two mononucleotide repeats (A)n, 10 dinucleotide repeats (CA)n, and 1 pentanucleotide repeat (AAAAT)n. The chromosomal location and related genes of the markers are shown in Table 1. In each of primer pairs, the 5' primer was synthesized with 6-carboxyfluorescein, tetrachloro-6-carboxyfluoroscein, or 2',4',5',7'-hexachloro-6-carboxyfluorescein fluorescent label. Amplifications were performed in a final volume of 20 µL by using 10 nM of each of the respective primers, 25 ng of normal or tumor DNA, 125 µM of each deoxynucleotide triphosphate, 1 U of AmpliTaq Gold DNA polymerase, (Applied Biosystems), 10 mM of Tris-HCl (pH 8.3), 50 mM of KCl, and 2 mM of MgCl2. Polymerase chain reaction (PCR) was performed in a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems) as follows: a 10-minute pre-PCR incubation step at 95°C; 30 cycles at 96°C for 10 seconds, 55°C for 30 seconds, and 70°C for 3 minutes; and a final extension at 70°C for 30 minutes.


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  		TABLE 1. Microsatellite marker panel
 
The individual amplification reactions were pooled, and 4 µL of the respective normal or tumor pooled panel was added to 5.5 µL of loading buffer consisting of recrystallized formamide, tetramethylrhodamine (TAMRA)-350 molecular weight marker, and blue dextran dye. Reactions were denatured at 95°C for 2 minutes and immediately placed on wet ice. Two microliters of each respective panel was loaded onto a denaturing polyacrylamide gel, and the data were collected by 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 the changes in peak height of each fluorescent marker via GeneScan analysis software (Applied Biosystems).

LOH index was calculated by the modified method described by Cawkwell et al.15 The LOH index was calculated for each paired normal and tumor sample, and then the tumor ratio was divided by the normal ratio, that is, T1:T2/N1:N2, where T1 and N1 are the peak heights of the tumor and normal samples, respectively, for the corresponding allele 1, and T2 and N2 are the peak heights of the tumor and normal samples for the corresponding allele 2. According to the manufacturer’s instructions, an LOH index of <=.67 or >=1.5 indicates allele loss. This is equal to a 33% decrease in the peak height of one of the tumor alleles as compared with the normal allele. The result of LOH analysis of a representative sample is shown in Fig. 2. The tumors that had LOH at more than two markers, or more than 50% of informative markers, were classified as the high-frequency LOH group; all others were classified as the low-frequency LOH group. Tumor samples that exhibited novel allele peaks, as compared with the corresponding normal sample, were classified as showing microsatellite instability (MSI) at that marker. Such markers were considered uninformative for the LOH study. The pattern of MSI of a representative sample is shown in Fig. 3. The analysis was performed twice if the data were controversial.



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  		FIG. 2. Representative results of GeneScan show loss of heterozygosity (LOH) at six genetic loci (NM23H1, APC, Tp53-Penta, DCC, hMSH2, and D7S501) in a 62-year-old man with stage III disease. The products of polymerase chain reaction (PCR) were labeled with 6-carboxyfluorescein, tetrachloro-6- carboxyfluoroscein, and 2',4',5',7'-hexachloro-6-carboxyfluorescein in (A), (B), and (C). *Size of the PCR product (base pairs); **fluorescence intensity of peak; {dagger}homozygosity.

 


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  		FIG. 3. Representative results of GeneScan show microsatellite instability at seven genetic loci (NM23H1, APC, D8S254, DCC, hMSH2, Tp53-Dint, and BAT26) in a 45-year-old man with stage II disease. The products of polymerase chain reaction were labeled with 6-carboxyfluorescein, tetrachloro-6-carboxyfluoroscein, and 2',4',5',7'-hexachloro-6-carboxyfluorescein in (A), (B), and (C). The arrowhead indicates novel alleles.

 
Statistical Analysis
Categorical variables were analyzed by using the {chi}2 test with Yates’ correction. The differences in quantitative variables between groups were compared by using Student’s t-test. P < .05 for the two-tailed test was considered statistically significant. The independent relationship between DNA aneuploidy and LOH of genetic loci was determined with multivariate binary logistic regression analysis (SPSS for Windows 9.0; SPSS Inc., Chicago, IL). All the data in the text and tables are expressed as mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Because only four patients had well-differentiated or poorly differentiated tumors, data about the degree of differentiation were not analyzed. Among the 112 colorectal adenocarcinomas subjected to DNA flow cytometry, 41 (36.6%) were diploid and 71 (63.4%) were aneuploid. As shown in Table 2, the status of ploidy and clinicopathologic variables were not significantly related. Aneuploid tumors tended to be located more commonly in the distal colon and rectum (66.7%) than in the proximal colon (33.3%) and more commonly showed expansive rather than infiltrative tumor growth, but these differences did not reach statistical significance (P = .180 and .051, respectively).


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  		TABLE 2. Correlation between DNA ploidy and clinicopathologic variables
 
LOH and MSI
Twenty-six tumors (23.2%) exhibited MSI in at least one genetic locus. The frequency of MSI exhibited a trend toward clustering in hMSH2 (9.8%), BAT25 (8%), and BAT26 (5.4%) (Table 3). According to international criteria for the determination of MSI,16 MSI in at least two loci (MSI-H) of five reference markers was noted in seven tumors (6.25%). Of the seven tumors with MSI-H, two tumors had DNA aneuploidy.


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  		TABLE 3. Frequency of loss of heterozygosity (LOH) and microsatellite instability (MSI) of tumor-suppressor genes in colorectal cancer
 
Those with detectable heterozygous alleles were defined as informative cases. Ninety-one tumors (81.3%) exhibited LOH in at least one tumor-suppressor gene. Of them, 23 had LOH at 2 loci, 24 at 3 loci, 7 at 4 loci, 8 at 5 loci, and 3 at 6 loci. The highest frequency of LOH in the tumor suppressor genes was Tp53-penta (76.7%), followed by DCC (64.4%), APC (50%), and D8S254 (49.1%) (Table 3).

The stage and location of the tumor were not related to the LOH status of any of the 13 genetic loci tested. The LOH of DCC locus was associated with older age (71.1 ± 6.8 years versus 65.0 ± 13.9 years; P = .015), female predominance (female versus male: 35.5% vs. 14.3%; P = .044), and infiltrative invasive pattern of tumor growth (infiltrative pattern versus expansive pattern: 18.4% vs. 0%; P = .036). The LOH of hMSH2 had a higher frequency of lymphovascular permeation (50% vs. 16.9%; P = .01). No other significant correlation was found between the clinicopathological characteristics and the LOH of other genetic loci.

Relationship of DNA Ploidy to LOH of Genetic Loci
The DNA aneuploidy was significantly associated with LOH of three loci: APC (P = .041), Tp53-penta (P = .001), and D8S254 (P = .016). High-frequency LOH also had a significant correlation with DNA aneuploidy (P < .001) (Table 4). In our results, the frequency of informative cases for various genetic loci ranged from 8.9% to 74.1% of total cases (Table 3), and multivariate analysis could not be performed. To determine which factor had a significant influence on DNA aneuploidy, a 2 x 1 binary logistic regression analysis was performed with four factors that had a significant correlation with DNA aneuploidy in the univariate analysis. As shown in Table 5, D8S254 had the most significant effect for DNA aneuploidy, followed by Tp53-penta, high-frequency LOH, and APC.


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  		TABLE 4. Relationship of DNA ploidy to the status of loss of heterozygosity (LOH) of specific genetic loci
 

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  		TABLE 5. Multivariate binary logistic regression analysis for DNA aneuploidy
 
Thirty-one patients who received curative resection developed distant metastasis within 2 years after surgery. The sites of distant metastases included 12 in the lung, 20 in the liver, and 4 in other areas. Combined with 21 patients who had distant metastasis at initial surgery, 52 patients had evidence of distant metastasis within 2 years after surgery. Of these patients, metastatic potential was significantly associated with high-frequency LOH (55.1% vs. 31.0%; P = .023), D8S254 (63.0% vs. 30.8%; P = .029), Tp53-penta (55.8% vs. 17.6%; P = .017), and DCC (63.2% vs. 28.6%; P = .022). The patients with DNA aneuploid tumors had a trend toward higher metastatic potential (50.7%) than those with diploid tumors (36.6%), but the difference did not reach statistical significance (P = .232).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The clinical use of DNA ploidy as a prognostic variable in colorectal carcinoma is still controversial.17,18 Conflicting results are explained by the lack of standardization of studies and the extent of DNA intratumoral heterogeneity.18,19 In our study, 63.4% (71 of 112) of tumors had an aneuploid DNA content, which is in agreement with findings in previous studies.18,20,21 However, in both our study and some previous studies,21 but in contrast to other findings,18,20 no significant association was found between DNA ploidy and pathologic stage. The seven tumors with MSI-H seemed to have a stable karyotype, and five (71.4%) had diploid DNA content. Of interest, another two MSI-H tumors with aneuploid DNA had LOH of Tp53-penta. This finding implies that p53 plays a major role in chromosome instability.

At the molecular level, aneuploidy is reflected in allelic imbalance and often is associated with LOH.22,23 In our study, 81.3% (91 of 112) of tumors exhibited LOH in at least 1 genetic locus. The DNA aneuploidy was significantly associated with LOH of the APC, p53, and 8p22 (D8S254) loci. The previous studies showed that in mitotic cells, the association of APC protein with microtubule-plus ends seems to affect the fidelity of chromosomal segregation.24,25 The association of APC protein with microtubules and its role in chromosome segregation may be important in colorectal tumorigenesis.

The p53 tumor-suppressor gene is frequently mutated in human tumors, and its loss or inactivation is correlated with genetic instability.26,27 The p53 protein has been implicated in cell-cycle checkpoints (cell-cycle arrest and cell death).28–30 The centrosome is a major microtubule-organizing center of animal cells and is essential for accurate chromosome transmission to daughter cells.31,32 Inactivation of the p53 gene is associated with the centrosome hyperamplification that was thought to act as the major contributor to chromosome instability and has been reported in various types of human cancers.33,34 A recent study showed that the p53 gene regulated the coordinated initiation of centrosome and DNA duplication by multiple pathways.35,36 However, the relationship between p53 and aneuploidy is not a simple one. Several reports have shown that aneuploidy has been observed in the absence of p53 overexpression, and chromosomal aberrations have been demonstrated in colorectal cancers with only wild-type p53.37,38 In our study, we also found that LOH in another p53 locus (Tp53-Dint) was not associated with aneuploidy. Such disparities may be partly explained by different mutations having different downstream effects in the p53 pathway.

Another putative tumor-suppressor gene located in the short arm of chromosome 8 (8p22), as compared with APC and p53 in our results, seemed to have the most significant association with DNA aneuploidy. In addition to colorectal cancer, several cancers show a high rate of allele loss on chromosome 8p.39,40 The most commonly deleted region in these cancers is 8p22, which is hypothesized to contain a tumor-suppressor gene. Several studies have shown that this novel tumor-suppressor gene locus was related to the progression of colorectal cancer.41,42 However, its mechanism of action has not been elucidated. Our results, suggesting that this novel tumor suppressor gene might be associated with chromosome segregation, may spur future investigation of this gene.

High-frequency LOH was more closely associated with DNA aneuploidy. Previous studies also showed that DNA diploid tumors, compared with aneuploid tumors, have few or no chromosomal or genetic alterations.43 However, this does not preclude the possibility that diploid colorectal cancers have subtle chromosomal number or DNA changes that are not detected with flow cytometry.4

In conclusion, there is strong association between DNA aneuploidy and LOH at specific genetic loci, including APC, p53, and a novel tumor-suppressor gene at the short arm of chromosome 8 (D8S254). Our findings also suggest that aneuploid tumors have more allelic loss. No single genetic marker is correlated with tumor stage. Using our experimental approach, we expect to learn more about the carcinogenesis of colorectal cancer.


   ACKNOWLEDGMENTS
 
ACKNOWLEDGMENTS

The acknowledgment are available online at www.annalssurgicaloncology.org.

Supported by grants from the Veterans General Hospital–Taipei (VGH90348) and the National Science Council (NSC92-2314-B-075-100).


   FOOTNOTES
 
Loss of heterozygosity (LOH) and DNA aneuploidy were determined with fluorescence polymerase chain reaction and DNA flow cytometry, respectively, in 112 colorectal cancer patients in Veterans General Hospital–Taipei. The DNA aneuploidy was significantly associated with LOH of Tp53-penta, D8S254, D5S346, and high-frequency LOH.

Received for publication December 17, 2002. Accepted for publication July 9, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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