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Clinical Cancer Research Vol. 11, 2180-2187, March 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Microsatellite Instability Testing in Colorectal Carcinoma: Choice of Markers Affects Sensitivity of Detection of Mismatch Repair–Deficient Tumors

Stephanie B. Hatch1, Harry M. Lightfoot, Jr.3, Christopher P. Garwacki4, Dominic T. Moore5,6, Benjamin F. Calvo3, John T. Woosley4, Janiece Sciarrotta4, William K. Funkhouser4,6 and Rosann A. Farber1,2,4,6

1 Curriculum in Genetics and Molecular Biology, Departments of 2 Genetics, 3 Surgery, 4 Pathology and Laboratory Medicine, and 5 Biostatistics, 6 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Requests for reprints: Rosann A. Farber, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, CB #7525 Brinkhous-Bullitt Building, Chapel Hill, NC 27599. Phone: 919-966-6920; Fax: 919-966-3630; E-mail: rfarber{at}med.unc.edu.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Microsatellite instability (MSI) is found in 10% to 15% of sporadic colorectal tumors and is usually caused by defects in DNA mismatch repair (MMR). In 1997, a panel of microsatellite markers including mononucleotide and dinucleotide repeats was recommended by a National Cancer Institute workshop on MSI. We investigated the relationship between instability of these markers and MMR protein expression in a cohort of sporadic colorectal cancer patients.

Experimental Design: Paraffin sections of normal and tumor tissue from 262 colorectal cancer patients were examined for MSI status by PCR amplification and for MMR protein expression using antibodies against hMLH1, hPMS2, hMSH2, and hMSH6.

Results: Twenty-six (10%) of the patients studied had tumors with a high level of MSI (MSI-H). The frequencies of MSI were the same in African-American and Caucasian patients. Each of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats and had loss of MMR protein expression, as did two tumors that had low levels of MSI (MSI-L). These two MSI-L tumors exhibited mutations in mononucleotide repeats only, whereas eight of the other nine MSI-L tumors had mutations in just a single dinucleotide repeat. There was not a statistically significant difference in outcomes between patients whose tumors were MMR-positive or MMR-negative, although there was a slight trend toward improved survival among those with MMR-deficient tumors.

Conclusions: The choice of microsatellite markers is important for MSI testing. Examination of mononucleotide repeats is sufficient for detection of tumors with MMR defects, whereas instability only in dinucleotides is characteristic of MSI-L/MMR-positive tumors.

Key Words: microsatellites • prognosis • immunohistochemistry


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal carcinoma is the second leading cause of cancer-related deaths in the Western world. There are ~110,000 new cases and 50,000 deaths due to the disease per year in the U.S. (1). A small fraction of patients (~5%) have a hereditary colorectal cancer syndrome (2). One of these syndromes, hereditary non-polyposis colorectal cancer (HNPCC), accounts for 1% to 2% of all colorectal carcinomas (3, 4). HNPCC is an autosomal dominant predisposition to colorectal carcinomas and to cancers at other sites, including the endometrium, stomach, small intestine, hepatobiliary and genitourinary tracts, and ovary (5–7). Instability of short tandem repeats, or microsatellites (MSI), is a characteristic of the tumors from these patients (8). In most HNPCC colorectal tumors, MSI has been shown to result from defects in DNA mismatch repair (MMR; ref. 9). Mutations in the hMLH1 or hMSH2 genes are the most common defects in these families; in equal proportions, these make up about 94% of the germ line mutations detected. Also, a few families have been found to have hPMS2 or hMSH6 mutations, accounting for the other 6% of mutations in HNPCC patients (2, 10).

About 10% to 15% of sporadic colorectal cancers also exhibit MSI, and loss of expression of one or more of the MMR proteins has been found in these tumors (11–13). Most sporadic MSI-positive tumors lack expression of hMLH1, as the result of promoter methylation (14). Loss of expression of hMSH2 is found in some apparently sporadic tumors, but because the hMSH2 promoter does not become methylated, it is likely that there is a germ line mutation in one allele in these patients (15). hMSH6 and hPMS2 defects seem to be rare in sporadic cases. In one study, loss of expression of either of these proteins was found in only 2% of tumors (12).

HNPCC tumors and sporadic MMR-deficient tumors may behave similarly because they share characteristics that differ from those of MSI-negative tumors. Sporadic MSI-positive and HNPCC tumors tend to be poorly differentiated and/or mucinous (16, 17). They are usually diploid, and p53 mutations, loss of heterozygosity at 18q, APC mutations, and KRAS mutations are found less frequently than in MSI-negative tumors (18, 19). Most of the genetic alterations that are common in MSI-negative but not in MSI-positive sporadic colorectal carcinoma have been linked to poor prognosis (20–23). MSI-positive sporadic colorectal carcinoma cases have been reported to have better outcomes in most, but not all, studies (24–27). HNPCC patients have also been reported to have more favorable prognoses than patients with sporadic colorectal cancer (28, 29) , but recent studies suggest that this observation may be due to ascertainment bias (30). Therefore, testing for MSI is important not only for the detection of patients with possible germ line mutations, but also for the study of sporadic MSI-positive tumors.

A meeting at the National Cancer Institute on MSI led to the recommendation of five microsatellite markers for the detection of MSI (31). It was also suggested that if one or more of the markers are unstable, a second panel of five markers should be examined (32, 33). If 40% or more of the markers tested are unstable, the tumor is categorized as having high levels of MSI (MSI-H).

We used these National Cancer Institute criteria to categorize 262 tumors from sporadic colorectal cancer patients from North Carolina, including 180 Caucasians and 82 African-Americans, into three groups: microsatellite-stable (MSS), those with low levels of MSI (MSI-L), or MSI-H. We also used immunohistochemistry to examine the expression of the MMR proteins hMLH1, hMSH2, hMSH6, and hPMS2. We found that the frequency of MMR-defective tumors was the same in Caucasian and African-American patients. We also observed that 28 of 29 tumors with mononucleotide repeat instability had loss of expression of one or more of the MMR proteins, whereas none of the tumors with only mutations in dinucleotide repeats showed loss of MMR. This finding indicates that the type of microsatellite examined affects the ability to detect tumors with MMR defects.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. All patients who had complete resection of colorectal cancer at University of North Carolina Hospitals from 1985 to 1995 were considered for the study. Patients were excluded if they had inflammatory bowel disease or hereditary colorectal carcinoma, if paraffin-embedded tissue was unavailable, or if they received preoperative chemotherapy or radiation. Of 275 cases, none were excluded because of hereditary colorectal carcinoma or inflammatory bowel disease, 8 were excluded because of tissue unavailability, and 5 were excluded because of preoperative chemotherapy or radiotherapy, resulting in a total of 262 eligible cases. Data collected from patient charts included stage (depth of tumor invasion, presence or absence of lymph node metastases, and presence or absence of distant metastases, using the tumor-node-metastasis system of the American Joint Committee on Cancer), grade, use of postoperative chemotherapy or radiation therapy, patient age, self-identified ethnicity, and sex. The tumors designated "proximal" were from the cecum, ascending colon, and transverse colon, whereas "distal" tumors were from the descending colon, sigmoid colon, and rectum (there were no tumors from the splenic flexure). We attempted to obtain date and cause of death from patient records and the UNC Hospitals Cancer Registry. If necessary, the date of death, county of last residence, or county of death was determined from the Social Security Death Index or from the web site http://www.ancestry.com/. Copies of official death certificates were obtained from North Carolina County Registers of Deeds offices.

The study was pre-approved by the Institutional Review Board of the UNC School of Medicine and is compliant with the Health Insurance Portability and Accountability Act. All patient identifiers were removed from the laboratory data set.

Detection of Microsatellite Instability. DNA samples for PCR were coded in order to avoid any possible bias in determination of MSI phenotypes due to foreknowledge of MMR protein expression or vice versa. MSI status was determined by PCR of genomic DNA isolated from formalin-fixed, paraffin-embedded normal and tumor tissues from each patient. Tissue sections were deparaffinized in xylene, digested with proteinase K (2 mg/mL) overnight at 55°C, and DNA was isolated using DNAzol reagent (Life Technologies, Carlsbad, CA). PCR was carried out in 10 µL reactions containing 1x manufacturer's PCR buffer, 1.5 mmol MgCl2, 200 µmol/L deoxynucleotide triphosphates, 0.5 units of Platinum Taq polymerase (Life Technologies), and 0.5 µmol/L of each primer; the forward primer had been end-labeled using {gamma}33P-ATP and polynucleotide kinase. The cycles were as follows: 8 minutes at 94°C, then 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. The products were subjected to electrophoresis in 8% denaturing polyacrylamide gels, which were subsequently dried and exposed to X-ray film (Kodak, BioMax) at room temperature overnight. To determine the extent of MSI, five microsatellite markers recommended by a National Cancer Institute workshop on MSI were examined (D5S346, BAT25, BAT26, D2S123, and D17S250; ref. 31). If one or more of these markers showed instability, or fewer than five of the markers were amplifiable, then a second panel of five markers was analyzed (BAT40, D10S197, D18S58, D18S69, and MYCL1; ref. 33). Microsatellites were judged to be unstable if one or more novel bands were present in the PCR product of the tumor sample compared with the PCR product of the normal tissue of the same individual. A tumor was considered to be MSI-H if 40% or more of the amplified markers were unstable, MSI-L if fewer than 40% of the markers were unstable, and MSI-negative or MSS if none of the first five markers were unstable. MSI status was scored independently by two investigators.

Immunohistochemistry. Staining was carried out on 5-µm-thick paraffin sections of normal and tumor tissue from each patient, using mouse monoclonal antibodies specific for each of the four human MMR proteins: hMLH1 (clone G168-15, 1:200, BD PharMingen, San Diego, CA), hMSH2 (clone FE11, 1:400, Calbiochem, La Jolla, CA), hMSH6 (clone 44, 1:400, BD Transduction Laboratories, San Diego, CA), and hPMS2 (clone A16-4, 1:50, BD Transduction Laboratories). Antigen retrieval was carried out for 30 minutes in a steamer in 1 mmol/L EDTA (pH 8), for hMLH1, hMSH2, and hMSH6, or in Citra buffer (BioGenex, San Ramon, CA) for hPMS2. Endogenous biotin was blocked using the avidin/biotin blocking kit (BioGenex). Tissue sections were incubated with the primary antibody at 4°C overnight. Anti-hMLH1, -hMSH2, and -hMSH6 primary antibodies were detected using a biotinylated anti-mouse secondary antibody, avidin-peroxidase complex (Vector Elite kit, Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine (DAKO, Carpinteria, CA) as the chromogen. Anti-hPMS2 antibody was detected using the Super Sensitive Detection System (BioGenex). Slides were counterstained with hematoxylin for ~5 to 10 seconds. MMR protein staining was considered negative when all of the tumor cell nuclei failed to react with the antibody. hMSH6 expression was seen in <100% of tumor nuclei in hMSH2/hMSH6-positive cases, although it was absent from all nuclei in hMSH2- and hMSH6-negative tumors. Adjacent normal tissue served as an internal control for positive staining in almost all tumor tissue sections (>99%). As a negative control, staining was carried out without the primary antibody. Protein expression was scored for signal strength and percentage of immunoreactive carcinoma nuclei by two independent investigators.

Statistics. Fisher's exact test was used to test for possible associations between variables of interest categorized into two-by-two contingency tables. The nonparametric Jonckheere-Terpstra method was used to test for differences among ordered categories, such as T, N, stage and grade (with this test, the null hypothesis is that the distribution of the response does not differ across ordered categories). Cox regression was used to perform multivariable analyses to evaluate the possible prognostic effect of covariates of interest on time to death and time to disease-specific death. For comparisons of the ages of different groups of patients, the P values were adjusted for multiple comparisons using the Bonferroni method. All statistical analyses were carried out using SAS statistical software, version 8.2, SAS Institute, Inc., Cary, NC.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 262 patients included in the study, 26 patients (10%) had tumors that were MSI-H, 11 (4%) were MSI-L, and 225 (86%) were MSS. Four patients had two primary tumors, none of which were MSI-positive. Nineteen (73%) of the 26 MSI-H tumors had loss of hMLH1 expression (Table 1). Each tumor that was negative for hMLH1 was also negative for hPMS2. Similarly, when hMSH2 was absent, expression of its partner hMSH6 was absent. Six (23%) of the 26 MSI-H tumors were negative for hMSH2 (and hMSH6) expression. The remaining MSI-H tumor (4%) had loss of hPMS2 expression only and was positive for hMLH1 expression (Fig. 1A-D). All of the MSI-H tumors had mutations in both mononucleotide and dinucleotide repeats (Table 2).


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  		Table 1 Characteristics of MSI+ tumors including MMR protein status

 


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  		Fig. 1 Immunohistochemical staining of the tumors lacking expression of only hPMS2 or hMSH6. Top, this tumor is positive for hMLH1 (A), hMSH2 (B), and hMSH6 (C), but negative for hPMS2 (D). Bottom, this tumor is positive for hMLH1 (E), hMSH2 (F), and hPMS2 (H), but negative for hMSH6 (G).

 

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  		Table 2 Microsatellite genotypes (stable vs. unstable)

 
Eleven (4%) of the 262 tumors examined had the MSI-L phenotype. This number represents a lower limit for the frequency of MSI-L tumors because the second panel of markers was examined only if at least one mutation was identified in the first panel (see Materials and Methods). Of the 11 MSI-L carcinomas, 1 (9%) was hMLH1-negative and 1 (9%) was hMSH6-negative. Immunohistochemical staining of the hMSH6-negative tumor is shown in Fig. 1E-H. The hMLH1-negative MSI-L tumor was unstable at BAT26 and BAT40 only, whereas the hMSH6-negative tumor was unstable at all three of the mononucleotide markers tested (BAT25, BAT26, and BAT40). The other nine (82%) MSI-L tumors expressed all four MMR proteins. Eight of these nine tumors had mutations in a single dinucleotide repeat; the ninth tumor had mutations in two mononucleotide repeats and one dinucleotide repeat (patient 15 in Table 2). In total, 28 tumors showed loss of expression of one or more of the MMR proteins and each had mutations in at least two of the mononucleotide repeats tested.

The only clinical feature of the tumors that was correlated with both MSI and absence of MMR protein expression was proximal location of the tumor (P < 0.0001; see Table 3). Higher grade was associated with tumors that were MSI-H, but when the two MSI-L/MMR-defective tumors were added, this association was no longer statistically significant. We observed a larger number of MMR-defective tumors in male patients (n = 16) than females (n = 10), but this difference was not statistically significant (P = 0.3). All six of the hMSH2-defective tumors were found in males (P = 0.03). African-Americans, who made up 31% of the total number of patients, had the same frequency of MSI-positive tumors as Caucasians. The average age of MSS patients was 66 years, whereas the average age of patients with hMLH1-negative tumors was 70 years, and that of patients with hMSH2-negative tumors was 53 years. Pairwise comparisons reveal that MSS and hMLH1-negative patients were equivalent in age (P = 0.3), but that the patients with hMSH2-negative tumors were younger than both the MSS patients (P = 0.048) and the hMLH1-negative patients (P = 0.042).


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  		Table 3 Characteristics of tumors based on MSI or MMR status

 
We also did survival analyses. Patients with both MSI-H and MSI-L tumors that lacked expression of one or more MMR proteins were categorized as MMR-deficient (n = 28), and these patients were compared with those who did not have MMR-defects (n = 234). The median follow-up time for survivors was 6.9 years. In this cohort, the presence of a MMR defect had no effect on overall patient survival (ignoring the cause of death; Fig. 2A). When the cause of death was taken into consideration (patients who died of causes other than colorectal cancer were censored at the time of death), there was a trend for better survival of patients with MMR-defective tumors, but the difference was still not significant (Fig. 2B). Individual significant predictors of survival were age (P = 0.0009), race (P = 0.01), TNM classification [tumor (P = 0.001), node (P < 0.0001), metastasis (P < 0.0001)], stage (P < 0.0001), and grade (P < 0.0001).



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  		Fig. 2 Kaplan-Meier curves comparing the survival of patients with MMR-deficient tumors to patients with MMR-proficient tumors. A, overall survival of patients with MMR-positive and of patients with MMR-negative tumors; B, disease-specific survival of patients with MMR-positive and of patients with MMR-negative tumors.

 
It has been suggested that other factors, such as patient sex, tumor location, and use of chemotherapy affect the survival of patients with MSI-positive tumors (34–36); therefore, we analyzed various subsets of patients. We did not find significantly longer cause-specific survival in either women or men with MMR-defective tumors or in patients with right-sided tumors, though there were trends toward longer survival in these patient groups. The response to adjuvant chemotherapy (5-fluorouracil and leucovorin or levamisole) could not effectively be examined because only a small number of patients with MMR-defective tumors were treated [5 (18%) of those with MMR-defective tumors and 72 (32%) of those with MMR-proficient tumors]; however, there was also no difference in outcomes when only patients who were not treated with 5-fluorouracil were examined. Caucasians had higher overall survival compared with African-Americans (P = 0.01), even taking into account all other covariates, but there was no correlation between MMR status and survival in either set of patients.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that if the original National Cancer Institute criteria (tumors are designated as MSI-H if two markers are unstable out of the first panel of five) had been used to select which tumors to stain for MMR expression, 3 of the 28 tumors (11%) with MMR defects would have been missed (Tables 1 and 2). The MSI-H tumor that was hPMS2-negative was stable at the BAT26 mononucleotide locus and at the three dinucleotide loci in the first panel of microsatellites. Two of the 11 MSI-L tumors had loss of expression of one of the MMR proteins and had frameshifts only in mononucleotide repeats. The MSI-L tumor that was hMLH1-negative showed instability in two of the three mononucleotide markers tested. It has been reported that the degree of instability in MSI-positive tumors increases with the transition from adenoma to carcinoma (37). This hMLH1-negative tumor was at a low stage, and the patient had no positive lymph nodes and no metastases. The hMSH6-negative tumor also showed only mononucleotide repeat instability; all three of the mononucleotides tested were unstable. The likely explanation for this observation is that the hMSH2/hMSH6 heterodimer preferentially corrects base-base mismatches and single-base insertion-deletion loops (38). Because only 3 of the 10 markers examined were mononucleotides, it is possible that if a higher fraction of the National Cancer Institute–recommended markers were mononucleotide repeats, these two tumors would have been categorized as MSI-H.

Nineteen of the 26 MSI-H tumors were missing hMLH1 expression, and all of these were also negative for hPMS2. The hMLH1 and hPMS2 proteins form a heterodimer, and it has been shown that when hMLH1 is missing the hPMS2 protein is degraded (39). Similarly, the hMSH2 and the hMSH6 proteins form a heterodimer, and it has been shown that when hMSH2 is missing, hMSH6 is degraded (40). We have shown that examination of hPMS2 and hMSH6 expression is useful in tumors that do not have defects in either hMLH1 or hMSH2, because 2 of the 28 MMR-negative tumors in this study had loss of one of these proteins. The patients with the hMSH6-deficient tumor and the hPMS2-deficient tumor may carry germ line mutations in these genes. Plaschke et al. (12) found mutations in hMSH6 in the germ lines of each of the three patients in their study who had sporadic tumors with loss of hMSH6 protein expression only. In another study, among 19 patients with hPMS2-negative tumors, germ line mutations were found in hPMS2 in 3 patients and in hMLH1 in 8 patients (41); therefore, it is possible that the patient with the hPMS2-negative tumor has a mutation in the hMLH1 gene, but that the mutant hMLH1 protein is still immunoreactive.

Eighty-two (31%) of the patients in our study were African-American, and MSI was present at equal frequencies in Caucasians and African-Americans. There has been one report that African-American patients have a significantly higher frequency of MSI-positive tumors (10 of 22 patients, or 45%) than has been reported among Caucasians (42). At least one other study has found the frequencies to be similar in the two groups (43). It is likely that the frequency of carriers of germ line mutations was lower among the African-American patients than among the Caucasians in our study population. One of the nine (11%) African-American patients with MMR-defective tumors was under age 50, whereas 5 of 17 (29%) of the Caucasian patients with MMR-defective tumors were age 50 or below. The African-Americans in our study had a significantly poorer prognosis, stage for stage, than the Caucasian patients, with worse overall survival (P = 0.012) and disease-specific survival (P = 0.003). This finding is consistent with previous reports and has been attributed to differences in stage at diagnosis and socioeconomic status (44).

We observed a trend for longer survival of patients with MMR-defective tumors when cause of death was taken into account, but the difference was not significant. Multiple publications have reported that patients with MSI-positive have better prognoses compared with patients with MSS tumors within certain subsets of patients; i.e., women, patients with right-sided tumors, or patients whose tumors have mutations in TGFBR2 (22, 34, 36). There have also been conflicting reports on the contribution of age and use of chemotherapy (24, 30, 34, 35). When we examined outcomes in terms of patient sex, tumor location, receipt of chemotherapy, tumor stage, or patient ethnicity, we still did not see a significant difference between the patients with MMR-negative tumors and the patients with MMR-positive tumors.

It is possible that the presence of germ line mutations in some of the patients in this study affected our ability to detect a significant difference in outcomes, even though none of the patients reported a family history of colorectal carcinoma. When we eliminated patients ≤50 years of age, who are more likely to harbor germ line mutations, there was no change in survival trends (data not shown). This was also true when we examined just the patients with hMLH1-defective tumors. It has been generally accepted that HNPCC patients with colorectal tumors survive longer than patients with sporadic colorectal cancer (45); however, very young patients (<30 years of age) with MSI-positive tumors have been reported to have relatively unfavorable outcomes (30), and the authors of that study have cautioned against using MSI alone as a predictor of survival. Given that none of our patients were <30 years of age, it is unlikely that the presence of a few individuals in our study with germ line mutations would have obscured a difference in prognosis. Other groups have reported no significant difference in outcomes among sporadic colorectal carcinoma patients with respect to MSI (26, 27, 36). We believe that the link between MSI and patient outcome is not completely straightforward, and that many factors may affect patient survival.

In conclusion, the existence of MMR-defective tumors that have only mononucleotide repeat instability suggests that a higher proportion of the markers used for MSI testing should be mononucleotide repeats. Indeed, at a more recent meeting at the National Cancer Institute, it was recommended that more mononucleotide markers should be included in the evaluation of MSI (46). Support for this conclusion comes from previous studies that have reported that mononucleotide-repeat instability is more specific for MSI-positive tumors than is dinucleotide-repeat instability (47, 48) and reports that the poly-A repeat BAT26 alone is sufficient for MSI testing (34, 49). In our study, all of the tumors with mutations in solely dinucleotide repeats were MMR-positive, although 28 of the 29 tumors with mutations in mononucleotides had loss of MMR protein expression. Examination of mononucleotide-repeat markers alone would have been sufficient for detecting all of the MMR-defective tumors; however, the use of BAT26 alone would have missed one MMR-defective tumor (Table 2, patient 116). Use of the appropriate microsatellite markers is important for the detection of MMR-defective tumors, not only in the search for patients with possible germ line mutations, but also for the analysis of sporadic colorectal carcinomas.


   ACKNOWLEDGMENTS
 
We thank Ji-Hyun Lee for assistance with the statistical analysis.


   FOOTNOTES
 
Grant support: NIH grant CA63264 (R. Farber) and UNC Lineberger Comprehensive Cancer Center grant (W. Funkhouser). S. Hatch was supported in part by NIH training grant GM07092.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 5/04; revised 12/14/04; accepted 12/17/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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