Microsatellite Instability and 8p Allelic Imbalance in Stage B2 and C Colorectal Cancers

Kevin C. Halling, Amy J. French, Shannon K. McDonnell, Lawrence J. Burgart, Daniel J. Schaid, Brett J. Peterson, Laurie Moon-Tasson, Michelle R. Mahoney, Daniel J. Sargent, Michael J. O'Connell, Thomas E. Witzig, Gist H. Farr, Jr., Richard M. Goldberg, Stephen N. Thibodeau

Affiliations of authors: K. C. Halling, A. J. French, L. J. Burgart, L. Moon-Tasson, S. N. Thibodeau (Departments of Laboratory Medicine and Pathology), S. K. McDonnell, D. J. Schaid, B. J. Peterson, M. R. Mahoney, D. J. Sargent (Health Sciences Research, Section of Biostatistics), T. E. Witzig (Internal Medicine, Division of Hematology), Mayo Foundation, Rochester, MN; M. J. O'Connell, G. H. Farr, Jr., R. M. Goldberg, North Central Cancer Treatment Group, Rochester.

Correspondence to: Stephen N. Thibodeau, Ph.D., Laboratory Genetics/HI 970, Mayo Clinic, 200 First St., SW, Rochester, MN 55905 (e-mail: sthibodeau{at}mayo.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Microsatellite instability (MSI) and allelic imbalance involving chromosome arms 5q, 8p, 17p, and 18q are genetic alterations commonly found in colorectal cancer. We investigated whether the presence or absence of these genetic alterations would allow stratification of patients with Astler-Coller stage B2 or C colorectal cancer into favorable and unfavorable prognostic groups. METHODS: Tumors from 508 patients were evaluated for MSI and allelic imbalance by use of 11 microsatellite markers located on chromosome arms 5q, 8p, 15q, 17p, and 18q. Genetic alterations involving each of these markers were examined for associations with survival and disease recurrence. All P values are two-sided. RESULTS: In univariate analyses, high MSI (MSI-H), i.e., MSI at 30% or more of the loci examined, was associated with improved survival (P = .02) and time to recurrence (P = .01). The group of patients whose tumors exhibited allelic imbalance at chromosome 8p had decreased survival (P = .02) and time to recurrence (P = .004). No statistically significant associations with survival or time to recurrence were observed for markers on chromosome arms 5q, 15q, 17p, or 18q. In multivariate analyses, MSI-H was an independent predictor of improved survival (hazard ratio [HR] = 0.51; 95% confidence interval [CI] = 0.31-0.82; P = .006) and time to recurrence (HR = 0.42; 95% CI = 0.24-0.74; P = .003), and 8p allelic imbalance was an independent predictor of decreased survival (HR = 1.89; 95% CI = 1.25-2.83; P = .002) and time to recurrence (HR = 2.07; 95% CI = 1.32-3.25; P = .002). CONCLUSIONS: Patients whose tumors exhibited MSI-H had a favorable prognosis, whereas those with 8p allelic imbalance had a poor prognosis; both alterations served as independent prognostic factors. To our knowledge, this is the first report of an association between 8p allelic imbalance and survival in patients with colorectal cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 
There are approximately 130 000 new cases of and 57 000 deaths from colorectal cancer annually in the United States (1). Early detection and improved surgical and medical therapies have led to modest improvements in the cure rates among these patients. An assessment of prognosis, based on features of the resected tumor, is useful information for the triage of patients into groups who may benefit from adjuvant therapy. Currently, staging is the most accurate predictor of prognosis. Patients with stage A disease and most patients with stage B1 disease usually are treated with surgery alone because of the 90% 5-year survival rates following surgical resection. Patients with stage D (metastatic) disease generally have incurable tumors and most often receive palliative therapy. Patients with stage C colon cancer and patients with stage B2 or C rectal cancer generally receive some form of adjuvant therapy (2,3). Whether adjuvant therapy benefits patients with stage B2 colon cancer, however, remains controversial (4,5). In addition, 30%-40% of patients with stage C cancer relapse despite adjuvant therapy. In the era before effective adjuvant therapy was identified, only 40% of patients with stage C disease were alive at 5 years from the time of diagnosis.

Identification of additional tumor characteristics to supplement standard clinical and pathologic staging could potentially subdivide both patients with stage B2 disease and patients with stage C disease into those at highest and lowest risk of relapse following surgery for colon or rectal cancer. This would facilitate better selection of high-risk patients (i.e., those who would benefit most from adjuvant therapy) and of low-risk patients (i.e., those with a lower likelihood of a treatment benefit, who instead would potentially benefit from avoiding the toxicity, expense, inconvenience, and risk of adjuvant therapy).

Substantial progress has been made in understanding the molecular events associated with malignant transformation. This process involves the stepwise accumulation of multiple genetic alterations including the activation of proto-oncogenes and the inactivation of tumor suppressor genes (6,7). Furthermore, colorectal cancer has been shown to arise through at least two distinct genetic pathways: one involving chromosomal instability and the other involving microsatellite instability (MSI) (8-10). Tumors characterized by chromosomal instability demonstrate a high frequency of allelic imbalance, the most common involved chromosomal arms being 5q, 8p, 17p, and 18q (7). The finding that tumor behavior is a reflection of these genetic events has stimulated numerous investigations into the possibility that specific genetic alterations may relate to prognosis. In colorectal cancer, initial studies suggested that MSI was associated with an improved prognosis (11,12), while allelic imbalance at certain loci, in particular 17p and 18q, appeared to be associated with a worse prognosis (13-15). Despite these observations, the clinical utility of such markers remains uncertain, largely because of the small numbers of patients on whom most reports have been based, and there remains a need for additional studies. To further define the clinical utility of molecular genetic alterations, we analyzed 508 patients with stage B2 or C colorectal cancer for MSI and allelic imbalance on chromosomal arms 5q, 8p, 15q, 17p, and 18q and tested these genetic alterations for associations with survival and time to disease recurrence.


    PATIENTS AND METHODS
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 
Patients

This retrospective study examined tumors from 508 of 2887 patients enrolled in one of seven different adjuvant chemotherapy protocols (3,16-21) of the North Central Cancer Treatment Group (NCCTG). NCCTG is a multidisciplinary cancer clinical trials organization comprised primarily of community clinics. Patient demographics are shown in Table 1.Go Five of the protocols treated colon cancer patients, and two protocols enrolled only rectal cancer patients. The 508 tumors that were analyzed for molecular genetic alterations were obtained in the following way: A letter requesting paraffin-embedded normal and tumor tissues for the 2887 patients was sent to the affiliated NCCTG institution where each patient was treated. From this request, paraffin-embedded tissue for 976 of the 2887 patients was submitted. DNA for polymerase chain reaction (PCR) analysis could be obtained from the paraffin-embedded normal and tumor tissues from 615 of these 976 patients. The molecular genetics results for 107 of the 615 patients were excluded from statistical analyses because fewer than five of the 11 markers could be amplified by PCR, leaving 508 patients suitable for statistical analyses. Common reasons for rejecting submitted blocks were inadequate tumor cell percentage (i.e., <60% tumor), absence of paired normal or tumor tissue, no primary tumor available, or a diagnosis of inflammatory bowel disease.


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Table 1. Demographics of patients evaluated for molecular genetic alterations (n = 508) versus all patients (n = 2887) in the seven treatment protocols

 
For the 508 patients, the median follow-up among the 266 patients alive as of their last follow-up was 8.4 years (range, 4.9-17.0 years). Tumors were staged according to the Astler-Coller modified Dukes' classification (22). Tumor grading was performed as described by Dukes and colleagues (23,24).

Microsatellite Analysis

Tumors were analyzed for MSI and allelic imbalance at 11 dinucleotide microsatellite markers on chromosomes 5q, 8p, 15q, 17p, and 18q as previously described (25). Markers included D5S346 and D5S107, localized near the APC gene on 5q; D8S254, located near a putative tumor suppressor gene on 8p; ACTC on 15q; D17S261 and TP53 on 17p at or near the P53 locus; and D18S34, D18S49, D18S35, D18S61, and D18S58, all localized on 18q both proximal and distal to the DCC gene. DNA extraction of paraffin-embedded normal and tumor tissues was performed as previously described (25,26). The tumors analyzed were composed of at least 60% tumor cells and had a minimum of five markers that successfully amplified for both normal DNA and tumor DNA. The median number of markers that gave a PCR product for both normal DNA and tumor DNA was nine (range, five to 11).

For MSI, tumors were classified into three groups: 1) microsatellite stable (MSS) with no MSI at any of the loci examined, 2) low instability (MSI-L, <30% of the loci demonstrating MSI), or 3) high instability (MSI-H, >=30% demonstrating MSI). The use of 30% as a breakpoint was based on previous work (25) that showed the following: (a) a bimodal distribution of tumors with one group of tumors exhibiting MSI at less than 30% of the loci and the other group of tumors exhibiting MSI at 30% or more of the loci and (b) distinct clinicopathologic differences between tumors having MSI at 30% or more of loci examined when compared with tumors with MSI at fewer than 30% of the loci examined.

Allelic imbalance was assessed by quantitation of the intensities of the upper and lower alleles in normal and tumor tissues with a phosphorimager using Image Quant software (Molecular Dynamics, Sunnyvale, CA). Tumors were classified as positive for allelic imbalance when the PCR assay for the control normal tissue showed heterozygosity of the microsatellite marker and the relative intensity of the two alleles in the tumor DNA differed from the relative intensity in the normal DNA by a factor of at least 1.5 (27).

Statistical Analysis

Survival was defined as the interval from surgery until the date of death from any cause. Time to recurrence was defined as the interval from surgery until the date of documented tumor recurrence. Time to recurrence was censored at time of death for patients dying of causes other than metastatic colon cancer. Follow-up for disease recurrence was according to protocol for 8 years from randomization and discretionary thereafter. For this analysis, all patients were censored for time to recurrence at 8 years. For the purpose of statistical analysis, the colon cancer treatments were grouped into three categories: 1) effective 5-fluorouracil (5-FU)-based therapy (5-FU plus levamisole [Lev], 5-FU plus leucovorin [LV], or 5-FU plus LV plus Lev), 2) therapies proven to have no clinical utility (Lev alone, 5-FU administered by portal vein infusion, or interferon gamma), and 3) untreated controls. The rectal cancer treatments were also broken down into three groups: 1) radiation therapy alone, 2) radiation therapy plus bolus 5-FU, and 3) radiation therapy and infusional 5-FU.

Distributions of survival times were compared with the use of the logrank test; survival distribution curves were estimated by the method of Kaplan and Meier (28). Multivariate analyses were performed with the use of the Cox proportional hazards model (29). All logrank tests and Cox models were stratified according to the treatment protocol to which the patient was randomly assigned. The patient's Dukes' stage, adjuvant treatment received, sex, and tumor grade were included as covariates in the multivariate Cox regressions. The assumption of proportional hazards was validated by graphical methods, and covariates that failed this assumption were included in the Cox proportional hazards model as stratification factors. All P values presented are two-sided, and a P value of less than .05 was considered statistically significant. Formal adjustment for multiple comparisons was not used, since the chromosomal loci studied were protocol specified on the basis of earlier studies implicating these specific loci in colorectal tumorigenesis (13,14,30). Statistical analyses were performed with the use of SAS software (SAS Institute, Inc., Cary, NC) (31).


    RESULTS
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 
Patient and tumor characteristics of the patients with usable blocks were compared with those with unusable blocks. These results are shown in Table 1Go. Patients with usable blocks were more likely to have Dukes' stage B2 disease and to have been randomly assigned to a control regimen than patients whose blocks were not used. Although these imbalances were statistically significant, because of the large number of patients involved, they were not judged sufficient to declare that the patient population studied was misrepresentative of the overall colon and rectal cancer patient population.

Of the 508 tumors, 335 (66%) exhibited MSS (i.e., no evidence of MSI), 97 (19%) exhibited MSI-L, and 76 (15%) exhibited MSI-H. In addition to lower stage, proximal site, female sex, and diploid or near-diploid DNA content (25), we also found the MSI-H phenotype to be strongly associated with high tumor grade (P = .001).

The frequency of allelic imbalance observed at individual loci and on the chromosomal arms for which more than one marker was assessed was as follows: D5S107, 98 (47%) of 210; D5S346, 170 (55%) of 310; 5q, 202 (57%) of 357; D8S254, 123 (54%) of 226; ACTC, 120 (42%) of 283; D17S261, 77 (65%) of 119; TP53, 221 (76%) of 292; 17p, 237 (74%) of 321; D18S49, 143 (66%) of 218; D18S34, 174 (74%) of 236; D18S35, 157 (76%) of 207; D18S58, 92 (80%) of 115; D18S61, 118 (72%) of 164; and 18q, 304 (79%) of 386. The presence of MSI at a microsatellite marker makes interpretation for allelic imbalance potentially inaccurate. Loci may falsely appear to have allelic imbalance when one of the two alleles in the tumor shows a greater degree of instability. Conversely, tumors may be falsely classified as lacking allelic imbalance if an unstable allele in the tumor comigrates with the imbalanced allele. For the purpose of data analysis, therefore, loci with MSI were considered noninformative for allelic imbalance. Furthermore, all loci, even those without obvious instability, were considered noninformative for allelic imbalance in MSI-H tumors. The frequency of allelic imbalance shown above reflects these considerations.

Univariate analyses for associations with survival and time to recurrence were performed for a number of variables, including tumor stage and grade, site, sex, DNA ploidy, MSI, and allelic imbalance (Table 2)Go. As expected, statistically significant associations were observed for stage and grade. The Kaplan-Meier plots for survival by stage and grade are shown in Fig. 1.Go


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Table 2. Univariate associations of tumor characteristics with survival and time to recurrence*

 



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Fig. 1. Overall survival, to 8 years, of patients with colorectal cancer according to Astler-Coller stage (A) and grade (B). Bars represent 95% confidence intervals. P values are two-sided and statistically significant when <.05.

 
Of the molecular markers, MSI status and 8p allelic imbalance demonstrated the strongest associations with survival. Patients with MSI-H tumors had a statistically significantly better survival (P = .02) and time to recurrence (P = .01) than patients with MSS tumors (Table 2;Go Fig. 2,Go A). There was no statistically significant difference in survival (P = .23) or time to recurrence (P = .49) for patients with MSI-L and MSS tumors. The MSI-H phenotype was present in 26% and 14% of the stage B2 or C tumors, respectively. Patients with stage C tumors that exhibited MSI-H had statistically significantly better survival (P = .02) and time to recurrence (P = .01) than patients with MSS stage C tumors (Fig. 2Go, B). The survival rate for patients with MSI-H stage C tumors was similar to that for patients with stage B2 tumors (with or without MSI). MSI status did not have any effect on survival for the patients with stage B2 tumors. Patients with MSI-H high-grade (i.e., grade 3 or 4) tumors had statistically significantly better survival (P = .001) and time to recurrence (P<.001) than patients with MSS high-grade tumors (Fig. 2Go, C). The survival and time to recurrence for patients with MSI-H high-grade tumors were similar to those for patients with low-grade tumors (with or without MSI). For the low-grade (i.e., grade 1 or 2) tumors, MSI status had a borderline statistically significant association with time to recurrence (P = .05) but not with survival (P = .11). Multivariate analysis adjusting for tumor stage, protocol, treatment, tumor grade, and sex revealed that MSI-H was independently associated with improved survival and time to recurrence. DNA ploidy data were not available for 58% of the patients and thus were not adjusted for in multivariate analyses. The hazard ratio (HR) for MSI-H relative to MSS was 0.51 (95% confidence interval [CI] = 0.31-0.82; P = .006) for survival and 0.42 (95% CI = 0.24-0.74; P = .003) for time to recurrence (Table 3).Go





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Fig. 2. Overall survival of patients according to microsatellite instability (MSI) status (A), MSI status by stage (B), and MSI status by grade (C). MSS = microsatellite stability; MSI-H = high microsatellite instability (at >=30% of the loci). Bars represent 95% confidence intervals. P values are two-sided and statistically significant when <.05.

 

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Table 3. Multivariate adjusted hazard ratios (HRs) for survival and time to recurrence*

 
For the allelic imbalance studies, no statistically significant associations with survival or time to recurrence were observed for markers on chromosomal arms 5q, 15q, 17p, and 18q (Table 2Go). However, 8p allelic imbalance was associated with a statistically significant worse survival (P = .02) and time to recurrence (P = .004) (Table 2Go; Fig. 3Go, A). Of the patients with stage B2 or C disease, 53% and 55%, respectively, exhibited 8p allelic imbalance. Patients with Astler-Coller stage C disease with 8p allelic imbalance had statistically significantly worse survival (P = .004) and time to recurrence (P = .002) than patients with stage C disease without 8p allelic imbalance (Fig. 3Go, B). The 8p allelic imbalance status was not associated with survival (P = .82) or time to recurrence (P = .39) in patients with stage B2 disease (Fig. 3Go, B). Patients with low-grade (grade 1 or 2) tumors with 8p allelic imbalance had statistically significantly worse survival (P = .04) and time to recurrence (P = .04) than patients with low-grade tumors without 8p allelic imbalance. Patients with high-grade (grade 3 or 4) tumors with 8p allelic imbalance had statistically significantly worse time to recurrence (P = .04) but not survival (P = .27) compared with patients with high-grade tumors without 8p allelic imbalance. Multivariate analysis adjusting for tumor stage, protocol, treatment, tumor grade, and sex revealed that 8p allelic imbalance was independently associated with decreased survival and time to recurrence, with HRs of 1.89 (95% CI =1.25-2.83; P = .002) and 2.07 (95% CI =1.32-3.25; P = .002), respectively (Table 3Go).




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Fig. 3. Overall survival of patients according to 8p allelic imbalance (AI) status (A) and 8p AI status by stage (B). P value represents the significance of the difference in survival for patients with or without 8p AI. In panel A, the survival curve for patients with high microsatellite instability tumors is shown for comparison. Bars represent 95% confidence intervals. P values are two-sided and statistically significant when <.05.

 
In several previous studies (13-15,32-34) that evaluated the prognostic significance of allelic imbalance, patients with MSI-H tumors were included, for a variety of reasons, with those lacking allelic imbalance in the statistical analyses. In some of these studies, tumors with the MSI-H phenotype were inadvertently included with the allelic imbalance-negative tumors because the technique used to assess allelic imbalance (Southern blot rather than microsatellite analysis) did not allow for the identification of tumor MSI (13,14,32-34). More recent studies (15,35-37), however, have utilized microsatellite analysis as the principal method to assess allelic imbalance. One of these studies (15) intentionally grouped MSI-H tumors with the allelic imbalance-negative group for the purpose of statistical analyses because 1) allelic imbalance is difficult to assess in the presence of MSI and 2) MSI-H tumors, when analyzed by Southern blot analysis, demonstrate a very low frequency of allelic imbalance (11,15). To facilitate comparisons with earlier work by other authors, we re-analyzed our data for the relationship between allelic imbalance and survival, but this time we assumed that the MSI-H tumors did not have allelic imbalance and included these tumors in the allelic imbalance-negative group for that marker. When the analyses were performed in this manner, statistically significant associations between genetic abnormalities and survival were evident (Table 4Go). Survival and time to recurrence were statistically significantly associated with allelic imbalance on 8p (P = .001 and P<.001, respectively), 17p (P = .02 and P = .003, respectively), and 18q (P = .02 and P = .007, respectively). The survival curves for patients with MSI-H tumors are included in Figs. 3Go and 4Go (survival curves for patients according to 8p, 17p, and 18q allelic imbalance status) to illustrate how the inclusion of MSI-H patients with the allelic imbalance-negative patients strengthens the apparent, and possibly misleading, prognostic significance of 17p and 18q allelic imbalance.


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Table 4. Univariate associations of tumor characteristics with survival and time to recurrence (MSI-H tumors scored as no AI)*

 



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Fig. 4. Overall survival of patients according to 17p (A) and 18q (B) allelic imbalance (AI) status. P values represent the significance of the difference in survival for patients with or without 17p AI and for patients with and without 18q AI. The survival curve for patients with high microsatellite instability (MSI-H) tumors is shown for comparison and to illustrate how the inclusion of MSI-H patients with the 17p or 18q AI-negative patients strengthens the apparent prognostic significance of 17p and 18q AI. Bars represent 95% confidence intervals. P values are two-sided and statistically significant when <.05.

 
Analyses were performed to determine the impact that MSI-H and 8p allelic imbalance have on survival and time to recurrence in patients receiving no treatment (n = 143). As with the entire dataset, the untreated group of patients also showed that 8p allelic imbalance was statistically significantly associated with decreased survival (P = .04) and time to recurrence (P = .02). The trend for untreated patients with MSI-H was similar to that for the overall group. In contrast, untreated patients with MSI-H did not show statistically significant associations with survival (P = .07) or time to recurrence (P = .25). However, the number of patients in these comparisons was small, and statistical power was therefore low (e.g., there were only four events for the MSI group).

Additional analyses were also conducted to determine whether MSI-H or 8p allelic imbalance identifies which patients may benefit the most from adjuvant chemotherapy. The survival and time to recurrence of patients treated with effective therapy were compared with those of patients treated with ineffective therapy or given no treatment within each of the following subgroups: MSS, MSI-L, MSI-H, 8p allelic imbalance, and no 8p allelic imbalance. No statistically significant benefit from adjuvant treatment was observed in any of the subgroups. However, these treatment comparisons have low power for the magnitude of benefit known to be conferred by adjuvant treatment. Hence, there is no evidence from this study that MSI or 8p allelic imbalance status is predictive of a benefit or a lack of a benefit of adjuvant therapy.


    DISCUSSION
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 
Results from this study confirm and extend earlier observations that certain genetic alterations can serve as prognostic markers in patients with colorectal cancer. Of any of the markers studied, 8p allelic imbalance exhibited the most striking association with survival. Patients with 8p allelic imbalance had statistically significantly worse survival and time to recurrence than patients without 8p allelic imbalance (Fig. 3Go). This was true for both univariate analyses (P = .02 and P = .004, respectively) and multivariate analyses (P = .002 and P = .002, respectively). In addition to tumor stage, tumor grade, and sex, the multivariate analyses were also adjusted for treatment and stratified on protocol. Among the patients with stage C disease, 8p allelic imbalance status divided the patients into better and worse prognostic groups (Fig. 3Go). Because of no apparent differences, this division was not possible with patients with stage B2 disease. To our knowledge, this is the first report of an association between 8p allelic imbalance and survival in patients with colorectal cancer. A previous study (38) has noted an association between 8p allelic imbalance and advanced stage of colorectal cancer, and another study (39) has noted an association between 8p allelic imbalance and tumor microinvasiveness. Microinvasion has been found to be a prognostic indicator in colorectal carcinoma that is independent of stage (40,41). An association between 8p allelic imbalance and poor survival has also been observed for patients with head and neck cancer (42) and for patients with prostate cancer (43).

Although 8p allelic imbalance is a frequently observed genetic abnormality in colorectal and other cancers (27,30,38,42,44,45), a candidate tumor suppressor gene has not yet been identified. More detailed mapping studies in colorectal and other cancers suggest that there may be up to three tumor suppressor genes on 8p (38,46-51). One of the three tumor suppressor gene loci maps to 8p22, the site of the marker used in our study (38).

The 8p allelic imbalance appears to be a relatively late event in colorectal tumorigenesis, since it is identified more frequently in colorectal cancers than in colorectal adenomas (52) and more frequently in metastatic than in primary tumors (53). This observation suggests that 8p allelic imbalance is associated with tumor progression. The strong effect that 8p allelic imbalance has on prognosis may be due, in part, to its "late" occurrence in the development of colorectal cancer. Genetic alterations that occur early in the evolution of a tumor, such as alterations in APC, would be expected to have less of an influence on prognosis. This is the most likely explanation for the lack of an association between 5q allelic imbalance and survival in this study. Of significance, allelic imbalance on chromosome arm 5q has consistently failed to show any associations with survival in other studies (13,14,34,54).

Contrasting with the unfavorable prognosis associated with 8p allelic imbalance is the favorable prognosis associated with the MSI-H phenotype. Patients with tumors exhibiting MSI-H had a significantly better survival (P = .02) and time to recurrence (P = .01) than patients with MSS tumors. Furthermore, the MSI-H phenotype identified a subset of patients with stage C or high-grade tumors who had a more favorable prognosis (Fig. 2Go, B and C).

Several smaller studies (11,12,55-57) have demonstrated a survival advantage for patients with sporadic colorectal cancer demonstrating MSI. Patients with hereditary nonpolyposis colon cancer with colorectal cancer have also been reported to have a better prognosis (58,59). Of interest, the MSI-H phenotype observed in the majority of tumors from patients with hereditary nonpolyposis colon cancer is a consequence of germline mutations in one of several DNA mismatch repair genes, primarily hMSH2 or hMLH1 (60,61). The MSI-H phenotype observed in sporadic colorectal cancer, however, now appears to be largely due to hypermethylation of the hMLH1 promoter (62-65). The MSI-H phenotype, therefore, appears to be associated with a good prognosis, irrespective of the mechanism giving rise to it.

A previous report (25) on the same group of patients included in this study showed that MSI-H (but not MSI-L or MSS) was associated with lower disease stage, female sex, proximal location, and diploidy. Our study also revealed an association between the MSI-H phenotype and high tumor grade. Nonetheless, MSI-H was a statistically significant prognostic factor for survival and time to recurrence after adjustment was made for stage, grade, site, protocol, treatment, and sex.

Our results show that 5q, 15q, 17p, and 18q allelic imbalances do not exhibit statistically significant associations with survival or time to recurrence. Several studies (13-15,34-36) have shown associations between 17p or 18q allelic imbalance and decreased survival in patients with colorectal cancer, whereas other studies (13,32-34,37,66,67) have not. One of these studies (34) showed a decreased survival for 18q allelic imbalance but not for 17p allelic imbalance. A possible explanation for the varying reports regarding the prognostic significance of 17p and 18q allelic imbalance is the manner in which allelic imbalance is scored in tumors with the MSI-H phenotype. In one study (15) that found a significant association between 18q allelic imbalance and poor survival, markers from the MSI-H patients were assumed to lack allelic imbalance and thus included with the 18q allelic imbalance-negative group. Because Southern blot analyses (11,15) have shown that MSI-H tumors show a very low frequency of allelic imbalance, we re-analyzed our data for associations between allelic imbalance (on each chromosomal arm) and survival, but we included tumors with a MSI-H phenotype in the allelic imbalance-negative group (Table 4)Go. When analyzed in this manner, 17p and 18q allelic imbalances then exhibited statistically significant associations with decreased survival (P = .02 and P = .02, respectively) and time to recurrence (P = .003 and P = .007, respectively) (Table 4)Go. It is apparent from Fig. 4Go that the mixed group of MSI-H patients and patients whose tumors lack allelic imbalance have a better survival and time to recurrence than patients whose tumors lack allelic imbalance alone. Future prognostic studies should be careful to identify and to separate for subset analysis the group of patients with MSI-H tumors.

The use of different 18q markers by various investigators may provide another explanation for the differing findings regarding the prognostic importance of 18q allelic imbalance. Several potential tumor suppressor genes have been mapped to 18q21. These genes include DCC, Smad4 (DPC4), and Smad 2 (68-70). Boland and co-workers (37) found no association between allelic imbalance and survival when they used five markers that were very close to the DCC gene. On the other hand, Martínez-López et al. (36) found an association between poor survival and allelic imbalance with an 18q marker proximal to DCC but not with a marker distal to DCC at 18q23. Our studies demonstrated a lack of an association across all of the markers tested.

At least two studies (13,14) have found that 17p allelic imbalance is associated with an adverse prognosis in colorectal cancer. Three other studies (32-34), however, have not found this association. The 17p allelic imbalance (at or near the P53 locus) is strongly associated with the presence of a point mutation on the remaining P53 allele (71,72) and is frequently accompanied by overexpression of the mutant p53 protein (73,74). Many studies [reviewed by Smith and Goh (75)] have examined the relationship between p53 overexpression and prognosis and between p53 mutation and prognosis. Studies of p53 overexpression have reached varied conclusions. A number of studies have found that p53 overexpression is associated with poor prognosis, but other studies have not [reviewed in (75)]. Several studies (76-78) have found that a P53 point mutation is strongly associated with an adverse prognosis and an independent prognosticator in multivariate analyses. With regard to this study, it is important to realize that 17p allelic imbalance does not necessarily imply the presence of a P53 mutation and that, while P53 mutations may negatively affect prognosis, 17p allelic imbalance may not (78).

The statistical analyses performed in this study involved multiple comparisons. We have not adjusted for multiple comparisons (Bonferroni correction) because the analyses performed were for a preselected and limited set of genetic loci that are implicated in colorectal tumorigenesis (13,14,30). The chromosomal regions 5q, 8p, 17p, or 18q examined in this study are sites of known or putative tumor suppressor genes. Chromosomal arm 15q was included in this study as a control, since a previous study (30) had not implicated this region in colorectal tumorigenesis. Hence, the analyses that were performed were protocol specified.

A secondary aim of this study was to identify patients who may be more likely to benefit from adjuvant chemotherapy. The survival and time to recurrence distributions of patients receiving effective 5-FU-based therapy were compared with those of patients receiving ineffective treatment or control (no therapy) in subgroups defined by MSI and 8p allelic imbalance status. Overall, a statistically significant benefit from adjuvant treatment was not observed for any of the subgroups examined. These results should be interpreted with caution, however, since these treatment comparisons had low power for the magnitude of benefit known to be conferred by adjuvant treatment. Because patients in this study received a variety of treatments over an extended period of time (13 years), this may not be the ideal setting for testing a marker's ability to predict a treatment effect. A better setting for such an analysis, for example, would be a single, large phase III randomized clinical trial, such as Cancer and Acute Leukemia Group B study C9581, testing the efficacy of 17-1A monoclonal antibody versus control in patients with Dukes' stage B2 colon cancer.

To our knowledge, this is the largest study to date exploring the role that allelic imbalance and MSI play in prognosis of colorectal cancer patients. Key attributes of this patient population include mature follow-up, protocolized prospective follow-up, and large sample size. The most significant findings of this study are confirmation that the MSI-H phenotype defines a good prognostic group and the novel finding that 8p allelic imbalance status defines a poor prognostic group. However, MSI or 8p allelic imbalance did not predict which patients would benefit the most from adjuvant therapy. Identification of the putative tumor suppressor gene on 8p may lead to an understanding of the biologic basis of the adverse prognosis associated with 8p allelic imbalance. Similarly, additional studies may elucidate the mechanism by which MSI-H leads to improved prognosis.


    NOTES
 
Editor's note: S. N. Thibodeau holds a patent on the use of microsatellite instability measurements for clinical purposes.

Supported by Public Health Service grants CA60117 and CA25224 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.


    REFERENCES
 Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received August 23, 1998; revised May 14, 1999; accepted June 8, 1999.


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