Colorectal carcinoma is the fourth most prevalent cancer

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1 GASTROENTEROLOGY 2006;131: Prognostic Impact of Microsatellite Instability and DNA Ploidy in Human Colon Carcinoma Patients FRANK A. SINICROPE,*, RAFAELA L. REGO,* KEVIN C. HALLING, NATHAN FOSTER, DANIEL J. SARGENT, BETSY LA PLANT, AMY J. FRENCH, JOHN A. LAURIE, RICHARD M. GOLDBERG, STEPHEN N. THIBODEAU, and THOMAS E. WITZIG *Divisions of Gastroenterology and Hepatology, Hematology and Oncology, Departments of Laboratory Medicine and Pathology, and Cancer Center Statistics, Mayo Clinic College of Medicine, Rochester, Minnesota Background & Aims: Genomic instability in colon cancers is a consequence of chromosomal instability characterized by aneuploidy or defective DNA mismatch repair (MMR) indicated by microsatellite instability (MSI). Given that high-frequency MSI (MSI-H) and diploidy are correlated, we determined whether they are independent prognostic variables. Methods: Astler Coller stage B2 and C colon cancers (N 528) from patients treated in 5-fluorouracil based adjuvant therapy trials were analyzed for MSI using 11 microsatellite markers. Immunostaining for hmlh1, hmsh2, and p53 proteins was performed. DNA ploidy was analyzed by flow cytometry. Associations with disease-free and overall survival were determined. Results: MSI-H was detected in 95 tumors (18%), and 70 (74%) of these were diploid. Tumors showing MSI-H (hazard ratio, 0.65; 95% confidence interval, ; P.023) or loss of MMR proteins (P.024) were associated with better overall survival. Improved disease-free and overall survival were found for diploid versus aneuploid/tetraploid tumors (overall survival: hazard ratio, 0.59; 95% confidence interval, ; P.0003). In the subgroups of MSI-H and microsatellite stable (MSS)/low-frequency MSI (MSI-L) tumors, diploidy was associated with better survival. The prognostic impact of ploidy was similar in stage B2 and C tumors. Ploidy did not predict the benefit of 5-fluorouracil based treatment. When ploidy, MSI, and MMR proteins were analyzed in the same multivariate model, only ploidy remained significant. Conclusions: DNA ploidy and MSI-H status were independent prognostic variables, yet ploidy was the strongest marker. Diploidy was associated with better survival in MSI-H and in MSS/MSI-L patient subgroups. Colorectal carcinoma is the fourth most prevalent cancer and the second leading cause of cancer-related death in the United States. 1 Despite decades of research, tumor stage is the only prognostic variable used in clinical decision-making despite considerable stage-independent variability in patient outcome. Prognostic markers are needed to enable stratification of patients into groups at high and low risk of recurrence and metastasis and thereby influence strategies for surveillance and adjuvant chemotherapy. Two pathways of genomic instability have been described during colonic tumorigenesis and include chromosomal instability and microsatellite instability (MSI). Tumors with chromosomal instability show frequent aneuploidy, 2,3 as well as allelic imbalance resulting in the inactivation of tumor suppressor genes and mutations in oncogenes. 2,4 MSI is due to defective DNA mismatch repair (MMR) resulting in an accumulation of somatic alterations in nucleotide repeat sequences called microsatellites, some of which are in gene promoter regions. 5 7 Recent data, however, suggest that a subset of sporadic tumors lack both allelic imbalance and MSI. 8 Up to 15% 20% of sporadic human colon cancers show high-frequency MSI (MSI-H), generally due to promoter hypermethylation of the hmlh1 gene Immunostaining for hmlh1 and hmsh2 proteins has been shown to accurately predict the molecular finding of MSI-H versus microsatellite stability or low-frequency MSI (MSS/MSI-L) in colorectal cancers. 12 MSI-H tumors have distinct clinical and pathologic features, including proximal colon predominance, diploid DNA content, and poor differentiation. 6,13 15 Furthermore, some studies have shown that MSI-H tumors have a more favorable prognosis relative to MSS and MSI-L tumors, 6,13,16 18 yet studies have often included multiple tumor stages, young patient age, and rectal cancers. The existence of alternative genetic pathways to colonic tumorigenesis suggests that cellular and molecular features of these pathways may be useful as prognostic or predictive markers. Alterations in tumor cell DNA content (ie, ploidy) have been shown to be prognostic in patients with colorectal cancer In a prior study in lymph node negative colon cancers, we found that the prognostic impact of DNA ploidy was stronger for proximal colon cancers, 25 yet this and most other ploidy studies were conducted before the routine analysis of MSI in archival material. Therefore, we sought to test the hypothesis that the better prognosis of patients with diploid sporadic colon cancers is due to the inclusion of MSI-H cases. To explore this hypothesis, we examined clinicopathologic and cellular/ molecular variables stratified by MSI and DNA ploidy in patients with resected stage B2 and C colon cancer treated in six 5-fluorouracil (5-FU) based adjuvant therapy trials. Patients and Methods Data were obtained from 6 randomized 5-FU based phase 3 adjuvant studies conducted by the North Central Cancer Treatment Group. Paraffin-embedded tumor tissue specimens were Abbreviations used in this paper: CI, confidence interval; CIMP, CpG island methylator phenotype; DFS, disease-free survival; 5-FU, 5-fluorouracil; HR, hazard ratio; MMR, mismatch repair; MSI, microsatellite instability; MSI-H, high-frequency microsatellite instability; MSI-L, lowfrequency microsatellite instability; MSS, microsatellite stability; OS, overall survival by the American Gastroenterological Association (AGA) Institute /06/$32.00 doi: /j.gastro

2 730 SINICROPE ET AL GASTROENTEROLOGY Vol. 131, No. 3 available from study participants. Tumors were staged according to the Astler Coller modification of the Dukes classification 26 and included stage B2 (n 158) and C (n 370) colon cancers (N 528). Tumor histologic grade was defined 27 as follows: grade 1, well differentiated; grade 2, moderately differentiated; grade 3, poorly differentiated; grade 4, undifferentiated. Grade 3 and 4 tumors were regarded as showing high histologic grade. Tumor site was defined relative to the splenic flexure, and tumors located at the splenic flexure were included in the distal category. Patients were censored at 5 and 8 years after randomization for disease-free survival (DFS) and overall survival (OS) data, respectively. Details of the completed, randomized 5-FU based adjuvant chemotherapy trials have been previously reported. 28 Of the 528 patients, 225 received observation alone or ineffective treatment (ie, levamisole alone or interferon gamma alone or portal venous 5-FU) and 303 patients received effective treatment consisting of modulated intravenous 5-FU. MSI Tumors (N 528) were analyzed for MSI using 11 dinucleotide microsatellite markers, as previously described. 15,16 We also determined the expression of MMR proteins (hmlh1, hmsh2) in a subset of these cases (n 329). In accordance with consensus definitions of the National Cancer Institute, tumors were classified into 3 groups: (1) 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% of the loci demonstrating MSI). 14,16 Given evidence indicating that MSI-L and MSS colon cancers are not biologically distinct, we grouped them together in all analyses. 12,14,29,30 DNA Content Analysis Analysis of DNA content in tumors was performed using paraffin-embedded tumor blocks as previously described. 23 Tissue sections were dewaxed to produce a single-cell suspension of nuclei; the nuclei were subsequently stained with propidium iodide as described by Hedley et al. 31 Samples were run on a flow cytometer with a minimum of 10,000 cells analyzed. DNA content was quantitated and a DNA histogram of the entire cell population created. DNA histograms were analyzed using Modfit software version 5.2 (Verity Software, Topsham, ME). Each histogram was reviewed by 2 investigators and assigned a ploidy classification (diploid, tetraploid, or aneuploid) as previously described. 23 Immunostaining for hmlh1, hmsh2, and p53 Immunohistochemistry for MMR proteins (hmlh1, hmsh2) (n 329) and p53 protein were performed. Immunostaining for hmlh-1 using the G clone (PharMingen, San Diego, CA) and hmsh-2 using the FE11 clone (Oncogene, Cambridge, MA) were performed as previously described. 32 Slides were scored as either positive or negative based on the presence or absence of staining for hmlh-1 or hmsh Immunostaining for p53 was performed in paraffin-embedded tissue sections using the p53-d07 primary antibody (Novocastra Laboratories Ltd, Newcastle upon Tyne, England) as previously described. 28 Nuclear p53 staining was regarded as positive when 10% or more of the malignant nuclei showed staining; all others were regarded as p53 negative. Statistical Analysis 2 tests were used to test for an association between prognostic markers. OS (censored at 8 years) was calculated as the number of days from random assignment to the date of death or last contact. DFS (censored at 5 years) was calculated as the number of days from random assignment to the date of disease recurrence or death. The distributions of OS and DFS were estimated using Kaplan Meier methodology. Cox proportional hazards models 33 were used to explore the association of clinical and laboratory parameters with OS and DFS. All of these models were stratified according to the patient s original treatment study and adjusted for histologic grade, stage, and treatment for OS and DFS. The likelihood ratio test was used to test for the significance of each covariate in all models run. Interaction test P values were calculated using a likelihood ratio test comparing a model with and without the interaction term included. Graphical methods were used to examine whether underlying model assumptions were satisfied (eg, proportional hazards). 34 Statistical tests were 2 sided, with P.05 considered significant. P values were not adjusted for multiple comparisons. Statistical analyses were performed using SAS (versions 8 9; SAS Institute, Inc, Cary, NC). Results The molecular and phenotypic features of colon cancers arising from the chromosomal instability and MSI pathways may be useful for prognostication. Colon cancers with MSI-H are frequently diploid and have been shown to have a better prognosis compared with MSS/MSI-L tumors. 6,13,16,17 Therefore, we sought to determine whether MSI and ploidy are independent prognostic variables or whether diploidy accounts for or contributes to the favorable outcome of MSI-H colon cancers. Clinicopathologic Features The study population (N 528) included 30% of patients with Astler Coller stage B2 (n 158) and 70% of patients with Astler Coller stage C (n 370) colon carcinomas; these patients were enrolled in six 5-FU based adjuvant chemotherapy trials. Clinicopathologic features of the study cases are shown in Table 1. The age distribution of the patients was as follows: 64 (12%) aged 50 years or younger, 48 (9%) aged years, 71 (13%) aged years, 202 (38%) aged years, 134 (25%) aged years, and 9 (2%) older than 80 years. Median duration of follow-up for patients who remain alive was 8 years. Molecular and Cellular Markers We found that 95 (18%) of Astler Coller stage B2 and C colon cancers were MSI-H and 433 (82%) were MSS/MSI-L (Table 1). A subset of these cases (n 329) that included 73 MSI-H tumors was subsequently studied for DNA MMR protein (hmlh1 or hmsh2) expression. Sixty-eight of 73 MSI-H tumors showed loss of either protein; of these, hmlh1 was absent in 61, hmsh2 was lost in 7 cases, and 3 cases were subsequently found to have loss of PMS2. All MSI-L (n 30) and MSS (n 244) tumors analyzed showed intact expression of both proteins. These results demonstrate a high level of concordance between MSI testing and MMR protein expres-

3 September 2006 DNA PLOIDY AND MSI IN COLON CANCER 731 Table 1. Tumor Markers Stratified by MSI Status Characteristics No. of patients MSI-H MSS/MSI-L P a Astler Coller stage (%) B2 158 (30) 35 (37) 123 (28).1040 C 370 (70) 60 (63) 310 (72) Age (y) Median (range) N/A 67 (33 86) 64 (25 88).0197 b Mean Sex (%) Male 274 (52) 33 (35) 241 (56).0002 Female 254 (48) 62 (65) 192 (44) Tumor site (%) Proximal 286 (54) 88 (93) 198 (46).0001 Distal 242 (46) 7 (7) 235 (54) Histologic grade (%) c 1/2 383 (73) 48 (51) 335 (77) /4 145 (27) 47 (49) 98 (23) DNA ploidy (%) Diploid 223 (42) 70 (74) 153 (35).0001 Aneuploid/tetraploid 305 (58) 25 (26) 280 (65) p53 staining (%) Negative 122 (42) 39 (68) 82 (35).0001 Positive 171 (58) 18 (32) 153 (65) Treatment (%) d Effective 303 (57) 55 (58) 248 (57).9119 None or ineffective 225 (43) 40 (42) 185 (43) a 2 P value. b Wilcoxon rank sum test P value. c 1/2, well/moderate; 3/4, poor/undifferentiated. d See Patients and Methods. sion, as has been previously reported. 12 Diploid DNA content was detected in 223 colon cancers (42%) (Table 1). Tumor Characteristics Stratified by MSI Status We compared clinicopathologic features and cellular and molecular marker data in MSI-H versus MSS/MSI-L colon cancers. Patients with MSI-H tumors were older (P.02) and more likely to be female (P.0002) compared with patients with MSS/MSI-L tumors. MSI-H tumors were more frequently diploid and located in the proximal colon, had higher-grade (ie, poor or undifferentiated) histology, and were more likely to be negative for p53 expression as compared with MSS/MSI-L tumors (all P.0001) (Table 1). Tumor Characteristics Stratified by DNA Ploidy We compared clinicopathologic features and prognostic marker data in diploid versus aneuploid/tetraploid colon cancers. For purposes of our analyses, we combined aneuploid and tetraploid tumors. Diploid tumors were more likely to be MSI-H (P.0001) and to be located in the proximal colon (P.0013) as compared with aneuploid/tetraploid tumors (Table 2). Aneuploid/tetraploid tumors were more likely to be Astler Coller stage C (P.03), have higher histologic grade (P.03), and to overexpress p53 (P.002) as compared with diploid tumors (Table 2). When MSI-H cases were excluded, no difference for histologic grade was found when comparing diploid and aneuploid/tetraploid MSS/MSI-L cases (P.74). Table 2. Tumor Markers Stratified by DNA Ploidy Tumor marker Diploid Aneuploid/tetraploid P a Astler Coller stage (%) B2 78 (35) 80 (26).0301 C 145 (65) 225 (74) Age (y) Median (range) 64 (29 88) 65 (25 84).5996 b Mean Sex (%) Male 115 (52) 159 (52).8985 Female 108 (48) 146 (48) Tumor site (%) Proximal 139 (62) 147 (48).0013 Distal 84 (38) 158 (52) Histologic grade (%) c 1/2 72 (32) 73 (24) /4 151 (68) 232 (76) MSI (%) High 70 (31) 25 (8).0001 Stable/low 153 (69) 280 (92) p53 staining (%) Positive 62 (48) 109 (66).0024 Negative 66 (52) 56 (34) Treatment (%) d Effective 125 (56) 178 (58).5965 None or ineffective 98 (44) 127 (42) a 2 P value. b Wilcoxon rank sum test P value. c 1/2, well/moderate; 3/4, poor/undifferentiated. d See Patients and Methods.

4 732 SINICROPE ET AL GASTROENTEROLOGY Vol. 131, No. 3 Table 3. Clinicopathologic Features and Patient Survival Parameter 5-year 5-year DFS (%) HR (95% Cl) P a OS (%) HR (95% Cl) P a Age (for any 1-year increase) N/A ( ).7580 N/A 1.01 ( ).1285 Sex Male ( ) ( ).0908 Female Astler Coller stage B ( ) ( ).0001 C Tumor site Proximal Distal ( ) ( ) Histologic grade b 1/ / ( ) ( ) Treatment c Effective ( ) ( ).0468 None or ineffective a Likelihood ratio P value adjusting for histologic grade, stage, and treatment and stratifying by study. b 1/2, well/moderate; 3/4, poor/undifferentiated. c See Patients and Methods. Patient and Tumor Characteristics in Relation to Survival Clinicopathologic variables found to be prognostic for DFS and OS included tumor stage, histologic grade, and treatment status, as shown in Table 3. Specifically, patients with Astler Coller B2 versus C tumors had better DFS and OS rates, as did tumors with better differentiation (grades 1 and 2). 27 Patients receiving 5-FU based adjuvant therapy showed better clinical outcome compared with those receiving observation or ineffective treatment (see Patients and Methods). Tumor site was prognostic for DFS (P.042) but not OS (P.14). DNA diploidy was associated with better DFS (P.0001) and OS (P.0003) rates as compared with aneuploid/tetraploid tumors (Table 4 and Figure 1A). Furthermore, diploidy was a favorable prognostic marker for OS in patients with stage B2 (hazard ratio [HR], 0.44; 95% confidence interval [CI], ; P.026) and patients with stage C tumors (HR, 0.61; 95% CI, ; P.002) (Figure 1B) (interaction P.70). Given that 74% of MSI-H tumors were diploid (Table 1), we determined whether the better outcome of diploid tumors is due to their strong correlation with MSI-H. When MSI-H cases were excluded, diploidy remained prognostic for DFS (P.002) and OS (P.006) in MSS/MSI-L tumors as compared with aneuploid/tetraploid cases (Figure 1C), indicating that the prognostic impact of ploidy is independent of MSI status. Patients with MSI-H tumors (n 95) had a better 5-year OS compared with patients with MSS/MSI-L tumors (74.7% vs 68.5%; P.023) (Table 4 and Figure 2). Similarly, loss of hmlh1 or hmsh2 proteins was also associated with better OS rates (P.034) (Table 4 and Figure 3). Survival curves for patients with MSI-H tumors versus diploid MSS/MSI-L cases were overlapping (P.80), indicating that diploidy identifies a patient subgroup with biologically less aggressive tumors and similar outcome as MSI-H cases. Table 4. Tumor Markers and Patient Survival Parameter 5-year 5-year DFS (%) HR (95% Cl) P a OS (%) HR (95% Cl) P a DNA ploidy Diploid ( ) ( ).0003 Aneuploid/tetraploid MSI High ( ) ( ).0229 Stable/low MMR proteins b Absent ( ) ( ).0344 Intact p53 staining Negative Positive ( ) ( ) a Likelihood ratio P value adjusting for grade, stage, and treatment and stratifying by study. b Refers to loss of either hmlh1 or hmsh2 proteins.

5 September 2006 DNA PLOIDY AND MSI IN COLON CANCER 733 Figure 2. OS in patients with Astler Coller stage B2 and C colon cancer by MSI status. Tumor stage was a stronger prognostic variable than ploidy, as shown by our finding that patients with Astler Coller stage C tumors did worse than aneuploid/tetraploid B2 cases (P.011) and that diploid stage C tumors showed worse OS compared with B2 (P.019) tumors. Nuclear p53 expression was not prognostic for OS (P.959). When DNA ploidy and MSI were included in the same multivariate model, only ploidy remained prognostic for OS and DFS in addition to tumor stage and grade (Table 6). Similar results were observed when loss of MMR protein expression (HR, 0.71; 95% CI, ; P.195) was used instead of MSI status, thereby confirming the MSI-H results (data not shown). When ploidy was omitted from this model, MSI became a significant independent prognostic variable (Table 7). Again, similar results were observed when loss of MMR protein expression (HR, 0.58; 95% CI, ; P.024) was used instead of MSI-H. Figure 1. (A) OS in patients with Astler Coller stage B2 and C colon cancer by DNA ploidy status. (B) OS in patients with Astler Coller stage B2 and C colon cancer by DNA ploidy status and tumor stage. (C) OS in patients with MSS/MSI-L Astler Coller stage B2 and C colon cancer by DNA ploidy status. Predictive Status of Tumor Markers The favorable effect of 5-FU treatment was found to be significant within all 6 studies (Table 3) and also in the 3 randomized studies that included an observation arm and an effective 5-FU treatment arm (DFS, P.092; OS, P.036). Within these 3 trials, patients with diploid tumors had significantly better OS as compared with aneuploid/tetraploid tumors in both the treated (HR, 0.46; P.04) and untreated (HR, 0.52; P.023) patient subgroups. Additionally, we analyzed the treatment effect within the diploid and aneuploid/tetraploid Within the MSI-H patient subset (n 95), there was a trend toward better outcome for diploid tumors (DFS; P.109) (Table 5). While this result did not achieve statistical significance given the limited sample size, the HRs for MSI-H diploid versus aneuploid/tetraploid tumors were 0.51 for DFS (95% CI, ) and 0.67 for OS (95% CI, ). In the patients with MSS/MSI-L tumors (n 433), the HRs for diploid versus aneuploid/tetraploid tumors were 0.57 for DFS (95% CI, ) and 0.63 for OS (95% CI, ) (Table 5). Given the similar HRs between groups and the observation that the DFS HR is smaller in the MSI-H group, we regard the diploid effect to be the same within MSS/MSI-L and MSI-H patient subsets. Furthermore, the interaction P value for DFS was.997 and for OS was.833, implying that the prognostic effect of diploidy is the same for both MSI groups. Figure 3. OS in patients with Astler Coller stage B2 and C colon cancer by loss of DNA MMR protein expression.

6 734 SINICROPE ET AL GASTROENTEROLOGY Vol. 131, No. 3 Table 5. Prognostic Impact of DNA Ploidy Within MSI-H and MSS/MSI-L Patient Subgroups Parameter DFS a HR (95% CI) P OS b HR (95% CI) P MSI-H (n 95) Diploid vs aneuploid/tetraploid 0.51 ( ) ( ).3211 MSS/MSI-L (n 433) Diploid vs aneuploid/tetraploid 0.57 ( ) ( ).0061 a Interaction P b Interaction P tumors separately and found that there was no evidence of a differential treatment effect (OS interaction, P.92; DFS interaction, P.71). After excluding the MSI-H cases, similar results were found for both OS (interaction P.43) and DFS (interaction P.24) within the MSS/MSI-L cases from the 3 trials outlined previously. The lack of significant interactions indicates that the treatment effect is the same in diploid and aneuploid/tetraploid groups. Therefore, the less aggressive behavior of diploid tumors cannot be explained by a differential response to 5-FU, indicating that ploidy is not a predictive marker for 5-FU based adjuvant therapy. Discussion The hallmark of the chromosomal instability pathway is aneuploidy and that of the MSI pathway is defective DNA MMR. We found that aneuploid/tetraploid DNA content was associated with higher tumor stage and poorer differentiation, distal tumor site, and nuclear p53 expression as compared with diploidy. In addition, these aneuploid/tetraploid tumors were generally MSS/MSI-L. Among patients with Astler Coller B2 and C colon cancers participating in these adjuvant studies, 18% of tumors were MSI-H and most are believed to be sporadic cases. Loss of hmlh1 expression was detected in 50 of 58 cases tested using immunohistochemistry, and based on the following study, 35 we can estimate the proportion due to hypermethylation of hmlh1. Among 257 patients with colorectal carcinomas referred to the Mayo Clinic for resection, 42 tumors were hmlh1 negative and 37 (88%) showed hmlh1 promoter methylation. 35 In our study, 4 of 58 MSI-H cases (6.8%) showed loss of hmsh2 proteins, suggesting a germline event. Based on these data, no more than 10 tumors in this series are likely to be hereditary nonpolyposis colorectal cancer (HNPCC). Of note, only 12% of patients were aged 50 years or younger at study enrollment. MSI-H tumors had distinct clinical and pathologic features, including frequent diploidy, proximal site, and poor/differentiation, consistent with the reported MSI-H Table 6. Multivariate Model for Tumor Markers in Relation to Patient OS (N 528) Variable HR (95% CI) P a MSI-H ( ).2025 Diploid ( ).0019 Astler Coller stage B ( ).0001 Treatment ( ).1086 Histologic grade (3/4) b ( ).0001 NOTE. Interaction P value between MSI-H and diploid was a Likelihood ratio P value. b Poor/undifferentiated. phenotype ,17,36 MSI-H tumors also showed a reduced frequency of p53 protein expression 32,36,37 and were more prevalent in women. In this regard, a higher frequency of hypermethylation of the hmlh1 gene promoter has been reported in colon cancers from women compared with men. 15,38,39 We determined the prognostic impact of DNA ploidy and MSI status and examined the relationship between these variables. Our data show that ploidy and MSI are independent prognostic variables in Astler Coller B2 and C colon cancers treated in 5-FU based adjuvant therapy trials. Specifically, patients with diploid tumors or those found to be MSI-H had a significantly better OS compared with aneuploid/tetraploid or MSS/MSI-L cases, respectively. To confirm our MSI data, DNA MMR proteins (hmlh1, hmsh2) were analyzed and tumors showing loss of expression of either protein had significantly better OS rates. Together, these results are consistent with other studies showing that MSI is a favorable prognostic variable in patients with colorectal cancer. 6,16,40,41 In contrast to our study population, however, most of these reports included all stages of colon cancer and also included patients with rectal cancer. 16,17,41 Better survival rates have also been reported for HNPCC cases. 42 Given that MSI-H colon cancers are generally diploid, as shown here and in other studies, 6,13,15 we determined whether MSI-H cases account for the better prognosis of diploid tumors When only MSS/MSI-L cases were analyzed, DNA ploidy remained prognostic for DFS (P.002) and OS (P.006). Therefore, inclusion of MSI-H cases does not account for the prognostic impact of ploidy. Of note, OS curves for patients with diploid MSS/MSI-L tumors overlapped with those for MSI-H cases. Importantly, the prognostic impact of ploidy was similar within B2 and C colon cancers (interaction P.70). This finding has important implications for the management of patients with B2 tumors who do not routinely receive adjuvant chemotherapy. Within the MSI-H cases, diploidy was associated with better DFS (P.109) compared with aneuploid/tetraploid MSI-H cases. Moreover, the HRs for diploidy in MSI-H cases were similar to those found in MSS/MSI-L tumors. The inter- Table 7. Multivariate Model for Tumor Markers in Relation to Patient OS (N 528) Variable HR (95% CI) P a MSI-H ( ).0229 Histologic grade (3/4) b ( ).0001 Astler Coller stage B ( ).0001 Treatment ( ).0543 a Likelihood ratio p value. b Poor/undifferentiated.

7 September 2006 DNA PLOIDY AND MSI IN COLON CANCER 735 action P values were not significant for either DFS or OS, implying that the prognostic effect of diploidy is the same for both MSI-H and MSS/MSI-L groups. Tumor stage, histologic grade, and adjuvant treatment status were found to be prognostic for DFS and OS, and adjustment was made for these known prognostic variables. Moreover, in a multivariate analysis, ploidy was a stronger stage-independent prognostic variable than was MSI-H status or loss of MMR protein expression. We also examined the predictive value of DNA ploidy. In patients treated in the three 5-FU based adjuvant studies that included both an observation and an effective treatment arm, treatment was associated with better DFS (P.092) and OS (P.036) compared with patients receiving observation or ineffective treatment. We did not detect any evidence of a differential treatment effect for diploid versus aneuploid/tetraploid tumors overall and within the patients with MSS/MSI-L tumors. Therefore, the less aggressive behavior of diploid tumors cannot be explained by a differential response to 5-FU, indicating that ploidy is a prognostic but not a predictive marker for 5-FU based adjuvant therapy. Given the limited number of MSI-H cases included in our study, we were unable to address the issue of differential sensitivity to 5-FU based on MSI status. However, a retrospective study that included cases from the North Central Cancer Treatment Group and National Cancer Institute of Canada showed that patients with MSI-H tumors did not benefit from 5-FU based adjuvant therapy in contrast to MSS/MSI-L tumors. 43 This result is consistent with data in MSI-H human colon cancer cell lines that exhibit resistance to 5-FU compared with cells with intact DNA MMR. 44 However, another retrospective study 40 found an opposite result whereby MSI-H tumors appeared to have a greater benefit from 5-FU based adjuvant treatment, yet patients receiving 5-FU were on average 13 years younger than those who did not. 40 More recently, that same group reported that the CpG island methylator phenotype (CIMP) independently predicted a survival benefit for 5-FU treatment in a retrospective analysis of patients with stage III colorectal cancer. 45 At present, the issue of MSI or CIMP status and 5-FU sensitivity remains controversial and unresolved. We found diploidy in 74% of MSI-H and in 35% of MSS/ MSI-L tumors. While we did not determine the methylation status of our cases as yet, we compared features of these cases with those reported for sporadic CIMP cases. In a large population-based study of colon cancers, CIMP was detected in 82% of MSI-H cases and in 24% of MSS cases. 46 Furthermore, CIMPhigh MSS cases showed older age, increased stage, and proximal tumor site, in addition to BRAF and KRAS2 mutations. Compared with aneuploid/tetraploid MSS/MSI-L cases, our diploid MSS/MSI-L cases were younger and less likely to express p53 but were similar with respect to stage, tumor site, and grade (data not shown). Among 28 tumors on which BRAF mutation (V599E) data were available, no relationship between BRAF and ploidy was found (P.624); however, BRAF mutation was associated with MSI-H status (P.0012). Samowitz et al 46 recently reported that MSS colon cancers with BRAF mutations are related to CIMP-high and show poor survival. However, our diploid MSS/MSI-L tumors showed improved survival rates. Goel et al 8 reported that a subset of colon cancers lack both MSI and chromosomal instability. Based on our cases where allelic imbalance results were available at chromosome 5q, 17p, and 18q, only 14 of 162 cases were found to lack allelic imbalance at all 3 loci, all were MSS/MSI-L, and 10 of these 14 cases were diploid. The survival of these 14 patients was similar to the larger group of diploid MSS/MSI-L cases. Therefore, our diploid MSS/MSI-L cases appear unlikely to significantly overlap with CIMP. Furthermore, we emphasize that diploid cases were not more chemosensitive compared with aneuploid/tetraploid tumors, which is also against their overlap with CIMP. In conclusion, DNA ploidy, MSI-H, and loss of MMR proteins were independent prognostic variables. However, DNA ploidy was a stronger prognostic variable than was MSI in patients with colon cancer treated in 5-FU based adjuvant therapy trials. Importantly, we found that diploidy was associated with better survival in MSI-H colon cancers and the magnitude of this effect was equivalent to that in MSS/MSI-L cases, as indicated by similar HRs. Therefore, diploidy may contribute to the better survival of MSI-H colon cancers. The prognostic impact of diploidy remained after removal of MSI-H cases, indicating that MSI-H does not account for the better outcome of diploid colon cancers despite their strong correlation. DNA ploidy was not predictive of treatment efficacy. Our results suggest that ploidy and MSI may influence clinical decisionmaking, including the selection of patients to receive adjuvant chemotherapy. Accordingly, these markers warrant prospective evaluation in clinical studies in patients with colon cancer. Ongoing studies by our group will evaluate and compare ploidy with nuclear morphometry 47 and centrosome amplification 48 in an effort to identify a potential surrogate marker for DNA content analysis. 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