Prognostic Impact of Microsatellite Instability and DNA Ploidy in Human Colon Carcinoma Patients

GASTROENTEROLOGY 2006;131:729 –737

FRANK A. SINICROPE,*,‡ RAFAELA L. REGO,* KEVIN C. HALLING,§ NATHAN FOSTER,储 DANIEL J. SARGENT,储 BETSY LA PLANT,储 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 储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, 0.44 – 0.96; 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, 0.43– 0.79; 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.

C

olorectal 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.9 –11 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.19 –24 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. © 2006 by the American Gastroenterological Association (AGA) Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.06.005

CLINICAL– ALIMENTARY TRACT

Prognostic Impact of Microsatellite Instability and DNA Ploidy in Human Colon Carcinoma Patients

730

SINICROPE ET AL

CLINICAL– ALIMENTARY TRACT

available from study participants. Tumors were staged according to the Astler–Coller modification of the Dukes’ classification26 and included stage B2 (n ⫽ 158) and C (n ⫽ 370) colon cancers (N ⫽ 528). Tumor histologic grade was defined27 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, ⱖ30% 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 G168-782 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-2.32 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.

GASTROENTEROLOGY Vol. 131, No. 3

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 models33 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 ⱕ .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 51–55 years, 71 (13%) aged 56 – 60 years, 202 (38%) aged 61–70 years, 134 (25%) aged 71– 80 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-

September 2006

DNA PLOIDY AND MSI IN COLON CANCER

731

Table 1. Tumor Markers Stratified by MSI Status

Astler–Coller stage (%) B2 C Age (y) Median (range) Mean Sex (%) Male Female Tumor site (%) Proximal Distal Histologic grade (%)c 1/2 3/4 DNA ploidy (%) Diploid Aneuploid/tetraploid p53 staining (%) Negative Positive Treatment (%)d Effective None or ineffective

No. of patients 158 (30) 370 (70) N/A

MSI-H 35 (37) 60 (63) 67 (33–86) 65.1

Pa

MSS/MSI-L 123 (28) 310 (72)

.1040

.0197b

64 (25–88) 62.6

274 (52) 254 (48)

33 (35) 62 (65)

241 (56) 192 (44)

.0002

286 (54) 242 (46)

88 (93) 7 (7)

198 (46) 235 (54)

⬍.0001

383 (73) 145 (27)

48 (51) 47 (49)

335 (77) 98 (23)

⬍.0001

223 (42) 305 (58)

70 (74) 25 (26)

153 (35) 280 (65)

⬍.0001

122 (42) 171 (58)

39 (68) 18 (32)

82 (35) 153 (65)

⬍.0001

303 (57) 225 (43)

55 (58) 40 (42)

248 (57) 185 (43)

.9119

a␹2

P value. rank sum test P value. c1/2, well/moderate; 3/4, poor/undifferentiated. dSee Patients and Methods. bWilcoxon

sion, as has been previously reported.12 Diploid DNA content was detected in 223 colon cancers (42%) (Table 1).

Table 2. Tumor Markers Stratified by DNA Ploidy Tumor marker

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).

Astler–Coller stage (%) B2 C Age (y) Median (range) Mean Sex (%) Male Female Tumor site (%) Proximal Distal Histologic grade (%)c 1/2 3/4 MSI (%) High Stable/low p53 staining (%) Positive Negative Treatment (%)d Effective None or ineffective a␹2

Diploid 78 (35) 145 (65) 64 (29–88) 62.7

Aneuploid/tetraploid 80 (26) 225 (74) 65 (25–84) 63.3

.0301

.5996b

115 (52) 108 (48)

159 (52) 146 (48)

.8985

139 (62) 84 (38)

147 (48) 158 (52)

.0013

72 (32) 151 (68)

73 (24) 232 (76)

.0337

70 (31) 153 (69)

25 (8) 280 (92)

⬍.0001

62 (48) 66 (52)

109 (66) 56 (34)

.0024

125 (56) 98 (44)

178 (58) 127 (42)

.5965

P value. rank sum test P value. c1/2, well/moderate; 3/4, poor/undifferentiated. dSee Patients and Methods. bWilcoxon

Pa

CLINICAL– ALIMENTARY TRACT

Characteristics

732

SINICROPE ET AL

GASTROENTEROLOGY Vol. 131, No. 3

Table 3. Clinicopathologic Features and Patient Survival Parameter CLINICAL– ALIMENTARY TRACT

Age (for any 1-year increase) Sex Male Female Astler–Coller stage B2 C Tumor site Proximal Distal Histologic gradeb 1/2 3/4 Treatmentc Effective None or ineffective

5-year DFS (%)

HR (95% Cl)

N/A

0.998 (0.984–1.012)

5-year OS (%)

HR (95% Cl)

.7580

N/A

1.01 (0.997–1.024)

.1285

Pa

Pa

61.2 66.8

1.19 (0.88–1.59) —

.2544

67.6 71.9

1.27 (0.96–1.67) —

.0908

79.6 57.1

0.40 (0.27–0.60) —

⬍.0001

83.3 63.8

0.46 (0.32–0.65) —

⬍.0001

66.8 60.3

— 1.36 (1.01–1.83)

.0424

71.6 67.3

— 1.23 (0.93–1.63)

.1401

68.3 51.8

— 1.66 (1.23–2.26)

.0015

73.6 59.1

— 1.75 (1.32–2.33)

.0002

64.5 63.1

0.68 (0.43–1.08) —

.0956

71.6 67.1

0.66 (0.43–1.00) —

.0468

aLikelihood

ratio P value adjusting for histologic grade, stage, and treatment and stratifying by study. well/moderate; 3/4, poor/undifferentiated. cSee Patients and Methods. b1/2,

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], 0.21– 0.92; P ⫽ .026) and patients with stage C tumors (HR, 0.61; 95% CI,

0.44 – 0.84; 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 DNA ploidy Diploid Aneuploid/tetraploid MSI High Stable/low MMR proteinsb Absent Intact p53 staining Negative Positive aLikelihood bRefers

5-year DFS (%)

HR (95% Cl)

Pa

5-year OS (%)

HR (95% Cl)

Pa

73.4 56.6

0.54 (0.39–0.74) —

⬍.0001

77.9 63.6

0.59 (0.43–0.79) —

.0003

68.7 62.7

0.72 (0.48–1.09) —

.1071

74.7 68.5

0.65 (0.44–0.96) —

.0229

69.2 61.4

0.67 (0.39–1.15) —

.1320

74.1 66.1

0.60 (0.36–0.99) —

.0344

65.4 60.9

— 1.19 (0.80–1.78)

.3830

70.4 67.4

— 0.99 (0.69–1.43)

.9594

ratio P value adjusting for grade, stage, and treatment and stratifying by study. to loss of either hMLH1 or hMSH2 proteins.

DNA PLOIDY AND MSI IN COLON CANCER

733

CLINICAL– ALIMENTARY TRACT

September 2006

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, 0.42–1.21; 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, 0.35– 0.96; P ⫽ .024) was used instead of MSI-H.

Predictive Status of Tumor Markers

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.

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, 0.23–1.13) and 0.67 for OS (95% CI, 0.31–1.46). 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, 0.39 – 0.82) and 0.63 for OS (95% CI, 0.45– 0.89) (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.

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

Figure 3.

OS in patients with Astler–Coller stage B2 and C colon cancer by loss of DNA MMR protein expression.

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

CLINICAL– ALIMENTARY TRACT

Parameter

DFSa HR (95% CI)

P

OSb HR (95% CI)

P

MSI-H (n ⫽ 95) Diploid vs aneuploid/tetraploid

0.51 (0.23–1.13)

.1086

0.67 (0.31–1.46)

.3211

MSS/MSI-L (n ⫽ 433) Diploid vs aneuploid/tetraploid

0.57 (0.39–0.82)

.0018

0.63 (0.45–0.89)

.0061

aInteraction bInteraction

P ⫽ .9972. P ⫽ .8332.

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)

Pa

MSI-H Diploid Astler–Coller stage B2 Treatment Histologic grade (3/4)b

0.772 (0.51–1.16) 0.620 (0.46–0.85) 0.484 (0.34–0.69) 0.712 (0.47–1.09) 1.966 (1.47–2.64)

.2025 .0019 ⬍.0001 .1086 ⬍.0001

NOTE. Interaction P value between MSI-H and diploid was .8332. ratio P value. bPoor/undifferentiated. aLikelihood

phenotype.13–15,17,36 MSI-H tumors also showed a reduced frequency of p53 protein expression32,36,37 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.19 –24 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)

Pa

MSI-H Histologic grade (3/4)b Astler–Coller stage B2 Treatment

0.647 (0.44–0.96) 1.900 (1.42–2.55) 0.472 (0.33–0.67) 0.668 (0.44–1.02)

.0229 ⬍.0001 ⬍.0001 .0543

aLikelihood

ratio p value.

bPoor/undifferentiated.

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 study40 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 al46 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 al8 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 imbal-

DNA PLOIDY AND MSI IN COLON CANCER

735

ance 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 morphometry47 and centrosome amplification48 in an effort to identify a potential surrogate marker for DNA content analysis. References 1. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ, American Cancer Society cancer statistics, 2004. CA Cancer 2004;54:8 –29. 2. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature 1997;386:623– 627. 3. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature 1998;392:300 –303. 4. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996;87:159 –170. 5. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558 –561. 6. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816 – 819. 7. Chung DC, Rustgi AK. DNA mismatch repair and cancer. Gastroenterology 1995;109:1685–1699. 8. Goel A, Arnold CN, Niedzwiecki D, Chang DK, Ricciardiello L, Carethers JM, Dowell JM, Wasserman L, Compton C, Mayer RJ, Bertagnolli MM, Boland CR. Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res 2003;63: 1608 –1614. 9. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, Thibodeau SN. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998;58:3455–3460. 10. Kuismanen SA, Holmberg MT, Salovaara R, Schweizer P, Aaltonen LA, de la Chapelle A, Nystrom-Lahti M, Peltomaki P. Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers. Proc Natl Acad Sci U S A 1999;96:12661–12666. 11. Kuismanen SA, Holmberg MT, Salovaara R, de la Chapelle A, Peltomaki P. Genetic and epigenetic modification of MLH1 ac-

CLINICAL– ALIMENTARY TRACT

September 2006

736

12.

CLINICAL– ALIMENTARY TRACT

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

SINICROPE ET AL

counts for a major share of microsatellite-unstable colorectal cancers. Am J Pathol 2000;156:1773–1779. Lindor NM, Burgart LJ, Leontovich O, Goldberg RM, Cunningham JM, Sargent DJ, Walsh-Vockley C, Petersen GM, Walsh MD, Leggett BA, Young JP, Barker MA, Jass JR, Hopper J, Gallinger S, Bapat B, Redston M, Thibodeau SN. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002;20:1043–1048. Lothe RA, Peltomaki P, Meling GI, Aaltonen LA, Nystrom-Lahti M, Pylkkanen L, Heimdal K, Andersen TI, Moller P, Rognum TO, et al. Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history. Cancer Res 1993;53: 5849 –5852. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:5248 –5257. Thibodeau SN, French AJ, Cunningham JM, Tester D, Burgart LJ, Roche PC, McDonnell SK, Schaid DJ, Vockley CW, Michels VV, Farr GH Jr, O’Connell MJ. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 1998;58:1713–1718. Halling KC, French AJ, McDonnell SK, Burgart LJ, Schaid DJ, Peterson BJ, Moon-Tasson L, Mahoney MR, Sargent DJ, O’Connell MJ, Witzig TE, Farr GF Jr, Goldberg RM, Thibodeau SN. Microsatellite instability and 8p allelic imbalance in stage B2 and C colorectal cancers. J Natl Cancer Inst 1999;91:1295–1303. Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB, Redston M, Gallinger S. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 2000;342:69 –77. Kakar S, Aksoy S, Burgart LJ, Smyrk TC. Mucinous carcinoma of the colon: correlation of loss of mismatch repair enzymes with clinicopathologic features and survival. Mod Pathol 2004;17: 696 –700. Bauer KD, Lincoln ST, Vera-Roman JM, Wallemark CB, Chmiel JS, Madurski ML, Murad T, Scarpelli DG. Prognostic implications of proliferative activity and DNA aneuploidy in colonic adenocarcinomas. Lab Invest 1987;57:329 –335. Schutte B, Reynders MM, Wiggers T, Arends JW, Volovics L, Bosman FT, Blijham GH. Retrospective analysis of the prognostic significance of DNA content and proliferative activity in large bowel carcinoma. Cancer Res 1987;47:5495–5496. Scott NA, Wieand HS, Moertel CG, Cha SS, Beart RW, Lieber MM. Colorectal cancer. Dukes’ stage, tumor site, preoperative plasma CEA level, and patient prognosis related to tumor DNA ploidy pattern. Arch Surg 1987;122:1375–1379. Armitage NC, Ballantyne KC, Evans DF, Clarke P, Sheffield J, Hardcastle JD. The influence of tumour cell DNA content on survival in colorectal cancer: a detailed analysis. Br J Cancer 1990;62:852– 856. Witzig TE, Loprinzi CL, Gonchoroff NJ, Reiman HM, Cha SS, Wieand HS, Katzmann JA, Paulsen JK, Moertel CG. DNA ploidy and cell kinetic measurements as predictors of recurrence and survival in stages B2 and C colorectal adenocarcinoma. Cancer 1991;68:879 – 888. Lanza G, Gafa R, Santini A, Maestri I, Dubini A, Gilli G, Cavazzini L. Prognostic significance of DNA ploidy in patients with stage II and stage III colon carcinoma: a prospective flow cytometric study. Cancer 1998;82:49 –59. Sinicrope FA, Hart J, Hsu HA, Lemoine M, Michelassi F, Stephens LC. Apoptotic and mitotic indices predict survival rates in lymph node-negative colon carcinomas. Clin Cancer Res 1999;5:1793–1804.

GASTROENTEROLOGY Vol. 131, No. 3

26. Astler VB, Coller J. The prognostic significance of direct extension of carcinoma of the colon and rectum. Ann Surg 1954;139:846 – 852. 27. Compton C, Fenoglio-Preiser CM, Pettigrew N, Fielding LP. American Joint Committee on Cancer Prognostic Factors Consensus Conference: Colorectal Working Group. Cancer 2000;88:1739 – 1757. 28. Allegra CJ, Parr AL, Wold LE, Mahoney MR, Sargent DJ, Johnston P, Klein P, Behan K, O’Connell MJ, Levitt R, Kugler JW, Tria Tirona M, Goldberg RM. Investigation of the prognostic and predictive value of thymidylate synthase, p53, and Ki-67 in patients with locally advanced colon cancer. J Clin Oncol 2002;20:1735–1743. 29. Laiho P, Launonen V, Lahermo P, Esteller M, Guo M, Herman JG, Mecklin JP, Jarvinen H, Sistonen P, Kim KM, Shibata D, Houlston RS, Aaltonen LA. Low-level microsatellite instability in most colorectal carcinomas. Cancer Res 2002;62:1166 –1170. 30. Halford S, Sasieni P, Rowan A, Wasan H, Bodmer W, Talbot I, Hawkins N, Ward R, Tomlinson I. Low-level microsatellite instability occurs in most colorectal cancers and is a nonrandomly distributed quantitative trait. Cancer Res 2002;62:53–57. 31. Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem 1983;31:1333–1335. 32. Garrity MM, Burgart LJ, Mahoney MR, Windschitl HE, Salim M, Wiesenfeld M, Krook JE, Michalak JC, Goldberg RM, O’Connell MJ, Furth AF, Sargent DJ, Murphy LM, Hill E, Riehle DL, Meyers CH, Witzig TE, North Central Cancer Treatment Group. Prognostic value of proliferation, apoptosis, defective DNA mismatch repair, and p53 overexpression in patients with resected Dukes’ B2 or C colon cancer: a North Central Cancer Treatment Group Study. J Clin Oncol 2004;22:1572–1582. 33. Cox DR. Regression models and life tables. J R Stat Soc (Series B) 1972;34:187–202. 34. Grambsch P, Therneau TM. Proportional hazards test and diagnostics based on weighted residuals. Biometrika 1994;81:515– 526. 35. Cunningham JM, Kim CY, Christensen ER, Tester DJ, Parc Y, Burgart LJ, Halling KC, McDonnell SK, Schaid DJ, Walsh Vockley C, Kubly V, Nelson H, Michels VV, Thibodeau SN. The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas. Am J Hum Genet 2001;69: 780 –790. 36. Kim H, Jen J, Vogelstein B, Hamilton SR. Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences. Am J Pathol 1994; 145:148 –156. 37. Samowitz WS, Curtin K, Ma KN, Edwards S, Schaffer D, Leppert MF, Slattery ML. Prognostic significance of p53 mutations in colon cancer at the population level. Int J Cancer 2002;99:597– 602. 38. Breivik J, Lothe RA, Meling GI, Rognum TO, Borresen-Dale AL, Gaudernack G. Different genetic pathways to proximal and distal colorectal cancer influenced by sex-related factors. Int J Cancer 1997;74:664 – 669. 39. Malkhosyan SR, Yamamoto H, Piao Z, Perucho M. Late onset and high incidence of colon cancer of the mutator phenotype with hypermethylated hMLH1 gene in women. Gastroenterology 2000;119: 598 (comments in Gastroenterology 2001;120:1309 –1310). 40. Elsaleh H, Powell B, Soontrapornchai P, Joseph D, Goria F, Spry N, Iacopetta B. p53 gene mutation, microsatellite instability and adjuvant chemotherapy: impact on survival of 388 patients with Dukes’ C colon carcinoma. Oncology 2000;58:52–59. 41. Hemminki A, Mecklin JP, Jarvinen H, Aaltonen LA, Joensuu H. Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology 2000;119:921–928.

42. Sankila R, Aaltonen LA, Jarvinen HJ, Mecklin JP. Better survival rates in patients with MLH1-associated hereditary colorectal cancer. Gastroenterology 1996;110:682– 687. 43. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, Hamilton SR, Laurent-Puig P, Gryfe R, Shepherd LE, Tu D, Redston M, Gallinger S. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 2003;349:247– 257. 44. Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB, Boland CR. Mismatch repair proficiency and in vitro response to 5-fluorouracil. Gastroenterology 1999;117:123–131. 45. Van Rijnsoever M, Elsaleh H, Joseph D, McCaul K, Iacopetta B. CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer. Clin Cancer Res 2003;9:2898 –2903. 46. Samowitz WS, Albertsen H, Herrick J, Levin TR, Sweeney C, Murtaugh MA, Wolff RK, Slattery ML. Evaluation of a large,

DNA PLOIDY AND MSI IN COLON CANCER

737

population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology 2005;129:837– 845. 47. Ikeguchi M, Sakatani T, Endo K, Makino M, Kaibara N. Computerized nuclear morphometry is a useful technique for evaluating the high metastatic potential of colorectal adenocarcinoma. Cancer 1999;86:1944 –1951. 48. D’Assoro AB, Lingle WL, Salisbury JL. Centrosome amplification and the development of cancer. Oncogene 2002;21:6146 – 6153.

Received February 13, 2006. Accepted May 18, 2006. Address requests for reprints to: Frank A. Sinicrope, MD, 200 First Street Southwest, Rochester, Minnesota 55905. e-mail: [email protected]; fax: (507) 266-0350. Supported in part by a grant from the National Institutes of Health/ National Cancer Institute CA 104683 (to F.A.S.).

CLINICAL– ALIMENTARY TRACT

September 2006