Imaging, Diagnosis, Prognosis

A Cohort Study of Cyclin D1 Expression and Prognosis in 602 Colon Cancer Cases Shuji Ogino,1, 2 , 3 Katsuhiko Nosho,1Natsumi Irahara,1Shoko Kure,1Kaori Shima,1Yo s hi f u m i B a b a , 1 SaoriToyoda,1Li Chen,2 Edward L. Giovannucci,3,4 Jeffrey A. Meyerhardt,1and Charles S. Fuchs1,4

Abstract Purpose: Cyclin D1 and cyclin-dependent kinases (CDK) are commonly activated in colorectal cancer. The activity of cyclin D1can be blocked by CDK inhibitors, including p27 (CDKN1B)and p21 (CDKN1A , which is induced by p53). However, prognostic significance of tumoral cyclin D1 remains uncertain, and no previous study has considered potential confounding effect of p53, p21, p27, and related molecular events [microsatellite instability (MSI), CpG island methylator phenotype, and LINE-1hypomethylation]. Experimental Design: Among 602 colon cancer patients (stage I-IV) in two prospective cohort studies, cyclin D1overexpression was detected in 330 (55%) tumors by immunohistochemistry. Cox proportional hazard models computed hazard ratios (HR) of colon cancer ^ specific and overall mortalities, adjusted for patient characteristics and tumoral molecular features, including p53, p21,p27, cyclooxygenase-2, fatty acid synthase, LINE-1methylation, CpG island methylator phenotype, MSI, BMI, KRAS,andBRAF. Results: Cyclin D1 overexpression was associated with a low cancer-specific mortality in Kaplan-Meier analysis (P = 0.006), and in both univariate Cox regression [unadjusted HR, 0.64; 95% confidence interval (CI), 0.47-0.88; P = 0.0063] and multivariate analyses (adjusted HR, 0.57; 95% CI, 0.39-0.84; P = 0.0048). Similar findings were observed for an overall mortality (adjusted HR, 0.74; 95% CI, 0.57-0.98; P = 0.036). Notably, the effect of cyclin D1on survival might differ by MSI status (Pinteraction = 0.008). Compared with tumors that were both cyclin D1^ negative and MSI-low/microsatellite stable, the presence of either cyclin D1or MSI-high or both seemed to confer better clinical outcome (adjusted HR point estimates, 0.10-0.65). Conclusions: Cyclin D1overexpression is associated with longer survival in colon cancer.

Cyclin D1 (CCND1, the official gene symbol) plays a key role Authors’ Affiliations: 1Department of Medical Oncology, Dana-Farber Cancer in cell cycle control, particularly in the transition from G to S 2 1 Institute and Harvard Medical School; Department of Pathology, Brigham and phase, which is regulated by cyclin-dependent kinases (1). The Women’s Hospital, Boston and Harvard Medical School; 3Department of Epidemiology, Harvard School of Public Health; and 4Channing Laboratory, ability of cyclin D1 to drive the cell cycle forward can be Department of Medicine, Brigham and Women’s Hospital and Harvard Medical blocked by cyclin-dependent kinase (CDK) inhibitors, such as School, Boston, Massachusetts p27 (CDKN1B) and p21 (CDKN1A, which is induced by Received12/29/08;revised3/16/09;accepted4/7/09;publishedOnlineFirst 6/23/09. p53; ref. 1). Cyclin D1 overexpression occurs in one-third or Grant support: U.S. NIH P01 CA87969 (S. Hankinson), P01 CA55075 (W. more of colorectal cancers (2–20). Cyclin D1 activation by Willett), P50 CA127003 (C.S. Fuchs), K07 CA97992 (J.A. Meyerhardt), and K07 CA122826 (S. Ogino); the Bennett Family Fund; and the Entertainment APC mutation/WNT signaling seems to contribute to colon Industry Foundation National Colorectal Cancer Research Alliance. K. Nosho neoplasia initiation (21, 22). was supported by a fellowship grant from the Japan Society for Promotion of Despite a well-established role of cyclin D1 in cell cycle Science. The content is solely the responsibility of the authors and does not progression, previous data on cyclin D1 and clinical outcome necessarily represent the official views of National Cancer Institute or NIH. Funding agencies did not have any role in the design of the study; the in colon cancer have been conflicting (4–20). Although cyclin collection, analysis, or interpretation of the data; the decision to submit the D1 expression has been associated with poor prognosis in two manuscript for publication; or the writing of the manuscript. studies (4, 5), another study showed good prognosis The costs of publication of this article were defrayed in part by the payment of associated with cyclin D1 expression (6), and most page charges. This article must therefore be hereby marked advertisement in studies revealed no independent prognostic value of cyclin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer D1 (7–20). However, most previous studies had limited Research Online (http://clincancerres.aacrjournals.org/). sample sizes, and only three studies (11, 15, 16) had sample Requests for reprints: ShujiOgino,CenterforMolecularOncologicPathology, sizes >170 (up to n = 363; ref. 16). In addition, cyclin D1 Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical expression in colon cancer is related with microsatellite School, 44 Binney Street, Room JF-215C, Boston, MA 02115. Phone: 617-632- 3978; Fax: 617-582-8558; E-mail: [email protected]. instability (MSI), the CpG island methylator phenotype F 2009 American Association for Cancer Research. (CIMP), and BRAF mutation (23). Although these molecular doi:10.1158/1078-0432.CCR-08-3330 features have been associated with patient outcome (24–26),

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underwent tumor resections (28). Tissue sections from all colon cancer Translational Relevance cases were reviewed and confirmed by a pathologist (S.O.). Tumor grade was categorized as high (V50% glandular area) or low (>50% Cyclin D1and cyclin-dependent kinase (CDK) activation glandular area). Based on availability of tissue samples, we included a has been shown to play an important role in carcinogenesis total of 602 stage I to IV colon cancer cases diagnosed up to 2002. There f in various organ systems including colon. CDK inhibitors were only 2.5% of Asians, Hispanics, and African Americans and the have been shown to be effective in cancer treatment.How- remaining 97.5% were non-Hispanic Caucasians. Written informed consent was obtained from all study subjects. This study was approved ever, the relation between cyclin D1expression in colon by the Human Subjects Committees at Brigham and Women’s Hospital cancer and patient survival has been controversial. We and the Harvard School of Public Health. have used the database of >600 colon cancer in two Measurement of mortality. Patients were observed until death or independent, prospective cohort studies, with available June 2006, whichever came first. Ascertainment of deaths included clinical information, adequate follow-up, and other reporting by the family or postal authorities. In addition, the names of important molecular events in colon cancers. To our persistent nonresponders were searched in the National Death Index. knowledge, this is the first large study to show influence The cause of death was assigned by physicians blinded to information of cyclin D1expression on clinical outcome independent of on life-style exposures and molecular changes in colon cancer. In rare related molecular events including p53, p21, p27, KRAS, patients who died as a result of colon cancer not previously reported, BRAF mutation, microsatellite instability, the CpG island we obtained medical records with permission from next of kin. More methylator phenotype, and LINE-1hypomethylation, all of than 98% of deaths in the cohorts were identified by these methods. DNA extraction, pyrosequencing of KRAS and BRAF,andMSI which are potential confounders. Thus, our findings are analysis. Genomic DNA from paraffin-embedded tissue was extracted, relevant to practice in oncology. and whole genome amplification was done (29). PCR and Pyrose- quencing targeted for KRAS codons 12 and 13 (29), and BRAF codon 600, were done (30). MSI status was determined using 10 microsatellite none of the previous studies (4–20) has considered con- markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, founding or modifying effect of MSI, CIMP, and BRAF. D18S56, D18S67, and D18S487; ref. 31). MSI-high was defined as the In this study, using a large number (n = 602) of stage I to IV presence of instability in z30% of the markers, MSI-low as the presence colon cancers in two independent cohort studies, we have of instability in <30% of the markers, and microsatellite stability (MSS) examined the effect of cyclin D1 expression in colon cancer on as no unstable marker. patient survival. Because we concurrently assessed other related Real-time PCR (MethyLight) to determine CIMP status. Sodium molecular variables including p53, p21, p27, KRAS, BRAF, MSI, bisulfite treatment on tumor DNA and subsequent real-time PCR (MethyLight) assays were validated and done as previously described CIMP, and LINE-1 hypomethylation, we could evaluate the (32). We quantified promoter methylation in 8 CIMP-specific independent effect of cyclin D1 after controlling for these genes (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, potential confounders. In particular, it is important to control RUNX3, and SOCS1; refs. 33–35). CIMP-high was defined as z6/ for the effect of MSI, CIMP, and LINE-1 hypomethylation 8 methylated promoters using the 8-marker CIMP panel, CIMP-low/0 because these molecular characteristics reflect genomic and as 0 to 5 methylated promoters, according to the previously established epigenomic status of cancer cells, and have been related with criteria (34). patient survival in colon cancer (24–27). Pyrosequencing to measure LINE-1 methylation. In order to accu- rately quantify relatively high LINE-1 methylation levels, we used Pyrosequencing as previously described (27). Materials and Methods Immunohistochemistry for cyclin D1, p53, p21, p27, cyclooxygenase-2 and fatty acid synthase. Tissue microarrays were constructed as Study population. We used the databases of two independent previously described (37). Two 0.6-mm tissue cores each from tumor and prospective cohort studies; the Nurses’ Health Study (N = 121,700 normal colonic mucosa were placed in each tissue microarrays block. Methods women followed since 1976) and the Health Professionals Follow-up of immunohistochemical procedures and interpretation were previous- Study (N = 51,500 men followed since 1986; ref. 28). Every 2 years, ly described as follows: cyclin D1 (23), cyclooxygenase (COX)-2 and participants have been sent follow-up questionnaires to update fatty acid synthase (FASN; ref. 28, 31), p21 and p27 (38), and p53 (39). information on potential risk factors and to identify newly diagnosed For cyclin D1, antigen retrieval was done, and deparaffinized tissue cancer and other diseases in themselves and their first-degree relatives. sections in Target Retrieval Solution (Dako) were treated with We calculated body mass index (BMI, kg/m2), using self-reported height microwave in a pressure cooker for 15 min. Tissue sections were from the baseline questionnaire and weight from the biennial incubated with 3% H2O2 (10 min) to block endogenous peroxidase, questionnaire that immediately preceded the diagnosis of colon cancer. and with 10% normal goat serum (Vector Laboratories) in PBS In validation studies in both cohorts, self-reported anthropometric (10 min). Primary antibody against cyclin D1 [rabbit monoclonal measures were well-correlated with measurements by trained techni- (SP4) to cyclin D1, 1:100 dilution; Abcam] was applied, and the slides cians (r > 0.96). On each biennial follow-up questionnaire, participants were maintained overnight at room temperature. This antibody has were asked whether they had a diagnosis of colon cancer during the been shown to be a robust antibody to detect cyclin D1 in paraffin previous 2 years. When a participant (or next of kin for decedents) tissue (40), and its specificity has been confirmed by Western blot (41). reported colon cancer, we sought permission to obtain medical records. In addition, CCND1 gene amplification has been correlated with Study physicians, while blinded to exposure data, reviewed all records protein expression level assessed by this antibody (42). Next, we related to colon cancer, and recorded American Joint Committee on applied an anti-rabbit IgG antibody (Vector Laboratories) for 30 min, Cancer tumor stage and tumor location. For nonresponders, we followed by an avidin-biotin complex conjugate (Vector Laboratories) searched the National Death Index to discover deaths and ascertain for 30 min. The immunochemical reaction was revealed by diamino- any diagnosis of colon cancer that contributed to death or was a benzidine (5 min) and methyl-green counterstain. Nuclear cyclin D1 secondary diagnosis. Approximately 96% of all incident colon cancer expression was recorded as no expression, weak expression, or cases were identified through these methods. We collected paraffin- moderate/strong expression, and the proportion of positive tumor cells embedded tissue blocks from hospitals where colon cancer patients was recorded. Moderate or strong staining for cyclin D1 in any fraction of

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Table 1. Clinical and molecular characteristics according to cyclin D1 status in colon cancer

Clinical or molecular feature All cases Cyclin D1 P Negative Positive Total n 602 272 330 Sex 0.19 Male (HPFS) 261 (43%) 110 (40%) 151 (46%) Female (NHS) 341 (57%) 162 (60%) 179 (54%) Mean age F SD 66.5 F 8.3 66.4 F 8.4 66.6 F 8.3 0.76 BMI 0.68 <30 kg/m2 482 (83%) 217 (82%) 265 (84%) z30 kg/m2 97 (17%) 48 (18%) 49 (16%) Family history of colorectal cancer in any 1st degree relative 0.35 Absent 454 (75%) 210 (77%) 244 (74%) Present 148 (25%) 62 (23%) 86 (26%) Year of diagnosis 0.25 Before 1990 91 (15%) 45 (17%) 46 (14%) 1990-1999 440 (73%) 190 (70%) 250 (76%) 2000-2002 71 (12%) 37 (14%) 34 (10%) Tumor location 0.080 Proximal (cecum to transverse) 344 (58%) 145 (54%) 199 (61%) Distal (splenic flexure to sigmoid) 249 (42%) 123 (46%) 126 (39%) AJCC tumor stage 0.054 I 123 (20%) 48 (18%) 75 (23%) IIA 187 (31%) 78 (29%) 109 (33%) IIB 18 (3.0%) 11 (4.0%) 7 (2.1%) IIIA 21 (3.5%) 8 (2.9%) 13 (3.9%) IIIB 80 (13%) 39 (14%) 41 (12%) IIIC 54 (9.0%) 31 (11%) 23 (7.0%) IV 78 (13%) 43 (16%) 35 (11%) Unknown 41 (6.8%) 14 (5.1%) 27 (8.2%) Tumor grade 0.40 Low 534 (89%) 238 (88%) 296 (90%) High 66 (11%) 33 (12%) 33 (10%) Mean LINE-1 methylation (%) F SD 60.9 F 9.6 59.9 F 9.5 61.8 F 9.6 0.014 MSI <0.0001 MSI-low/MSS 487 (82%) 242 (89%) 245 (75%) MSI-high 109 (18%) 29 (11%) 80 (25%) CIMP 0.0003 CIMP-low/0 488 (81%) 238 (88%) 250 (76%) CIMP-high 114 (19%) 34 (12%) 80 (24%) BRAF mutation 0.006 (-) 489 (84%) 234 (88%) 255 (80%) (+) 95 (16%) 31 (12%) 64 (20%) KRAS mutation 0.98 (-) 377 (63%) 171 (63%) 206 (63%) (+) 221 (37%) 100 (37%) 121 (37%) p53 expression 0.38 (-) 366 (61%) 160 (59%) 206 (63%) (+) 232 (39%) 110 (41%) 122 (37%) p21 <0.0001 Expressed 125 (21%) 32 (12%) 93 (29%) Lost 460 (79%) 229 (88%) 231 (71%) p27 0.023 Nuclear expression 117 (20%) 42 (16%) 75 (24%) Cytoplasmic expression or loss of nuclear expression 458 (80%) 218 (84%) 240 (76%) COX-2 0.80 (-) 100 (17%) 44 (16%) 56 (17%) (+) 500 (83%) 227 (84%) 273 (83%) Fatty acid synthase (FASN) 0.022 (-) 511 (86%) 240 (90%) 271 (83%) (+) 81 (14%) 27 (10%) 54 (17%)

NOTE: % indicates the proportion of tumors with a specific clinical or molecular feature in cyclin D1- (or cyclin D1+) tumors. Abbreviations: AJCC, American Joint Commission on Cancer; LINE-1, long interspersed nucleotide element-1.

tumor cells was interpreted as positive. Appropriate positive and negative sample of 160 tumors were reexamined by a second observer (K.S.) controls were included in each run of immunohistochemistry. All unaware of other data. The concordance between the two observers was immunohistochemically-stained slides for cyclin D1 were interpreted 0.83 (n = 0.64; P < 0.0001), indicating substantial agreement. For the by one of the investigators (K.N.) unaware of other data. A random other markers, a random selection of 108 to 246 cases was reexamined

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KRAS, BRAF, p53, p21, p27, COX-2, and FASN. In addition, we also did unconditional Cox regression analysis to assess the unadjusted, main effect of cyclin D1 expression on mortality. For analyses of colon cancer–specific mortality, death as a result of colon cancer was the primary end point and deaths as a result of other causes were censored. To adjust for potential confounding, age, year of diagnosis, and LINE-1 methylation were used as continuous variables, and all of the other covariates were used as categorical variables. We dichotomized family history (present versus absent), BMI (<30 kg/m2 versus z30 kg/m2), tumor location (proximal versus distal), tumor grade (high versus low), CIMP (high versus low/0), MSI (high versus low/MSS), p53, p21, p27, COX-2, FASN, KRAS, and BRAF. For cases with missing tumor location (1.5% missing) or p27 (4.5% missing), we assigned a separate (‘‘missing’’) indicator variable and included those cases in the multivariate analysis models. For missing information in other covariates [including BMI (3.8% missing), tumor grade (0.3% missing), MSI (1.0% missing), KRAS (0.7% missing), BRAF (3.2% missing), p53 (0.7% missing), p21 (2.8% missing), COX-2 (0.3% missing), and FASN (1.7% missing)], we included those cases in a majority category in the particular missing variable, to minimize the number of ‘‘missing’’ indicator variables and maximize the efficiency of multivariate Cox regression analyses. We confirmed that excluding cases with missing information in any of the covariates did not substantially alter results (data not shown). An interaction was assessed by including the cross product of the cyclin D1 variable and another variable of interest in a multivariate Cox model, and the likelihood ratio test was done. Considering multiple hypotheses testing, P values for interactions were conservatively interpreted and higher level of significance (i.e., P < 0.01) was considered to be statistically significant. To assess an interaction of cyclin D1 and stage, we treated stage as a binary variable (I-II versus III-IV) as well as an ordinal categorical variable (I-IV). To confirm the relation between cyclin D1 expression and survival, we computed HR according to the proportion of tumor cells positive for cyclin D1 (as a continuous variable), nonparametrically with restricted cubic splines (43), which was independent of predetermined Fig. 1. Kaplan-Meier curves for colon cancer ^ specific survival (top)andoverall categorization of cyclin D1 status. survival (bottom) according to cyclin D1status in colon cancer. The Kaplan-Meier method was used to describe the distribution of colon cancer-specific and overall survival time, and the log-rank test was m2 for each marker by a second pathologist (p53 and FASN by K.N.; p21 done. The test was used to examine an association of cyclin D1 with and p27 by K.S.; COX-2 by R. Dehari, Kanagawa Cancer Center, Japan) any of the categorical variables. The t test assuming unequal variances unaware of other data, and concordance rates and n coefficients was done to compare mean age and mean LINE-1 methylation level. between the two pathologists were as follows: 0.87 (n = 0.75; n = 118) All analyses used SAS version 9.1 (SAS Institute) and all P values were for p53; 0.93 (n = 0.57; n = 246) for FASN; 0.83 (n = 0.62; n = 179) two-sided. for p21; 0.94 (n = 0.60; n = 114) for p27; 0.92 (n = 0.62; n = 108) for COX-2, indicating good to substantial agreement. Statistical analysis. We used stage-matched, conditional Cox Results proportional hazard models to calculate hazard ratios (HR) of death according to tumoral cyclin D1 status, adjusted for age, sex, year of Cyclin D1 expression in colon cancer and patient survival. diagnosis, BMI, family history of colorectal cancer in any first-degree Among 602 patients with stage I to IV colon cancer, cyclin D1 relative, tumor location, stage, grade, and status of MSI, CIMP, LINE-1, overexpression was observed in 330 (55%) tumors by

Table 2. Cyclin D1 expression in colon cancer and patient mortality

Total n Colon cancer – specific mortality Overall mortality Deaths/ Univariate Stage- Multivariate Deaths/ Univariate Stage- Multivariate person- HR matched HR HR person- HR matched HR HR years (95% CI) (95% CI) (95% CI) years (95% CI) (95% CI) (95% CI) Cyclin D1 (-) 272 83/2080 1 (Reference) 1 (Reference) 1 (Reference) 124/2080 1 (Reference) 1 (Reference) 1 (Reference) (45%) Cyclin D1 (+) 330 70/2855 0.64 0.66 0.57 135/2855 0.81 0.80 0.74 (55%) (0.47-0.88) (0.46-0.93) (0.39-0.84) (0.63-1.03) (0.61-1.04) (0.57-0.98) P 0.0063 0.018 0.0048 0.084 0.090 0.036

NOTE: The multivariate, stage-matched (stratified) conditional Cox regression model included age, year of diagnosis, sex, family history of colorectal cancer, BMI, tumor location, grade, KRAS, BRAF, p53, p21, p27, COX-2, FASN, LINE-1 methylation, MSI, and CIMP.

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Fig. 2. Smoothing spline plot of unadjusted HRs for colon cancer ^ specific (left)and overall mortality (right) according to the proportion of tumor cells positive for cyclin D1(with0%asareference).Hatched lines, 95% CI.

immunohistochemistry (Supplementary Figure). We assessed negative tumors (83% versus 73%, long rank P = 0.006; Fig. 1). clinical and molecular characteristics of colon cancers, accord- In univariate Cox regression analysis, compared with patients ing to tumoral cyclin D1 status (Table 1). Compared with with cyclin D1–negative tumors, those with cyclin D1–positive cyclin D1–negative tumors, cyclin D1–positive tumors were tumors experienced a significantly lower cancer-specific more likely to show MSI (MSI-high, P < 0.0001), CIMP (CIMP- mortality [HR, 0.64; 95% confidence interval (CI), 0.47-0.88; high, P = 0.0003), BRAF mutation (P = 0.006), p21 expression P = 0.0063; Table 2]. In the multivariate Cox model adjusting (P < 0.0001), p27 nuclear expression (P = 0.023), and FASN for potential predictors of patient outcome, cyclin D1 was expression (P = 0.022). associated with a significantly lower colon cancer–specific During follow-up, there were 259 deaths, including 153 colon mortality (adjusted HR, 0.57; 95% CI, 0.39-0.84; P = 0.0048) cancer–specific deaths. We assessed the influence of cyclin D1 and overall mortality (adjusted HR, 0.74; 95% CI, 0.57-0.98; on patient survival. In Kaplan-Meier analysis, 5-year colon 0.036). No major confounder was present. cancer–specific survival probability among patients with cyclin To confirm the relation between cyclin D1 expression and D1–positive tumors was higher than patients with cyclin D1– survival, we also visually estimated the proportion of tumor

Table 3. Combined MSI/cyclin D1 category and patient mortality in colon cancer

Combined Total n Colon cancer – specific Overall MSI/cyclin mortality mortality D1 category Deaths/ Univariate Stage- Multivariate Deaths/ Univariate Stage- Multivariate person- HR matched HR HR person- HR matched HR HR years (95% CI) (95% CI) (95% CI) years (95% CI) (95% CI) (95% CI) MSS/MSI-low Cyclin D1 (-) 242 82/1769 1 (Reference) 1 (Reference) 1 (Reference) 118/1769 1 (Reference) 1 (Reference) 1 (Reference) Cyclin D1 (+) 245 57/2199 0.61 0.60 0.52 101/2199 0.71 0.69 0.65 (0.43-0.85) (0.42-0.87) (0.35-0.78) (0.54-0.92) (0.52-0.91) (0.48-0.87) MSI-high

Cyclin D1 (-) 29 1/303 0.080 0.14 0.10 6/303 0.31 0.42 0.29 (0.011-0.58) (0.02-1.04) (0.01-0.78) (0.13-0.70) (0.18-0.97) (0.12-0.70) Cyclin D1 (+) 80 13/591 0.45 0.67 0.40 34/591 0.86 1.09 0.65 (0.25-0.80) (0.36-1.25) (0.18-0.86) (0.58-1.26) (0.73-1.63) (0.39-1.10)

NOTE: The multivariate, stage-matched (stratified) Cox model included the combined MSI/cyclin D1 category, age, year of diagnosis, sex, family history of colorectal cancer, BMI, tumor location, grade, KRAS, BRAF, p53, p21, p27, COX-2, FASN, LINE-1 methylation, and CIMP.

Pinteraction (cyclin D1 and MSI) = 0.061 for colon – cancer specific mortality; Pinteraction (cyclin D1 and MSI) = 0.008 for overall mortality.

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Fig. 3. Stratified analysis of cyclin D1 status and colon cancer mortality. Loge(adjusted HRs) with 95% CI for colon cancer ^ specific mortality in cyclin D1+ tumors (versus cyclin D1^ negative tumors) in various strata are shown. A Pinteraction value indicates statistical significance of an interaction between cyclin D1and a given variable in a multivariate Cox model. *,‘‘p27 abnormal’’ is defined as cytoplasmic localization or loss of nuclear expression. HPFS, Health Professionals Follow-up Study; NHS, Nurses’ Health Study.

cells positive for cyclin D1 (although not primarily used status (Table 3). Compared with tumors that were both MSS/ for categorization of cyclin D1 status). Then, we computed MSI-low and cyclin D1-negative, tumors that were either MSI- HR according to the proportion of positive cells as a high or cyclin D1-positive seemed to be associated with lower continuous variable, nonparametrically with restricted cubic cancer-specific mortalities (adjusted HR point estimates, 0.10- splines (Fig. 2; ref. 43). This method allowed us to assess the 0.52) and overall moralities (adjusted HR point estimates, relationship between cyclin D1 expression and survival, 0.29-0.65). besides any predetermined categorization of cyclin D1 status. Stratified analysis of cyclin D1 and mortality. We further It was evident that, as the proportion of positive cells examined the influence of cyclin D1 overexpression on colon increased, the HR for colon cancer-specific and overall cancer–specific mortality across strata of other potential mortality decreased. predictors of patient survival (Fig. 3). Considering multiple Effect of MSI on the relation between cyclin D1 and hypotheses testing, there was no evidence for significant effect mortality. We found a modifying effect of MSI on the modification by any of the variables (all Pinteraction z 0.03). relation between cyclin D1 and patient mortality (Pinteraction Notably, the effect of cyclin D1 did not significantly differ = 0.061 for colon cancer-specific mortality and Pinteraction = between the two independent cohort studies (Pinteraction = 0.008 for overall mortality). This was not unexpected, 0.19). The effect of cyclin D1 might differ according to stage (I-II considering a potential pathogenetic link between cyclin D1 versus III-IV; Pinteraction = 0.030; ordinal scale from I-IV; Pinteraction expression and MSI in colon cancer (23). Thus, we stratified = 0.032); however, considering multiple hypothesis testing, this could patients into four categories based on MSI and cyclin D1 be a chance event.

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Discussion In addition, we found a potential modifying effect of MSI on the relation between cyclin D1 and clinical outcome in colon We conducted this study to examine the relation between cancer. Specifically, compared with tumors that were both MSI- cyclin D1 expression and patient survival in stage I to IV colon low/MSS (microsatellite stable) and cyclin D1–negative, cancer. Cyclin D1 (CCND1, the official gene symbol) activation tumors with either cyclin D1–positive or MSI-high seemed to has been implicated in colon carcinogenesis (1, 21, 22), among be associated with lower mortalities. We have previously shown many other molecular changes. We found that cyclin D1 that cyclin D1 expression is associated with MSI, independent overexpression was associated with longer survival, indepen- of CIMP status (23). Although the mechanistic link between dent of patient characteristics and other related molecular MSI and cyclin D1 has not been elucidated, it is conceivable variables including p53, p21, p27, KRAS, BRAF,LINE-1 that cyclin D1 may exert different effects on tumor behavior methylation, MSI, and the CIMP. All of these characteristics according to tumoral MSI status. are potential confounders in analysis of tumoral cyclin D1 There are advantages in using the database of the two status and patient survival. Our results indicate that cyclin D1 independent prospective cohort studies, the Nurses’ Health expression in colon cancer is associated with superior Study and Health Professionals Follow-up Study. Exposure prognosis. and other clinical information were prospectively collected, and It is very common to hypothesize that oncogene activation entered into the database blinded to patient and tumoral (or tumor suppressor inactivation) is associated with aggressive features, and outcome. Data were updated every 2 years. Cohort tumor behavior. However, this hypothesis does not always hold participants who developed colon cancer were treated at true. This is well exemplified by the association between MSI hospitals throughout the United States. Tumor specimen and good prognosis (24). MSI is known to cause inactivation of procurement rate has been f60%, and there were no a number of tumor suppressors (including TGFBR2, BAX, and significant demographic difference between cases with tumor many others); yet MSI is associated with better patient tissue analyzed and those without tumor tissue analyzed (28). outcome. This probably reflects a fundamental molecular However, a limitation of this study is that data on cancer difference between MSI-high tumors and non–MSI-high treatment were limited. Nonetheless, it is unlikely that tumors. Our current findings also imply a molecular difference chemotherapy use differed according to tumoral cyclin D1 between cyclin D1–positive and cyclin D1–negative tumors. It status because such data were not available to patients or is well known that colon cancers develop through accumula- treating physicians. In addition, beyond cause of mortality, data tion of multiple genetic and epigenetic events. Some tumors on cancer recurrences were not available in these cohorts. activate cyclin D1, whereas others do not. In order to acquire Nonetheless, given the median survival for metastatic colon malignant characteristics, cyclin D1–negative cancers might cancer was f10 to 12 months during much of the time period have bypassed the necessity of cyclin D1 activation, which of this study (27), colon cancer–specific survival should be a might cause more aggressive behavior than cyclin D1–activated reasonable surrogate for cancer-specific outcomes. cancers. Thus, an aberration of a given oncoprotein such as There are currently no standardized methods to assess cyclin cyclin D1 or tumor suppressor can be associated with indolent D1 in colon cancer. Nonuniformity in methods to evaluate behavior. tumoral cyclin D1 expression may contribute to the inconsis- Examining molecular changes and prognostic factors is tent results in the previous studies. Nonetheless, our method important in colon cancer research (44–51). Previous studies yielded highly significant associations between cyclin D1 have examined the relationship between tumoral cyclin D1 expression and other related molecular variables (including expression and clinical outcome in colon cancer (4–20). MSI, CIMP, BRAF, and p21; see Table 1). Moreover, any However, those studies have yielded inconsistent results. random misclassification of tumors in terms of cyclin D1 Although two studies (4, 5) have shown that cyclin D1 expression would drive our results on patient outcome toward expression has been associated with poor prognosis, most the null hypothesis. studies have shown no independent prognostic value of cyclin In summary, our large cohort study suggests that cyclin D1 D1 (7–20), and one study has shown good prognosis expression is independently associated with good prognosis in associated with cyclin D1 expression (6). A study has reported colon cancer. Our findings may have considerable clinical associations of cyclin D1 expression with MSI, CIMP, and BRAF implications. Future studies are needed to confirm this mutation (23), and CIMP, MSI, and BRAF mutation in colon association as well as to elucidate exact mechanisms by which cancer have been related with clinical outcome (25, 26, 44). cyclin D1 affects tumor behavior. Thus, CIMP, MSI, and BRAF are potential confounders in analysis of cyclin D1 and clinical outcome. However, none of Disclosure of Potential Conflicts of Interest the previous studies (4–20) have examined confounding or modifying effect of these tumoral molecular events. Moreover, No potential conflicts of interest were disclosed. most previous studies were limited by small sample sizes (n < 170), and only 3 studies (11, 15, 16) examined >170 cases Acknowledgments (up to n = 363; ref. 16). In the current study, we concurrently examined a number of molecular events, which have been We thank the Nurses’ Health Study and Health Professionals Follow-up related with both tumoral cyclin D1 expression and patient Study cohort participants who have generously agreed to provide us with biological prognosis. In addition, our study had adequate statistical power specimens and information through responses to questionnaires; hospitals and pathology departments throughout the United States for providing us with tumor with a large number (n = 602) of stage I to IV colon cancers, tissue materials; Frank Speizer, Walter Willett, Susan Hankinson, Graham Colditz, and our results have been consistent across the two indepen- Meir Stampfer, and many other staff members who implemented and have dent cohort studies. maintained the cohort studies.

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References 1. Alao J. The regulation of cyclin D1degradation: roles D1 and Rb protein expression and their correlation large population-based sample. J Mol Diagn 2007;9: in cancer development and the potential for therapeu- with prognosis in patients with colon cancer. World 305^14. tic invention. Mol Cancer 2007;6:24. J Surg Oncol 2006;4:5. 35.Weisenberger DJ, Siegmund KD, Campan M, et al. CpG 2. Arber N, Hibshoosh H, Moss SF, et al. Increased 18. Lyall MS, Dundas SR, Curran S, Murray GI. Profiling island methylator phenotype underlies sporadic microsa- expression of cyclin D1 is an early event in multi- markers of prognosis in colorectal cancer. Clin Cancer tellite instability and is tightly associated with BRAF muta- stage colorectal carcinogenesis. Gastroenterology Res 2006;12:1184 ^ 91. tion in colorectal cancer. Nat Genet 2006;38:787 ^ 93. 1996;110:669^74. 19. Schmitz KJ,WohlschlaegerJ, Alakus H, et al. Activa- 36. Ogino S, KawasakiT,Nosho K, et al. LINE-Ihypome- 3. Palmqvist R, Stenling R, Oberg A, Landberg G. tion of extracellular regulated kinases (ERK1/2) but thylation is inversely associated with microsatellite in- Expression of cyclin D1 and retinoblastoma pro- not AKT predicts poor prognosis in colorectal carci- stability and CPG island methylator phenotype in tein in colorectal cancer. Eur J Cancer 1998;34: noma and is associated with k-ras mutations. Virch- colorectal cancer. Int JCancer 2008;122:2767 ^ 73. 1575 ^ 81. ows Arch 2007;450:151 ^ 9. 37. Ogino S, Brahmandam M, Kawasaki T, Kirkner GJ, 4. MaedaK,ChungYS,KangSM,etal.Overexpression 20. TheocharisS,GiaginisC,ParasiA,etal.Expression Loda M, Fuchs CS. Combined analysis of COX-2 and of cyclin D1and p53 associated with disease recur- of peroxisome proliferator-activated receptor-g in co- p53 expressions reveals synergistic inverse correla- rence in colorectal adenocarcinoma. Int J Cancer lon cancer: correlation with histopathological parame- tions with microsatellite instability and CpG island 19 97;74 :310 ^ 5. ters, cell cycle-related molecules, and patients’ methylator phenotype in colorectal cancer. Neoplasia 5. Bahnassy AA, Zekri AR, El-Houssini S, et al. Cyclin survival.DigDisSci2007;52:2305^11. 2006;8:458 ^ 64. A and cyclin D1 as significant prognostic markers in 21. Shtutman M, ZhurinskyJ, Simcha I, et al. The cyclin 38. Ogino S, Meyerhardt JA, Cantor M, et al. Molecular colorectal cancer patients. BMCGastroenterol D1gene is a target of the h-catenin/LEF-1pathway. alterations in tumors and response to combination 2004;4:22. Proc Natl Acad Sci U S A1999;96:5522 ^ 7. chemotherapy with gefitinib for advanced colorectal 6. Holland TA, Elder J, McCloud JM, et al. Subcellular 22. Tetsu O, McCormick F. Beta-catenin regulates ex- cancer. Clin Cancer Res 2005;11:6650 ^ 6. localisation of cyclin D1protein in colorectal tumours pression of cyclin D1in colon carcinoma cells. Nature 39. Ogino S, Kawasaki T, Kirkner GJ, Yamaji T, Loda M, is associated with p21(WAF1/CIP1) expression and 1999;398:422^6. Fuchs CS. Loss of nuclear p27 (CDKN1B/KIP1) in co- correlates with patient survival. Int J Cancer 2001;95: 23. Nosho K, Kawasaki T, Chan AT, et al. Cyclin D1is lorectal cancer is correlated with microsatellite insta- 302^6. frequently overexpressed in microsatellite unstable bility and CIMP. Mod Pathol 2007;20:15^ 22. 7. Handa K,Yamakawa M,Takeda H, Kimura S,Takahashi colorectal cancer, independent of CpG island methyl- 40. Cheuk W,Wong KO,Wong CS, Chan JK. Consistent T. Expression of cell cycle markers in colorectal carci- ator phenotype. Histopathology 2008;53:588 ^ 98. immunostaining for cyclin D1 can be achieved on a noma: superiority of cyclin A as an indicator of poor 24. Popat S, Hubner R, Houlston RS. Systematic Re- routine basis using a newly available rabbit monoclo- prognosis. Int J Cancer 1999;84:225 ^ 33. view of Microsatellite Instability and Colorectal Can- nal antibody. Am J Surg Pathol 2004;28:801 ^ 7. 8. Bukholm IK, Nesland JM. Protein expression of p53, cer Prognosis. J Clin Oncol 2005;23:609 ^ 18. 41. Fleming IN, Hogben M, Frame S, McClue SJ, Green p21 (WAF1/CIP1), bcl-2, Bax, cyclin D1and pRb in hu- 25. SamowitzWS, Sweeney C, HerrickJ, et al. Poor sur- SR. Synergistic inhibition of ErbB signaling by com- man colon carcinomas. Virchows Arch 2000;436: vival associated with the BRAF V600E mutation in bined treatment with seliciclib and ErbB-targeting 224 ^ 8. microsatellite-stable colon cancers. Cancer Res agents. Clin Cancer Res 2008;14:4326^35. 9. Bhatavdekar JM, Patel DD, Chikhlikar PR, et al. 2005;65:6063^9. 42. Gibcus JH, Mastik MF, Menkema L, et al. Cortactin Molecular markers are predictors of recurrence 26. Ogino S, Nosho K, Kirkner GJ, et al. CpG island expression predicts poor survival in laryngeal carcino- and survival in patients with Dukes B, Dukes C. methylator phenotype, microsatellite instability, BRAF ma. Br J Cancer 2008;98:950 ^ 5. colorectal adenocarcinoma. Dis Colon Rectum mutation and clinical outcome in colon cancer. Gut 43. Durrleman S, Simon R. Flexible regression models 2001;44:523^ 33. 2009;58:90 ^ 6. with cubic splines. Stat Med 1989;8:551 ^ 61. 10. Pasz-Walczak G, Kordek R, Faflik M. P21 (WAF1) 27. Ogino S, Nosho K, Kirkner GJ, et al. A cohort study 44. Ogino S, Goel A. Molecular classification and corre- expression in colorectal cancer: correlation with of tumoral LINE-1hypomethylation and prognosis in lates in colorectal cancer. JMol Diagn 2008;10:13^27. P53 and cyclin D1 expression, clinicopathological colon cancer. J Natl Cancer Inst 2008;100:1734 ^ 8. 45. Zlobec I,Terracciano LM, Lugli A. Local recurrence in parameters and prognosis. Pathol Res Pract 2001; 28. Chan AT, Ogino S, Fuchs CS. Aspirin and the risk of mismatch repair-proficient colon cancer predicted by an 197:683 ^ 9. colorectal cancer in relation to the expression of infiltrative tumor border and lack of CD8+ tumor-infil- 11. McKay JA, Douglas JJ, Ross VG, et al. Analysis of COX-2. N Engl J Med 2007;356:2131^ 42. tratinglymphocytes. Clin Cancer Res 2008;14:3792^7. key cell-cycle checkpoint proteins in colorectal 29. Ogino S, Kawasaki T, Brahmandam M, et al. Sensi- 46. Morris M, Platell C, Iacopetta B. Tumor-infiltrating tumours. J Pathol 2002;196:386 ^ 93. tive sequencing method for KRAS mutation detection lymphocytes and perforation in colon cancer predict 12. Cheah PY, Choo PH,Yao J, Eu KW, Seow-Choen F. by Pyrosequencing. J Mol Diagn 2005;7:413^ 21. positive response to 5-fluorouracil chemotherapy. A survival-stratification model of human colorectal 30. Ogino S, Kawasaki T, Kirkner GJ, Loda M, Fuchs Clin Cancer Res 2008;14:1413^ 7. carcinomas with h-catenin and p27kip1. Cancer CS. CpG island methylator phenotype-low (CIMP- 47. Zlobec I, Baker K,Terracciano LM, Lugli A. RHAMM, 2002;95:2479 ^ 86. low) in colorectal cancer: possible associations with p21 combined phenotype identifies microsatellite in- 13. Bondi J, Bukholm G, Nesland JM, Bukholm IR. male sex and KRAS mutations. J Mol Diagn 2006;8: stability-high colorectal cancers with a highly adverse Expression of non-membranous h-catenin and g- 582^8. prognosis. Clin Cancer Res 2008;14:3798 ^ 806. catenin, c-Myc and cyclin D1 in relation to patient 31. Ogino S, Brahmandam M, Cantor M, et al. Distinct 48. Suehiro Y,Wong CW, Chirieac LR, et al. Epigenetic- outcome in human colon adenocarcinomas. APMIS molecular features of colorectal carcinoma with signet genetic interactions in the APC/WNT, RAS/RAF, P53 2004;112:49 ^ 56. ring cell component and colorectal carcinoma with pathways in colorectal carcinoma. Clin Cancer Res 14. Bondi J, Husdal A, Bukholm G, Nesland JM, Bakka mucinous component. Mod Pathol 2006;19:59 ^ 68. 2008;14:2560 ^ 9. A, Bukholm IR. Expression and gene amplification of 32. Ogino S, Kawasaki T, Brahmandam M, et al. Preci- 49. French AJ, Sargent DJ, Burgart LJ, et al. Prognostic primary (A, B1, D1, D3, and E) and secondary (Cand sion and performance characteristics of bisulfite con- significance of defective mismatch repair and BRAF H) cyclins in colon adenocarcinomas and correlation version and real-time PCR (MethyLight) for V600E in patients with colon cancer. Clin Cancer Res with patient outcome. JClin Pathol 2005;58:509 ^ 14. quantitative DNA methylation analysis. J Mol Diagn 2008;14:3408^15. 15. Knosel T, Emde A, Schluns K, et al. Immunoprofiles 2006;8:209 ^ 17. 50. ChengYW, Pincas H, Bacolod MD, et al. CpGisland of 11biomarkers using tissue microarrays identify 33. Ogino S, Cantor M, Kawasaki T, et al. CpG island methylator phenotype associates with low-degree prognostic subgroups in colorectal cancer. Neoplasia methylator phenotype (CIMP) of colorectal cancer is chromosomal abnormalities in colorectal cancer. 2005;7:741 ^ 7. best characterised by quantitative DNA methylation Clin Cancer Res 2008;14:6005 ^ 13. 16. Hilska M, CollanYU,VJ OL, et al.The significance of analysis and prospective cohort studies. Gut 2006; 51. Weichert W, Roske A, Niesporek S, et al. Class I his- tumor markers for proliferation and apoptosis in pre- 55:1000 ^ 6. tone deacetylase expression has independent prog- dicting survival in colorectal cancer. Dis Colon Rectum 34. Ogino S, Kawasaki T, Kirkner GJ, Kraft P, Loda M, nostic impact in human colorectal cancer: specific 2005;48:2197 ^ 208. Fuchs CS. Evaluation of markers for CpG island meth- role of class I histone deacetylases in vitro and in vivo. 17. KouraklisG,TheocharisS,VamvakasP,etal.Cyclin ylator phenotype (CIMP) in colorectal cancer by a Clin Cancer Res 2008;14:1669 ^ 77.

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Shuji Ogino, Katsuhiko Nosho, Natsumi Irahara, et al.

Clin Cancer Res 2009;15:4431-4438.

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