BMJ Open. A Nationwide Danish Cohort Study challenging the Categorization into Right Sided and Left Sided Colon Cancer

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1 A Nationwide Danish Cohort Study challenging the Categorization into Right Sided and Left Sided Colon Cancer Journal: BMJ Open Manuscript ID: bmjopen Article Type: Research Date Submitted by the Author: -Jan-0 Complete List of Authors: Jess, Per; Roskilde Hospital, Department of Surgery; University of Copenhagen, Faculty of Health Sciences Hansen, Iben; University of Copenhagen, Faculty of Health Sciences Gamborg, Michael; Copenhagen University Hospital, Institute of Preventive Medicine Jess, Tine; Statens Serum Institut, Department of Epidemiology Research <b>primary Subject Heading</b>: Oncology Secondary Subject Heading: Oncology, Gastroenterology and hepatology, Surgery Keywords: Gastrointestinal tumours < GASTROENTEROLOGY, Colorectal surgery < SURGERY, EPIDEMIOLOGY BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

2 Page of BMJ Open ORIGINAL ARTICLE A Nationwide Danish Cohort Study challenging the Categorization into Right Sided and Left Sided Colon Cancer Per Jess,, Iben Onsberg Hansen, Michael Gamborg, Tine Jess. On behalf of the Danish Colorectal Cancer Group. Department of Surgery, Roskilde Hospital, Roskilde, Denmark. Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. Institute of Preventive Medicine, Copenhagen University Hospital, Copenhagen, Denmark. Department of Epidemiology Research, Statens Serum Institut, National Health Surveillance and Research, Copenhagen, Denmark Correspondance: Per Jess, M.D., Dr.Med.Sci. Department of Surgery, Roskilde Hospital, DK-000 Roskilde, Denmark; address: pjss@regionsjaelland.dk ; Telephone: +0; Fax: + 00 Keywords: Colon neoplasia; Colon subsites; Epidemiology Word count: 0 BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

3 Page of Author Contributions: The study was conceived and designed by Per Jess, Iben Onsberg Hansen, Michael Gamborg, and Tine Jess. Statistical analyses were performed by Michael Gamborg. All authors were involved in the interpretation of data. Per Jess drafted the manuscript, which was critically revised by all authors, who also approved the final version of the manuscript. Conflicts of interest: The authors have no conflicts to disclose Funding: Not relevant Trial registration: Not relevant No additional data available. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

4 Page of BMJ Open Article Summary Article Focus. Colon cancer is normally classified into right sided and left sided colon cancer.. It has been suggested that there are differences in epidemiology, pathology and prognosis between patients with cancer in the right and left side of the colon due to different embryologic development of the two segments of the colon which may result in different molecular biological patterns of the tumors.. Recent studies have challenged this simple dichotomization of colon cancer into right sided and left sided colon cancer. Key Messages. Our nationwide cohort study of, colon cancer patients confirms that patients with right sided colon cancer are older, more often females, and have more advanced tumors and a worse prognosis than patients with left sided cancer.. However, more detailed colon subsite analysis reveals a much more complex picture. Highest ASA scores are observed in patients with cancer of the transverse and descending colon and of both colon flexures. The most advanced colon cancers are those of the descending colon, splenic flexure, and cecum. The highest mortality is seen in patients with splenic flexure cancer. Strengths and Limitations. The strength of the present study is the population-based patient cohort with nearly complete (-%) inclusion and follow-up of all Danish patients diagnosed with colon cancer during the last years.. The study has potential limitations: Despite detailed information on patients, data on cancer recurrence has not been collected. Hence, we were only able to compare overall survival rather than cancer free survival. However, this would not influence the already BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

5 Page of observed marked differences in clinical characteristics and survival by specific colon cancer subsites. Further, we did not have genetic or biomarker information that could have served to further qualify the observations of these differences, and this merits further study. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

6 Page of BMJ Open ABSTRACT Objectives: The categorization of colon cancer (CC) into right sided (RCC) and left sided (LCC) disease may not capture more subtle variances in etiology and prognosis. In a nationwide study, we investigated differences in clinical characteristics and survival of RCC versus LCC and of the complete range of CC subsites. Design: Prospective nationwide cohort study. Setting: The database of the Danish Colorectal Cancer Group (DCCG). Participants:, CC patients. Outcome measures: Clinical data on age, sex, ASA score (the American Society of Anaesthesiologists score), tumor location and stage, number of lymph nodes harvested at operation, number of lymph nodes with metastases, and presence of distant metastases. as well as survival data using hazard ratios (HRs) for mortality according to CC localization using Cox proportional hazard models with adjustment for possible confounders. Results: Patients with RCC had a higher median age at diagnosis (. years) than patients with LCC (. years) (p<0.000) and the proportion of women increased the more proximal the tumor, except for hepatic flexure cancers. Although, RCC patients had higher ASA scores than LCC patients (p<0.000), highest ASA scores were observed in patients with cancer in the transverse and descending colon and at both colon flexures. While RCCs overall were more advanced than LCCs (p<0.000), the most advanced CCs were those of the descending colon, splenic flexure, and cecum. RCC mortality was higher than LCC mortality only during the first two years (women: HR,.; % CI,.0-.0; men: HR,.; % CI,.0-.), and relative to mortality from sigmoid colon cancer, the highest mortality was observed from splenic flexure cancer (HR,.; % CI,.-.00). Conclusions: The present data challenge the simple categorization of CC into RCC and LCC. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

7 Page of INTRODUCTION Colorectal cancer is one of the most commonly diagnosed cancers worldwide with over. million new cancer cases and 0,00 deaths estimated to occur annually []. In 0, Bufill was the first to propose that colon cancer (CC) located to the right (RCC) and left (LCC) side of colon, respectively, may rise from different biological pathways []. Subsequently, it has been suggested, that being two different disease entities, RCC and LLC also differ in terms of patient characteristics, pathology, and prognosis []. A recent systematic review of the sparse literature on the topic supports that such differences exist []. The reason for this is uncertain, but a suggested explanation is the different embryologic development of the two segments of the colon resulting in different molecular biological tumor patterns [,]. However, recent studies have challenged the simple dichotomization of CC into RCC and LCC, since factors as tumor stage [], genetic alterations [] and molecular features [] vary markedly between colonic subsites irrespective of their localization in the right or left site of colon. Such possible differences might have consequences for planning of screening and for the treatment of patients with CC. The aim of the present investigation was to examine differences in patient characteristics, cancer pathology, and survival according to RCC vs. LCC localization of CC and according to more specific colon subsite localization, using unique prospectively collected nationwide data from years BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

8 Page of BMJ Open MATERIALS AND METHODS On May, 00 the Danish Colorectal Cancer Group (DCCG) established a nationwide database [], and since then all patients with a first-time diagnosis of colorectal adenocarcinoma from all Danish surgical departments treating such patients have been prospectively registered in the database. Patients are identified by their Danish Civil Registration Number, a unique -digit person number given to all Danish citizens at birth and ascribed to every Danish person since []. The data reported to DCCG from the surgical departments in Denmark include demographics, clinical manifestation, tumor location and stage (UICC; Union for International Cancer Control), diagnostic and therapeutic procedures, and postoperative complications. Data on vital status are updated monthly through linkage to the Danish Civil Registration System []. The completeness of data collection in DCCG is estimated annually and has increased from to % in the period 00-0 []. The present study covers all Danish patients (n=,) in DCCG with a diagnosis of CC from January, 00 trough December, 0. From DCCG, we specifically obtained data on age, sex, ASA score (the American Society of Anaesthesiologists score), tumor location and stage, number of lymph nodes harvested at operation, number of lymph nodes with metastases, and presence of distant metastases. Localization of CC was treated both according to specific sub-sites and according to the suggested categorization into RCC (n=,; caecum, ascending colon, hepatic flexure, or transverse colon) and LCC (n=,; from the splenic flexure to the sigmoid colon, both BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

9 Page of included; the border between the sigmoid colon and rectum defined as cm from the anal verge). Statistical analyses Non-parametric statistics were used for description of the patient material. P-values <0.0 were considered statistically significant. Patients were followed from date of colon cancer diagnosis to emigration, death, or end of the study (October, 0) whichever occurred first. Overall survival among patients with RCC vs. LCC was illustrated by Kaplan-Meier plots for, respectively, women and men and were compared by log-rank test. Mortality from RCC relative to mortality from LCC was estimated by hazard ratios (HRs) obtained by performing Cox proportional hazard regression analysis with time since diagnosis as the underling time axis, stratified on gender and adjusted for age. Next, the impact of ASA score, number of lymph nodes harvested at operation, number of lymph nodes with metastases, presence of distant metastases, and UICC stage on the relative difference in mortality from RCC and LCC was investigated by performing adjusted analyses. Finally, analyses were performed comparing the survival among patients with cancer of, respectively, caecum, ascending colon, hepatic flexure, transverse colon, splenic flexure, and descending colon, with the survival among patients with sigmoid colon. All analyses were performed using STATA version.. Ethics The Danish National Committee on Biomedical Research Ethics and the Danish Data Protection Agency have approved the use of the DCCG database for scientific purposes. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

10 Page of BMJ Open RESULTS Age, gender and ASA score The median age at diagnosis of RCC was. years (range.-. years) versus. years (range.-0.) for LCC (p<0.000) (Table ). Table Demographic, clinical and pathological characteristics among, patients with right sided colon cancer (RCC) versus left sided colon cancer (LCC), Denmark 00-0 RCC LCC p-value N=, N=, Age (median years),, <0.000 Sex (% women)..0 <0.000 ASA score (% score >) 0.. <0.000 UICC stage (% III+IV).. <0.000 Distant metastases (%).. 0. Number of lymph nodes Harvested (median) <0.000 The same pattern was seen when examining CC subsites (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

11 Page of Table Demographic, clinical and pathological characteristics according to specific colon cancer localization among, patients, Denmark 00-0 Caecum Ascending Hepatic Transverse Splenic Descending Sigmoid colon Colon flexure colon flexure colon N=, N=, N=, N=, N=, N=,00 N=, Age (median years) Sex (% women) ASA score (% score >) UICC stage (% III+IV) Distant metastases (%) Number of lymph nodes harvested (median) on December 0 by guest. Protected by copyright. BMJ Open: first published as./bmjopen on May 0. Downloaded from

12 Page of BMJ Open A significantly higher proportion of RCC (.%) than LCC (%) patients were women (p<0.000) (Table ). Likewise, when examining CC subsites, the proportion of women increased with proximal tumor localization, except for hepatic flexure cancers, where women were not in excess (Table ). A modestly higher ASA score was observed in RCC as compared to LCC patients (p<0.000), with 0. % of RCC patients having ASA scores of > compared to. % of LCC patients (Table ). However, focusing on CC subsites, ASA scores of > were most common in patients with CC located to the hepatic flexure, the transverse colon as well as the splenic flexure and the descending colon (Table ). Pathology Although RCC patients overall had significantly more advanced UICC stage than patients with LCC (p=0.00) (Table ), CC subsite analyses revealed that caecal, splenic flexure and descending colon cancers were of most advanced stages (Table ). The median number of lymph nodes harvested was slightly higher in patients with RCC (n=) than in patients with LCC (n=) (p<0.000), which corresponded to the observation for CC subsites (Table ). However, a similar proportion of RCC (.%) and LCC (.%) patients were found to have distant metastases (p=0.) and CC subsite analysis showed that the highest proportion of distant metastases was observed in patients with CC of the cecum, splenic flexure, or descending colon (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

13 Page of Survival Patients were followed for up to. years (median months) from diagnosis of CC. Kaplan-Meier plots of -year survival in women (Figure ) and men (Figure ) showed survival to be poorer in patients with RCC than in patients with LCC (p<0.000). This was in age-adjusted Cox-regression analyses found to be explained by excess mortality in patients with RCC relative to patients with LCC within the first two years after diagnosis (HR women,.; % CI,.0-.0; p<0.000; HR men,., % CI,.0-.; p<0.000) (Table ). In the following - years after CC diagnosis, mortality tended to be lower in patients with RCC than in patients with LCC (HRwomen, 0.; % CI, 0.-0.; p=0.0; HR men, 0., % CI, 0.-.0; p=0.) (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

14 Page of BMJ Open Table Hazard ratios (HR) for mortality from right sided colon cancer relative to mortality from left sided colon cancer among, patients, Denmark 00-0 Women Men HR (% CI) HR (% CI) Overall Years -. (.0-.0). (.0-.) Years - 0. (0.-0.) 0. (0.-.0) Adjusted for ASA score Years -. (.0-.). (.-.) Years - 0. (0.-0.) 0. (0.-.0) Adjusted for no. of harvested lymph nodes Years -. (.-.). (.-.) Years - 0. (0.-.0).0 (0.-.) Adjusted for no. of lymph nodes with metastases Years -. (.0-.).0 (.0-.0) Years - 0. (0.-0.) 0. (0.-.) Adjusted for presence of distant metastases Years -. (.-.). (.-.) Years - 0. (0.-.0).0 (0.-.) Adjusted for UICC stage Years -. (.-.). (.-.0) Years - 0. (0.-0.) 0. (0.-.) Cox proportional hazard models adjusted for age with additional adjustment for the mentioned factors one by one BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

15 Page of Adjustment for ASA score did not change the presented estimates (Table ). Adjustment for number of removed lymph nodes resulted in a stronger association between localization and mortality (especially in the first two years; Table ), which was found to be explained by a positive association between RCC and number of lymph nodes removed and an inverse association between number of lymph nodes removed and mortality. Adjustment for number of lymph nodes with metastases had little impact on the difference in mortality from RCC relative to LCC (Table ). However, further analyses revealed an interaction between CC localization and number of lymph nodes with metastases, which was most pronounced in women (p<0.00) and the excess - year mortality from RCC relative to LCC among female patients was only present among women with one or more lymph nodes with metastases whereas the mortality was decreased in RCC women relative to LCC women without lymph node metastases (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

16 Page of BMJ Open Table Hazard ratios (HR) for mortality from right sided colon cancer relative to mortality from left sided colon cancer according to number of lymph nodes with metastases Women Men Years - Years - Years - Years - HR (% CI HR (% HR (% HR (% CI) CI) CI) Lymph nodes with metastases 0. (0.-0.) (0.-0.) (.0-.) (0.-.) 0. (0.-.) (.-.) (.-.) (0.-.). (0.-.). 0. >. (.-.) (.- (0.-.).) Cox proportional hazard models adjusted for age BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

17 Page of Presence of distant metastases and UICC stage did not explain the difference in mortality between RCC and LCC (Table ). Analyses were repeated for CC subsites, using sigmoid colon localization as reference (Table ). Relative mortality was higher for all other CC subsites than sigmoid CC both in women and men during the first two years after CC diagnosis (Table ). Interestingly, mortality from cancer of the caecum and ascending colon were closest to mortality from sigmoid cancer (Table ), whereas mortality was -0% increased for cancer of the hepatic flexure, -% increased for cancer of the transverse colon, and, notably, -% increased for cancer of the splenic flexure relative to mortality from sigmoid cancer (Table ). During years - following cancer diagnosis, mortality for the investigated CC subsites were not different from mortality from sigmoid colon cancer except for a significantly lower relative mortality from cancer of the ascending colon (Table ). Adjustment for ASA score, number of harvested lymph nodes, number of lymph nodes with metastases, presence of distant metastases, and UICC stage had minor impact on these observations (data not shown). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

18 Page of BMJ Open Table Hazard ratios (HR) for mortality according to colon subsites (using sigmoid colon as reference) among, patients, Denmark 00-0 Ascending Hepatic Transverse Splenic Descending Sigmoid Caecum Colon flexure colon flexure colon colon Females Years - Years - Males Years - Years - HR (% CI). (.-.) 0. (0.-.). (.-.). (0.-.) HR (% HR CI).) 0.).) 0.) (% CI)..0 (0.- (.-.) (0.- (0.-.0).0. (.0- (.-.) (0.- (0.-.0) HR HR (% HR (% CI) (% HR (% CI) CI) CI).).0).).).. (.0- (.-.00) (0.- (0.-.).. (.- (.-.)..0 (0.- (0.-.). (.-.).0 (reference). (0.-.0.) (reference). (.0-.0.) (reference) 0. (0.-.0.) (reference) Cox proportional hazard models adjusted for age. Further adjustment for ASA score, number of harvested lymph nodes, lymph nodes with metastases, and UICC stage had minor impact on estimates (please see text). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

19 Page of DISCUSSION Our nationwide cohort study of, prospectively followed CC patients revealed that although patients with RCC overall were older, more often of female gender, had modestly higher ASA scores, and more advanced UICC stages than patients with LCC, the picture was indeed more complex when assessing the underlying CC subsites. Mortality from RCC was only relatively higher than mortality from LCC during the first two years after CC diagnosis and in analyses of CC subsites, the highest relative mortality was from cancer of the splenic flexure (i.e. belonging to the LCC category). The differences in mortality from CC of different colon subsites was only to a minor extent explained by ASA scores, number of harvested lymph nodes, number of lymph nodes with metastases, presence of distant metastases, and UICC stage. Overall, our observations challenge the simple categorization of CC into RCC and LCC. The primary strength of our study was the population-based patient cohort with nearly complete (-%) inclusion and follow-up of all Danish patients diagnosed with CC during the last years. Using the -digit personal identification number given to all Danish citizens at birth, complete information on vital status could be obtained from the Danish Central Person Registry. Further, detailed information on patient characteristics, surgical procedures including harvesting of lymph nodes, lymph node and tumor pathology, and UICC stages were available. The study also had potential limitations to consider. Despite detailed information on patients, data on cancer recurrence had not been collected. Hence, we were only able to compare overall survival rather than cancer free survival between RCC and LCC patients or by CC subsite. However, this would not influence the already observed marked differences BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

20 Page of BMJ Open in clinical characteristics and survival by specific CC subsites. Further, we did not have genetic or biomarker information that could have served to further qualify the observations of these differences, and this merits further study. In the present study we observed an almost linear relationship between tumor location and age; that is, the more proximal the tumor was located, the higher was the median age among patients. This did, obviously, result in a higher median age among RCC patients than among LCC patients. However, the same would have been the case had the division between groups been made at any other place along the colon (comparing proximal cancers with distal cancers), and it may not necessarily support the theory of RCC and LCC being two separate disease entities. The observed age distribution may be explained by diagnostic delay of cancers of the right site of the colon due to weaker symptoms. The sojourn time of preclinical colorectal cancer is estimated to range from. to. years [] and the difference in median age of patients with cancer in the caecum and cancer in the sigmoid colon was only. years, hence likely reflecting diagnostic delay. Another part of the explanation could be, that the diagnostic accuracy of colonoscopy is lower in the right site than in the left site of the colon [] and lower in women, which would further explain the observed gender differences between patients with RCC and LCC []. In accordance with a few earlier studies [,], we observed higher ASA scores among RCC than LCC patients. However, more detailed analyses of CC subsites revealed that ASA scores > were most often seen in patients with cancer of the hepatic flexure, transverse colon as well as of the splenic flexure and descending colon, i.e. cancers belonging to both the RCC and LCC category, hence again questioning the simple dichotomization of CC. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

21 Page 0 of We observed patients with RCC to have significantly more advanced cancer stages than patients with LCC in accordance with a previous systematic literature review []. However, CC subsite analyses again revealed a more complex picture with the most advanced stages and distant metastases observed for cancers of the cecum, splenic flexure and descending colon. This is in accordance with a study of Benedix et al [] who in an analysis of, CC patients found the highest rate of prognostically favourable UICC stages (I or II) among carcinomas of the descending colon (.%) and ascending colon (.%), whereas the lowest rates were found for the splenic flexure (.0%) and the cecum (.%). Further cancers of the cecum and splenic flexure (i.e. representing both RCC and LCC) were most advanced with regard to metastatic spread (UICC stage IV). In the present study, overall survival was poorer in patients with RCC than in patients with LCC, in accordance with the previous systematic review []. However, the review also revealed that when eliminating confounding by differences in patient or tumor characteristics, the prognostic picture became more complex in most studies. Suttie et al [] found that age, tumor stage, and acute presentation had a significant impact on differences in survival between patients with RCC and LCC, in accordance with Faivre- Finn et al [] who also found tumor location per se to associate with survival. This has, however, been questioned by others [,]. We observed mortality from RCC (relative to LCC) to be increased only during the first two years after CC diagnosis and to be most pronounced in men. ASA score, UICC stage and presence of distant metastases had little impact on the difference in mortality from RCC and LCC, whereas an interaction with number of lymph nodes with metastases was observed. However, when assessing CC subsites and using sigmoid colon localization as reference, the highest relative mortality was observed in patients with splenic flexure cancer. This is in accordance with one [] BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

22 Page of BMJ Open but in contrast to another previous study [], and again challenges the concept of RCC and LCC as two distinct disease entities. It remains unknown why patient characteristics, cancer pathology, and survival differ between CC subsites []. However, it has recently been shown that biological features, such as rate of cancer gene mutations in form of microsatellite instability (MSI), K-RAS and BRAF mutations differ along colonic subsites [,] and, hence, do not fit the theory of CC simply arising from the embryological right or left site of colon [0]. These observations fit well with results of the present study and thereby underscore the need for further studies of colorectal neoplasia by CC subsite at the molecular, pathological and epidemiological level [,]. In conclusion, the present nationwide population-based cohort study of more than 0,000 prospectively followed CC patients revealed that - overall - patients with RCC may differ from patients with LCC in terms of age, gender, ASA scores, cancer stage, and survival. However, these findings cover a much more complex pattern of disease with especially cancer of the splenic flexure showing poor stage and prognosis. Our study strongly challenges the simple classification of CC into right sited versus left sited disease and underscores the need for a more subtle CC classification, in accordance with recent molecular biological findings. A better classification will enhance our possibilities to provide optimal surveillance and treatment of CC. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

23 Page of REFERENCES. Jemal A, Bray F, Center MM, et al. Clobal Cancer Statistics. CA Cancer J Clin 0;: -0.. Bufill JA. Colorectal cancer: Evidence for distinct genetic categories based on proximal or distal location. Ann Int Med 0;:-.. Benedix F, Kube R, Meyer F, et al. Comparison of, patients with right- and left- sided colon cancer: differences in epidemiology, perioperative course, histology, and survival. Dis Colon Rectum 0;:-.. Hansen IO, Jess P. Possible better long-term survival in left versus right-sided colon cancer a systematic review. Dan Med J 0;():A.. Elnatan J, Gosh HS, Smith DR. C-KI-RAS activation and the biological behaviour of proximal and distal colonic adenocarcinomas. Eur J Cancer ;A:-.. Hutchins G, Southward K, Handley K, et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol 0;:-0.. Benedix F, Schmidt U, Mroczkowski P, et al. Colon carcinoma Classification into right and left sided cancer or according to colonic subsites? Analysis of patients. EJSO 0; :-.. Benedix F, Meyer F, Kube,R et al. Influence of anatomical subsite on the incidence of microsatellite instability, and KRAS and BRAF mutation rates in patients with colon carcinoma. Pathol Res Pract 0 Oct ;http//dx.doi.org/./j.prp Yamauchi M, Morikawa T, Kuchiba A, et al. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut 0;:-.. Danish Colorectal Cancer Group (DCCG) BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

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25 Page of colon cancers based on clinicophatological, molecular and and protein profiles. Int J Oncol 0;:0-.. Prichard CC, Grady WM. Colorectal cancer molecular biology moves into clinical practice. Gut 0;0:-.. Ogino S, Chan AT, Fuchs CS. Molecular pathological epidemiology of colorectal neoplasia: an emerging transdisciplinary and interdisciplinary filed. Gut;0:-. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

26 Page of BMJ Open FIGURE LEGENDS Figure. Kaplan-Meier plot of survival according to time since diagnosis of right sided (thin line) and left sided (black line) colon cancer in, Danish women (00-0) Figure. Kaplan-Meier plot of survival according to time since diagnosis of right sided (thin line) and left sided (black line) colon cancer in, Danish men (00-0) BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

27 Page of Figure. Kaplan-Meier plot of survival according to time since diagnosis of right sided (thin line) and left sided (black line) colon cancer in, Danish women (00-0) x0mm ( x DPI) BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

28 Page of BMJ Open Figure. Kaplan-Meier plot of survival according to time since diagnosis of right sided (thin line) and left sided (black line) colon cancer in, Danish men (00-0) x0mm ( x DPI) BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

29 A Nationwide Danish Cohort Study challenging the Categorization into Right Sided and Left Sided Colon Cancer Journal: BMJ Open Manuscript ID: bmjopen r Article Type: Research Date Submitted by the Author: -Apr-0 Complete List of Authors: Jess, Per; Roskilde Hospital, Department of Surgery; University of Copenhagen, Faculty of Health Sciences Hansen, Iben; University of Copenhagen, Faculty of Health Sciences Gamborg, Michael; Copenhagen University Hospital, Institute of Preventive Medicine Jess, Tine; Statens Serum Institut, Department of Epidemiology Research <b>primary Subject Heading</b>: Oncology Secondary Subject Heading: Oncology, Gastroenterology and hepatology, Surgery Keywords: Gastrointestinal tumours < GASTROENTEROLOGY, Colorectal surgery < SURGERY, EPIDEMIOLOGY BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

30 Page of BMJ Open ORIGINAL ARTICLE A Nationwide Danish Cohort Study challenging the Categorization into Right Sided and Left Sided Colon Cancer Per Jess,, Iben Onsberg Hansen, Michael Gamborg, Tine Jess. On behalf of the Danish Colorectal Cancer Group. Department of Surgery, Roskilde Hospital, Roskilde, Denmark. Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. Institute of Preventive Medicine, Copenhagen University Hospital, Copenhagen, Denmark. Department of Epidemiology Research, Statens Serum Institut, National Health Surveillance and Research, Copenhagen, Denmark Correspondance: Per Jess, M.D., Dr.Med.Sci. Department of Surgery, Roskilde Hospital, DK-000 Roskilde, Denmark; address: pjss@regionsjaelland.dk ; Telephone: +0; Fax: + 00 Keywords: Colon neoplasia; Colon subsites; Epidemiology Word count: BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

31 Page of Author Contributions: The study was conceived and designed by Per Jess, Iben Onsberg Hansen, Michael Gamborg, and Tine Jess. Statistical analyses were performed by Michael Gamborg. All authors were involved in the interpretation of data. Per Jess drafted the manuscript, which was critically revised by all authors, who also approved the final version of the manuscript. Conflicts of interest: The authors have no conflicts to disclose Funding: Not relevant Trial registration: Not relevant No additional data available. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

32 Page of BMJ Open Article Summary Article Focus. Colon cancer is normally classified into right sided and left sided colon cancer.. It has been suggested that there are differences in epidemiology, pathology and prognosis between patients with cancer in the right and left side of the colon due to different embryologic development of the two segments of the colon which may result in different molecular biological patterns of the tumors.. Recent studies have challenged this simple dichotomization of colon cancer into right sided and left sided colon cancer. Key Messages. Our nationwide cohort study of, colon cancer patients confirms that patients with right sided colon cancer are older, more often females, and have more advanced tumors and a worse prognosis than patients with left sided cancer.. However, more detailed colon subsite analysis reveals a much more complex picture. Highest ASA scores are observed in patients with cancer of the transverse and descending colon and of both colon flexures. The most advanced colon cancers are those of the descending colon, splenic flexure, and cecum. The highest mortality is seen in patients with splenic flexure cancer. Strengths and Limitations. The strength of the present study is the population-based patient cohort with nearly complete (-%) inclusion and follow-up of all Danish patients diagnosed with colon cancer during the last years.. The study has potential limitations: Despite detailed information on patients, data on cancer recurrence has not been collected. Hence, we were only able to compare overall survival rather than cancer free survival. However, this would not influence the already BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

33 Page of observed marked differences in clinical characteristics and survival by specific colon cancer subsites. Further, we did not have genetic or biomarker information that could have served to further qualify the observations of these differences, and this merits further study. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

34 Page of BMJ Open ABSTRACT Objectives: The categorization of colon cancer (CC) into right sided (RCC) and left sided (LCC) disease may not capture more subtle variances in etiology and prognosis. In a nationwide study, we investigated differences in clinical characteristics and survival of RCC versus LCC and of the complete range of CC subsites. Design: Prospective nationwide cohort study. Setting: The database of the Danish Colorectal Cancer Group (DCCG). Participants:, CC patients. Outcome measures:overall survival (Kaplan Meier plots) and mortality (hazard ratios [HR] from Cox proportional hazards regression analysis) according to CC localization. For adjustment and stratification, we used age, sex, ASA score (the American Society of Anaesthesiologists score), tumor location and stage, number of lymph nodes harvested at operation, number of lymph nodes with metastases, and presence of distant metastases. Results: Patients with RCC had a higher median age at diagnosis (. years) than patients with LCC (. years) (p<0.000). Overall, the proportion of patients who were women increased the closer the tumour site was to the small intestine. Although, RCC patients had higher ASA scores than LCC patients (p<0.000), highest ASA scores were observed in patients with cancer in the transverse and descending colon and at both colon flexures. While RCCs overall were more advanced than LCCs (p<0.000), the most advanced CCs were those of the descending colon, splenic flexure, and cecum. RCC mortality was higher than LCC mortality only during the first two years (women: HR,.; % CI,.0-.0; men: HR,.; % CI,.0-.), and relative to mortality from sigmoid colon cancer, the highest mortality was observed from splenic flexure cancer (HR,.; % CI,.-.00). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

35 Page of Conclusions: The present data challenge the simple categorization of CC into RCC and LCC. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

36 Page of BMJ Open INTRODUCTION Colorectal cancer is one of the most commonly diagnosed cancers worldwide with over. million new cancer cases and 0,00 deaths estimated to occur annually []. In 0, Bufill was the first to propose that colon cancer (CC) located to the right (RCC) and left (LCC) side of colon, respectively, may rise from different biological pathways []. Subsequently, it has been suggested, that being two different disease entities, RCC and LCC also differ in terms of patient characteristics, pathology, and prognosis []. A recent systematic review of the sparse literature on the topic supports that such differences exist []. The reason for this is uncertain, but a suggested explanation is the different embryologic development of the two segments of the colon resulting in different molecular biological tumor patterns [,]. However, recent studies have challenged the simple dichotomization of CC into RCC and LCC, since factors as tumor stage [], genetic alterations [] and molecular features [] vary markedly between colonic subsites irrespective of their localization in the right or left site of colon. Such possible differences might have consequences for planning of screening and for the treatment of patients with CC. The aim of the present investigation was to examine differences in patient characteristics, cancer pathology, and survival according to RCC vs. LCC localization of CC and according to more specific colon subsite localization, using unique prospectively collected nationwide data from years BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

37 Page of MATERIALS AND METHODS On May, 00 the Danish Colorectal Cancer Group (DCCG) established a nationwide database [], and since then all patients with a first-time diagnosis of colorectal adenocarcinoma from all Danish surgical departments treating such patients have been prospectively registered in the database. Patients are identified by their Danish Civil Registration Number, a unique -digit person number given to all Danish citizens at birth and ascribed to every Danish person since []. The data reported to DCCG from the surgical departments in Denmark include demographics, clinical manifestation, tumor location, stage (UICC; Union for International Cancer Control), diagnostic and therapeutic procedures, and postoperative complications. Data on vital status are updated monthly through linkage to the Danish Civil Registration System []. The completeness of data collection in DCCG is estimated annually and has increased from to % in the period 00-0 []. The present study covers all Danish patients (n=,) in DCCG with a diagnosis of CC from January, 00 trough December, 0. From DCCG, we specifically obtained data on age, sex, ASA score (the American Society of Anaesthesiologists score), tumor location and stage (UICC), number of lymph nodes harvested at operation, number of lymph nodes with metastases, and presence of distant metastases. Localization of CC was treated both according to specific sub-sites and according to the suggested categorization into RCC (n=,; caecum, ascending colon, hepatic flexure, or transverse colon) and LCC (n=,; from the splenic flexure to the sigmoid colon, both included; the border between the sigmoid colon and rectum defined as cm from the anal verge). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

38 Page of BMJ Open Statistical analyses Non-parametric statistics (median, range, chi-square test, and Mann-Whitney test) were used for description of the demographic, clinical and pathological characteristics of the patient population. P-values <0.0 were considered statistically significant. Patients were followed from date of colon cancer diagnosis to emigration, death, or end of the study (October, 0) whichever occurred first. Overall survival among patients with RCC vs. LCC was illustrated by Kaplan-Meier plots for, respectively, women and men and were compared by log-rank test. Mortality of any course from RCC relative to mortality from LCC was estimated by hazard ratios (HRs) obtained by performing Cox proportional hazard regression analysis with time since diagnosis as the underling time axis, stratified on gender and adjusted for age. Next, the impact of ASA score, number of lymph nodes harvested at operation, number of lymph nodes with metastases, presence of distant metastases, and UICC stage on the relative difference in mortality from RCC and LCC was investigated by performing adjusted analyses. Finally, analyses were performed comparing the survival among patients with cancer of, respectively, caecum, ascending colon, hepatic flexure, transverse colon, splenic flexure, and descending colon, with the survival among patients with sigmoid colon. All analyses were performed using STATA version.. Ethics The Danish National Committee on Biomedical Research Ethics and the Danish Data Protection Agency have approved the use of the DCCG database for scientific purposes and for the present study, also. BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

39 Page of RESULTS Age, gender and ASA score The median age at diagnosis of RCC was. years (range.-. years) versus. years (range.-0.) for LCC (p<0.000). The same pattern was seen when examining CC subsites (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

40 Page of BMJ Open Table Demographic, clinical and pathological characteristics according to right sided colon cancer versus left sided colon cancer and specific colon cancer localization among, patients, Denmark 00-0 RCC LCC Caecum Ascending Hepatic Transverse Splenic Descending Sigmoid colon flexure colon flexure colon colon N=, N=. p-value N=, N=, N=, N=, N=, N=,00 N=, p-value Age (median years).. < <0.000 (First and third quartile) Sex (% women)..0 < <0.000 ASA score 0.. < <0.000 (% score >) UICC stage (% III+IV).. < <0.000 Distant metastases (%) <0.000 Number of Lymph nodes harvested <0.000 <0.000 on December 0 by guest. Protected by copyright. BMJ Open: first published as./bmjopen on May 0. Downloaded from

41 Page of (median, first and third quartile) RCC: Right sided colon cancer LCC. Left sided colon cancer on December 0 by guest. Protected by copyright. BMJ Open: first published as./bmjopen on May 0. Downloaded from

42 Page of BMJ Open A significantly higher proportion of RCC (.%) than LCC (%) patients were women (p<0.000). Likewise, when examining CC subsites, the proportion of patients who were women increased the closer the tumour site was to the small intestine. However, among patients with hepatic flexure cancers, women were not in excess (Table ). A modestly higher ASA score was observed in RCC as compared to LCC patients (p<0.000), with 0. % of RCC patients having ASA scores of > compared to. % of LCC patients. However, focusing on CC subsites, ASA scores of > were most common in patients with CC located to the hepatic flexure, the transverse colon as well as the splenic flexure and the descending colon (Table ). Pathology Although RCC patients overall had significantly more advanced UICC stage than patients with LCC (p=0.00), CC subsite analyses revealed that caecal, splenic flexure and descending colon cancers were of most advanced stages (Table ). The median number of lymph nodes harvested was slightly higher in patients with RCC (n=) than in patients with LCC (n=) (p<0.000), which corresponded to the observation for CC subsites. However, a similar proportion of RCC (.%) and LCC (.%) patients were found to have distant metastases (p=0.) and CC subsite analysis showed that the highest proportion of distant metastases was observed in patients with CC of the cecum, splenic flexure, or descending colon (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.

43 Page of Survival Patients were followed for up to. years (median months) from diagnosis of CC. Kaplan-Meier plots of -year overall survival in women and men (Figure ) showed overall survival to be poorer in patients with RCC than in patients with LCC (p<0.000). This was in age-adjusted Cox-regression analyses found to be explained by excess mortality in patients with RCC relative to patients with LCC within the first two years after diagnosis (HR women,., p<0.000; HR men,., p<0.000) (Table ). In the following - years after CC diagnosis, mortality tended to be lower in patients with RCC than in patients with LCC (HRwomen, 0., p=0.0; HRmen, 0., p=0.) (Table ). BMJ Open: first published as./bmjopen on May 0. Downloaded from on December 0 by guest. Protected by copyright.