IJC International Journal of Cancer

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1 IJC International Journal of Cancer Risk factors for cancers of unknown primary site: Results from the prospective EPIC cohort Rudolf Kaaks 1, Disorn Sookthai 1, Kari Hemminki 2,3, Alwin Kr amer 4,5, Heiner Boeing 6, Elisabet Wirf alt 7, Elisabete Weiderpass 8,9,10,11, Kim Overvad 12, Anne Tjïnneland 13, Anja Olsen 13, Petra H. Peeters 14,15, H. B(as) Bueno-de-Mesquita 15,16,17, Salvatore Panico 18, Valeria Pala 19, Paolo Vineis 15,20, J. Ramon Quiros 21, Eva Ardanaz 22,23, Marıa-Jose Sanchez 23,24, Maria-Dolores Chirlaque 23,25, Nerea Larra~naga 23,26, Paul Brennan 27, Dimitrios Trichopoulos 28,29,30, Antonia Trichopoulou 28,30, Pagona Lagiou 29,30,31,G oran Hallmans 32, Kay-Tee Khaw 33, Timothy J. Key 34, Elio Riboli 15 and Federico Canzian 35 1 Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Center for Primary Health Care Research, Lund University, Malm o, Sweden 4 Clinical Cooperation Unit, Molecular Hematology and Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany 6 Department of Epidemiology, German Institute of Human Nutrition (DIfE) Potsdam-Rehbr ucke, Nuthetal, Germany 7 Department of Clinical Sciences, Lund University, Malm o, Sweden 8 Department of Community Medicine, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsï, Norway 9 Department of Research, Cancer Registry of Norway, Oslo, Norway 10 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 11 Samfundet Folkh alsan, Helsinki, Finland 12 Department of Public Health, Section for Epidemiology, Aarhus University, Aarhus, Denmark 13 Danish Cancer Society Research Center, Copenhagen, Denmark 14 Department of Epidemiology, Julius Center for Health Sciences and Primary Care, University Medical Center, Utrecht, The Netherlands 15 Department of Epidemiology and Biostatistics, School of Public Health, Imperial College, London, United Kingdom 16 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 17 Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands 18 Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy 19 Department of Preventive & Predictive Medicine, Nutritional Epidemiology Unit, Fondazione IRCCS Istituto, Nazionale dei Tumori, Milan, Italy 20 Human Genetics Foundation (HuGeF), Torino, Italy 21 Public Health Directorate, Asturias, Spain 22 Navarre Public Health Institute, Pamplona, Spain 23 CIBER Epidemiology and Public Health CIBERESP, Spain 24 Escuela Andaluza de Salud Publica, Instituto de Investigacion Biosanitaria de Granada (Granada.ibs), Granada, Spain 25 Department of Epidemiology, Murcia Regional Health Authority, Murcia, Spain 26 Public Health Division of Gipuzkoa, Department of Health of the regional Government of the Basque Country, San Sebastian, Spain Key words: cancer of unknown primary site (CUP), prospective cohort study, smoking, alcohol, obesity, waist circumference Disclosure: The authors have declared no conflicts of interest. Grant sponsors: European Commission (DG-SANCO); International Agency for Research on Cancer, Danish Cancer Society (Denmark); Ligue contre le Cancer, Mutuelle Generale de l Education Nationale, Institut National de la Sante et de la Recherche Medicale (France); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum and Federal Ministry of Education and Research (Germany); Hellenic Health Foundation (Greece); Italian Association for Research on Cancer (AIRC) and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); Norwegian Research Council, Norwegian Cancer Society, Nordforsk, Nordic Centre of Excellence programme on Food, Nutrition and Health (Norway); Health Research Fund (FIS), Catalan Institute of Oncology, Swedish Cancer Society, Swedish Scientific Council and Regional Government of Skåne and V asterbotten (Sweden); Cancer Research UK, Medical Research Council, Stroke Association; British Heart Foundation, Department of Health, Welcome Trust (United Kingdom); Grant sponsor: European Research Council; Grant number: ERC-2009-AdG ; Grant sponsor: Spanish Ministry of Health; Grant number: ISCIII RETICC RD06/0020/0091; Grant sponsor: Regional Governments of Andalucıa, Asturias, Basque Country, Murcia and Navarra; Grant number: ISCIII RETIC (RD06/0020; Spain); Grant sponsor: Deutsche Krebshilfe; Grant numbers: EU FP7/ , (260715) DOI: /ijc History: Received 6 Dec 2013; Accepted 4 Mar 2014; Online 2 Apr 2014 Correspondence to: Rudolf Kaaks, Department of Cancer Epidemiology, German Cancer Research Center (DKFZ) Heidelberg, Im Neuenheimer Feld 581, D Heidelberg, Germany, Tel: , Fax: , r.kaaks@dkfz.de

2 2476 Risk factors for cancers of unknown primary site 27 Section of Genetics, International Agency for Research on Cancer (IARC), Lyon, France 28 Hellenic Health Foundation, Athens, Greece 29 Department of Epidemiology, Harvard School of Public Health, Boston, MA 30 Bureau of Epidemiologic Research, Academy of Athens, Athens, Greece 31 Department of Hygiene, Epidemiology and Medical Statistics, WHO Collaborating Center for Food and Nutrition Policies, University of Athens Medical, Greece 32 Department of Public Health and Clinical Medicine/Nutritional Research, Umeå University, Umeå, Sweden 33 School of Clinical Medicine, University of Cambridge, Cambridge, United Kingdom 34 Cancer Epidemiology Unit, Nuffield Department of Clinical Medicine, University of Oxford, United Kingdom 35 Genomic Epidemiology Group, German Cancer Research Center (DKFZ), Heidelberg, Germany Cancer of unknown primary site (CUP) may be called an orphan disease, as it is diagnosed when metastases are detected while the primary tumor typically remains undetected, and because little research has been done on its primary causes. So far, few epidemiological studies, if any, have addressed possible risk factors for CUP. We analyzed data from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (N 5 476,940). During prospective follow-up, a total of 651 of incident of CUP were detected (ICD-O-2 code C809). Proportional hazards models were conducted to examine the associations of lifetime history of smoking habits, alcohol consumption, levels of education and anthropometric indices of adiposity with risk of being diagnosed with CUP. Risk of being diagnosed with CUP was strongly related to smoking, with a relative risk of 3.66 [95% C.I., ] for current, heavy smokers (261 cigarettes/day) compared to never smokers (adjusted for alcohol consumption, body mass index, waist circumference and level of education) and a relative risk of 5.12 [ ] for with CUP who died within 12 months. For alcohol consumption and level of education, weaker associations were observed but attenuated and no longer statistically significant after adjusting for smoking and indices of obesity. Finally, risk of CUP was increased by approximately 30 per cent for subjects in the highest versus lowest quartiles of waist circumference. Our analyses provide further documentation, in addition to autopsy studies, that a substantial proportion of cancers of unknown primary site may have their origin in smoking-related tumors, in particular. What s new? When cancer appears as metastatic disease but no primary tumor can be observed, it s called cancer of unknown primary site. Little is known about the risk factors for this type of cancer. This study analyzed data from a European cohort and discovered a strong association between smoking and these cancers. Other risk factors they identified were drinking alcohol and being fat. This is the first epidemiological study of these type of cancers, and it strengthens the observations from autopsy studies that many of these cancers of unknown primary site stem from smoking-related tumors. Cancer of unknown primary site (CUP) has been called an orphan disease because it is diagnosed when one or more metastases are detected while the primary tumor typically remains undetected. 1 It accounts for about 3 5% of cancer occurrences overall, but generally has a highly aggressive clinical course associated with very short (pre-)clinical history and short survival times, and ranks fourth to fifth among causes of cancer deaths. 2,3 Autopsy studies could identify primary tumors in about 50 75% of with CUP, and showed that the lung, liver, pancreas and gastrointestinal tract (esophagus, colorectum) are common sites for primary tumors. 1 Histologically, CUP includes essentially adenocarcinomas (40 60%), and undifferentiated carcinomas (30%), and a small remaining proportion of poorly differentiated tumors, squamous cell carcinomas and small cell carcinomas. The predominant theory regarding CUP is that it may represent a family of tumor forms that form metastases more rapidly than usual tumors at the same organ site of origin, and that these metastases have a stronger than usual growth potential in comparison to the primary tumor. A parallel theory is that the primary tumors giving rise to CUP have a reduced growth, or even regress, for example as a result of immune suppression. A recent study within the Swedish Family-Cancer Database demonstrated familial clustering of CUP and the association of CUP with occurrence of many other cancers, in large part tumors originating from organs suspected to be responsible for many CUP diagnoses such as cancers of the lung, colorectum and liver, but also tumors of the ovary and kidney. 4 The latter results suggest that the primary tumor types underlying CUP might share some common genetic predisposition factors. A further reason why CUP is appropriately called an orphan disease is that comparatively little basic research has been done on its primary causes. Hardly anything is

3 Kaaks et al known about epidemiological risk factors for CUP. A study of Swedish cancer registry data has shown roughly equal incidence rates for CUP among men and women, and a slowly increasing time trend to about years , followed by a sharp decline of about 25%. 2 The authors could only speculate, however, about the reasons for these temporal incidence patterns. The few epidemiologic reports so far that addressed possible risk factors showed an increased risk of CUP among low socioeconomic population groups 5 and in relation to occupations linked with tobacco exposures. 6 In the present article, we report the associations of three basic lifestyle-related risk factors smoking, alcohol consumption, obesity and level of education with risk of CUP within the European Prospective Investigation into Cancer and Nutrition EPIC cohort. Material and Methods Study population The EPIC study is an ongoing multicenter population-based cohort study to investigate the relation between diet, nutritional and metabolic characteristics, lifestyle factors, and subsequent cancer incidence and cause specific mortality. The cohort consists of 521,448 participants (367,993 women and 153,455 men), enrolled into the cohort between 1992 and 2000, in 23 study centers across 10 different countries (i.e. Denmark, France, Germany, Greece, Italy, the Netherlands, Norway, Spain, Sweden and United Kingdom). At recruitment, the majority of participants were aged between 35 and 70 years 7 with the noticeable exception of a subcohort in the United Kingdom, which included individuals in the age range of (EPIC-Norfolk, Cambridge) and years (health conscious subcohort, Oxford; php). Detailed information about the selection of the studypopulation, data collection and follow-up procedures have been reported previously. 7 All participants of the EPIC cohort gave written informed consent, and the study was approved by the International Agency for Research on Cancer ethical review committee and by the local committees at the participating centers. At baseline, questionnaires and computer-guided, face-toface interviews were used to collect information about basic demographic variables, past illnesses, use of medications and lifestyle. Questionnaire information on lifestyle covered lifetime history as well as current habits (at the time of recruitment) of smoking and alcohol consumption, and detailed questions on diet and physical activity. Baseline physical examinations included measurements of height, weight and waist circumferences following standardized procedures. Although weight and height were available for all participants, only 77.55% of participants had waist circumference measurements. A total of 44,508 participants who had reported a prevalent or previous cancer at the time of baseline recruitment or who had no follow-up data were excluded, leaving a total of 476,940 cohort participants for the present analyses. Prospective ascertainment of incident cancer and vital status; coding of cancer endpoints In all countries except France, Germany, Greece and Naples (Italy) incident cancer are identified using record linkage with cancer and pathology registries. In France, cancer occurrence is prospectively ascertained through linkages with health insurance records, and in Germany and Greece through regular direct contacts with participants and their next of kin. In all of the three latter countries, self-reported disease occurrences are systematically verified against clinical and pathological records and coded by specialized study physicians. Information on vital status is collected from regional or national mortality registries. Cancer incidence data are coded according to the International Classification of Diseases for Oncology (ICD-O-2), and mortality data are coded according to the 10th Revision of the International Statistical Classification of Diseases, Injuries and Causes of Death (ICD-10). The latest updates of endpoint data for cancer incidence and vital status were between 2005 and 2010, depending on the study center. Up to this latest update, a total of 1050 cancer endpoints were identified with the ICD-O-2 code (C809) for CUP. Of these 1050, 318 additionally had morphology codes for lymphoma (N 5 141), skin cancers (N 5 32) or other/miscellaneous histology N 5 145), and these were excluded from the analyses. Furthermore, as 80 of all 81 of CUP in France had missing morphology status, the French subcohort was excluded entirely from the present analyses. Thus, after excluding a total of 399 subjects amongst coded C809, a core set of 651 tumors of primary origin remained, which had morphology codes indicating the tumor was an adenocarcinoma (N 5 273), a carcinoma (N 5 140), malignant neoplasm (N 5 210), or other codes for diverse, undifferentiated tumors (N 5 28) and these formed the set of tumors of unknown primary origin retained for our present analyses. For 522 of the 651 of CUP in this core set additional information on 12-month survival was available. Statistical analyses Proportional hazards models were used to estimate relative risks (hazard ratios and their 95% confidence intervals) of CUP by levels of body mass index (BMI), waist circumference, alcohol consumption, education and smoking. Attained age was used as the underlying time scale and the time of origin of the study was age at recruitment, and study participants were considered at risk from their age at recruitment until the age at which either CUP was diagnosed or censoring was applied because of the occurrence of death, diagnosis of another type of cancer, loss to follow up or closure of the latest follow-up assessment. The proportional hazards assumption was checked by examining time-by-covariate interaction terms to test whether the hazard ratio was constant over time, no strong violation of this assumption were noted. Participants were divided into quartile categories of BMI and waist circumference with gender specific quartile cut-

4 2478 Risk factors for cancers of unknown primary site Table 1. Characteristics of CUP by country and gender Survival time Histological Subtypes 12 > 12 Adenocarcinoma Carcinoma Neoplasm Other CUP as proportion of all Country Total months (%) months (%) (%) (%) (%) (%) cancer occurrences (%) Italy (64.9) 13 (35.1) 21 (56.8) 9 (24.3) 7 (18.9) 1.0 Spain (75.0) 9 (25.0) 21 (58.3) 7 (19.4) 5 (13.9) 3 (8.3) 1.3 United (90.1) 21 (9.9) 50 (23.5) 68 (31.9) 92 (43.2) 3 (1.4) 3.3 Kingdom The (77.4) 12 (22.6) 37 (69.8) 3 (5.7) 8 (15.1) 5 (9.4) 1.7 Netherlands Greece (93.8) 1 (6.3) 4 (25.0) 12 (75.0) 1.5 Germany (63.3) 11 (36.7) 17 (56.7) 2 (6.7) 8 (26.7) 3 (10.0) 0.7 Sweden (77.6) 28 (22.4) 44 (35.2) 23 (18.4) 53 (42.4) 5 (4.0) 1.9 Denmark (79.5) 26 (20.5) 69 (54.3) 27 (21.3) 24 (18.9) 7 (5.5) 1.8 Norway 14 6 (42.9) 8 (57.1) 10 (71.4) 1 (7.1) 1 (7.1) 2 (14.3) 0.6 Gender Male (86.1) 37 (13.9) 105 (39.4) 54 (20.2) 97 (36.4) 11 (4.0) 1.8 Female (76.1) 92 (23.9) 168 (43.6) 86 (22.4) 113 (29.5) 17 (4.5) 1.4 Total (80.2) 129(19.8) 273 (41.9) 140 (21.5) 210 (32.3) 28 (4.3) 1.5 points based on the full study cohort. With regard to smoking-related variables, study participants were categorized according to smoking status at the time of recruitment (never, past, current). Subjects who reported current or past smoking at baseline were further categorized by lifetime average number of cigarettes smoked per day (1 15, 16 25, 261), and past smokers were also further categorized by time since quitting (<510 years, >10 years). Categories of lifetime alcohol included lifetime nonconsumers (0 g/day), former consumers and current consumers (at the time of recruitment) drinking an average of >0 12, >12 24, >24 60 and >60 g/day. Levels of formal education were categorized into primary school or less, technical and professional school, secondary school and longer education including university. Univariate models, to assess the effects of single risk factors at a time, were stratified only for study center, sex and age at recruitment in 2-year categories. Trend tests across levels of exposure were assessed using continuous covariates. In addition, in multivariable models the associations of risk with lifetime smoking history and intensity, alcohol consumption habits, anthropometric indices of adiposity and levels of education were estimated with additional, mutual adjustments between these risk factor variables. Tests for interaction between risk factors were done by adding the multiplicative terms (cross-product term) in the model. Finally, sensitivity analyses were conducted to explore whether risk factors differed for CUP with survival times of up to 12 months (N 5 522). Results An overview of the 651 incident of CUP by the nine countries retained for this analysis is in Table 1. The vast majority of the CUP were coded as adenocarcinomas not otherwise specified (N 5 273) neoplasm not otherwise specified (N 5 210), or carcinoma not otherwise specified (N 5 140), and the remainder (labeled other ) had morphology codes for mucinous/mucin-producing adenocarcinoma (N 5 16), signet ring cell carcinoma (N 5 7), solid carcinoma (N 5 2), small cell carcinoma (N 5 1) and tubular cell adenocarcinoma (N 5 2). The ratio of tumors coded adenocarcinomas to carcinomas not otherwise specified varied between the nine countries, from 0.74 to CUP represented 1.5 per cent of the overall cancer occurrence, and this proportion varied from 0.7 to 3.3 per cent across the EPIC countries. Overall, the occurrence of CUP was roughly equal among men and women (rate ratio [RR] [95% C.I ]). The median age at diagnosis was 66.6 years (range years). The median duration of survival after diagnosis, all countries combined, was 2 months (range months), and of all 651 CUP, 522 (80%) had a survival time of less than 12 months (Table 1). The longest median survival time was noted in Norway and the shortest was in the United Kingdom and Greece. A higher level of formal education was associated with modest reduction in risk for CUP, although this inverse association was weakened after adjustments for smoking, alcohol consumption and adiposity (Table 1). Relative risks (RR) for CUP by level of major risk factors are presented in Table 2. Increased risks were observed among current smokers, with a relative risk of 4.05 [ ] (unadjusted model) (RR [ ], after additional adjustments for alcohol consumption, level of education, and adiposity) for current smokers of 261 cigarettes a

5 Kaaks et al Table 2. Hazard ratios and 95% confidence intervals for CUP in relation to smoking, alcohol consumption, levels of education and anthropometric indices of obesity CUP CUP with survival time 12 months Smoking intensity 3 Never smoked 219 Referent Referent 166 Referent Referent 1 15 cigarettes/ day (1.44,2.40) 1.81 (1.39,2.34) (1.65,2.93) 2.10 (1.57,2.80) cigarettes/ day 261 cigarettes/ day Former smokers, quit 10 years Former smokers, quit >10 years pipe or cigar Average lifetime alcohol consumption (g/day) (2.63,4.56) 3.25 (2.46,4.30) (2.88,5.39) 3.61 (2.63,4.95) (2.49,6.58) 3.66 (2.24,5.97) (3.53,9.54) 5.12 (3.09,8.47) (1.04,1.87) 1.34 (0.99,1.80) (1.10,2.14) 1.46 (1.04,2.03) (0.88,1.38) 1.08 (0.86,1.36) (0.90,1.50) 1.14 (0.88,1.47) (1.02,2.28) 1.49 (1.00,2.23) (1.00,2.48) 1.52 (0.97,2.40) Former (0.80,1.78) 1.05 (0.70,1.58) (0.88,2.04) 1.17 (0.76,1.79) Referent Referent 235 Referent Referent > (0.83,1.39) 1.04 (0.80,1.35) (0.84,1.49) 1.07 (0.81,1.43) > (1.04,1.93) 1.26 (0.93,1.72) (1.13,2.18) 1.37 (0.98,1.91) > (1.02,3.23) 1.42 (0.79,2.53) (0.88,3.24) 1.27 (0.66,2.45) P linear trend Body mass index 3 Quartile1 144 Referent Referent 123 Referent Referent Quartile (0.71,1.13) 0.92 (0.73,1.16) (0.66,1.10) 0.88 (0.68,1.13) Quartile (0.77,1.21) 0.98 (0.78,1.23) (0.67,1.11) 0.88 (0.68,1.14) Quartile (0.83,1.32) 1.06 (0.84,1.33) (0.80,1.33) 1.04 (0.80,1.34) P linear trend Waist circumference 3 Quartile1 129 Referent Referent 105 Referent Referent Quartile (0.70,1.16) 0.91 (0.71,1.16) (0.66,1.15) 0.87 (0.66,1.15) Quartile (0.81,1.31) 1.02 (0.80,1.30) (0.74,1.27) 0.95 (0.73,1.25) Quartile (1.06,1.71) 1.29 (1.02,1.65) (1.08,1.84) 1.34 (1.03,1.75) P linear trend < < Highest education level 3 Primary school or 238 Referent Referent 193 Referent Referent less Technical or professional school (0.82,1.25) 1.06 (0.86,1.31) (0.78,1.25) 1.04 (0.82,1.32)

6 2480 Risk factors for cancers of unknown primary site Table 2. Hazard ratios and 95% confidence intervals for CUP in relation to smoking, alcohol consumption, levels of education and anthropometric indices of obesity (Continued) CUP CUP with survival time 12 months Secondary school (0.59,1.06) 0.82 (0.61,1.10) (0.56,1.09) 0.82 (0.58,1.14) Longer education (including university) (0.58,0.98) 0.81 (0.62,1.06) (0.50,0.92) 0.75 (0.55,1.01) 1 Stratified by center, sex and age at recruitment. 2 Smoking intensity and average lifetime alcohol consumption were adjusted for levels of education, body mass index and waist circumference, and vice versa; smoking intensity was further adjusted for average lifetime alcohol consumption and vice versa; levels of education were adjusted for smoking intensity, average lifetime alcohol consumption, body mass index and waist circumference. 3 Case numbers do not add up to the total of 651 incident CUP, due to missing information of smoking intensity (N 5 32), average lifetime alcohol consumption (N 5 141), BMI (N 5 17), waist circumference (N 5 51) and levels of education (N 5 76). day versus never smokers. In univariate analyses increased risks were also observed for lifetime alcohol consumption [p trend ], up to a relative risk of 1.81 [ ] for those reporting an average lifetime consumption of >60 g/ day; after adjustment for smoking and anthropometric indices of adiposity; however, these estimates were attenuated and no longer statistically significant (p trend ). When restricting the analysis to CUP who survived only 12 months or less, the relative risk for smoking, in particular, was more pronounced (RR [ ] for 261 cigarettes/day versus never (unadjusted risk model); RR [ ] after additional adjustments for alcohol consumption, level of education and anthropometry); RR [ ] for >40 years versus <25 years of smoking duration among current smokers. No statistically significant interaction between smoking intensity and average lifetime alcohol consumption was observed (P interaction ). Regarding anthropometric indices of adiposity, multivariable risk models adjusting for smoking, level of education and alcohol drinking showed a statistically significant tendency toward increasing risks of CUP with increasing waist circumference (p trend ; RR51.29 [ ] for top vs. bottom quartiles), and no significant association BMI (p trend ). Discussion To our knowledge, this is the first epidemiologic study reporting associations between basic lifestyle-related risk factors and the development of cancers of unknown primary site. Major findings of this prospective study are significant risk associations of CUP with smoking and alcohol consumption, as well as an association of risk with indices of adiposity. The association of risk with smoking was relatively strong, particularly when the analysis was restricted to CUP that had survived only 12 months or less. This relatively strong association suggests that a large proportion of CUP originates from smoking-related primary tumors. In a previous analysis of the EPIC cohort, 8 strong associations were found between smoking and risk of cancers of the lung, larynx and oropharynx with relative risks of 5.95 [ ], 16.0 [ ] and 13.6 [ ], respectively, for current versus never smokers. Moderately strong associations were also seen for cancers of the lower urinary tract (3.54 [ ]). In terms of population attributable fraction, over 80 per cent of tumors of the lung, and around one third of cancers of lower urinary tract could be related to smoking. These observations, combined with a 4 6-fold relative risk of CUP in relation to smoking, strongly suggest that a large part of CUP may have its origins in primary tumors of the lung or lower urinary tract. This observation is in line with those of autopsy findings, 1 microarray profiling studies on likely primary origin of CUP metastatic cells 9 and findings from the Swedish Family-Cancer Database study 10 which, in addition to familial risk associations between CUP and cancers of the lung, kidney, liver, ovary and colorectum also showed associations with cancers of the upper aerodigestive tract and bladder. The relatively strong association of CUP with smoking history might provide some explanation for the sharp downward trend in CUP incidence rates starting around the year 2000, observed in Swedish cancer registry data, as in Sweden there has been a steady decline in the prevalence of smoking since the late 1980 s ( tobacco/media/en/sweden.pdf). While all the above observations would make it likely that smoking is indeed a major lifestyle risk determinant of CUP, some degree of confounding is also theoretically possible. For some of the classified with CUP, the absence of diagnosis of a primary site may have been due to poor general health and advanced stage of disease at first diagnosis, precluding an in-depth diagnostic work-ups and poor clinical condition at first diagnosis could also be associated with smoking status. With regard to our findings on alcohol consumption, it is important to mention that squamous cell tumors not otherwise specified (N 5 37) were deliberately excluded from our analyses, since many of these are identified as metastases in cervical lymph nodes and very likely to have upper aerodigestive tract tumors as a primary site. We therefore speculate

7 Kaaks et al that the moderate increase in risk of CUP at higher levels of alcohol consumption could be indicative of tumors of liver, pancreas and colon as primary sites. The association of risk with adiposity (after adjustment for smoking and alcohol consumption) has no known parallel with lung cancer (for lung cancer, if anything, risk is inversely related to adiposity). However, an elevated waist circumference is generally associated with higher risk of adenocarcinomas of the esophagus and pancreas, for example, as well as colon. A major strength of the present study is the prospective cohort design, which avoids biases that could arise in classical case control comparisons. A possible limitation of the study is that the classification of cancer into CUP may have been based on somewhat heterogenous criteria and practices, as reflected by the between-country variation in the ratio CUP with morphology codes for adenocarcinoma versus undifferentiated carcinomas and by the between-country variations in survival. A further limitation, in this regard, is also that we had no complete data about the location (organ site or lymph node) at which the CUP metastasis had been initially diagnosed. Treatment of CUP is guided by the suspected origin of the primary tumor, and therefore extensive diagnostic workup is generally applied in order to identify the tissue-oforigin. In spite of many new methods that have been introduced for the characterization of CUP, including immunohistochemical and gene expression profiling and advanced imaging techniques, the primary tumor still often evades identification, probably because the primary tumor remains dormant or has disappeared. Our analyses provide further documentation, in addition to autopsy studies, that a substantial proportion of cancers of unknown primary site may have their origin in smoking-related tumors, in particular. References 1. Kr amer A, Gattenl ohner S, Neben K. CUP-Syndrom. Pathologe 2009;30: Randen M, Rutqvist LE, Johansson H. Cancer patients without a known primary: incidence and survival trends in Sweden Acta Oncol 2009;48: Morris GJ, Greco FA, Hainsworth JD, et al. Cancer of unknown primary site. Semin Oncol 2010; 37: Hemminki K, Ji J, Sundquist J, et al. Familial risks in cancer of unknown primary: tracking the primary sites. J Clin Oncol 2011;29: Luke C, Koczwara B, Karapetis C, et al. Exploring the epidemiological characteristics of cancers of unknown primary site in an Australian population: implications for research and clinical care. Aust N Z J Public Health 2008;32: Pukkala E, Martinsen JI, Lynge E, et al. Occupation and cancer follow-up of 15 million people in five Nordic countries. Acta Oncol 2009;48: Bingham S, Riboli E. Diet and cancer the European prospective investigation into cancer and nutrition. Nat Rev Cancer 2004;4: Agudo A, Bonet C, Travier N, et al. Impact of cigarette smoking on cancer risk in the european prospective investigation into cancer and nutrition study. J Clin Oncol 2012;30: Pentheroudakis G, Golfinopoulos V, Pavlidis N. Switching benchmarks in cancer of unknown primary: from autopsy to microarray. Eur J Cancer 2007;43: Hemminki K, Bevier M, Sundquist J, et al. Cancer of unknown primary (CUP): does cause of death and family history implicate hidden phenotypically changed primaries? Ann Oncol 2012;23: Morawietz L, Floore A, Stork-Sloots L, et al. Comparison of histopathological and gene expression-based typing of cancer of unknown primary. Virchows Arch 2010;456: Monzon F, Medeiros F, Lyons-Weiler M, et al. Identification of tissue of origin in carcinoma of unknown primary with a microarray-based gene expression test. Diagn Pathol 2010;5: Handorf CR, Kulkarni A, Grenert JP, et al. A multicenter study directly comparing the diagnostic accuracy of gene expression profiling and immunohistochemistry for primary site identification in metastatic tumors. Am J Surg Pathol 2013; 37: Pavlidis N, Pentheroudakis G. Cancer of unknown primary site. Lancet 2012;379: