Postmenopausal Hormone Therapy and Colorectal Cancer Risk by Molecularly Defined Subtypes and Tumor Location

Abstract Background Postmenopausal hormone therapy (HT) is associated with a decreased colorectal cancer (CRC) risk. As CRC is a heterogeneous disease, we evaluated whether the association of HT and CRC differs across etiologically relevant, molecularly defined tumor subtypes and tumor location. Methods We pooled data on tumor subtypes (microsatellite instability status, CpG island methylator phenotype status, BRAF and KRAS mutations, pathway: adenoma-carcinoma, alternate, serrated), tumor location (proximal colon, distal colon, rectum), and HT use among 8220 postmenopausal women (3898 CRC cases and 4322 controls) from 8 observational studies. We used multinomial logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CIs) for the association of ever vs never HT use with each tumor subtype compared with controls. Models were adjusted for study, age, body mass index, smoking status, and CRC family history. All statistical tests were 2-sided. Results Among postmenopausal women, ever HT use was associated with a 38% reduction in overall CRC risk (OR =0.62, 95% CI = 0.56 to 0.69). This association was similar according to microsatellite instability, CpG island methylator phenotype and BRAF or KRAS status. However, the association was attenuated for tumors arising through the serrated pathway (OR = 0.81, 95% CI = 0.66 to 1.01) compared with the adenoma-carcinoma pathway (OR = 0.63, 95% CI = 0.55 to 0.73; Phet =.04) and alternate pathway (OR = 0.61, 95% CI = 0.51 to 0.72). Additionally, proximal colon tumors had a weaker association (OR = 0.71, 95% CI = 0.62 to 0.80) compared with rectal (OR = 0.54, 95% CI = 0.46 to 0.63) and distal colon (OR = 0.57, 95% CI = 0.49 to 0.66; Phet =.01) tumors. Conclusions We observed a strong inverse association between HT use and overall CRC risk, which may predominantly reflect a benefit of HT use for tumors arising through the adenoma-carcinoma and alternate pathways as well as distal colon and rectal tumors.


Description of Study Populations
The Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) is a collaborative effort comprised of a coordinating center and scientific researchers from wellcharacterized cohort and case-control studies conducted in North America and Europe. Summary descriptions and study participant inclusions/exclusion criteria for each of the studies included in our analysis are detailed below. For all studies, only participants with available tumor molecular characterization were included in analysis.

Cancer Prevention Study II (CPS II)[1, 2]
The CPS II Nutrition cohort is a prospective study of cancer incidence and mortality in the United States, established in 1992. At enrollment, participants completed a mailed selfadministered questionnaire including information on demographic, medical, diet, and lifestyle factors. Follow-up questionnaires to update exposure information and to ascertain newly diagnosed cancers were sent biennially starting in 1997. Reported cancers were verified through medical records, state cancer registry linkage, or death certificates. Controls were matched on race, gender, and age. The Emory University Institutional Review Board approves all aspects of the CPS II Nutrition Cohort.
Colon Cancer Family Registry (CCFR) [3] The CCFR (www.coloncfr.org) is a National Cancer Institute-supported consortium consisting of six centers dedicated to the establishment of a comprehensive collaborative infrastructure for interdisciplinary studies in the genetic epidemiology of colorectal cancer. The CCFR includes data from approximately 42,500 total subjects (10, 500 case probands and 26,900 unaffected and affected relatives, 4,280 unrelated population-based controls, and 920 spouse).
Cases and unaffected controls, age 20 to 74 years, were recruited at the six participating centers beginning in 1998. All participants self-completed a standardized questionnaire that asked about established and suspected risk factors for colorectal cancer, including questions on medical history and medication use, reproductive history (for female participants), family history, physical activity, demographics, alcohol and tobacco use, and dietary factors. Participants from three participating centers (Seattle, Australia, Ontario) were included in this study.
Darmkrebs: Chancen der Verhütung durch Screening (DACHS) [4,5] This German study was initiated as a large population-based case-control study in 2003 in the Rhine-Neckar-Odenwald region (southwest region of Germany) to assess the potential of endoscopic screening for reduction of CRC risk and to investigate etiologic determinants of disease, particularly lifestyle/environmental factors and genetic factors. Briefly, cases with a first diagnosis of invasive CRC (International Classification of Diseases 10 codes C18-C20) who were at least 30 years of age, German speaking, resident in the study region, and mentally and physically able to participate in a one-hour interview, were recruited by their treating physicians either in the hospital a few days after surgery, or by mail after discharge from the hospital. Cases were confirmed based on histologic reports and hospital discharge letters following diagnosis of CRC. All hospitals treating CRC cancer patients in the study region participated. Communitybased controls were randomly selected from population registries, employing frequency matching with respect to age (5-year groups), sex, and county of residence. Controls without a history of CRC were contacted by mail and follow-up calls. During an in-person interview, data were collected on demographics, medical history, family history of CRC, and various lifestyle factors, as were blood and mouthwash samples.
Diet, Activity, and Lifestyle Study (DALS) [6] DALS was a population-based, case-control study of colon cancer. Participants were recruited between 1991 and 1994 from 3 locations: the Kaiser Permanente Medical Care Program of Northern California, an 8-county area in Utah, and the metropolitan Twin Cities area of Minnesota. Eligibility criteria for cases included age at diagnosis between 30 and 79 years, diagnosis with first primary colon cancer (International Classification of Disease for Oncology, Second Edition, 18.0 and 18.2-18.9) between October 1, 1991, and September 30, 1994, English speaking, and competency to complete the interview. Individuals with cancer of the rectosigmoid junction or rectum were excluded, as were those with a pathology report noting familial adenomatous polyposis, Crohn's disease, or ulcerative colitis. A rapid-reporting system was used to identify all incident cases of colon cancer, resulting in the majority of cases being interviewed within 4 months of diagnosis. Controls from the Kaiser Permanente Medical Care Program were selected randomly from membership lists. In Utah, controls younger than 65 years of age were selected randomly through random-digit dialing and driver license lists. Controls 65 years of age and older were selected randomly from Health Care Financing Administration lists. In Minnesota, controls were identified from Minnesota driver license or state identification lists.

European Prospective Investigation into Cancer (EPIC) -Sweden[7]
EPIC is an on-going multicenter prospective cohort study designed to investigate the associations between diet, lifestyle, genetic and environmental factors and various types of cancer. Briefly, 521,448 participants (~70% women) mostly aged 35 years or above were recruited between 1992 and 2000. Participants were recruited from 23 study centers in ten European countries. The current study included participants from Sweden. All study participants provided written informed consent, and ethical approval for the EPIC study was obtained from the review boards of IARC and local participating centers. Cases were identified by linkage with the essentially complete Cancer Registry of Northern Sweden and were verified by a gastrointestinal pathologist. Controls were selected from the full cohort of individuals who were alive and free of cancer (except non-melanoma skin cancer) at the time of case diagnosis.
Melbourne Collaborative Cohort Study (MCCS) [8,9] The MCCS is a prospective study that recruited 41,514 healthy adult participants aged between 27 and 76 years (99% aged 40-69) from the Melbourne metropolitan area between 1990 and 1994. The goal of this study was to examine the role of lifestyle factors in the risk of cancer and heart disease. Incident cases of CRC were identified through linkage to population-based cancer registries in Australia. Cases include participants with a histopathological diagnosis of invasive colorectal adenocarcinoma following the baseline study visit, informed consent, and sufficient FFPE material for somatic testing. Study protocols were approved by the Human Research Ethics Committee at the Cancer Council Victoria.

Nurses' Health Study (NHS)[10]
The NHS cohort began in 1976 when 121,700 married female registered nurses age 30-55 years returned the initial questionnaire that ascertained a variety of important health-related exposures.1 Since 1976, follow-up questionnaires have been mailed every 2 years. Colorectal cancer and other outcomes were reported by participants or next-of-kin and followed up through review of the medical and pathology record by physicians. Overall, more than 97% of selfreported colorectal cancers were confirmed by medical-record review. Information was abstracted on histology and primary location. The rate of follow-up evaluation has been high: as a proportion of the total possible follow-up time, follow-up evaluation has been more than 92%.
Colorectal cancer cases were ascertained through June 1, 2008.
Northern Swedish Health and Disease Study (NSHDS) [11,12] NSHDS, which has been described in more detail elsewhere, comprises over 110,000 Programme, in which all residents of Västerbotten County were invited to a health examination upon turning 30 (some years), 40, 50 and 60 years of age. Extensive measured and self-reported health and lifestyle data were collected at the health exam.

Microsatellite Instability (MSI) Status
In addition to polymerase chain reaction as outlined in the manuscript and Supplemental Table 1, MSI testing was conducted using standardized immunohistochemical (IHC) detection of deficiency for mismatch repair gene proteins MLH1, MSH2, MSH6, and PMS2 for NSHDS and EPIC [11] as well as a subset of CCFR [13,14] and MCCS samples without PCR-based MSI characterization. MSI status was also determined using a mononucleotide marker panel [15] (DACHS) and a panel of two mononucleotides (BAT26 and TGFβRII) and 10 tetranucleotide repeats [16][17][18] (DALS). Both the methods used in DACHS and DALS have been shown to have high concordance with the Bethesda Consensus Panel. [19] Tumoral and normal DNA were PCR amplified with these 12 primer sets, and MSI was defined as > 1 new PCR product either smaller or larger than those produced from normal DNA. For BAT26, the PCR product from tumor had to be >4 base pairs smaller than that from germline. For the tetranucleotide repeat panel, MSI status was based on >30% markers showing instability and MSI-L/MSS if <30% of repeats were unstable.

Harmonization of Environmental Data
We carried out a multi-step data harmonization procedure, reconciling each study's unique protocols and data-collection instruments at the GECCO coordinating center (Fred Hutchinson Cancer Research Center). First, we defined common data elements (CDEs). We examined the questionnaires and data dictionaries for each study to identify study-specific data elements that could be mapped to the CDEs. Through an iterative process, we communicated with each data contributor to obtain relevant data and coding information. The data elements were written to a common data platform, transformed via a SQL programming script, and combined into a single dataset with common definitions, standardized permissible values, and standardized coding. The mapping and resulting data were reviewed for quality assurance, and range and logic checks were performed to assess data distributions within and between studies.
Outlying samples were truncated to the minimum or maximum value of established range for each variable.
For CCFR, DALS, NHS, and NSHDS, post-menopausal hormone therapy use was ascertained as use at the reference period. The reference period time was defined as 2 years preceding enrollment for CCFR and DALS, use in 1990 for NHS, and current use for NSHDS.
For CPS-II, DACHS, EPIC, and MCCS, post-menopausal hormone therapy use was ascertained as ever vs. never use.

Supplementary Tables
Supplementary Table 1 Table 4. Baseline characteristics of 1) 8,637 post-menopausal women, comparing women in our analytical population to women who were excluded due to missing postmenopausal hormone therapy (HT) data; 2) 8,220 postmenopausal women with HT data, comparing women with formulation-specific HT data to those without; 3) 3,898 postmenopausal CRC cases, comparing women who could be classified by pathway to those who could not