The earliest reports of high incidence of esophageal cancer (EC) in the northern parts of Iran date back to the early 1970s. A population-based cancer registry was established in 1969 as a joint effort between Tehran University and the International Agency for Research on Cancer (IARC).  This registry confirmed the high incidence of EC in the eastern portion of the Caspian Sea littoral, in the area which is now known as Golestan Province.  The highest incidence rates were reported from the semi-desert plain settled mainly by people of Turkmen ethnicity in Gonbad and Kalaleh counties, with estimated incidence rates of 109/10⁵ among men and 174/10⁵ among women (adjusted to the 1970 World Standard Population). The registry also showed low incidence of EC in the nearby Gilan province, 300 km to the west of Golestan, with incidence rates of 15/10⁵ and 5.5/10⁵ among men and women, respectively.  A series of studies were conducted in the region in the 1970s, but they were not conclusive in explaining the very high rates.  However, they pointed to several factors, including: (i) a diet deficient in fruits and vegetables; (ii) low socioeconomic status; (iii) thermal injury from consumption of very hot tea; and (iv) carcinogen exposure from lifestyle factors including opium consumption. The high incidence of EC in Golestan was confirmed by a recent screening study.

Etiological hypotheses related to diet and life style can be best addressed in prospective cohort studies, in which measurement error can be reduced and recall bias is minimal. From 2002 to 2003, a pilot study of 1057 subjects was conducted by the Digestive Disease Research Center (DDRC) of Tehran University of Medical Sciences in collaboration with IARC and the US National Cancer Institute (NCI) to evaluate the logistical aspects of establishing a prospective study in Golestan.  The aims of the pilot study were to assess the response rate of the study population, to develop valid and reliable methods for assessing nutritional, anthropometric and life style factors, to develop follow-up methods to ascertain mortality and cancer incidence among the enrolled subjects, and to establish efficient procedures for collecting and storing biological samples.  Results of the pilot study confirmed the feasibility of conducting a prospective cohort study in Golestan. Subsequently, the Golestan Cohort Study (GCS) was launched in January 2004.  The study protocol and the informed consent used for this study were approved by the ethical review committees of DDRC, IARC, and NCI.  In June 2008, the accrual goal of 50 000 subjects was reached and enrollment was closed. 

The primary aims of the GCS are:

(i) To identify risk factors for EC by a comprehensive assessment of ethnicity, occupational history, socioeconomic status, past medical history, family history of cancers, gastrointestinal symptoms and signs, tobacco, opium and alcohol use, oral health, anthropometric characteristics, physical activity, and tea drinking habits, including tea temperature. Nutritional patterns are also evaluated using a food frequency questionnaire (FFQ) specifically developed for this population and validated during the pilot study. The FFQ covers 116 food items, including bread and cereals, meat and dairy products, oils, sweets, legumes, vegetables, fruits, and condiments, as well as cooking methods.

(ii) To establish bio-specimen banks for blood, urine, hair, and nail samples to be used in molecular and genetic studies of cross-sectional or nested case-control design.

(iii) To provide a model for population-based studies in a country in economic and social transition based on collaboration between local health workers, local health authorities, national research centers, national government, and international research institutions.

The study population is a sample of the Golestan population, aged 40-75 years.  The primary goal was to establish a cohort of 50 000 healthy individuals, with equal numbers of men and women, 20% from urban areas, and 80% of Turkmen ethnicity.  We planned to enroll the urban participants from Gonbad City, the second largest city of Golestan with 126 797 inhabitants (28 102 aged 40-75), and the rural participants from villages in Gonbad, Kalaleh, and Aq-Qala counties (Figure 1), with 347 683 inhabitants (53 121      aged 40 – 75). 

A total of 16 599 urban inhabitants older than 40 years were selected randomly from five areas of Gonbad City by systematic clustering based on the household number.  The selected inhabitants were contacted at home by specially-trained health workers and invited to visit the Golestan Cohort Study Center, a research center specifically established for this project in Gonbad, and to participate in the study.  A total of 10 032 urban participants were enrolled from Gonbad, with participation rates of approximately 70% for women and 50% for men.

In rural areas, recruitment took advantage of the network of health houses, primary health care centers present in each group of villages, which are typically staffed by two auxiliary health personnel (locally called the Behvarz).  The Behvarz are in charge of vaccination programs, family planning, reporting births, deaths, and major communicable diseases, and initial primary care treatment. All residents of all villages in the study catchment area who were eligible for this study were invited to participate.  Temporary recruitment centers were established in the health houses of 198 selected villages, and the Behvarz accompanied the GCS research team to contact the selected subjects at their homes.  The invitation group thoroughly explained the purpose and procedures of the study to the eligible subjects and invited them to participate in the study.  If the study participant did not fully understand the procedures, he/she was invited to visit the study center and observe all steps of the study in person.  A total of 40 013 participants were enrolled from 326 villages, with participation rates of 84% for women and 70% for men.

Exclusion criteria were: (i) unwillingness to participate at any stage of the study for any reason, (ii) being a temporary resident, and (iii) having a current or previous diagnosis of an upper gastrointestinal (UGI) cancer. Before interview, a written informed consent was obtained from each participant.

Each subject was interviewed by a trained general physician and a trained nutritionist, either in the local language (Turkmen) or in the national formal language (Persian), depending on the participant’s preference.  Two structured questionnaires were administered, a lifestyle questionnaire and a food frequency questionnaire.  Following the questionnaires and a limited physical examination, samples of blood (10 mL), urine (4.5 mL), hair (3 cm from the base of scalp) and nail (trimmings from all 10 toenails) were collected by a trained technician.  In the urban area, all biological samples were immediately processed in the central laboratory at the Golestan Cohort Study Center.  In the rural areas, blood samples were kept in refrigerators (+4° C), until they were transferred within cooling boxes to the central laboratory; the maximum duration between blood collection and final processing was 8 hours. The blood samples were centrifuged and aliquoted in 500 μL straws (8 straws of plasma, 4 straws of buffy coat, and 2 straws of red blood cells) and stored at -80° C.  Urine samples were stored at -20° C, and hair and nail samples were stored at room temperature.  Half of the frozen blood samples were subsequently transferred on dry ice to DDRC in Tehran, and then shipped at regular intervals to IARC in Lyon, France, where they are stored in nitrogen vapour (approximately -135° C).

All participants received a personal GCS identification card at the time of enrollment which allows them to come to Atrak Clinic if they experience any gastrointestinal symptoms.  Atrak Clinic is a specialized gastrointestinal clinic established by DDRC in the main hospital in Gonbad, and provides free services for the GCS participants.

Table 1 shows the composition of the cohort.  The distribution by ethnicity and place of residence is close to the initial goal; however, because of a higher response rate, the number of women in the cohort (n = 28 804) is higher than that of men (n = 21 241).

Table 1. Age, sex, ethnicity, and place of residence of the 50 045 participants in the Golestan Cohort Study (2004 – 2008)

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