Cases
Five hundred and ninety-seven (597) histologically confirmed cases of myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), leukaemia, lymphoma and plasmacell disease (PcD) were collected from the medical records of the two hospitals within a two-years period. The FAB (French American British) and REAL (Revised European-American Lymphoma) classifications were used, since both were used by the attending physicians during the study period.
During the data collection period, 35 cases died before entering the study. There were also new cases enrolled (16), and a number (102) of cases could not be identified due to data missing from the medical records (incorrect address or phone number etc). Out of the 476 available prevalent cases, 354 agreed to participate in the study and completed the questionnaire (response rate 74.36%). Moreover, 325 cases provided blood samples for further studies.
Questionnaire
To ascertain information from both cases and controls a structured questionnaire was developed and pre-tested (see Additional file
1).
The questionnaire included six different sections: 1) demographics, 2) residence, 3) occupation, 4) exposure and agricultural practices, 5) family history and 6) habits and medical history. The demographic section included the hospital where treatment was obtained, level of education and type of insurance. The residence section included full address, chronological residential changes with duration of stay and residential level (urban, suburban or rural). The assignment of the residential level and the coding of the residence were performed using lists from the Ministry of Internal Affairs of Greece. In the occupational section, detailed information on previous employment and other activities were reported.
Subjects who reported at least one year of work in farming were asked to complete the part of the questionnaire on pesticide exposure. The information collected included type of crop, duration of farming, surface area of the farm, characteristics of the adjacent farm and crop infestations. The participants were asked to provide commercial names of pesticide products, number of applications per year, total years of use and type of application (spraying, during planting and usage of treated seeds). To provide help to the participants in recalling the pesticides used, a list of the most common brand names was provided. Moreover, questions were included regarding adherence to application guidelines and familiarity with caution signs/first aid help. The type and purpose of the use of PPE (e.g. uniforms, masks, boots etc) the use of equipment machinery, the frequency of consulting experts, aspects of product handling (e.g. location of storage) and the existence of an integrated farm system were also investigated. In addition, information on certain habits such as changing clothes before entering the house, smoking, eating and accidents (e.g. accidental poisoning), was also collected. Finally, details of work with animals and pet ownership were considered.
In the family history section, all types of cancers, LHC and immunological disorders as well as the family proximity (first, second and third degree relatives) were recorded. Among the immunological disorders rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, inflammatory bowel diseases, sarcoidosis, psoriasis and atopic conditions were included.
Categories for smoking habit and alcohol consumption according to the Centers for Diseases Control and Prevention (CDC) 2002 classifications were adopted [
21]. Information regarding the subject's medical history included other cancers, cardiovascular disorders, respiratory diseases, immunological disorders, inflammatory diseases, endocrine disorders, diabetes mellitus and other metabolic disorders.
The questionnaire was pilot tested among both cases and controls aged 27 to 73 years and modifications were made according to the results obtained. The time needed to fill the questionnaire was reasonable with the only delay at the section regarding the specific pesticides used. The interviews were conducted by a trained occupational physician and the date of diagnosis was used as a reference date for both cases and controls.
Due to changes in pesticide exposure over time, an expert (phytopathologist) evaluated each questionnaire and the data collected regarding pesticide exposure and handling practices, as well as commercial names of pesticides and types of application. All active ingredients were grouped according to the target pest into three major categories: 1) insecticides (organophosphates, carbamates, organochlorines, pyrethroids, avermectins etc), 2) fungicides (inorganic composition e.g. sulfur, disulfide carbamates, benzo-imidazolic composition etc) and 3) herbicides (phenoxy acids, urea products, triazines, bipyridine, nitroanilines etc). According to the chemical composition, the active ingredients were grouped into another three categories: 1) carbamates (carbaryl, propoxur, mancozeb, maneb, thiram, ziram etc), 2) organophosphates (monocrotophos, dimethoate, methamidophos, parathion, chlorpyrifos, methidathion, phosmet etc) and 3) organochlorines (dicofol, dieldrin, heptachlor, endrin, endosulfan etc) [
22].
A new variable was developed for total pesticide exposure (residential, environmental and occupational) for all subjects, in order to classify them in the "Low/No", "Medium" or "High" category. Participants who had never worked in the agricultural sector as their main or secondary occupation and declared urban or suburban residences were classified in the "Low/No" exposure category. For the "Medium" and "High" exposure categories the following equation for total pesticide exposure was developed: number of treatments per year multiplied by total lifetime years of use multiplied by area cultivated (Km2). To classify participants in the "Medium" or "High" exposure category, a cutoff point of 60,000 y·Km2 was set, which is approximately the mean value for total pesticide exposure (59,900 y·Km2). Moreover for participants close to the cut-off value, the following exposure modifying factors were taken into consideration before final categorization in the "Medium" or "High" category: 1) application of pesticides by the person, which possibly enhanced exposure, was considered to be the major exposure modifying factor and, for this reason, the value obtained from the above equation was multiplied by the factor 1.3 2) the type of equipment used during application, which possibly reduced exposure, was considered the second most important exposure modifying factor and, for this reason, the value obtained from the above equation was multiplied by 0.8 3) the use of PPE was considered of minor importance in our assessment for the classification of participants into the "Medium" or "High" exposure category given the improper use of PPE and, in this case, the value obtained from the above equation was multiplied by 0.9.
In order to avoid reporting exposure that occurred after LHC diagnosis and to allow a minimum latency period, the period preceding one year before the reference date for both cases and controls was used for recorded exposure.
Statistical analysis
Standard statistical procedures were carried out using the epidemiological software Epi-info (version 3.4.3.) and the Statistical Package for Social Sciences (SPSS) (version 15.0). Data were validated for completeness before statistical analysis was performed. Descriptive analysis was conducted for each variable including frequencies, ranges, means ± SD, median values and interquartile ranges (IQR) for both cases and controls. The chi-square or Fisher exact test was used to analyze qualitative data, whereas the student
t-test or Mann-Whitney test was used for quantitative data. Total pesticide exposure and smoking habit as well as smoking and eating during farming were made categorical (yes/no) variables. Agricultural occupations were grouped together (farmer, pesticide applier, seasonal farm worker, animal breeder) and compared to the group of other occupations (e.g. teacher, trader, public servant, unemployed etc.) Backward conditional logistic regression analysis was used to control for confounders and to identify independent risk factors. Exposure to pesticides was an independent categorical variable. Each histological group and the group comprising all LHC cases were analyzed separately. To assess the dose-response relationship between exposure and disease, the chi square test for trend was used. Moreover, age, sex, smoking and family history (all types of cancer, LHC and immunological disorders) were included as confounders. Afterwards, a multinomial logistic regression (polytomous regression) model was used to evaluate whether the risk factors differ across the histological groups and the total LHC group [
23]. In this model LHC was the dependent variable, and pesticide exposure was the independent categorical variable. Smoking during application, age and sex were confounders. All control subjects were included in the statistical analysis of each histological group. The analysis was conducted separately for the five histological groups (Group 1: MPD, Group 2: MDS, Group 3: leukaemia, Group 4: lymphoma and Group 5: PcD) and for all the LCH cases. Differences were considered statistically significant when the two-sided p value was ≤ 0.05.