RCMD is the most common type of MDS in China,11 and involves cytopenia and dysplasia of multiple cell lineages. It comprises 71% of all MDS cases in this series. The ORs for EG2 to EG4 were each statistically significant in both crude and adjusted models, with adjusted ORs of 3.19 (95% CI: 1.25, 8.12), 5.38 (95% CI: 1.36, 21.3), and 3.75 (95% CI: 1.53, 9.21), respectively (see Fig. 1 and Table 4). The linear trend was significant (P < 0.01). The confounding by farm residence observed in the all-MDS analysis persisted in the RCMD analysis to an even larger extent, resulting in a 15% to 16% lowering of ORs in EG3 and EG4. An alternative multivariable analysis combining EG3 and EG4 generated the following results: EG1 OR = 1.72 (95% CI: 0.84, 3.50); EG2 OR = 3.15 (95% CI: 1.24, 7.99); EG3&4 OR = 4.20 (95% CI: 1.98, 8.90).
RCMD was also a driver for elevated benzene-related risks observed for all multilineage subtypes combined (Table S4, http://links.lww.com/JOM/A328) which were 92% RCMD. Cases in the EG3 and EG4 exposure groups were over-represented by rubber/plastic manufacturing (6 cases/1 control) and agriculture (7 cases/5 controls) occupations. Of the 53 RCMD cases exposed to benzene, 26 (49%) were last exposed more than 10 years before diagnosis, while only 30% of other MDS diagnoses were exposed more than 10 years prediagnosis.
RAEB and RAEB Stages
RAEB represents the most advanced stage of MDS, and poses a relatively higher risk of advancing to acute myeloid leukemia (AML). Benzene exposure by EG showed no clear relationship with RAEB, although the precision of the effect estimates was less than that for RCMD (see Fig. 1 and Table 5). An ever/never benzene parameterization resulted in an essentially null finding (OR = 1.10; 95% CI 0.46, 2.65). There were too few cases to analyze RAEB I and RAEB II separately. A history of ever smoking was observed to be a statistically significant and independent risk factor (OR = 2.96; 95% CI: 1.37, 6.39) for RAEB. Other adjustment covariates were: exposure to fertilizer, BMI, and diabetes; the latter was negatively associated with RAEB (OR = 0.28; 95% CI 0.10, 0.77).
RCUD is a combined category comprised of refractory anemia (RA, 39 cases) and refractory cytopenia with unilineage dysplasia (RCUD) other than RA (15 cases). For the 54 combined cases, nine were exposed to benzene (see Fig. 1 and Table S5, http://links.lww.com/JOM/A328although seven of these nine were from the smaller subgroup—that is, RCUD other than RA. Furthermore, seven of the nine exposed cases were exposed to more than 3 ppm (EG3 and EG4). Since there were no matched controls exposed above 3 ppm, risks for benzene exposure are not estimable (eg, infinite) for total RCUD via logistic regression. Approximate ORs can be calculated based on Gart and Zweifel13; for exposures above 3 ppm, RA shows an OR of 9.9 (95% CI: 0.46 to 212), while RCUD other than RA shows an OR of 39.4 (95% CI: 1.97 to 787). Thus, the statistical association between benzene and all types of RCUD was driven by a relatively high proportion (7/15, or 46%) of non-RA cases of RCUD exposed to benzene. However, these findings are imprecise based on the wide confidence intervals.
This hospital-based case-control study of benzene exposure and major subtypes of MDS indicate a consistent relationship between benzene exposure and aggregate MDS which is more pronounced for RCMD. Notably, there was no suggestive relationship with benzene exposure for refractory anemias, a grouping which includes RAEB, the subtype with the highest propensity to progress to AML. Despite the relatively low number of RCUD cases and the accompanying statistical imprecision, evidence for benzene effects above 3 ppm was also suggestive for the non-RA fraction. Some of the rarest MDS subtypes (eg, RARS, MDS with 5q-, MDS-U) precluded informative statistical analyses.
One common feature of RCMD and non-RA RCUD is the presence of non-erythroid dysplasia (not necessarily at the exclusion of erythroid dysplasia). While the specific mechanism in which benzene interacts with myeloid cells in the bone marrow to produce MDS is not known, these statistical associations show a preference for benzene to ultimately result in MDS involving lineages that are not predominantly erythroid.
Our study suggests that benzene (as well as other agents) may affect different WHO subtypes differently. Most notably, besides the observed effect of benzene on subtypes which are not predominantly erythroid, the elevated risk for smoking shows the opposite effect, as it appears to be restricted to all refractory anemias, including RAEB.
Farm Residence and Agricultural Exposures
Although the primary focus of this study was on benzene exposure, our results are in concert with several previous studies which report that MDS is likely influenced by several endogenous and environmental factors. Farm residence, past or present, was an independent risk factor for most of the MDS subtypes and groupings, and was the only substantial confounder among the covariates. However, it had limited inferential impact on the benzene-MDS relation given the strength of the ORs for benzene exposure. In a previous US study of similar design, Strom et al4 observed a positive exposure–response gradient between total MDS and agricultural chemical exposures in men in their unadjusted results for RA/RARS and for RAEB in multivariable analyses. Exposure to farm/agrichemical exposures has also been reported to decrease survival in some MDS patients.14
Smoking was not positively associated with total MDS or RCMD; however, it showed a positive association with RAEB, especially advanced RAEB (RAEB II, OR = 3.2) and all RAs, OR = 2.6, both of which were statistically significant. Du et al15 conducted a meta-analysis of 10 case-control studies that examined the relation between smoking and MDS overall, finding an overall risk of 1.45 (95% CI 1.21 to 1.74); all studies except one reported a positive association. One of those studies4 examined this relation by MDS subtype, observing independent positive associations between smoking and RA/RARS and with RAEB which were consistent with the present study. Smoking is also a well-known risk factor for AML.16 The specific association of cigarette smoking with RA and RAEB in our study has a biologic basis since these subtypes have a greater propensity for clonal cytogenetic abnormalities versus other MDS subtypes, and cigarette smoke contains genotoxic substances such as PAH's and formaldehyde.
Diabetes was a remarkably persistent protective factor, as fewer cases had had diabetes versus controls. No clear associations between diabetes and MDS have been previously reported.17–19 Since diabetes was not excluded from control diagnoses, recurring hospitalizations among diabetic controls due to a myriad of potential complications could likely have produced a spurious protective association. Whether the reduced risk of MDS for those with a history of diabetes has a biologic basis or is related to the use of a hospitalized control population cannot be resolved further.
MDS risk is seemingly reduced 3% to 10% for every increased unit of BMI, depending on subtype. Since obesity is associated with diabetes and other chronic diseases, the negative BMI association could, like diabetes, be biased low due to the use of hospitalized controls. Hainer and Aldhoon-Hainerova20 suggest that a reduced BMI risk for serious diseases (such as MDS) may be due to controls being more robust (ie, having higher BMIs), since illnesses for which control individuals were hospitalized were likely, on average, to be of lesser clinical severity than MDS. This explanation is further supported considering that BMI showed the most protective relationship with RAEB, the subtype with the worst prognosis.
Educational attainment was associated with all MDS subtypes and with RCMD alone. The educational gradient showed evidence of positive linear trend (P < 0.01) from the lowest to highest attainment levels, and also showed a non-confounded independent relation with MDS. Education has frequently been used as a surrogate for socioeconomic position which is often a reliable predictor of many health outcomes.
MDS and Benzene
While benzene has historically been linked to acute leukemia,21 and acute myeloid leukemias,22 its link to MDS has been investigated only recently. MDS was formally classified as a disease in 1982, and became reportable in cancer registries as late as 1999. One of the first studies reporting a potential link between benzene and MDS reported excess risk for the combined diagnoses of AML and MDS.1 More recently, an international pooled analysis in petroleum distribution workers reported significant OR's for MDS in workers exposed to peak benzene exposures more than 3 ppm; these same workers were predominantly exposed to average concentrations less than 1 ppm.3 More recently, two cohort studies in similar workers have neither confirmed nor refuted these observations.23,24
Irons et al2 using the same database, compared benzene “signal” MDS cases with high (more than 20 ppm) average benzene exposure to other MDS cases with only background benzene exposure. Signal cases were 11.1 times more likely to have MDS-U (based on the 2001 WHO nomenclature) versus non-signal cases. However, this study likely over-represented MDS-U cases that were provisionally diagnosed based on the limited diagnostic information available at case presentation. Based on the more complete follow-up information used in the present study, MDS-U diagnoses were markedly less prevalent: only five of 604 cases in the present study, versus 29 of 649 previously.
Previous studies have revealed that MDS cases with a history of benzene exposure exhibited distinct clinical, immunologic and cytogenetic features.2,25 Bone marrow findings included evidence of inflammation and eosinophilic dysplasia in addition to multilineage dysplasia. This was accompanied by increased CD8+ T cells with clonal and oligoclonal TCR gene rearrangements in T cell populations.2,25,26 These findings suggest a role for an autoimmune pathogenesis for typically low-risk MDS cases that is frequently associated with RA and a subset of RCMD with immunologic features. Several authors have demonstrated that a proximate cause of progressive bone marrow cytotoxity in some cases of MDS is the presence of activated CD8+ lymphocytes which produce persistent inflammation, apoptosis, and cytotoxicity via production of TNF-α.27–31 These findings are also consistent with those of Kordasti et al32 in which cytotoxic +CD8+ T cells predominate in low risk MDS, while increased expression of Foxp3+ regulatory CD4+ T cells correlate with progression of high risk MDS, that is, RAEB.
Benzene has been shown to be non-discriminatory on specific cell line lineages in terms of its non-neoplastic hematotoxic effects. It has long been known to produce pancytopenia,33 while more recent studies34,35 show that all prominent cell types (leukocytes, erythrocytes, platelets) are reduced in workers when there is sufficient benzene exposure. This is due to benzene's effect on bone marrow function rather than specific circulating cells per se. The association reported here with RCMD, which also involves multiple lineages, is consistent with previous hematotoxicity observations.
RAEB is the MDS subtype that has the highest rate of progression to AML, and benzene has been linked to AML more consistently than any other hematopoietic cancer.36 Accordingly, one might have predicted that RAEB would be the subtype most likely to show a relationship with benzene. The fact that our data do not suggest this relationship may point to distinct etiologies for benzene-induced AML and MDS, but this is not necessarily so. In fact, recent data suggest a similar pattern of cytogenetic abnormalities in benzene-induced AML, MDS and de novo cases.26 Given the limited follow-up data, we could not ascertain whether RCMD cases progressed to either RAEB and/or AML. It is possible that benzene-induced MDS is the stronger response when compared with benzene-induced AML, and spontaneous transformation of benzene-induced MDS to AML (if it occurs at all) may be governed more by stochastic mechanisms rather than continued benzene exposure. Addressing this hypothesis would require sustained follow-up of populations exposed to appreciable levels of benzene.
Study Strengths and Limitations
Strengths include the large number of cases, standardized diagnosis by a single reference laboratory, well-matched controls, and collection of extensive information on occupational exposure, lifestyle, and other individual-level characteristics. Robust retrospective benzene exposure assessment methods were employed in a blinded fashion, with estimates validated by an expert panel. The ability to examine MDS subtypes using the most current WHO diagnostic criteria made concurrent with treatment is another distinctive aspect of this study. Likewise, the high level of post-collection quality checks on the research dataset enhances the validity of the findings.
The primary study limitation is our use of hospital-based controls, a practice that could result in control group that does not represent the base population that gave rise to the cases. However, the controls were matched on age and sex, were admitted close to the case's admission date, and were patients from the same hospitals as the cases, in toto reducing the opportunity for selection bias.37 QA/QC procedures documented the absence of blood and lymphoid diseases among controls and confirmed that the most frequent hospital departments for controls were cardiovascular, respiratory, internal medicine, and endocrine. Benzene risk estimates would be unaffected unless the control diagnoses were somehow related to a higher or lower opportunity for benzene exposure. Controls were significantly less likely than cases to have ever lived on a farm (34% vs. 48%, P < 0.01), which was an independent MDS risk factor in these data. Nevertheless, the potential impact from such a selection bias was lessened by control for farm residence (as a confounder) in the calculation of benzene risk estimates, and by the fact that the study covered a demographically fluid period in China. Specifically, between 2000 and 2010, the rural population of China decreased from 64% to 50%,38 while the Shanghai population increased from 13.3 million to 16.4 million.39 While we cannot totally rule out selection bias, the consistency with previous findings on benzene and key covariates provides some degree of certainty and credence to our findings.
Another limitation is the use of questionnaires, subject to recall bias or socially acceptable responses, to collect data on several co-exposures, and lifestyle factors. In addition, we limited this assessment to long-term benzene concentrations and did not study the duration of such exposures, although most workers were long-term workers with very few job changes. While the exposure estimating process was rigorous and subject to validation,9 some misclassification still likely occurred although the ordinal exposure groupings likely reduced the impact of such errors.
In this study population, we observed statistically significant associations between benzene and MDS, which were largely driven by RCMD, in which statistically significant associations were observed for concentration estimates of 0.3 to 3 ppm, 3 to 12 ppm, and more than 12 ppm. No associations between benzene exposure and RAEB, the MDS subtype most likely to progress to AML, were observed. Another relationship with higher uncertainty was found for RCUD not classified as RA; together these findings suggest that benzene affects MDS cell types that are not predominantly of erythroid cell lineages. While selection bias is a potential threat to the magnitude of our benzene-specific findings, it would not be expected to differ by MDS cell-type. The findings suggesting benzene's effect on non-erythroid MDS may provide important clues toward uncovering a more precise mechanistic model for benzene-induced MDS.
We thank Lauren Green for final quality control checks on the data and Gail Jorgensen for assembling the data and programming.
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