This manuscript describes a case-control replication study in which, due to a change in the standard of care, the majority of cases received an immune-modulating factor: temozolomide. The inverse association of self-reported allergy and glioma (including a dose-response) here was nearly exactly the same as our previous series, indicating a robust association5, 7
. IgE levels were also inversely related to glioma although far more weakly than we previously observed and most of the inverse relationship was confined to analyses including only cases who had received temozolomide as a treatment. This inverse association may also be impacted by length of temozolomide treatment (). Despite this introduction of temozolomide as the standard of care, the prevalence of elevated IgE levels among cases was paradoxically increased
in the current series (Series 3), reducing IgE case control differences observed previously7
. Self-reported allergies were more frequent in the current series among cases (20% in Series 3 vs. 13% in Series 2), suggesting some potential cohort differences between the Series, which may also impact IgE levels. Both new treatments and a potential new cohort of individuals in Series 3 have then complicated this replication study with regards to IgE as a variable impacting glioma status.
During the early stages of glioma treatment, temozolomide is given daily, typically over the same time course as radiation treatments. Following initial treatment, temozolomide is prescribed in 5-day “on” and 23 day “off” recovery periods. It appears to be during this subsequent period that IgE levels were lower, and case-control odds ratios become significant (, Supplementary Table 3
). As these were cross-sectional data, the possibility remains that the lower levels were due to other unmeasured factors. A similar loss of immune function parameters (CD4 T-helper cells) was found in a study of melanoma patients treated with temozolomide over a longer period13
. CD4 cells were not significantly lost after the first treatment cycle but became more severe with subsequent cycles13
, mirroring the lymphopenia effect noticed in glioblastoma patients9
and the fall in IgE shown here (). Whether temozolomide may actively suppress IgE levels cannot be answered with the current data series and requires a study of repeat measurements during therapy. The one patient with repeat measurements here indicated that IgE levels are stable over a one month period during later stages of treatment, and are not sensitive to changes in T-regulatory cells among other cell types (Supplementary Table 4
). This patient does not inform what happens to IgE during initial therapy, which is a question that will require additional studies.
We did not detect case-control differences in IgE levels with cases not treated with temozolomide, who comprised 37% of the patient cohort. No significant differences in gender, income, ethnicity, or histopathological diagnosis were found among patients who did not take temozolomide compared to those who did (). However, there was a strong inverse association of temozolomide therapy and age – older individuals were less likely to be prescribed temozolomide. In our previous series the relationship between IgE and case-control status was weakest in the oldest tertile (Supplementary Table 2
). Assuming a similar profile for the current Series 3, we would expect less of an IgE difference between older cases and controls, which constitute the bulk of the patients not
treated with temozolomide. This factor, combined with unknown clinical considerations directing the decision not to treat with temozolomide, make it difficult to compare the temozolomide treated and untreated patients. Like our previous study, we did not detect significant effects from dexamethosone on IgE levels, nor other medications outside of the cytotoxic chemotherapeutics.
A remaining possible explanation on IgE differences between the two cohorts may be that the patients themselves are fundamentally different in Series 3, having grown up in the post-World War II era. Interestingly, the prevalence of childhood allergies is quite different between these groups, reported at 13% in Series 2 and 20% in Series 3 (P = 0.02). This corresponds to the rise of suburbanization in the United States with concomitant rise in the prevalence of allergies14–16
. Indeed, the average of 50 years of age for those with IgE measurements in Series 3 corresponds to birthdates between 1950 and 1954, which puts the bulk of participants at a post-WWII birth when modern trends including reduced exposure to microbes and endotoxins, intestinal parasites, and increased use in antibiotics, affected the prevalence of allergies and potentially affected the allergic axis in the interaction with brain tumors. The increases in allergies are attributed to the “hygiene hypothesis” which posits that the immune system has not had a normal early modulation from frequent infections, making it vulnerable to overreact to other environmental antigens17
. This enhanced predisposition to overreact is sometimes referred to as the “missing immune deviation” hypothesis, since humans are born with a strong Th2-allergic-phenotype which is poised to develop balanced immunity in response to frequent infections early in life18
. If the immune deviation to a balanced Th1/Th2 immune system does not occur early in life, then lifetime risk of allergic disease is increased. Our control population had the same IgE distribution in the current series as the prior series7
, indicating that profound population shifts have not taken place; however, our cases had higher IgE levels in the current series. It may be possible that these cases are reacting to their tumor or to temozolomide treatment (initially) with higher IgE levels due to an enhanced predisposition to allergies due to their birth in the modern era. Further studies on a possible cohort effect are warranted.
For the current series we obtained sera from a higher proportion of cases than for our prior series – and ascertained them somewhat closer to diagnosis. We were also able to transport the blood on average one day faster (one vs. two days) from the field to the laboratory. We assessed whether time since diagnosis and transit time for blood was related to IgE levels, with no significant relationship found (when excluding patients who took temozolomide). We also performed a reconstruction experiment in the laboratory to assess whether IgE levels are affected by storing whole blood at room temperature. IgE levels did not perceptibly change during a four day period that blood was stored at room temperature. We also note that the improvement in transport time affected both cases and controls equally, so is unlikely to play a role in case-control differences. It is therefore highly unlikely that our improvement of sampling techniques would explain any differences between Series 2 and 3.
While we have emphasized the differences between the series, it is also important to note that many results were similar. Respiratory IgE had similar odds ratios in both series and reported respiratory allergy odds ratios were also similar and significant. In both series we observed much stronger odds ratios among proxy-compared to self-reported individuals; in both Series this is likely the result of reporting bias. Proxies may not know about allergies, amplifying the odds ratios; therefore self-report odds ratios are likely to be closer to the truth (). Case-control ORs for self-reported food allergies also did not differ between the series; however, one key difference was found in the relationship of food IgE between the two series (OR = 0.12 vs 0.88, respectively, Series 2 and 3). The Series 2 result may be a false positive finding due to small numbers. The relationship of reported respiratory allergens is more robust; it exhibited virtually the same relationship in both reports. This finding should refocus attention on allergens and allergic responses that enter via the nasal route, and away from the digestive route, which was not confirmed in the current analysis (Series 3). Interestingly, cytokines and other peptides administered intranasally can enter the brain directly19
, presenting a possible direct effect on intracranial immune responses from respiratory allergy pathophysiology.
In sum, our current results on reported allergy and glioma are quite similar to our and others previous reports. Although we still observed overall that cases had lower levels of IgE than controls, the effect was only apparent in the temozolomide treated patients, and our cross-sectional analysis suggests that the lower levels of IgE in the patients could possibly be due to temozolomide treatment. To address differences between the series to the extent possible, we considered recruitment and sampling changes and differences in the population ascertained, including the later birth cohort of the Series 3 population. Our efforts do not fully explain the differences between the series, and cast some uncertainty on the capacity of IgE as an adequate biomarker to illuminate an immunologic mechanism that suppresses glioma, at least in the current temozolomide era. Less clear is the reason for an overall higher IgE levels in glioma patients compared to the previous series, which occurred despite the temozolomide IgE suppression that may take place over the course of treatment. This IgE suppression mirrors the loss of T-helper cell function as studied in a temozolomide-treated melanoma cohort13
and serves as a warning to those developing immune-mediated glioma treatments. Definitive evaluation of the role of IgE as a biomarker of glioma risk awaits results from a large cohort study that collects serum before diagnosis. Additionally, the complete effects of temozolomide and other therapies on IgE await the analysis of serial samples obtained before and throughout glioma therapy.