Inhibitor formation in non-severe hemophilia A is a major complication, transforming a manageable disease to one with substantial morbidity. Although much has been learned over the past decade about risk factors for inhibitor formation in those with severe hemophilia A, very little is known about risk factors for inhibitor formation in non-severe hemophilia A. In addition to confirming that intensive exposure to FVIII is a risk factor for inhibitor development, this is the first study to identify an interaction between age and intensive FVIII treatment. This interaction was present even after adjustment for a lifetime exposure to FVIII of < 50 days. Therefore, the impact of intensive treatment in adults does not appear to be the result of either less FVIII exposure prior to adulthood nor because intensive FVIII treatment is their first exposure to FVIII.
Intensive FVIII treatment occurred relatively equally in those < 30 years of age and in those ≥ 30 years of age (24% and 35%, respectively, P
= 0.22), however, the indication for intensive FVIII treatment in each age group was different. In those cases that were 30 years of age and older, surgery was the indication for intensive FVIII treatment in 14 out of the 17 subjects with 78.6% of these surgeries for orthopedic indications. Both severe traumatic bleeding and surgery require intensive FVIII treatment and have been hypothesized to potentiate inhibitor development through signals that cause antigens to be perceived as foreign and dangerous thereby promoting an antibody response [13
]. If danger signals are considered to be important for inhibitor development in mild and moderate hemophilia A, then these results may indicate that in patients < 30 years of age there are mechanisms for danger signals to occur that are unrelated to intensive FVIII treatment, such as infections or immunizations. Alternatively, danger signals may be less pathophysiologically important for inhibitor development in young persons with non-severe hemophilia A. Although the present study is the first to report on the interaction between age and intensive FVIII treatment, a similar pattern was seen in the cohort reported by Eckardht et al
]. In their study, 5 out of the 10 subjects with an inhibitor were older than 30 years of age. Inhibitor development was associated with intensive FVIII treatment in 100% of older subjects and only 40% of younger subjects. In the present study inhibitor development was associated with intensive FVIII treatment in 68% of older subjects and 29% of younger subjects. Although we can speculate that the risk of inhibitor development associated with intensive exposure may be strongly influenced by the indication for intensive treatment and the types of surgeries performed in older patients (i.e. orthopedic surgery), it is clearly multifactorial and this study was not designed to evaluate the independent effect of surgery or a specific type of surgery.
There have been previous reports of the R593C mutation and its association with inhibitor formation in patients with mild hemophilia A [14
]. Most recently, in a cohort of subjects from the Netherlands, 8 out of 10 subjects with an inhibitor had the R593C mutation [3
]. However, because this mutation was determined to be a founder effect it may have been over-represented in this population. In our more diverse US population, the missense mutation R593C was again associated with inhibitor development on univariate analysis but not multivariate analysis. In the present study, the R593C mutation was seen in subjects from five different states and only two of the ten subjects were known to be related. The N1922S mutation was the second most common mutation seen. Outside of these two specific missense mutations, there did not appear to be a location on the FVIII gene where mutations clustered, as has been previously hypothesized [8
It is remarkable that 41.7% of case subjects developed their inhibitor after more than 50 prior days of FVIII exposure (). In severe hemophilia A, inhibitors develop after a median of 9–14 FVIII exposure days [15
]. In the cohort study reported by Eckhardt et al
] the total cohort of 138 mild and moderate hemophiliacs was significantly less treated than our population with a median of 10 FVIII exposure days (IQR 23 days). Accordingly, the effect of more or < 50 prior FVIII exposure days on inhibitor development could not be assessed. The present study cannot exclude any association between prior FVIII exposure and risk of inhibitor development, as it is not adequately powered to detect weak associations. However, it does not appear that the risk of inhibitor development is as significantly different before and after 50 days of FVIII exposure in persons with mild and moderate hemophilia A as it is in those with severe hemophilia A.
The subset analysis suggested that among those who received intensive FVIII treatment, both surgery and longer durations of FVIII treatment were associated with inhibitor formation. Continuous infusion as a method of delivery of intensive FVIII treatment was not significantly associated with inhibitor development in the present study. Although a small effect of continuous infusion cannot be excluded, the strength of the association is less than that between surgery and inhibitor formation. This result is in contrast to the study by Sharathkumar et al
] who reported that continuous infusion was associated with inhibitor formation. Additionally, in the cohort study reported by Eckhart et al
. continuous infusion was significantly associated with inhibitor formation after adjusting for the R593C missense mutation, receipt of ones first intensive FVIII treatment at the time of surgery and FVIII product change (RR 13; 95% CI 1.9–86). The different results may be due in part to the different study populations: our subjects were older when compared with those studied by Sharathkumar et al
. and had more prior FVIII treatment compared with those studied by Eckhardt et al
. In addition, we were limited to a very small subset of our population for those analyzes and our findings should be considered preliminary.
There are several limitations of this study. First, the sample size is small leading to a lack of power to detect weak associations. Second, the independent effect of surgery or continuous infusion on inhibitor formation could not be evaluated because these variables were only determined in subjects that received intensive FVIII treatment. Third, the inclusion of related subjects could impair the ability to detect the independent influence of FVIII genotype vs. other genetic modifiers. However, only one related-pair of subjects had the N1922S and the R593C mutation, respectively. Family history of inhibitor was the only variable that was influenced by the choice of which member of a related pair was included in the analysis. As a result, the univariate association between family history of an inhibitor and inhibitor formation could be inflated. As the multivariate model was not altered significantly with the inclusion or exclusion of the variable family history of inhibitor, we felt that inclusion of the related subjects without additional adjustment was appropriate. Fourth, we enrolled subjects based on their reported FVIII activity but their inclusion was re-evaluated when several intron-22 inversions were detected. In order to have a cohort of subjects that clearly represented non-severe hemophilia A, 10 subjects with the FVIII genotype associated only with severe disease in the HAMSTeRS database were excluded from the analysis [12
]. However, when these 10 were included, the association with intensive FVIII treatment was not substantially changed (OR, 4.29; 95% CI, 1.81–10.25). Lastly, the number of FVIII exposure days in one-third of subjects was based predominantly on patient interview and recall. However, the use of recall alone was equal amongst cases and controls (33.3% and 35.5%, respectively) and the distribution of prior exposure days was similar amongst those that used patient recall and those that did not regardless of case–control status. Therefore, we do not believe that a significant source of bias was introduced with the use of patient recall to estimate prior FVIII exposure days when complete medical records or calendars were not available.
Overall, the present study has demonstrated that intensive FVIII treatment is strongly associated with inhibitor development in patients with non-severe hemophilia A. The risk is greatest in those 30 years of age or older. Importantly, the risk of inhibitor development does not appear to be as significantly influenced by the number of prior FVIII exposure days as seen in patients with severe disease. Further clinical investigation is needed to understand the effect of age on the risk of inhibitor development associated with intensive FVIII treatment, and what aspects surrounding intensive FVIII treatment promote inhibitor formation. Specifically, it will be important to determine which factors exert the greatest influence: (i) surgical indications and if so, what types of surgery, (ii) method of FVIII delivery, (iii) dose of FVIII, or (iv) duration of FVIII treatment.