High rate of spontaneous inhibitor clearance during the long term observation study of a single cohort of 524 haemophilia A patients not undergoing immunotolerance

A cohort of patients from one centre (Castelfranco Veneto Haemophilia Centre) where surgical synovectomy was adopted, in the 70s and 80s, as the main treatment method and where inhibitor patients had never undergone ITI.

This was a database originated research. Data were extracted anonymously from the database of clinical and laboratory data of the Hemophilia Center in Castelfanco Veneto. A generic consent to store information for research purposes was obtained from the patients over time, but, for the need of gathering a complete inception cohort, no specific consent was sought for this specific retrospective study protocol.

Haemophilia A was diagnosed and classified according to factor VIII plasma levels [14]. Inhibitor titre was assessed by the Bethesda assay based on measuring the amount of FVIII inactivated by patient plasma mixed in equal proportions with pooled normal plasma as a source of FVIII [15]. For the purpose of this analysis inhibitors were considered 'low' if the titre was < 5 BU/ml and 'high' if it was > 5 BU/ml and patients accordingly defined as low and high responders respectively.

The cohort consisted of all consecutive patients admitted to the centre from 1973–2010 who were tested for inhibitors the first time and at least 5 more times during a minimum 24-month follow up period and 3-month minimum interval between each test. Patients were regularly tested for inhibitors before and 2 weeks after surgical synovectomy.

Patients with less than 5 tests were excluded. Exposure days before inclusion in the study cohort ranged from 0 to >200 while all the inhibitor negative patients reached 200 exposure days during follow up. The entire cohort was treated with factor concentrates approved and available at the time, i.e. cryoprecipitate at the beginning, then plasma derived concentrates manufactured from Italian plasma (by FarmaBiagini and subsequently Kedrion, as Koate, Uman-Cry and Emoclot) and lately recombinant concentrates (used in a minority of patients). Bypassing agents were used to treat bleedings in patients with high inhibitor titre. When patients reached a low inhibitor level (= < 5 BU), they were treated with FVIII concentrates for spontaneous bleeding or for surgery. In these cases, FVIII was dosed calculating a neutralizing dose (BU × 40 × kg/bw) and adding it to the dose needed for the targeted increment in the patient plasma factor VIII level (frequently in the range of 30–50 IU kg/bw). When treated, patients were closely monitored for any anamnestic response by laboratory testing every other day for 1 or 2 weeks. Most of the increases in titer occurred within an average of 6 days (range 3–10).

Inhibitors were classified as follows: 'transient' if the inhibitor disappeared spontaneously within 6 months and did not reappear when the patient was exposed to factor VIII; 'slowly resolving' if the inhibitor disappeared spontaneously after 6 months and did not reappear when exposed to factor VIII; 'persistent' if the inhibitor remained positive or could not be detected in the absence of factor VIII treatment but reappeared when factor VIII was re-administered.

From the Italian Registry of Haemophilia A Mutations [16], it was possible to identify patient mutations, classified accordingly as, inversions (int1inv and int22inv), null mutations (large deletions, nonsense and frameshift ins/del) or not null mutations (all the others).

To estimate and compare the probability of inhibitor resolution, we have used a model of clinical outcome (class of inhibitor) as predicted by immunological response (high versus low) and gene mutation (inversion, null, not null) using a multinomial logistic regression.

The total number of patients was initially 543. They were all tested for inhibitors at least once but 19 patients, with less than 5 tests, were excluded from the analysis (Figure 1).

In total, 7,779 test results were available with a median of 15 tests per patient (Figure 2). The mean (+/− SD) age at the onset of inhibitors was 17+/−8 yrs (range 3–47). The mean (+/− SD) follow up was 24+/−12 yrs (range 8–36).

The cumulative inhibitor incidence total was 34% (180/524), 39% (168/434) in severe cases, 16% (10/60) in moderate ones and 7% (2/30) in mild patients. The distribution of inhibitors in persistent, slowly resolving and transient according to haemophilia severity and inhibitor characteristics is reported in Figure 1 and Table 1. 29 inhibitor patients were found positive when first tested, of these, 22 were high responders and 7 low.

Mutations were identified in 335/524 patients (64%). Transient, slowly resolving, persistent inhibitor distribution and titre is shown in Table 2. The presence of inversions/null mutations was higher in the persistent and high responding inhibitor groups. The median persistence time of spontaneously clearing slowly resolving inhibitors is reported in Table 3.

The characteristics of the models and analysis details are reported in Table 4 and its legend. Since underlying gene mutation data was known in only a proportion (131/180) of inhibitor patients the probability of developing a transient, slowly resolving or permanent inhibitor was estimated in two different ways. If available, the probability associated with both the initial inhibitor titre and the gene mutation was assessed, otherwise only the inhibitor titre was used.

The relative risk ratio between a high and low responder in having a slowly resolving inhibitor is 3.63 (95% CI 1.39 – 9.47) and a permanent inhibitor 62 (95% CI 21.12 – 182.00). In the full model - inhibitor titre, gene mutation and their interactions, the effect of the titre was not statistically significant when considered alone, whereas, in patients with inversions compared to those with non null mutations a statistically significant and clinically relevant increase in the risk for permanent inhibitors in patients with high titre inhibitors (21.7, 95% CI 1.80 – 263) was found. It was not possible to assess the effect of large mutations in this study due to their relatively low incidence.

The patients were tested every time they underwent surgical synovectomy or exposure to factor VIII for bleeding at the optimal time of two weeks after FVIII administration making the monitoring ideal. The strict inhibitors testing was necessary to avoid the risk of uncontrolled bleeding during synovectomy. The results of this close observation has made feasible a study, based on a cohort subjected to stringent follow up and well-documented inhibitor incidence from as far back as the 70s and 80s, when inhibitor testing was not routinely performed in all Haemophilia Centres.

Due to the early implementation of this strategy, it has been possible to detect all the patients with inhibitors, including low titre as well as transient ones and to identify subgroups of patients on the basis of different time-dependent inhibitor clearance. On the opposite, inhibitor surveillance programs started worldwide as recombinant products became available and were not as suitable to assess the natural history of inhibitors because in the meanwhile ITI has become widespread used.

The value of this retrospective analysis has to be considered mainly exploratory, and some unaccounted confounders might have played a role. Furthermore, as studies on ITI do not have an internal control group of patients not undergoing ITI, any inference from the natural history of inhibitors to ITI treated patients and vice-versa can only be considered as indirect. Some characteristics of the population deserve mention. First, the high rate of surgical synovectomy that occurred in this study might have affected the rate of inhibitor appearance and disappearance. Second, the mean patient age at inhibitor development was rather high, which is explained by the fact that the study in question goes back to the 70s and 80s, a period when concentrates were still not widely available or extensively used. Third, the intensity of treatment spun over a very high range and was not used to adjust the analysis. How these population characteristics could have affected the results and impair their external validity remains to be assessed.

The cumulative inhibitor incidence in the cohort appears relatively high (34%), particularly so if compared with reports from the same period, but it is in keeping with similar studies conducted later on in other settings [17‐19] and substantially overlapping the results from the recent prospective RODIN study [20].

The possibility of a higher incidence of inhibitors in our cohort because of the surgery cannot be excluded as it has been hypothesized that tissue damage would trigger the immune system, raising the risk of developing inhibitors [21], but the same hypothesis has not been confirmed in another case–control study [22].

Another possible explanation for this higher incidence is that the inhibitor rate reported in previous studies was incorrectly low. The low inhibitor rate in the 1980s could actually be explained, as suggested by Iorio et al. [18], by the combined effect of the year of the studies (the older the study, the lower the rate, and no study before the 1980s was included) and the testing frequency. As discussed above, the cohort underwent very frequent inhibitor testing, even when clinical inefficacy was not suspected.

In addition to 38% of transient inhibitors, 26% of slowly resolving inhibitors disappeared in a median of 3 to 6 years, respectively, in low and high responders. The characteristics of the patients spontaneously clearing their inhibitors are of interest, particularly when contrasted with available evidence from ITI studies [7, 23‐25]. First, the upper bound of ITI duration needed to obtain tolerance is considered to be up to 33 months, close to the 3-year median time period for spontaneous resolution of the inhibitors among the low responders in our study cohort. Second, a low inhibitor titre has been recognized as significant predictive factor for success of ITI, as it has shown to be in our subgroup of slowly disappearing inhibitors, in which 90% of low titre patients spontaneously lose the inhibitor. Third, underlying haemophilia genotype has been associated with the risk of anti-FVIII inhibitor development [26‐28] and the probability of ITI success [29, 30]; most patients with large deletions undergoing ITI fail to respond whereas patients with int22inv have an intermediate and variable success rate. The logistic regression analysis performed in our study, though limited by the small number of available patients and incomplete accounting for all potential confounders, seems to fit this picture.