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Keywords:

  • danger theory;
  • early prophylaxis;
  • FVIII inhibitors;
  • haemophilia;
  • immunological danger signals;
  • PUPs

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

Summary.  The most problematic complication of haemophilia A treatment is the development of inhibitors to FVIII. The highest risk of developing inhibitors is during the first 20 exposure days (EDs). If the patient can be brought through this high risk period without inhibitor development, the subsequent risk is low. Therefore, as a pilot project, we developed a prophylaxis regimen for the first 20–50 EDs specifically designed to induce tolerance to the administered FVIII and to minimize inhibitor development by avoiding immunological danger signals. Twenty-six consecutive previously untreated patients (PUPs) with severe haemophilia A were treated with the new prophylaxis regimen and the incidence of inhibitor development in this group was compared with that in a historical control group of 30 consecutive PUPs treated with a standard joint protection prophylaxis regimen (40–50 IU kg−1, three times a week). There were no significant differences between the study and control groups in patient-related inhibitor risk factors such as ethnicity (all Caucasian), severity of haemophilia (all <1% FVIII), severity of FVIII gene mutation (< 0.0006) nor in some treatment-related factors such as product type, age at first exposure, vaccination regimen or the need for surgery. 14 of 30 subjects given standard prophylaxis but only one of the 26 subjects given the new regimen developed an inhibitor (= 0.0003, odds ratio 0.048, 95% CI: 0.001–0.372). Our results indicate that minimizing danger signals during the first 20 EDs with FVIII may reduce the risk of inhibitor formation. These results should be confirmed in a larger prospective clinical study.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

Today, the most problematic and costly complication of the treatment of haemophilia A that remains to be overcome is the development of inhibitory antibodies (FVIII inhibitors) to FVIII replacement therapy, particularly in previously untreated patients (PUPs). It is now becoming clear that inhibitor development is a complex, multi-factorial immune response involving both patient-specific and treatment-related factors [1–3]. It has been shown that patients with severe defects in the FVIII gene, such as large deletions, inversions (most commonly intron 22 inversion) and stop mutations are significantly more likely to develop inhibitors than are those with more minor defects such as missense mutations, small deletions or insertions and splice site mutations [1].

Severe mutations in the FVIII gene are predicted to cause a complete deficit of any endogenous FVIII production. In these circumstances, FVIII cannot be presented to the immune system during negative selection of high-affinity autoreactive T cells in the thymus [4,5] and central immune tolerance against FVIII cannot establish itself. FVIII in FVIII products that are given for replacement therapy to patients who carry such mutations would be seen as a foreign protein by their immune system. Why some of these patients develop FVIII inhibitors while others do not is far from clear. For many years immunologists believed that the immune system’s primary goal was to discriminate between self and non-self [6,7]. Matzinger introduced the concept that the primary driving force of the immune system is the need to detect and protect against danger [8]. If a foreign or a self-antigen is not dangerous, immune tolerance is the expected outcome [8]. In recent years, it has been suggested that the ability of the immune system to sense danger is part of a more general surveillance, defence and repair system that enables multicellular organisms to control whether their cells are alive or dead and to recognize when micro-organisms intrude [9–12]. Danger is transmitted by various signals that are associated either with pathogens or with tissue and cell damage [9–12]. Pathogens express pathogen-associated molecular patterns (PAMPS) that are recognized by pattern recognition receptors such as toll-like receptors (TLR), Nod1-like receptors (NLRs) or Rig-I like receptors (RLRs) that are expressed on a range of cells of the innate and the adaptive immune system. Once these receptors are triggered, several signaling pathways are activated that can induce inflammatory responses and the activation of specific anti-pathogen immune responses. Evidence is accumulating that trauma, ischemia and tissue damage can cause inflammatory responses that are very similar to responses induced by pathogens [9–12]. Damaged cells release so called damage-associated molecular patterns (DAMPs) that recruit and activate receptor-expressing cells of the innate immune system, including dendritic cells, granulocytes, monocytes or eosinophils, and thus directly or indirectly promote adaptive immune responses [9–12].

Based on the increasing evidence that both pathogen-associated as well as cell-damage associated molecules present danger signals that can stimulate inflammatory responses of the innate immune system and thereby up-regulate antibody responses, we asked whether the prevention of such danger signals during treatment with FVIII products could decrease the risk for the development of FVIII inhibitors in PUPs with severe haemophilia A. We minimized the exposure to immunological danger signals by avoiding first treatment with FVIII in a bleeding situation or during infection, by avoiding surgery during the first 20 exposure days (EDs) and by avoiding vaccinations on the same day as FVIII treatments. Furthermore, any bleeds that did occur were treated early by giving higher doses immediately, thereby avoiding long and intensive treatment and shortening the time of tissue damage.

Our results indicate that minimizing danger signals during the first 20 EDs with FVIII might indeed reduce the risk of inhibitor formation. However, these results should be interpreted as hypothesis generating and need to be confirmed in a larger prospective clinical study.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

Twenty six PUPs in two centers in Germany with severe haemophilia A (all <1% FVIII baseline activity) with a variety of FVIII gene mutations, the majority high risk, were treated with a prophylaxis regimen designed to induce immune tolerance by avoiding immunological danger signals. The incidence of inhibitor development in this group was compared with that in a historical control group of 30 children treated with a standard joint protection prophylaxis regimen. To avoid selection bias both study and control group consists of consecutive PUPs with severe haemophilia A (<1% FVIII) as they appeared in the respective haemophilia center during a given time period. Based on the immunological danger theory and their potential impact on FVIII inhibitor development the new prophylaxis regimen was prospectively planned and implemented as standard of care by January 2001 in center A (Bremen) and by January 2005 in center B (Munich).

Study aim

The overall risk of developing inhibitors to FVIII during the first 150 EDs is 20–30% for PUPs [13]. Of those developing inhibitors, 50% will do so within the first 20 days and 95% during the first 50 days [13]. If the patient can be brought through this high risk period without inhibitor development, the subsequent risk is low [14].

We therefore decided to test the efficacy in overcoming the high risk of the first 50 EDs of a prophylaxis regimen specifically designed to induce tolerance to the administered FVIII and to minimize inhibitor development.

Treatment

According to the German haemophilia treatment guidelines prophylaxis in children with haemophilia is standard of care [15]. Patients in the study group were treated with low dose prophylaxis, starting with 250 IU once a week (corresponds to approximately 25 IU kg−1 week−1) as soon as a bleeding tendency manifested, either through soft tissue and muscle bleeds or a significant tendency for haematomas (Fig. 1). It was also introduced for ‘safety’ reasons as bleed prophylaxis after child-felt trauma (i.e. typical head trauma without bleeding signs). Prophylaxis was initiated without insertion of a Port-A-Cath after a minimal number of on-demand FVIII exposures. In patients with early joint bleeds prophylaxis was introduced at the higher frequency of 25 IU kg−1 twice a week, and in those with early severe joint or life threatening bleeds at 25–50 IU kg−1 three times a week. When required by the severity of the bleeding tendency the frequency was increased from one per week to two per week or three per week. For tolerization (as also known from ITI programs in inhibitor patients) it seems to be important to give prophylactic FVIII doses always on the same weekday and to avoid interrupting the prophylaxis regimen even when additional on-demand FVIII doses to manage bleeds are given.

image

Figure 1.  Treatment scheme for PUPs receiving the new prophylaxis regimen.

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During this ‘tolerization’ period, immunological danger signals were minimized by avoiding giving first FVIII in a severe bleeding situation or during an infection, avoiding surgery during the first 20 EDs, avoiding giving vaccinations on the same day as FVIII and giving all vaccinations subcutaneously rather than intramuscularly. Any bleeds that did occur were treated early by giving a higher than the prophylactic dose immediately, thereby avoiding long or intensive treatment. Patients in the study group were tested for inhibitors every 3–4 EDs.

Patients in the control group were treated with a standard joint-protection prophylaxis regimen of 40–50 IU kg−1 FVIII three times a week, starting at or after the first joint or other severe bleed. Please note that some of the patients in the control group (= 8) developed their inhibitors already during on-demand therapy before they entered a standard prophylaxis program. The vaccination guidelines have been the same for both the study and the control group.

Statistical analysis

Differences in inhibitor development between the study group and the historical control group were analysed by Fisher’s exact test and odds ratios (OR).

The effect of potential determinants on inhibitor risk such as FVIII gene mutation and type of product (recombinant vs. plasma-derived FVIII) was evaluated for the two groups in a logistic regression model.

Differences between the two study groups of treatment-related parameters such as median EDs before prophylaxis and age at start of prophylaxis were assessed by Wilcoxon test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

Fifty six of the 58 subjects studied had more than 100 EDs to FVIII therapy. Data from these were analysed for inhibitor development and both patient-related and treatment-related factors which might have affected inhibitor development.

There were no significant differences between the study and control groups in any patient-related factors (Table 1), nor in the majority of treatment-related factors (Table 2). In a logistic regression model for inhibitor development with factors for study group (standard vs. new regimen prophylaxis), genetic risk for inhibitor development (low vs. high), and type of factor concentrate (recombinant vs. plasma-derived), only the type of prophylaxis regimen had a significant effect (= 0.005). Logistic regression analysis was not performed for the risk of high responder inhibitors due to lack of events in patients given the new regimen.

Table 1.   Patient-related risk factors for inhibitor development in the study group compared with the control group.
 Control group (standard prophylaxis regimen) (= 30)Study group (new prophylaxis regimen) (= 26)Statistical significance
  1. *Categorization of genetic risk according to Oldenburg J, Pavlova A. Genetic risk factors to inhibitors against FVIII and IX. Haemophilia 2006; 12 (Suppl. 6): 1–8.

Demographics – Bremen
Born betweenMarch 1995–December 2000January 2001–July 2007Not significant
EthnicityAll Caucasian (= 15)All Caucasian (= 13)
Demographics – Munich
Born betweenJanuary 2002–September 2004January 2005–October 2007Not significant
EthnicityAll Caucasian (= 15)All Caucasian (= 13)
Genetic factors
Severity of haemophilia AAll <1% FVIII activityAll <1% FVIII activityNot significant
FVIII mutation type*:
High risk (%)24 (80)18 (69)Not significant
Low risk (%)5 (17)8 (31)Not significant
Unknown (%)1 (3) 
Table 2.   Treatment-related risk factors for inhibitor development in the study group compared with the control group.
 Control group (standard prophylaxis regimen) (= 30)Study group (new prophylaxis regimen) (= 26)Statistical significance
  1. *As on August 2009.

Product type
rFVIII (%)16 (53)15 (58)Not significant
pdFVIII (%)14 (47)11 (42)
Age at first exposure
Median (months)9.88.1Not significant
Range (months)0.1–220–21
Reason for first exposure
Bleed (%)21 (70)12 (46)Not significant:= 0.103
Safety (%)9 (30)14 (54)
Total EDs*>100>100Not significant
Surgery
Within the first 20 EDs32Not significant
Vaccination
Given i.m.1All noNot significant
Given on a FVIII dayAll noAll no

There were however highly significant differences between groups for the prophylaxis-related factors: age at start of prophylaxis and the number of EDs before the introduction of prophylaxis (Table 3). Whereas the new prophylaxis regimen was started after a median of 1 FVIII EDs at a median age of 10.7 months compared to the historical control group were high dose prophylaxis was started later after a median of 30 FVIII on-demand EDs at a median age of 19 months (< 0.006).

Table 3.   Prophylaxis-related factors for inhibitor development in the study group compared with the control group.
 Control group (standard prophylaxis regimen) (= 30)Study group (new prophylaxis regimen) (= 26)Statistical significance
Age at start of prophylaxis (= 23)(= 26) 
Median (months)1910.7 Highly significant:P < 0.0006
Range (months)0.8–870.5–24.5
EDs before prophylaxis
Median301 Highly significant:P < 0.0001
Range1–infinity0–14

Age at start of prophylaxis was available for 23 of the 30 subjects in the standard prophylaxis group and all 26 subjects given the new regimen. The median age at start of prophylaxis was 19 months (range 0.8–87) for those given standard prophylaxis and 10.7 months (range 0.5–24.5) for those given the new regimen. This difference is highly significant (< 0.0006).

Standard prophylaxis had been introduced after a median of 30 EDs (range 1–infinity) whereas the new regimen was introduced after a median of 1 ED (range 0–14). This difference too is highly significant (< 0.0001).

Fourteen of the 30 subjects given standard prophylaxis and one of the 26 subjects given the new prophylaxis regimen developed an inhibitor. The difference between the groups was highly significant (= 0.0003, OR 0.048, 95% CI: 0.001–0.372) (Table 4).

Table 4.   Inhibitor development in the study group compared with the control group.
 Control group (standard prophylaxis regimen) (= 30)Study group (new prophylaxis regimen) (= 26)Statistical significance
Inhibitors (%)14 (47)1 (3.8) Highly significant: P = 0.0003 OR 0.048 (95% CI: 0.001–0.372)
High responders (%)8 (27)0 Highly significant: = 0.005 OR of high response 0.00 (95% CI: 0.00–0.57)
Low responders (%)6 (20)1 (3.8)

Eight subjects given standard prophylaxis but none of those given the new regimen were high responders. The difference between groups was again significant (= 0.005, OR for high response 0.00, 95% CI: 0.00–0.57) (Table 4). Inhibitors in the control group developed after a median of 11 EDs (range: 3–170 EDs) which is well in agreement with a recent international study [16].

The cumulative inhibitor incidence in the study group on the new prophylaxis regimen was reduced by 95% (OR 0.048) as compared to the control group on a standard protocol (= 0.0003, 95% CI: 0.001–0.372) (Fig. 2).

image

Figure 2.  Cumulative inhibitor incidence with increasing number of EDs: control vs. study group.

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As a post-hoc analysis, these results should be interpreted as hypothesis generating. Confirmation in a prospectively planned, historically controlled study would be warranted.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

It may be considered that the overall risk of developing an inhibitor reflects the level of danger signals perceived by the patient’s immune system. It is not, therefore, surprising that on-demand treatment which is, by definition, given in the presence of bleeding should cause inhibitor development more frequently than prophylaxis.

The value of prophylactic factor replacement therapy in the prevention of severe joint bleeds and arthropathy is now well established [17], and is increasingly being adopted as the standard approach to treatment of haemophilia A. However, even in those countries, such as Sweden, where prophylaxis is virtually universal there has been no reduction in the overall incidence of inhibitors in PUPs [18]. The prophylaxis regimens employed have been designed for joint protection, with relatively high doses of concentrate such as 50 IU kg−1 three times per week. Because they are usually introduced at or just after the first significant joint bleed, the FVIII is being introduced at a time when there are strong immunological danger signals present, to an immune system which has already been ‘primed’ by previous on-demand therapy. Therefore, prophylaxis might start too late to prevent inhibitor formation.

An effective prophylactic regimen for the treatment of PUPs without the development of inhibitors must take into account and avoid known danger signals, such as bleeding associated with tissue damage, immunological challenges such as vaccination, or infection. This would permit the immune system to develop tolerance to the foreign protein in a ‘non-threat’ situation. The results of this study demonstrate that this approach with an early start of low dose prophylaxis once weekly might have the capacity to dramatically reduce the incidence of inhibitors, even in high-risk patients, from the normally expected level, which in PUPs has been around 30% [1,13].

It remains difficult to judge which parameters of the new prophylaxis regimen were of major influence on inhibitor development: the low number of on-demand exposures before prophylaxis, the low dose/frequency of the prophylaxis regimen, the young age at start of prophylaxis or a combination of some or of all of them. The avoidance of first FVIII exposure during a severe bleeding episode might be a direct protector from inhibitor development whereas the age, however, might play only an indirect role as the earlier prophylaxis is started the more likely the PUP can reach >50 ‘tolerizing’ EDs without the need for intensive treatment due to a severe joint bleed.

However future studies will have to evaluate the significance of single treatment-related factors and further refine the optimal regimen for inducing immunotolerance.

We are aware of the fact that our results can only be considered as hypothesis generating and need to be confirmed in a larger prospective clinical study.

Our results also suggest that early introduction of FVIII is a more satisfactory way of avoiding inhibitors than attempting to delay the use of FVIII, for example by treating bleeds with rFVIIa [19]. Starting with prophylaxis early in life, in our study at a median age of 10.7 months, was not associated with an increased inhibitor risk, a finding that is well in line with other recent studies [20,21].

A low dose, escalating regimen may also provide a better long-term outcome for patients, with less frequent joint bleeds and better joint scores, due to the earlier start on prophylaxis. The beneficial effect on joint outcomes is hard to explain, since a weekly prophylaxis regimen cannot maintain FVIII levels above 1%. Nevertheless, benefit from a regimen similar to ours has been demonstrated in a 10-year study into the Canadian tailored primary prophylaxis regimen [22]. This regimen differs from our proposed regimen in using higher doses, introducing prophylaxis only after a joint bleed has occurred and stepping up only after inadequacy of dosage is demonstrated by several joint bleeds or development of a target joint. This beneficial effect should be the subject of further study.

As well as its key role in preventing inhibitor development, the new prophylaxis regimen offers a number of other advantages. With once a week administration, it is not necessary to insert a Port-A-Cath, thereby avoiding surgery. If the initial dosage proves inadequate, it may still be possible to avoid the need for a Port-A-Cath by increasing the individual dose rather than the frequency of dosing. Avoiding the need for a Port-A-Cath is probably a major advantage for the induction of immune tolerance to FVIII because any surgical procedure is likely to be associated with some form of tissue damage together with the generation of danger signals.

Once weekly administration is also simpler for parents, requiring only one visit to the haemophilia center each week, so that concordance is easier to achieve with a consequent improvement in control.

There is also a pharmacoeconomic benefit in that lower doses and less frequent treatments can allow considerable cost savings compared with standard prophylactic regimens.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

Summarizing our results, we conclude that early start of prophylaxis associated with minimizing immunological danger signals during the first 20 EDs with FVIII should be considered for future therapy of patients with severe haemophilia A to reduce the risk of inhibitor formation. Once the patients have developed tolerance to FVIII, usually after about 20–50 EDs on the low dose regimen, and venous access permitted, prophylaxis might be changed to the normal three times weekly regimen for optimal joint protection (Fig. 1).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

The authors thank Baxter for support in development of this manuscript. The Munich centre thanks Martin Olivieri and Susan Jenkins for valuable support on patient care and data collection. It also acknowledges greatly the work of the coagulation laboratory of Prof. Dr W. Schramm. The Bremen centre thanks Dr Julia Johne and Dr David Overberg for intensive support on data collection.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References

G. Auerswald, K. Kurnik and C. Bidlingmaier have been reimbursed for attending and/or speaking at and/or organizing several symposia on the behalf of several pharmaceutical industries.

K. Kurnik received funding for research by Baxter, CSL Behring, Bayer, Wyeth/Pfizer; C. Bidlingmaier by CSL Behring, Bayer, and Wyeth/Pfizer, and G. Auerswald by Baxter, CSL-Behring and NovoNordisk.

B. Reipert, W. Engl and H. Chehadeh are Baxter employees.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. Disclosures
  10. References