Materials and methods
Description of study
This was a multicentre, open-label clinical study in PUPs and MTPs with severe or
moderately severe haemophilia A (baseline FVIII ≤2%). Subjects underwent an evaluation
of the immunogenicity, efficacy, and safety of rAHF-PFM according to one of the following
three treatment regimens: on-demand treatment, a standard prophylactic regimen consisting
of 25 to 50 IU/kg body weight, 3–4 times per week, or a modified prophylactic regimen
(with dose and frequency at the discretion of the investigator). rAHF-PFM was also
used for perioperative management, if necessary. The treatment regimen was determined
by the investigator and could be changed at any time. Subjects who developed a confirmed
high-titre inhibitor (>5 Bethesda Units [BU/ml]) or who developed a low-titre inhibitor
and in whom bleeding could not be adequately managed by prophylactic or on-demand
treatment with rAHF-PFM were eligible to undergo immune tolerance induction (ITI)
with rAHF-PFM, according to regimens that were at the discretion of the investigators.
Subjects were to be followed for 75 EDs or three years, whichever occurred first.
The sponsor, on the recommendation of the study’s Data Safety Monitoring Board, could
have stopped the study at any time if there were unacceptable safety risks to study
participants. Consistent with the Committee for Proprietary Medicinal Products (CPMP)
guideline on studies of recombinant FVIII products regarding PTPs greater than 12
years old, the number of subjects intended for enrollment in this study was approximately
50 ([7]).
The primary safety endpoint was the percentage of subjects who developed an inhibitor
to FVIII. Secondary endpoints were the percentage of subjects who had adverse events
(AEs) deemed possibly or probably related to treatment with rAHF-PFM, and the percentage
of subjects who developed antibodies to Chinese hamster ovary (CHO) cell protein,
murine immunoglobulin G (IgG), or human von Willebrand factor (VWF). Also analysed
were potential risk factors for inhibitor development.
Efficacy outcome measures were the number of rAHF-PFM infusions required to achieve
adequate haemostasis for all BEs; the overall haemostatic efficacy rating of rAHF-PFM
for all treated BEs; the annualised bleeding rate, incremental recovery of rAHF-PFM
determined at study visits, intra- and post-operative haemostatic efficacy rating,
and the percentage of actual to predicted blood loss during surgery.
Efficacy assessments
Haemostatic efficacy for BEs was evaluated by the subject’s legally authorised representative
(for home infusion) or the investigator (for infusions at the clinic). Intraoperative
and postoperative assessments of efficacy were evaluated by the operating surgeon
and the investigator, respectively. For efficacy rating scales, refer to online supporting
information.
Laboratory assessments
Inhibitor testing by the Nijmegen assay at the Baxter Central Laboratory was to be
performed at screening, then after 5 ± 1, 10 ± 1, 15 ± 1, and 20 ± 1 EDs, and thereafter
every 10 ± 3 EDs or 3 months ± 7 days from the last inhibitor test, whichever came
first. If inhibitor formation was observed, epitope mapping of the inhibitory antibody
was performed on remaining plasma. For additional information on epitope mapping,
refer to the online supplemental material (available online at www.thrombosis-online.com). Subjects were to be evaluated for in vivo incremental recovery (IR) throughout the study. Part way through the study it was
noted that IRs determined at the Baxter Laboratory in Round Lake were markedly lower
than expected. Following an in-depth investigation and with the concurrence of the
US Food and Drug Administration (FDA), it was decided to have the subsequent testing
of FVIII activity conducted at the Department of Medical and Chemical Laboratory Diagnostics,
Medical University Vienna, Austria. Due to the systematic underestimation of the peak
levels, the results obtained at the Baxter Round Lake Laboratory are not meaningful
and cannot be used for the interpretation of recoveries. Due to the small number of
evaluable IR values from the Medical University Vienna Laboratory, IR results will
not be presented in this report.
FVIII gene mutation and human leukocyte antigen (HLA) genotype testing was performed
in the central laboratory at DRK-Blood Donor Service, Institute of Transfusion-Medicine/Immunology/
Haematology, Frankfurt, Germany. The laboratory techniques used for mutation analysis
are described in the online supplemental material (available online at www.thrombosis-online.com).
Serum was assayed for the presence of antibodies to heterologous proteins which are
present in trace quantities in the study product (i.e. CHO protein, murine IgG, and
VWF) using proprietary enzyme immunoassays.
Subjects
Eligible subjects were PUPs and MTPs <6 years of age with severe (FVIII level < 1%)
or moderately severe haemophilia A (FVIII level 1–2%) determined at baseline. Subjects
with a detectable inhibitor to FVIII, a known hypersensitivity to rAHF-PFM, or a history
of exposure to FVIII other than rAHF-PFM were excluded from the study. Subjects may
have had up to three infusions of rAHF-PFM within 28 days prior to enrolment to treat
a BE and three infusions between enrolment and the first IR infusion. Infusions of
rAHF-PFM received prior to the start of the study were factored into the calculation
of EDs.
Statistical methods
Efficacy and safety data were summarised by medians (min and max) and means (standard
deviations [SD]). Incidence of inhibitor development was summarised by percentages
(95% confidence intervals [CI]), and the median time to inhibitor development was
calculated. The cumulative incidence of inhibitor development, including all, high
and low titre, was presented in a post-hoc plot showing percentage inhibitor development
by the number of EDs prior to inhibitor incidence. Individual analyses of incidence
of inhibitor development for the PUPs and MTPs were provided as post-hoc analyses.
Immunogenicity to heterologous protein was analysed by linear regression with antibody
titre (Y) as the dependent variable and time (X) as the independent variable per subject.
A Fisher exact test was used for a post-hoc calculation to assess association between
geographic location with regards to prescription of a prophylactic regimen and age
of enrolment. The examination of initial treatment regimens and calculations of annualized
bleeding rates for all subjects and by age of enrolment into the study were conducted
as post-hoc analyses.
Risk factor analysis
To investigate potential risk factors of inhibitor development, post-hoc logistic
regression analyses for putative risk factors were performed. The analysis of risk
factors was performed on an immunogenicity analysis set (N=50) which included all
those developing an inhibitor and all those who were inhibitor-free with at least
10 EDs.
Genetic risk factor information such as family history of inhibitors and race/ethnicity
were collected at the time of enrolment. FVIII mutations considered high risk for
inhibitor development were large deletion; intron inversion; nonsense; splice site;
and frameshift mutations (small deletions/insertions) resulting in stop codons with
no prior documentation of partial correction of reading frame. Mutations considered
low risk were missense; in-frame (small deletion/insertions); and frameshift mutations
(small deletions/insertions) at or near an A run previously documented to have partial
correction of the reading frame (details provided in online supplemental material,
available online at www.thrombosis-online.com). High-risk HLA type was defined as having any combination of DR15 and/or DQ06 HLA
genotypes ([11]).
Non-genetic risk factor analysis took the following potential risk factors into account
at exposures to rAHF-PFM of ≤10 EDs, ≤20 EDs, and ≤30 EDs: surgery, port placement,
intensive treatment (at least five consecutive study days of treatment), intensive
treatment at high dose (five consecutive study days of a mean infusion dose of rAHF-PFM
>50 IU/kg), and age at first exposure to rAHF-PFM. For inhibitor subjects, the risk
factor must have been captured prior to development of inhibitor.
Results
The Institutional Review Boards or Ethics Committees representing each investigational
site approved the study protocol. The first subject entered the study on April 1,
2004. The last subject exited on September 11, 2009.
Subjects
Of 66 subjects enrolled (consented) from 24 international sites, 55 (18 PUPs and 37
MTPs) received at least one infusion of rAHF-PFM during the study interval, including
one screen failure (►[Fig. 1A]), and 44 subjects completed the protocol.
Figure 1: Disposition of subjects (A) and distribution of bleeding episodes (B). BE=bleeding episodes, Hgb=haemoglobin, n=number of subjects. aOf the 55 subjects dosed, 44 subjects received treatment for at least one BE and the
remaining 11 subjects received rAHF-PFM for other reasons (e.g. recovery study infusion,
surgical prophylaxis, prophylactic regimen, etc.) One subject did not meet eligibility
criteria but was inadvertently dosed and subsequently withdrawn.
Eleven subjects did not receive investigative product: six subjects were screen failures,
one was considered lost to follow-up and terminated by the investigator after missing
two site visits, three withdrew from the study, and one had low haemoglobin prior
to enrolment and was withdrawn by the physician. There were no screen failures due
to inhibitors.
Eleven of the 55 subjects who received at least one infusion of rAHF-PFM on study
did not complete the study per protocol: one terminated participation in the study
after developing an inhibitor, in order to enroll in the international ITI study ([12]), one was withdrawn by the investigator for administration of non-study rAHF PFM
considerably outside the permitted 28-day window prior to screening, one was lost
to follow-up, one was withdrawn during ITI treatment by the investigator, one was
a screen failure inadvertently dosed with one infusion of rAHF-PFM; and six withdrew
consent for non-product related reasons[
1
].
For the 55 subjects who received at least one infusion of rAHF-PFM, the median age
at enrolment was seven months (range: 14 days-16 months); 21 (38%) subjects were <6
months old, 26 (47%) were 6–12 months old, and 8 (15%) were ≥13 months old. All of
the subjects were male. Thirty-seven (67%) subjects were Caucasian, and 18 (33%) were
non-Caucasian, including: nine (16%) Hispanic, five (9%) Black, two (4%) Caucasian/Black,
one (2%) Indian (East), and one (2%) Asian/Caucasian. The baseline FVIII level at
enrolment was <1% in 53 (96%) subjects, 1% to ≤2% in one (2%) subject, and >2% in
one (2%) subject (considered a screen failure and subsequently withdrawn). Seventeen
(31%) subjects had a family history of FVIII inhibitor and 35 (64%) did not, while
three (6%) subjects had unknown family history. The gene mutations for all 55 subjects
were analysed; 45 (82%) had FVIII mutations considered high risk for inhibitor development
and 10 (18%) had low-risk FVIII mutations (see Materials and methods and ►Suppl. Table 2, available online at www.thrombosis-online.com).
Treatment
The MTPs received a median of 1 (range: 1–4) infusions of non-study rAHF-PFM prior
to the start of the study which were factored into the calculation of EDs (administered
for haemostatic control of traumatic head BEs, joint BEs, soft tissue BEs, and surgical
prophylaxis for circumcision). While these subjects are termed MTPs, they only received
a single FVIII replacement product, rAHF-PFM, had extensive records of medical histories,
and were infused within a well defined timeframe prior to enrollment according to
protocol.
Of the 55 subjects treated with rAHF-PFM, the initial regimen prescribed was on-demand
in 47 (85.5%), standard prophylaxis in three (5.5%), and modified prophylaxis in five
(9.1%) subjects. An analysis of the treatment regimens of 52/55 subjects (three subjects
exited the study too early to be included in this analysis) revealed that the predominant
treatment regimen, defined as at least 80% of the time on study, was mixed in 29 (56%)
subjects, on-demand in 15 (29%), modified prophylaxis in three (6%), standard prophylaxis
in three (6%), and ITI in two (4%). Subjects initially prescribed on-demand treatment
were frequently switched to prophylactic treatment. An examination of prescribed regimens
in the same 52 subjects demonstrated a statistically significant association between
the choice of treatment modality and geographic region of the prescribing physician.
Although the majority of subjects were either on an on-demand or a mixed regimen for
at least 80% of the time, a significantly higher proportion of subjects in Western
Europe (100% [15/15]) than in North America (54% [20/37]) were prescribed prophylaxis
at least once (Fisher exact p-value=0.001). An examination of the age at enrolment
between geographies in the same 52 subjects described above, showed that although
the median age at enrolment was somewhat higher in the EU than in North America (1.02
vs. 0.78 year), this difference was not statistically significant.
During the study, 27 subjects underwent surgical procedures, including 22 port placements
combined with circumcision in six subjects and a herniotomy in one subject, four circumcisions,
and one venous fistula. Not included in the analysis were two circumcisions, which
were conducted prior to enrolment and recorded in medical history, for which one and
two infusions of non-study rAHF-PFM were administered. Neither of these subjects subsequently
developed inhibitors.
Efficacy
Haemostatic efficacy in bleeding episodes
Fifty subjects experienced a total of 633 BEs, of which 517 in 44 subjects were treated.
In some cases it was the opinion of the treating physician and/or the legal representative
that a BE did not require an infusion of rAHF-PFM. Children may often experience superficial
BEs, such as bruises, which are not treated. Of the 517 treated BEs, 466 received
efficacy ratings and 51 had an unknown efficacy rating. All but one (50/51) of these
unrated treatments were recorded at a single study site and were not included in the
efficacy rating analysis. Some of the infusions rated as “unknown” efficacy were performed
pre-emptively for head trauma, in accordance with NHF MASAC recommendations ([13]). Subsequent head MRI or CT assessments did not reveal a BE, and therefore, an efficacy
analysis of these BEs was not possible of these BEs. A flowchart of the distribution
of BEs is presented in ►[Figure 1B].
Haemostatic efficacy in 93% of rated BEs was considered excellent (258 [55%]) or good
(177 [38%]) (►[Table 1A]). To achieve adequate haemostasis, 90% of BEs were treated with one or two infusions;
one infusion was administered in 356/517(69%) BEs in 42 subjects and two infusions
were administered in 107 (21%) BEs in 34 subjects (►[Table 1B]). Fifty-four BEs were treated with three or more infusions; of these, haemostatic
efficacy was rated excellent in 22 BEs and good in 22 BEs. Those BEs requiring three
or more infusions were: a tongue BE, forehead bruise, mouth/frenulum BE, psoas BE,
chest wall BE, cut, antecubital BE, and joint BEs. Of 517 BEs, 42/96 (44%) joint BEs
were treated with one infusion compared to 314/421 (75%) of non-joint BEs.
Table 1
Haemostatic efficacy of rAHF-PFM. A) Haemostatic efficacy in bleeding episodes. B) Number of infusions administered
to manage bleeding episodes. C) Haemostatic efficacy in surgical procedures.
|
A) Overall efficacy of rated treatments
|
|
Rating
|
Number of
unique subjects
|
Number of BEs
|
BEs
(%)
|
|
Excellent
|
42
|
258
|
55.4
|
|
Good
|
36
|
177
|
38.0
|
|
Fair
|
11
|
30
|
6.4
|
|
None
|
1[a]
|
1
|
0.2
|
|
All rated
|
44
|
466
|
100.0
|
BEs=bleeding episodes.
a The BE for this rating of “none” had three evaluations for three infusions (spontaneous
BE of the buttocks). The first and second of the three infusions was rated “none”
and the third infusion was rated “good.” Haemostatic efficacy in 93.4% of rated BEs
was considered excellent or good. Of 517 BEs treated with rAHF-PFM, 466 BEs were rated
and 51 had an unknown efficacy rating.
BEs=bleeding episodes. Of 517 treated BEs, 89.6% were controlled with one or two infusions.
N=number of surgical procedures. There were a total of 27 surgeries in 27 subjects.
Intraoperative: 22 subjects were rated, two did not receive product, and in three
subjects, assessment was not done by the surgeon. Postoperative: 25 subjects were
rated, and two were not.
|
B) Number of infusions administered to manage bleeding episodes
|
|
Infusions
|
Number of
unique subjects
|
Number of BEs
|
Bes
(%)
|
|
1
|
42
|
356
|
68.9
|
|
2
|
34
|
107
|
20.7
|
|
3
|
23
|
35
|
6.8
|
|
≥4
|
14
|
19
|
3.7
|
|
All
|
44
|
517
|
100.0
|
BEs=bleeding episodes.
a The BE for this rating of “none” had three evaluations for three infusions (spontaneous
BE of the buttocks). The first and second of the three infusions was rated “none”
and the third infusion was rated “good.” Haemostatic efficacy in 93.4% of rated BEs
was considered excellent or good. Of 517 BEs treated with rAHF-PFM, 466 BEs were rated
and 51 had an unknown efficacy rating.
BEs=bleeding episodes. Of 517 treated BEs, 89.6% were controlled with one or two infusions.
N=number of surgical procedures. There were a total of 27 surgeries in 27 subjects.
Intraoperative: 22 subjects were rated, two did not receive product, and in three
subjects, assessment was not done by the surgeon. Postoperative: 25 subjects were
rated, and two were not.
|
C) Haemostatic efficacy in surgical procedures
|
|
Intraoperative
(Operating
Surgeon)
|
Postoperative
(Study Site
Investigator)
|
|
N
|
%
|
N
|
%
|
|
Excellent
|
18
|
81.8
|
23
|
92.0
|
|
Good
|
4
|
18.2
|
2
|
8.0
|
|
Total
|
22
|
100.0
|
25
|
100.0
|
BEs=bleeding episodes.
a The BE for this rating of “none” had three evaluations for three infusions (spontaneous
BE of the buttocks). The first and second of the three infusions was rated “none”
and the third infusion was rated “good.” Haemostatic efficacy in 93.4% of rated BEs
was considered excellent or good. Of 517 BEs treated with rAHF-PFM, 466 BEs were rated
and 51 had an unknown efficacy rating.
BEs=bleeding episodes. Of 517 treated BEs, 89.6% were controlled with one or two infusions.
N=number of surgical procedures. There were a total of 27 surgeries in 27 subjects.
Intraoperative: 22 subjects were rated, two did not receive product, and in three
subjects, assessment was not done by the surgeon. Postoperative: 25 subjects were
rated, and two were not.
There were a total of 96 joint BEs and 421 non-joint BEs during the study requiring
treatment. While BEs were not rated as major or minor, none of the BEs that were reported
as SAEs were considered life threatening. The median annualised bleeding rate was
4.83 (range: 0.00–33.71) BEs/year/subject for all 55 subjects. The median annualised
bleeding rate tended to increase with the age of enrolment; 4.22 (range: 0.00–17.90)
BEs/year/subject for 21 subjects enrolled at <6 months old, 5.90 (range: 0.00–33.71)
BEs/year/ subject for 26 subjects enrolled at 6–12 months old, and 10.16 (range: 2.40–15.98)
BEs/year/subject for eight subjects enrolled at ≥13 months old. Although these differences
were not statistically significant they highlight the possibility that bleed rates
in very young children may not be constant over time. No conclusions could be drawn
from analysis of annualised bleeding rate by regimen in the PUP/MTP population as
the study was not designed to make this comparison. In addition, the low number of
subjects (three each) receiving modified or standard prophylaxis as a predominant
treatment as compared to 15 subjects treated predominantly on-demand does not provide
the statistical power to make a comparison between treatments, and the majority of
subjects switched treatment regimen during the course of the study. Furthermore, there
was great variety in dosing and frequency of prophylactic infusions.
Perioperative management
Intraoperative haemostatic efficacy, as judged by the operating surgeon in 22/27 subjects,
was rated excellent in 18 (82%) subjects and good in four (18%) subjects (efficacy
was unrated or not applicable in 5/27 subjects) (►[Table 1C]). Post-operative haemostatic efficacy, as judged by the investigator in 25/27 subjects,
was rated excellent in 23 (92%) subjects and good in two (8%) subjects (efficacy was
not rated in 2/27 subjects). The actual blood loss as a percentage of predicted maximum
blood loss was equal to or less than 100% for all procedures (median; 20%; range:
0.01–100%).
Safety[
2
]
Throughout the entire study, 3,877,140 IU of rAHF-PFM were administered to 55 subjects
at a median dose of 49.2 (range: 22.6–112.5) IU/kg/subject and a median exposure of
76 (range: 1–414) EDs. This includes rAHF-PFM consumed during ITI treatment. Excluding
the 11 subjects who received ITI treatment, 1,403,473 IU were administered at a median
dose of 45.7 (range: 22.6–110.1) IU/kg/subject and a median exposure of 75 (range-1–87)
EDs. Throughout the entire study, the median duration of a subject’s participation
was 498 (range: 82–1360) days including ITI, and 549 (range: 82–1,360) days excluding
subjects who received ITI.
Adverse events
During the study period, 53 subjects experienced 931 AEs, the majority of which were
non-serious. Of 885 non-serious AEs, 14 events in eight subjects were considered product-related,
none of which were rated severe: the following AEs were rated as moderate (one case
of each): diarrhoea, vomiting, peripheral oedema, infection, and urticaria. Five cases
of rash occurred in four subjects: one rated as moderate, and four in three subjects
rated as mild. Four cases of pyrexia in four subjects were moderate and mild in two
subjects each. Two of the cases of pyrexia (one moderate and one mild) occurred shortly
after port placement surgery, and were of short duration.
The most commonly reported non-serious non-related AEs in this study are typically
seen in the age group investigated: pyrexia, nasopharyngitis, cough, rhinorrhea, diarrhoea,
ear infection, vomiting, upper respiratory tract infections, nasal congestion, rash,
anaemia, conjunctivitis, procedural pain, diaper dermatitis, otitis media, and wheezing.
All other non-serious AEs occurred in less than 10% of subjects.
There were 46 serious adverse events (SAEs) in 28 subjects. Sixteen product-related
SAEs in 16 subjects were all cases of inhibitor development. Further, there were 11
complications/infections in six subjects potentially associated with the placement
of a port/venous access device. Six of these complications were SAEs, (five catheter-related
infections and one catheter site haematoma).
Inhibitor development
The primary safety evaluation in this study addressed inhibitor development. Inhibitory
antibodies to FVIII developed in 16/55 (29.1%; 95% CI: 17.1%-41.1%) subjects. Calculations
of the incidence of inhibitor development for MTPs (11/37 [29.7%; 95% CI: 15.0%-44.5%])
and PUPs (5/18 [27.8%; 95% CI; 7.1%-48.5%]) were post-hoc analyses. All the subjects
who developed inhibitors had severe haemophilia (FVIII <1%). At the time of inhibitor
diagnosis, seven subjects were categorised with high-titre inhibitors (>5 BU/ml confirmed
with a new blood sample) and nine subjects were categorised with low-titre inhibitors
(<5 BU/ml). There was a similar incidence of inhibitor development for confirmed high-titre
inhibitors (12.7%; 95% CI: 3.9%, 21.5%) and for confirmed low-titre inhibitors (16.4%;
95% CI: 6.6%, 26.1%). Eight of 16 inhibitor subjects experienced a peak high-titre
inhibitor (>5 BU/ ml) at least once during the course of the study (►[Table 2]). One peak high titre was an unconfirmed first high titre which occurred prior to
ITI treatment without an anamnestic response during ITI, and the confirmation of low
titre occurred during ITI. Of these eight peak high-titre subjects, seven underwent
ITI on study, four of whom were successfully tolerised, and one exited the study to
join the International ITI Study ([12]). Four subjects with low-titre inhibitors also underwent ITI and were successfully
tolerised. Of those four subjects who did not undergo ITI, one fulfilled the criteria
for a transient inhibitor while remaining primarily on an ondemand regimen, and the
other three subjects were switched from on-demand to prophylactic infusions ranging
from 50–100 IU/kg 1–3 times weekly. No inhibitors were detected at study completion
in these three subjects, and in two of these three subjects the last two inhibitor
determinations were negative.
Table 2
Characteristics of inhibitor subjects.
|
Subject
ID
|
Peak inhibitor
titre (BU/ml)
|
EDs prior
to
inhibitor
|
ITI
|
Race/
Ethnicity
|
Family history
of inhibitor
|
Intensive treatment
at high
dose[a] (Y/N)
≤10, 20,or 30 EDs
|
Surgery (Y/N)
≤10, 20, or 30 EDs
|
High risk
HLA[b]
|
Gene mutation classification
(high/low risk)
|
|
1
|
9.3
|
6
|
N
|
Caucasian
|
Unknown
|
N:N:N
|
N:N:N
|
N
|
frameshift resulting in stop codon[c] (High)
|
|
4
|
1.8
|
20
|
Y
|
Hispanic
|
N
|
N:N:N
|
N:N:N
|
N
|
Nonsense (High)
|
|
5
|
24.0
|
8
|
Y
|
Black
|
N
|
N:N:N
|
N:N:N
|
N
|
frameshift resulting in stop codon[c] (High)
|
|
9
|
1.96
|
13
|
N
|
Caucasian
|
Y
|
N:N:N
|
Y:Y:Y
|
Y
|
inversion; intron 22 (High)
|
|
11
|
21.6
|
12
|
Y
|
Hispanic
|
Y
|
Y:Y:Y
|
Y:Y:Y
|
Y
|
inversion; intron 22 (High)
|
|
12
|
1.4
|
15
|
Y
|
Caucasian
|
Y
|
Y:Y:Y
|
Y:Y:Y
|
N
|
inversion; intron 22 (High)
|
|
17
|
183.9
|
7
|
Y
|
Hispanic
|
N
|
N:N:N
|
N:N:N
|
N
|
inversion; intron 22 (High)
|
|
19
|
3.6
|
15
|
Y
|
Black
|
Y
|
Y:Y:Y
|
Y:Y:Y
|
Y
|
inversion; intron 1 (High)
|
|
27
|
1.5
|
9
|
N
|
Caucasian
|
Y
|
N:N:N
|
Y:Y:Y
|
Y
|
inversion; intron 22 (High)
|
|
28
|
12.8
|
18
|
Y
|
Caucasian
|
N
|
Y:Y:Y
|
Y:Y:Y
|
N
|
inversion; intron 22 (High)
|
|
29
|
1.0
|
26
|
N
|
Caucasian
|
N
|
Y:Y:Y
|
Y:Y:Y
|
Y
|
frameshift at or near A run[d] (Low)
|
|
35
|
3.6
|
11
|
N
|
Indian
|
N
|
N:N:N
|
N:N:N
|
Y
|
inversion; intron 22 (High)
|
|
38
|
44.8
|
17
|
Y
|
Caucasian/ Asian
|
N
|
N:N:N
|
N:N:N
|
Y
|
inversion; intron 22 (High)
|
|
39
|
21.6
|
16
|
Y
|
Hispanic
|
Y
|
N:Y:Y
|
N:N:N
|
Y
|
inversion; intron 22 (High)
|
|
40
|
38.4
|
13
|
Y
|
Hispanic
|
Y
|
N:N:N
|
Y:Y:Y
|
N
|
splice site (High)
|
|
47
|
4.8
|
10
|
Y
|
Caucasian
|
Y
|
N:N:N
|
N:N:N
|
Y
|
inversion; intron 22 (High)
|
ID= identification; ITI=immune tolerance induction; Y=yes; N=no; BU/ml= Bethesda Units;
EDs=exposure days.
a Intensive treatment at high dose is defined as five consecutive study days of treatment
at a mean infusion dose of rAHF-PFM >50 IU/kg within ≤10 EDs, ≤20 EDs, or ≤30 EDs.
b High risk HLA type: defined as DR15 and/or DQ06.
c With no prior documentation of partial correction of reading frame.
d Previously documented to have partial correction of reading frame.
The median time to inhibitor formation was 13 (range: 6–26) EDs. The median time to
inhibitor development was also 13 EDs for both low-titre (range: 9–26) and high-titre
(range: 6–18) subjects. A cumulative inhibitor incidence plot demonstrating time to
inhibitor development is presented in ►[Figure 2]. The FVIII epitopes targeted by inhibitor subjects’ antibodies were identified by
affinity selection and clustered mainly in the C2 domain (nine subjects), the A2 domain
(five subjects), and the A3 domain (three subjects). Epitope analysis results and
discussion are provided in online supplemental material (available online at www.thrombosis-online.com).
Figure 2: Cumulative incidence of inhibitor development
Risk factor analysis
An immunogenicity analysis set of 50 subjects was used for the calculation of the
odds ratio (OR) of risk factors for inhibitor development, which included all 16 subjects
with inhibitors and subjects without inhibitors who had at least 10 EDs (►[Table 3]). Univariate analysis identified statistically significant OR results for three
risk factors: family history of inhibitor (4.95 [95% CI: 1.29–19.06]), non-Caucasian
ethnicity (4.18, [95% CI: 1.18–14.82]), and intensive treatment at high dose within
the first 20 EDs (4.50, 95% CI: 1.05–19.25). Intensive treatment at high dose in all
but one subject, where it was administered for a BE, consisted of perioperative infusions
in the context of port placement. In the 10 subjects who had intensive treatment at
high dose, the four subjects who did not develop inhibitors had a median product exposure
of 80 (range: 75–82) EDs throughout the study. Intensive treatment at high dose tended
to be administered very early in the study, after a median of 3.5 (range: 0–13) EDs.
Intensive treatment alone was not associated with an increased risk. Neither surgery
nor port placement alone were found to be risk factors for inhibitor development;
however, most subjects who received intensive treatment at high dose, which was a
significant risk factor, were treated because of surgery. In some risk factor categories,
the number of subjects was very small and consequently the analysis lacked statistical
power to generate a meaningful result. Other putative risk factors not found to impart
a statistically significantly increased risk of inhibitor development were FVIII gene
mutations, the DR15 and DQ06 HLA genotypes, and age at first exposure.
Table 3
Summary of risk factors for all subjects in the immunogenicity data set (N=50).
|
Risk factor
|
Inhibitor
negative
(N=34)
|
Inhibitor
positive
(N=16)
|
Odds ratio (95% CI)
|
|
N
|
N
|
|
Family history of inhibitors
|
Yes
|
6
|
8
|
4.95 (1.29, 19.06)
|
|
Unknown
|
2
|
1
|
|
No (ref.)
|
26
|
7
|
|
Race/Ethnicity
|
Non-Caucasian
|
8
|
9
|
4.18 (1.18, 14.82)
|
|
Caucasian (ref.)
|
26
|
7
|
|
FVIII gene mutation[a]
|
High risk
|
26
|
15
|
4.62 (0.52, 40.58)
|
|
Low risk (ref.)
|
8
|
1
|
|
High risk HLA[b]
|
Yes
|
15
|
9
|
1.63 (0.49, 5.39)
|
|
No (ref.)
|
19
|
7
|
|
Intensive treatment at high dose[c] (≤20 EDs +)
|
Yes
|
4
|
6
|
4.50 (1.05, 19.25)
|
|
No (ref.)
|
30
|
10
|
|
Intensive treatment[d] (≤30 EDs +)
|
Yes
|
10
|
7
|
1.87 (0.54, 6.40)
|
|
No (ref.)
|
24
|
9
|
|
Surgery (≤30 EDs)[e]
|
Yes
|
13
|
8
|
1.62 (0.49, 5.36)
|
|
No (ref.)
|
21
|
8
|
|
Port placement (≤30 EDs)[e]
|
Yes
|
11
|
6
|
1.25 (0.36, 4.34)
|
|
No (ref.)
|
23
|
10
|
|
Age at first exposure (months)
|
6–12 months
|
16
|
7
|
0.44 (0.10, 2.01)
|
|
13–18 months
|
5
|
5
|
0.31 (0.06, 1.64)
|
|
<6 months (ref.)
|
13
|
4
|
|
The immunogenicity data set included all those developing an inhibitor and all those
who were inhibitor-free with at least 10 EDs.
a Gene mutation was categorised as follows: large deletion, inversion, nonsense, splice
site, frameshift resulting in stop codon, with no prior documentation of partial correction
of reading frame were assessed as high risk. Missense, inframe, frameshift at or near
a run previously documented to have partial correction of reading frame were assessed
as low risk.
b High risk HLA type: defined as DR15 and/or DQ06. (DR15 and DQ06 analysed individually
were not significant risk factors).
c Intensive treatment and high dose: defined as five consecutive study days of a mean
infusion dose of FVIII >50 IU/kg. (≤20 EDs and ≤30 EDs were significant risk factors,
but ≤10 EDs was not).
d Intensive treatment: defined as at least five consecutive days of FVIII treatment.
( ≤10 EDs, ≤20 EDs, and ≤30 EDs were not significant risk factors).
e ≤10 EDs, ≤20 EDs, and ≤30 EDs were not significant risk factors. ref.=reference
class, + Refers to Advate: non-study or investigational product
Inhibitor incidence in low-risk subpopulations of subjects who lacked the risk factors
confirmed in this study was examined. While 16/55 (29.1%) subjects in the entire study
population developed inhibitors, 7/37 (18.9%) subjects who were Caucasian and 2/25
(8.0%) subjects who were both Caucasian and had no family history developed inhibitors.
In the 20 subjects who were Caucasian, had no family history of inhibitors, and did
not receive intensive treatment at high dose (lacked all three risk factors) there
was no inhibitor development (0/20 [0.0%]). These 20 subjects had a median of 77 (range:
1–82) EDs: 16/20 subjects had ≥30 EDs, 2/20 had <20 to ≥10 EDs and 2/20 subjects had
<10 EDs. Throughout the study, these 20 subjects had a median of 10.0 (range: 0.0
to 29.0) BEs.
A linear regression analysis of antibody formation to heterologous proteins demonstrated
that five subjects had a slight increase over time in antibodies against CHO or murine
IgG proteins, but no statistically significant increases could be demonstrated and
therefore the percentage of subjects who developed antibodies is 0% for all three
heterologous proteins. No clinically relevant correlation could be found, as assessed
by an examination of AEs of allergic reaction (including those considered related
to rAHF-PFM [i.e. rash, urticaria]) or elevated eosinophil counts in temporal relationship
with infusions.
Discussion
In this clinical study of 55 PUPs and MTPs with severe and moderately severe haemophilia
A, haemostatic efficacy of rAHF-PFM was confirmed in the treatment of BEs and surgical
prophylaxis. One or two infusions of rAHF-PFM were used to manage 90% of BEs, and
the efficacy was considered excellent or good in 93% of treated BEs that were rated,
consistent with previously reported values for rAHF-PFM ([1], [2]) and related rFVIII products ([14]–[16]). Likewise, intraoperative and postoperative haemostatic efficacies are comparable
to previously reported values ([3]).
In 22 BEs treated with three or more infusions, the haemostatic efficacy was rated
as excellent, although the protocol definition for excellent haemostatic efficacy
only allowed a single infusion. The most likely explanation for this discrepancy could
be the serious nature of the BEs where additional infusions were administered for
maintenance of haemostasis.
Although more subjects were prescribed on-demand treatment than prophylaxis as the
first treatment modality (47 vs. 8), on-demand subjects tended to be switched to a
prophylactic regimen at a later date, when they became more physically active, following
increasing numbers of BEs, or at a standard age determined by the investigative site.
Patients <2 years of age generally have fewer BEs than children >2 years and thus
tend to be initially prescribed an on-demand regimen, but switched to prophylaxis
later ([17]–[19]). Investigators in the EU prescribed prophylaxis (either as first treatment option
or switched from on-demand treatment) in a statistically significantly greater proportion
of subjects than did the investigators in North America.
Approximately 30% of PUPs with severe haemophilia A develop inhibitory antibodies
that diminish the efficacy of FVIII replacement therapy ([4]–[6]). The results obtained in the current study are concordant with these published
results: inhibitors developed in 16/55 (29.1%) subjects. Notably, 8/16 (50.0%) inhibitor
subjects never experienced a peak high-titre inhibitor (>5 BU/ml). BEs in low-titre
subjects can still be managed with FVIII concentrate as demonstrated in the four subjects
with low-titre inhibitor not undergoing ITI who were successfully managed with rAHF-PFM.
In the ITI part of the study, all four low-titre subjects were successfully tolerised,
compared to 4/7 high-titre subjects (unpublished results). Of the 16 subjects who
developed inhibitors, 11 went on to ITI treatment in the ITI part of the study, eight
of whom were successful[
3
]. One subject who developed an inhibitor was withdrawn to undergo ITI in the International
ITI study ([12]), from where he was withdrawn again and eventually tolerised with rAHF-PFM[
4
]. Inhibitor development was similar between MTPs (29.7%) and PUPs (27.8%), indicating
that no bias was introduced into the study by using subjects who had limited previous
exposure to rAHF-PFM.
Our finding of a median time to FVIII inhibitor development of 13 (range: 6–26) EDs
is consistent with values reported in other previously untreated cohorts ([14], [20]). Epitope analysis in this study agreed with published findings that inhibitory
antibodies from patients with haemophilia A are most commonly directed against epitopes
in the A2 and C2 domains of FVIII ([21]).
Subject characteristics were evaluated as risk factors for inhibitor development.
The OR of risk for developing inhibitors was significantly higher in subjects with
a family history of inhibitor (4.95 [95% CI: 1.29–19.06]). This is in agreement with
the findings of the Malmö International Brother Study and an analysis of a subset
of 332 subjects from the CANAL cohort which report ORs of 3.2 (95% CI: [2.1–4.9])
and 3.7 (95% CI: [1.5–9.2]), respectively ([22], [23]). Santagostino et al. reported that family history of inhibitors was more frequent
in patients with inhibitors compared with inhibitor-free controls (20% vs. 2%) in
an Italian case-control study of children with haemophilia ([24]).
Non-Caucasian ethnicity (4.18, [95% CI: 1.18–14.82]) was also found to be a significant
risk factor for inhibitor development in the current study. Maclean et al. report
non-Caucasian ethnicity to be a significant risk factor (OR: 4.7 [95% CI: 1.5–14.7])
([25]). In a retrospective cohort study, Gouw et al. demonstrated that patients of African
and Hispanic descent had a relative risk of inhibitor formation of 2.4 and 2.5, respectively,
compared to Caucasians ([26]). Aledort also reported that the prevalence of inhibitors in African-Americans and
Latinos is greater than that of Caucasians ([27]), and Carpenter et al. report an increased inhibitor incidence in Mexican Hispanic
patients (OR: 1.5 [95% CI: 1.1–1.9]) ([28]).
The third significant risk factor identified in the study was exposure to intensive
treatment at high dose, defined as five consecutive days of a mean infusion dose >50
IU/kg/day, within ≤20 EDs, with an OR of 4.50 [95% CI 1.05–19.25]. In the CANAL cohort
study of 366 PUP subjects, it was found that intensive treatment at first exposure
was associated with a 3.3-fold higher incidence of inhibitor development, and intensive
treatment during the first 50 EDs was associated with a two fold higher incidence
of inhibitor formation ([20]). Ter Avest et al. reported an OR of 7.7 (95% CI: 3.8,15.2) for five consecutive
days of treatment at first treatment in a PUP population ([23]).
To examine the impact of risk factors on inhibitor development, inhibitor incidence
in subjects who lacked the high risk factors confirmed in this study were examined.
As subpopulations were defined with successively lower risk (Caucasian, no family
history of inhibitors, and no intensive treatment at high dose), inhibitor incidence
progressively decreased from 29.1%, to 18.9%, to 8.0% and ultimately to 0.0%. This
progressive decrease and the absence of inhibitor development in the lowest risk population
confirm the influence of these risk factors on inhibitor development in the present
study.
In several analyses of data from PUPs, high-risk mutations of the FVIII gene were
associated with increased inhibitor risk: Maclean et al. ([25]) report an OR of 5.1 (95% CI: 1.9–13.7) in 143 subjects with genetic analysis, and
Gouw et al. ([20]) report an OR of 2.8 (95% CI: 1.5–5.0) in 312 patients with genetic analysis. However,
in the current study, which had a considerably smaller sample size, high-risk mutations
of the FVIII gene were not found to be statistically significant risk factors, although
the majority of subjects (15/16) who developed inhibitors had a high-risk mutation
such as inversion, nonsense, or splice site mutation. In this study, intensive treatment
at high dose was employed almost exclusively in the context of port-placement. Therefore,
any surgical intervention requiring intensive treatment at high dose including port
placement should only be considered after careful evaluation of the potential risks
and benefits.
In order to confirm the increasing importance of avoiding immunologic danger signals
during prophylactic treatment of PUPs, as described by Kurnik et al. ([29]), a prospective, historically controlled clinical study has been initiated by Baxter
to evaluate an early low-dose prophylactic regimen. The study is planned to avoid
immunological danger signals, such as tissue and cell damage (e.g. surgical procedures,
BEs), which activate inflammatory responses, at the time of rAHF-PFM infusion.
The incidence of inhibitor formation observed in the current study is aligned with
previous reports as are the findings of significant associations of inhibitor development
with non-Caucasian ethnicity, family history of inhibitors, and intensive treatment
at high dose within ≤20 EDs. In summary, the results of this study confirm an already
established overall record of safety and haemostatic efficacy of rAHF-PFM for the
routine clinical management and perioperative coverage of patients with severe to
moderately severe haemophilia A in PUPs and MTPs.
