Rheumatology Department, AP-HP, Henri Mondor University Hospital, 51 avenue du Mal de Lattre de Tassigny, 94010 Créteil, France
Rheumatology Department, Jean Minjoz University Hospital, 2 boulevard Fleming, 25030 Besançon, France
Rheumatology Department, Gabriel Montpied University Hospital, 58 rue Montalembert, 63000 Clermont-Ferrand, France
Rheumatology Department, Robert Ballanger Hospital, boulevard Robert Ballanger, 93602 Aulnay sous Bois, France
Rheumatology Department, Hôtel Dieu University Hospital, place Alexis Ricordeau, 44093 Nantes, France
Rheumatology Department, University Hospital, Grenoble, Hôpital Sud, 19 avenue de Kimberley BP 185, 38130 Echirolles, France
CIC-Biotherapy 506, St Jacques Hospital University Hospital, 2 boulevard Fleming, 25030 Besançon France
Rheumatology Department, Trousseau University Hospital, 37044 Tours Cedex 1, France
Rheumatology Department, Conception University Hospital, 147 boulevard Baille, 13385 Marseille, France
Rheumatology Department, Saint-Antoine Hospital, Pierre et Marie Curie University, 184 rue du faubourg Saint Antoie, 75012 Paris, France
INSERM Unit 955, AP-HP, Henri Mondor University Hospital, 51 avenue du Mal de Lattre de Tassigny, 94010 Créteil, France
Pharmacy Department, AP-HP, Henri Mondor University Hospital, 51 avenue du Mal de Lattre de Tassigny, 94010 Créteil, France
LIC EA4393, University Paris Est, 8 avenue du Général Sarrail, 94000 Créteil, France
Abstract
Introduction
The aim of this study was to evaluate, under real-life conditions, the safety and efficacy of tocilizumab in patients having failed anti-TNFα therapy for spondyloarthritis.
Methods
French rheumatologists and internal-medicine practitioners registered on the Club Rhumatismes et Inflammations website were asked to report on patients given tocilizumab (4 or 8 mg/kg) to treat active disease meeting Assessment of SpondyloArthritis International Society (ASAS) criteria for axial or peripheral spondyloarthritis, after anti-TNFα treatment failure. Safety and efficacy after 3 and 6 months were assessed retrospectively using standardised questionnaires.
Results
Data were obtained for 21 patients, 13 with axial spondyloarthritis (46% men; median age, 42 years; disease duration, 11 years; HLA-B27-positive, 92.3%) and eight with peripheral spondyloarthritis (25% men; median age, 40 years; disease duration, 10 years; HLA-B27-positive, 62.5%). No patients with axial disease had at least a 20 mm decrease in the BASDAI, nor a BASDAI50 response or major ASAS-endorsed disease activity score improvements after 3 or 6 months; an ASAS-endorsed disease activity score clinically important improvement was noted at month 3 in five of 13 patients and at month 6 in one of four patients. A good DAS28 response was achieved in four patients with peripheral disease, including one in EULAR remission at month 3. Four patients were still taking tocilizumab at month 6, including one in EULAR remission and one with a good DAS28 response. Tocilizumab was well tolerated, with no serious adverse events. Initially elevated acute-phase reactants declined during tocilizumab therapy.
Conclusion
In patients having failed anti-TNFα therapy, tocilizumab decreased acute-phase reactants but failed to substantially improve axial spondyloarthritis and was inconsistently effective in peripheral spondyloarthritis.
Introduction
The introduction of TNFα antagonists (anti-TNFα) has revolutionised the management of patients with spondyloarthritis (SpA). Randomised controlled trials demonstrated substantial efficacy of anti-TNFα therapy in ankylosing spondylitis (AS)
In experimental studies, serum IL-6 levels were elevated in patients with AS and PsA and correlated with disease activity
Tocilizumab is a humanised monoclonal antibody to the human IL-6 receptor
Materials and methods
Case ascertainment
All of the 1,400 French rheumatologists and internists belonging to the CRI were asked, through the CRI website
Among reported cases, we selected the patients who met at least the Assessment of SpondyloArthritis International Society (ASAS) criteria for axial SpA
Assessment
For each patient, data were collected by the referent rheumatologist on a standardised case-report form available on the CRI website
Data analysis
Completed case-report forms were sent to the coordinating centre (Henri Mondor Teaching Hospital). Patients meeting the study inclusion criteria were identified. Missing data on these patients were obtained by contacting the treatment centres.
Effectiveness was assessed based on data obtained at baseline (M0), after 3 months (M3), and after 6 months (M6). Patients with axial disease were classified as responders if they had at least a 20 mm decrease in the BASDAI or at least a 50% improvement in the BASDAI (BASDAI50 response) at M3 or M6, as proposed for assessment of efficacy of an anti-TNFα drug
Safety was assessed based on the rates of adverse events, serious adverse events, and infections, as well as on laboratory test results at each tocilizumab infusion.
Results
Baseline characteristics
We identified 21 patients meeting our inclusion criteria, 13 with axial disease and eight with peripheral disease, according to ASAS classification criteria; one of those patients has been reported elsewhere
Of the 13 patients with axial disease, nine also met Amor's criteria
Main baseline features of the 13 patients meeting ASAS criteria
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Type of SpA
AS
AS
AS
AS
AS
AS
AS
Sacroiliitis
Grade 4
Grade 4
Grade 3
Grade 4
Grade 3
Grade 3
Grade 4
HLA-B27
+
+
+
+
+
+
+
Gender
Female
Female
Female
Male
Male
Female
Male
Disease duration (years)
9
27
11
6
4
24
19
Main location of inflammatory pain
Back, neck, chest, peripheral enthesitis (Achilles tendon, knee)
Neck, chest, peripheral enthesitisa
Back, buttock, chest
Neck
Back, neck, buttock
Back, neck, chest
Back, neck, peripheral enthesitis (Achilles tendon)
Concomitant NSAIDs/steroids (dosage)
Yes/no
Yes/no
Yes/no
No/no
No/no
Yes/no
Yes/no
Concomitant DMARDs
No
No
No
Sulfasalazine
No
No
No
Previous biologics other than anti-TNF
Abatacept
Abatacept
Abatacept
No
No
No
No
Patient 8
Patient 9
Patient 10
Patient 11
Patient 12
Patient 13
Type of SpA
AS and PsA
uSpA
uSpA
uSpA
uSpA
uSpA
Sacroiliitis
Grade 3
Grade 1
Grade 0
Grade 0
Grade 0
Grade 0
HLA-B27
-
+
+
+
+
+
Gender
Male
Female
Female
Male
Female
Male
Disease duration (years)
15
11
9
25
6
12
Main location of inflammatory pain
Back, neck, arthritis
Back, neck, buttock, chest
Back, buttock
Back, neck, buttock
Back, buttock, chest, peripheral enthesitis (Achilles tendon)
Back, neck
Concomitant NSAIDs/steroids (dosage)
No/no
Yes/no
Yes/no
No/no
Yes/no
No/yes (25 mg/day)
Concomitant DMARDs
No
No
No
No
No
No
Previous biologics other than anti-TNF
Rituximab
Abatacept
Abatacept
No
No
No
AS, ankylosing spondylitis meeting modified New York criteria
Of the eight patients with peripheral disease, seven also met Amor's criteria or the European Spondyloarthropathy Study Group criteria, four met modified New York criteria for AS, and two met the Classification Criteria for Psoriatic Arthritis. Seven patients had failed treatment with three anti-TNFα agents, and the remaining patient had failed treatment with two anti-TNFα agents (Table
Main baseline features in the eight patients meeting ASAS criteria
Patient 14
Patient 15
Patient 16
Patient 17
Patient 18
Patient 19
Patient 20
Patient 21
Type of SpA
AS
AS
AS
AS
uSpA
uSpA
PsA
PsA
Sacroiliitis
Grade 2
Grade 3
Grade 3
Grade 3
Grade 0
Grade 1
Grade 0
Grade 0
HLA-B27
+
-
+
+
+
+
-
-
Gender
Male
Female
Male
Female
Female
Female
Female
Female
Disease duration (years)
8
15
19
6
10
10
10
20
Main articular and extraarticular symptoms
Coxitis, arthritis
Arthritis
Arthritis, uveitis
Peripheral enthesitis (Achilles tendon, knee, elbow), arthritis, chest pain
Arthritis
Arthritis, IBD, uveitis
Arthritis, dactylitis, psoriasis, uveitis
Arthritis, psoriasis
Concomitant NSAIDs/steroids (dosage)
Yes/no
No/yes (20 mg/day)
Yes/yes (5 mg/day)
Yes/yes (15 mg/day)
Yes/yes (8 mg/day)
No/yes (10 mg/day)
Yes/yes (20 mg/day)
Yes/yes (20 mg/day)
Concomitant DMARDs
Methotrexate
No
Methotrexate
Leflunomide
Methotrexate
Methotrexate
Methotrexate
Methotrexate
Previous biologics other than anti-TNF
No
Anakinra
No
Abatacept
No
No
No
No
AS, ankylosing spondylitis meeting modified New York criteria
Effectiveness
The effectiveness criteria at M0, M3, and M6 are reported in Table
Baseline and 3-month and 6-month tocilizumab therapy criteria in 13 patients with axial spondyloarthritis
Pt 1
Pt 2
Pt 3
Pt 4
Pt 5
Pt 6
Pt 7
Pt 8
Pt 9
Pt 10
Pt 11
Pt 12
Pt 13
BASDAI, 0 to 100
M0
78
90
72
42
77
63
80
48
49
59
48
72
57
M3 (change from M0; %)
+6
-19
+1
-15
+1
-1.6
+3.8
-2
+4
-5
+43
-10
-12
M6 (change from M0; %)
NA
NA
NA
-17
NA
NA
NA
NA
-6
-15
+17
NA
NA
ASDAS
M0
4.87
3.35
3.24
3.37
5
5.36
3.8
2.98
2.68
2.41
2.94
3.2
4.12
M3 (change from M0; units)
-1.6
-0.8
-0.75
-0.04
-1.5
-1.7
-0.3
-0.24
-1.36
-0.43
-0.35
+0.02
-1.96
M6 (change from M0; units)
NA
NA
NA
-1.17
NA
NA
NA
NA
-1.02
-0.61
-0.23
NA
NA
BASFI, 0 to 100
M0
86
95
63
32
72
MD
MD
59.5
50
57
59
28
36
M3 (change from M0; %)
-8
-8
+16
+3
+28
MD
MD
-6
+6
-7
+30
-29
+47
M6 (change from M0; %)
NA
NA
NA
+22
NA
MD
MD
NA
0
-7
-34
NA
NA
Pain VAS, 0 to 10
M0
9
10
7
4
7
7
8
4
5
6
5
7
4
M3 (change from M0; units)
+0.5
-3
+1
0
0
-0.2
+1
+3
+1
-2
0
-1
0
M6 (change from M0; units)
NA
NA
NA
-1
NA
NA
NA
NA
+1
-2
+2
NA
NA
CRP (mg/dl)a
M0
29
0
2
46
35
96
10
10.4
1
1
1.8
3
35.2
M3 (absolute value)
0.3
0
0
NA
3
4
1
< 5
NA
NA
NA
3
1
M6 (absolute value)
NA
NA
NA
6
NA
NA
NA
NA
0
1
0.6
NA
NA
ESR (mm)a
M0
71
9
8
47
35
104
21
12
9
8
1
11
15
M3 (absolute value)
10
2
4
NA
2
2
1
3
NA
NA
NA
3
1
M6 (absolute value)
NA
NA
NA
5
NA
NA
NA
NA
4
1
1
NA
NA
BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; ASDAS, Assessment of SpondyloArthritis International Society-endorsed disease activity score; BASFI, Bath Ankylosing Spondylitis Functional Index; VAS, visual analogue scale; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; M0, month 0 (baseline); M3, month 3; M6, month 6; MD, missing data; NA, not available; Pt, Patient. aFor CRP and ESR, the table shows only the values at baseline (M0) and at last follow-up (M3 or M6).
Baseline and 3-month and 6-month tocilizumab therapy evaluation criteria in eight patients with peripheral spondyloarthritis
Patient 14
Patient 15
Patient 16
Patient 17
Patient 18
Patient 19
Patient 20
Patient 21
DAS28
M0
4.02
4.52
5
4.58
5.41
6.2
5.23
4.92
M3 (absolute value)
2.82
3.85
4.3
2.88
3.18
1.1
5.1
4.70
M3 EULAR response (yes/no)
Yes (good)
No
Yes (moderate)
Yes (good)
Yes (good)
Yes (remission)
No
No
M6 (absolute value)
1.95
4.01
5.34
3.12
NA
NA
NA
NA
M6 EULAR response (yes/no)
Yes (remission)
No
No
Yes (good)
NA
NA
NA
NA
ASDAS
M0
2.41
4.03
MD
MD
MD
3.8
3
MD
M3 (absolute value)
1.50
2.64
MD
MD
MD
1.35
3.1
MD
M6 (absolute value)
1.03
2.79
MD
MD
MD
NA
NA
MD
SJCa
M0
7
2
2
0
3
9
4
7
M3 (absolute value)
NA
NA
NA
NA
7
0
3
6
M6 (absolute value)
0
6
1
0
NA
NA
NA
NA
TJCa
M0
7
6
5
5
0
15
7
12
M3 (absolute value)
NA
NA
NA
NA
0
0
6
11
M6 (absolute value)
2
4
3
8
NA
NA
NA
NA
BASDAI, 0 to 100
M0
42
75
NA
79.4
67
48
4.7
NA
M3 (change from M0; %)
-31
-6.7
NA
-9
+6
NA
-2.1
NA
M6 (change from M0; %)
-59.5
-4.7
NA
-32
NA
NA
NA
NA
BASFI, 0 to 100
M0
40
72
MD
MD
54
70
NA
NA
M3 (change from M0, %)
-17.5
-26
MD
MD
+44
NA
NA
NA
M6 (change from M0, %)
-12.5
-12.5
MD
MD
NA
NA
NA
NA
Pain VAS, 0 to 10
M0
1.5
7
5
7
7
5
6
8
M3 (absolute value)
2
8
NA
6
8
1
6.5
6
M6 (absolute value)
1
9
6
7.5
NA
NA
NA
NA
PGA, 0 to 10
M0
1
6
7
7.5
7
5.2
6.5
8
M3 (absolute value)
3.5
6
MD
MD
7
1
6.5
8
M6 (absolute value)
1.2
6
8
MD
NA
NA
NA
NA
CRP (mg/dl)a
M0
20.2
17
119
12
112.3
65
4
1
M3 (absolute value)
NA
NA
NA
NA
9.5
1
5
1
M6 (absolute value)
0.4
0.8
128
1
NA
NA
NA
NA
ESR (mm)a
M0
39
22
67
26
103
60
27
7
M3 (absolute value)
NA
NA
NA
NA
8
4
7
8
M6 (absolute value)
5
7
70
2
NA
NA
NA
NA
ASDAS, Assessment of SpondyloArthritis International Society endorsed disease activity score; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; CRP, C-reactive protein; DAS28, Disease Activity Score in 28 joints; ESR, erythrocyte sedimentation rate; EULAR, European League Against Rheumatism; M0, month 0 (baseline); M3, month 3; M6, month 6; NA, not available; MD, missing data; PGA, patient's global assessment of disease activity; SJC, swollen joint count; TJC, tender joint count; VAS, visual analogue scale. aFor SJC, TJC, CRP, and ESR, the table shows only the values at baseline (M0) and at last follow-up (M3 or M6).
In the group of patients with axial disease, no patient at M3 had achieved at least a 20 mm decrease in BASDAI, nor a BASDAI50 response (Table
Of the eight patients with AS in the axial disease group (Patients 1 to 8), none had at least a 20 mm decrease in BASDAI or a BASDAI50 response at M3, three had an ASDAS clinically important improvement, none had an ASDAS major improvement, and only one patient continued tocilizumab therapy until M6, when the response was moderate.
Of the five patients (out of the total 21) with a very high disease activity at baseline according to the ASDAS (ASDAS > 3.5), four reached an ASDAS clinically important improvement.
In the group with peripheral disease, the DAS28 at baseline was ≥ 4 in all eight patients. At M3, four patients had a good DAS28 response, including one who met EULAR remission criteria, and another patient had a moderate DAS28 response. The remaining three patients had an insufficient response. At last follow-up, the SJC had decreased in five of the seven patients with joint swelling at baseline, including two patients who had no clinical synovitis; in the remaining two patients, the SJC had increased. At least a 20% SJC decrease was noted in four patients. The ASDAS was available at M3 for four patients and showed a clinically important improvement in one patient and a major improvement in one patient. These two patients were those that had a very high baseline disease activity according to the ASDAS (> 3.5). No patient had a BASDAI50 response. The VAS pain score decreased slightly in two patients and increased in four patients. Of the four patients treated for 6 months, two had a good DAS28 response at M6, including one who was in remission, and two had an insufficient DAS28 response. The ASDAS was available at M6 in two patients and showed a clinically important improvement with no major improvement in both. At M6, the VAS pain score was increased in three of four patients and diminished by 0.5 point in one patient.
Of the eight patients with peripheral disease, seven were taking glucocorticoid therapy at baseline. The glucocorticoid dosage was increased during tocilizumab therapy in three patients, left unchanged in two patients, and decreased in two patients. Two patients received three and two local corticosteroid injections, respectively.
Of the four patients with peripheral AS (Patients 14 to 17), two had a good DAS28 response and one had a moderate DAS 28 response. Neither of the two patients with PsA (Patients 20 and 21) achieved a clinically relevant improvement.
Acute-phase reactants
Of the seven axial disease patients with CRP elevation at baseline, six had normal CRP values at M3; the M3 CRP level was missing in the remaining patient. Similarly, the ESR decreased substantially in the patients with ESR elevation at baseline (Table
Of the six peripheral disease patients with CRP elevation at baseline, two patients had normal CRP values at M3 and four patients at M6. The CRP level had increased in one patient at M6. The ESR decreased substantially, except in the patient whose CRP value had increased by M6.
Safety
No serious adverse events occurred in either group. The following adverse events were reported: axillary abscess (Patient 1), toe blister (Patient 1), allergic reaction (Patient 2), oral ulcers (Patients 14 and 17), nasopharyngitis (Patient 15), Q fever (Patient 16), vasospasm of the extremities (Patient 17), liver enzyme elevation < 3N (Patient 8), and cholesterol and triglyceride elevation (Patient 8).
Flares of anterior uveitis occurred in two patients (Patients 7 and 16) with previous episodes of uveitis, and
Discussion
We obtained detailed descriptions of 3-month and 6-month outcomes of tocilizumab therapy in 21 patients with SpA who had failed anti-TNFα therapy. In the 13 patients meeting the ASAS criteria for axial SpA
Tocilizumab provided no clinically relevant effects in the patients with axial disease. The effects were mixed in the group with peripheral disease. Tocilizumab was ineffective in both the patients with axial AS and in those with axial undifferentiated SpA. The numbers of patients with peripheral SpA were too small for subgroup analyses by diagnosis; however, the good M6 response in the two patients with peripheral AS is worth noting.
We found four previous case reports of tocilizumab therapy for SpA published in 2009 and 2010
The absence of meaningful clinical improvements in our study contrasted with decreases in acute-phase reactants during tocilizumab therapy. These decreases indicate that IL-6 blockade was effective in our patients. Our results demonstrate that the markers for systemic inflammation can return to normal despite no clinical improvement. Consequently, ESR and CRP values cannot be used as isolated surrogate markers for efficacy in SpA patients, even for those with elevated ESR and CRP values at baseline. On the other hand, incorporating ESR or CRP into composite scores such as the ASDAS can improve the assessment of patients with SpA, and perhaps improve the selection of candidate patients for other biologics after several failures. In line with this hypothesis we can note that, within axial patients with a very high disease activity at baseline according to the ASDAS (ASDAS > 3.5), we observed four out of five (80%) patients with an ASDAS clinically important improvement.
Our study has several limitations. Firstly, we should highlight the retrospective design and the limited number of patients. Our purpose was not to conduct a therapeutic trial, however, but instead was to learn from the patients who had been treated under real-life conditions in France. Case ascertainment was incomplete, because not all rheumatologists and internists in France belong to the CRI; on the other hand, those who do belong to the CRI are used to providing detailed case reports - indeed, we excluded only one case because of missing data. We selected patients fulfilling recent ASAS criteria for axial SpA
Secondly, BASDAI alone is not specific enough to evaluate the inflammatory activity of axial SpA in these highly refractory patients. However, the BASDAI and expert opinion are the criteria recommended in France to start biologics after failure of non steroidal anti-inflammatory drugs. There is clearly a need to find new, more stringent, criteria for refractory patients failing treatment with at least two biologics. The ASDAS is a good candidate for these new criteria. It should be argued that some of our patients might actually have other causes of back pain than SpA; however, they all had significant morning stiffness and more than one-half of the axial patients had an increased baseline CRP level, suggesting that SpA itself was still active in those patients. In addition, doses and methods of administration of tocilizumab are those used in RA, a disease with different pathogenesis. Further therapeutic approaches different from those used for RA are needed, reaching as far as research on the new pathogenesis hypothesis in SpA.
Conclusion
Despite a significant decrease of acute-phase reactants, IL-6 blockade did not improve the clinical status of patients with axial SpA who had previously failed anti-TNFα therapy. Tocilizumab does not seem to hold promise for the management of axial SpA refractory to anti-TNFα agents, although only the ongoing randomised controlled trials with this drug in AS could confirm this supposition. There is a need for studies of other drugs
Abbreviations
anti-TNFα: TNFα antagonists; AS: ankylosing spondylitis; ASAS: Assessment of SpondyloArthritis International Society; ASDAS: Assessment of SpondyloArthritis International Society-endorsed disease activity score; BASDAI: Bath Ankylosing Spondylitis Disease Activity Index; BASDAI50: 50% improvement in the Bath Ankylosing Spondylitis Disease Activity Index; BASFI: Bath Ankylosing Spondylitis Functional Index; CRI: Club Rhumatismes et Inflammation; CRP: C-reactive protein; DAS28: disease activity score in 28 joints; ESR: erythrocyte sedimentation rate; EULAR: European League Against Rheumatism; IL: interleukin; M0, month 0 (baseline); M3: month 3; M6: month 6; PsA: psoriatic arthritis; RA: rheumatoid arthritis; SJC: swollen joint count; SpA: spondyloarthritis; TNF: tumour necrosis factor; VAS: visual analogue scale.
Competing interests
DW has received consulting fees, speaking fees and/or honoraria from Abbott, Amgen, Wyeth-Pfizer, Roche, Chugai, Bristol-Myers Squibb, and Schering-Plough (< $10,000 each), and was investigator for Roche Chugai, Sanofi Aventis, and Novartis. MS has received consulting fees, speaking fees, and/or honoraria from Roche, Pfizer (Wyeth), MSD (Schering Plough), BMS, and Abbott. JMB has received consulting fees, speaking fees, and/or honoraria from Roche (< $10,000 each). PGa has received consulting fees, speaking fees and/or honoraria from Abbott, Wyeth-Pfizer, and Roche Chugai (< $10,000 each). PGo has received consulting fees, speaking fees and/or honoraria from Abbott, Wyeth-Pfizer, Roche, Chugai, Bristol-Myers Squibb, Schering-Plough, and MSD (< $10,000 each), and was investigator for Abbott, Wyeth-Pfizer, Roche Chugai, and Bristol-Myers Squibb. TP has received consulting fees, speaking fees and/or honoraria from Abbott, Amgen, Wyeth-Pfizer, Roche, Chugai, Bristol-Myers Squibb, and Schering-Plough (< $10,000 each), and was investigator for Roche Chugai, Sanofi Aventis, and Novartis. JS has received consulting fees, speaking fees, and/or honoraria from Pfizer (Wyeth), MSD (Schering Plough), and Roche France (< $10,000 each). PC has received consulting fees, speaking fees and/or honoraria from Abbott, Wyeth-Pfizer, Roche, Chugai, Bristol-Myers Squibb, Schering-Plough, and UCB (< $10,000 each), and was investigator for Roche Chugai, Sanofi Aventis, Abbott, Wyeth-Pfizer, and BMS (< $10,000 each). FKL, CP, MDB, ET, RB, MP, and VF declare that they have no competing interests.
Authors' contributions
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. PC had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. FKL, CP, DW, TP, JS, MP, and PC were involved in the study conception and design. FKL, CP, DW, MS, MDB, JMB, PGa, ET, PGo, TP, JS, RB, MP, VF, and PC were involved in the acquisition of data. FKL, CP, and PC were involved in the analysis and interpretation of data.
Acknowledgements
The authors thank Dr Antoinette Wolfe for help with English review of this manuscript and the French Institutional Review Board for accepting the project.