Is intralymphatic immunotherapy ready for clinical use in patients with grass pollen allergy?
2013; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2013.07.033
ISSN1097-6825
AutoresMarianne Witten, Hans‐Jørgen Malling, Lars Blom, Britta C. Poulsen, Lars K. Poulsen,
Tópico(s)Dermatology and Skin Diseases
ResumoAllergen-specific subcutaneous immunotherapy confers significant clinical benefits in patients with grass pollen–induced allergic rhinoconjunctivitis. Nevertheless, treatment is costly; it lasts up to 5 years and can occasionally lead to systemic adverse reactions.1Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (172) Google Scholar Improving immunotherapy by means of direct administration of the allergen extract into a lymph node with 3 intralymphatic injections has been attempted in 3 previous studies. Two of the studies have shown efficacy in nasal provocations for grass pollen2Senti G. Prinz Vavricka B.M. Erdmann I. Diaz M.I. Markus R. McCormack S.J. et al.Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial.Proc Natl Acad Sci U S A. 2008; 105: 17908-17912Crossref PubMed Scopus (298) Google Scholar and cat dander.3Senti G. Crameri R. Kuster D. Johansen P. Martinez-Gomez J.M. Graf N. et al.Intralymphatic immunotherapy for cat allergy induces tolerance after only 3 injections.J Allergy Clin Immunol. 2012; 129: 1290-1296Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar One small study combining patients with grass and birch pollen allergy has demonstrated an improved global evaluation of seasonal symptoms.4Hylander T. Latif L. Petersson-Westin U. Cardell L.O. Intralymphatic allergen-specific immunotherapy: an effective and safe alternative treatment route for pollen-induced allergic rhinitis.J Allergy Clin Immunol. 2013; 131: 412-420Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar However, no double-blind, placebo-controlled studies have yet been published looking at efficacy based on seasonal symptom and medication scores, as proposed by the US Food and Drug Administration and the European Medical Agency (EMA).5Godicke V. Hundt F. Registration trials for specific immunotherapy in Europe: advanced guidance from the new European Medical Agency guideline.Allergy. 2010; 65: 1499-1505Crossref PubMed Scopus (5) Google Scholar, 6Canonica G.W. Baena-Cagnani C.E. Bousquet J. Bousquet P.J. Lockey R.F. Malling H.J. et al.Recommendations for standardization of clinical trials with Allergen Specific Immunotherapy for respiratory allergy. A statement of a World Allergy Organization (WAO) taskforce.Allergy. 2007; 62: 317-324Crossref PubMed Scopus (186) Google Scholar To study the efficacy based on patients' seasonal diaries, we performed a randomized, double-blind, placebo-controlled study testing 3 and 6 preseasonal injections of intralymphatic immunotherapy (ILIT) with alum-adsorbed Phleum pratense. A detailed methodology is given in the Methods section (in this article's Online Repository at www.jacionline.org), but briefly, 45 adult subjects with a clinical history of grass pollen–induced rhinoconjunctivitis were recruited. Grass pollen allergy was confirmed by means of skin prick tests (SPTs) and specific IgE measurements. Patients with contraindications to immunotherapy were excluded.1Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (172) Google Scholar Additional sensitizations were permitted as long as the patient did not have symptoms overlapping the grass pollen season. By using the minimization technique,7Ivers N.M. Halperin I.J. Barnsley J. Grimshaw J.M. Shah B.R. Tu K. et al.Allocation techniques for balance at baseline in cluster randomized trials: a methodological review.Trials. 2012; 13: 120Crossref PubMed Scopus (7) Google Scholar patients were randomized into 3 groups receiving 6 injections of 1000 standard quality units (SQ-U; x6 group), 3 injections of 1000 SQ-U followed by 3 injections of placebo (x3 group), or 6 injections of placebo. The primary efficacy measure was the combined symptom and medication score (SMS) during the grass pollen season After the treatment, as proposed by the EMA.5Godicke V. Hundt F. Registration trials for specific immunotherapy in Europe: advanced guidance from the new European Medical Agency guideline.Allergy. 2010; 65: 1499-1505Crossref PubMed Scopus (5) Google Scholar, 6Canonica G.W. Baena-Cagnani C.E. Bousquet J. Bousquet P.J. Lockey R.F. Malling H.J. et al.Recommendations for standardization of clinical trials with Allergen Specific Immunotherapy for respiratory allergy. A statement of a World Allergy Organization (WAO) taskforce.Allergy. 2007; 62: 317-324Crossref PubMed Scopus (186) Google Scholar Additionally, 2 assessments were made: a global seasonal assessment and assessment with the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). Forty-five patients were allocated to treatment, 43 started treatment, and 38 were available for evaluation of the primary efficacy parameter (see Fig E1 in this article's Online Repository at www.jacionline.org). The groups were well balanced (see Table E1 in this article's Online Repository at www.jacionline.org). On the basis of the primary outcome, the combined mean SMS for the entire grass pollen season, no significant clinical efficacy of ILIT could be demonstrated (Fig 1). On the contrary, the x3 group displayed a slightly negative outcome, with a 12% higher total SMS than the placebo group (P = .612). During the peak season, both treatment groups had a higher SMS than the placebo group. For the x3 group, the SMS was significantly higher than in the placebo group (76%, P = .009). Week-per-week comparison of the SMS showed a significant difference between the x3 and placebo groups for weeks 22 and 23 in favor of placebo treatment. No other weeks showed a significant difference. Comparing all treated patients (ie, combining the 2 treatment groups; Fig 1 and see Table E2 in this article's Online Repository at www.jacionline.org) with the placebo-treated patients did not reveal any significant difference in the mean SMS (P = .856, t test). The 2 treatment groups experienced no significant change in global assessment or RQLQ scores as opposed to the placebo group, who improved significantly from pretreatment to posttreatment (P = .0014); however, no significant differences between groups were found (Fig 1 and Table E2). In most cases, treatment was painless. Only 1 patient withdrew because of discomfort during the examination and treatment of side effects. All adverse events were mild; no moderate, severe, or life-threatening symptoms (grade 2-41Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (172) Google Scholar) occurred (see Table E1). Local reactions at the injection site were seen in 83% of the actively treated patients (P < .0001). Intradermal test readings after 24 hours (Fig 2) and grass pollen SPTs showed significantly reduced reactions after treatment in all 3 groups compared with those before treatment but without differences between the groups. The allergen-specific IgE level has a natural decrease out of season and an increase during the season.8Shamji M.H. Ljorring C. Francis J.N. Calderon M.A. Larche M. Kimber I. et al.Functional rather than immunoreactive levels of IgG4 correlate closely with clinical response to grass pollen immunotherapy.Allergy. 2012; 67: 217-226Crossref PubMed Scopus (235) Google Scholar The IgE level for the placebo group followed this pattern. For the treated patients, an increase in IgE levels after treatment was apparent, and the IgE ratio (post/pre) was significantly 1.5- and 1.4-fold increased for the x6 and x3 groups. Actively treated patients had a significant increase in IgG4 levels after treatment (x6: P = .0099, x3: P = .0128). In addition, a significant 2.8-fold increase (P = .0005) was seen in the x6 group and a 2.9-fold increase was seen in the x3 group for IgG4 levels, indicating a recognition/modulation of the immune system in the treated patients. There was no apparent change in results of the grass pollen–induced histamine release test for any group. T-cell cytokines showed a significantly lower IFN-γ level in the x3 group in comparison with the placebo group and a tendency toward higher IL-10 levels. No group differences were seen in IL-4 and forkhead box protein 3 (FoxP3) expression (Fig 2). Concluding on our study, the ILIT for grass pollen allergy revealed no improvement in patients' registered symptoms and use of rescue medication or in quality-of-life or intradermal test scores in spite of weak signs of immune modulation of IgE and IgG4. The reason that ILIT appears to be effective in some studies2Senti G. Prinz Vavricka B.M. Erdmann I. Diaz M.I. Markus R. McCormack S.J. et al.Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial.Proc Natl Acad Sci U S A. 2008; 105: 17908-17912Crossref PubMed Scopus (298) Google Scholar, 3Senti G. Crameri R. Kuster D. Johansen P. Martinez-Gomez J.M. Graf N. et al.Intralymphatic immunotherapy for cat allergy induces tolerance after only 3 injections.J Allergy Clin Immunol. 2012; 129: 1290-1296Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 4Hylander T. Latif L. Petersson-Westin U. Cardell L.O. Intralymphatic allergen-specific immunotherapy: an effective and safe alternative treatment route for pollen-induced allergic rhinitis.J Allergy Clin Immunol. 2013; 131: 412-420Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar but did not show efficacy in ours might be related to the different outcome parameters. The appropriate efficacy measures for specific immunotherapy can be discussed; nevertheless, it would seem logical that the combined symptom and medication registration should be a key factor in the primary end points, as recommended by the World Allergy Organization.6Canonica G.W. Baena-Cagnani C.E. Bousquet J. Bousquet P.J. Lockey R.F. Malling H.J. et al.Recommendations for standardization of clinical trials with Allergen Specific Immunotherapy for respiratory allergy. A statement of a World Allergy Organization (WAO) taskforce.Allergy. 2007; 62: 317-324Crossref PubMed Scopus (186) Google Scholar Considerations as to which parameters could enhance efficacy are not straightforward. The amount of allergen could be increased because in immunotherapy the allergen dose is linked to efficacy generally.1Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (172) Google Scholar However, on the basis of our study, it is not recommendable to increase the amount of allergen without an updosing regimen because systemic side effects were seen in 9% of the patients. Perhaps the ILIT treatment could be effective in combination with hypoallergenic recombinant allergens because higher amounts of allergen can be used. Consequently, on the basis of guidelines from regulatory agencies with respect to accepted primary outcomes, efficacy still has to be documented for this treatment. Even though the number of treated patients in our study exceeds the sum of the 2 previous placebo-controlled studies, it was small, and further adequately performed large-scale, double-blind, placebo-controlled studies focusing on optimizing allergen extracts, dose regimens, or both are required before ILIT is ready for clinical use. The study was randomized, double-blind, and placebo-controlled and performed at the Allergy Clinic, Copenhagen University Hospital, Gentofte, Denmark. Male and female subjects aged 18 to 60 years with a clinical history of grass pollen–induced rhinoconjunctivitis for the last 2 years were recruited primarily by means of advertisement. Grass pollen allergy was confirmed by using SPTs (SPT wheal diameter ≥3 mm with Soluprick SQ 10 HEP Phleum pratense; ALK-Abelló, Hørsholm, Denmark) and specific IgE measurement (Timothy grass pollen–specific antibody [IgE] class ≥2; Phadia CAP-FEIA, Uppsala, Sweden). Patients with additional sensitizations were permitted as long as they did not have symptoms overlapping the grass pollen season. Patients with severe or uncontrolled seasonal asthma or grass pollen immunotherapy within the last 5 years were excluded, as were patients with conditions contraindicatory to immunotherapy.E1Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (399) Google Scholar The group allocation by minimizationE2Ivers N.M. Halperin I.J. Barnsley J. Grimshaw J.M. Shah B.R. Tu K. et al.Allocation techniques for balance at baseline in cluster randomized trials: a methodological review.Trials. 2012; 13: 120Crossref PubMed Scopus (156) Google Scholar and the preparation of study medicine were performed by persons not involved in the trial. Staff administering medication and assessing outcomes were blind to the treatment. One doctor performed all treatments and assessments. Blinding was maintained until all assessments and recordings were completed. The study was conducted in accordance with good clinical practice guidelinesE3ICH harmonized tripartite guideline: guideline for good clinical practice.J Postgrad Med. 2001; 47: 45-50PubMed Google Scholar with written informed consent and was monitored by the Danish GCP Unit, Bispebjerg University Hospital, Copenhagen, Denmark. The protocol was approved by the Danish Medicines Agency and the local ethics committee (no, H-2-2010-020) and complies with the Consort 2010 guidelines for reporting randomized trials.E4Schulz K.F. Altman D.G. Moher D. CONSORT 2010 statement: Updated guidelines for reporting parallel group randomised trials.J Pharmacol Pharmacother. 2010; 1: 100-107Crossref PubMed Google Scholar The patients were treated 6 times with an aseptic ultrasound-guided intralymphatic injection of 0.1 mL of 10,000 SQ-U/mL of depot grass pollen extract (Alutard, Phleum pratense, ALK-Abelló), which corresponds to 0.2 μg of Phl p 5, or saline. Placebo and active medication were identical in color and amount. The 6 treatments were conducted with a minimal interval of 14 days from the beginning of February until April 2011. All patients were observed for 1 hour after the injection. Treatment was postponed in cases of peak flow of less than 80% of normal value or increased allergy symptoms. All injections were performed in the left groin. Lymph nodes were easily detected by using the NanoMaxx ultrasound system (transducer L38N) with color Doppler measurements. The correct placement of the fluid was documented by means of photography. The primary efficacy measure was the combined SMS during the grass pollen season after treatment. The patients recorded nasal and eye symptoms on a 4-point scale (symptom score [SS]) and daily medication (medication score [MS]), as proposed by the EMA and the US Food and Drug Administration guidelines.E5Godicke V. Hundt F. Registration trials for specific immunotherapy in Europe: advanced guidance from the new European Medical Agency guideline.Allergy. 2010; 65: 1499-1505Crossref PubMed Scopus (16) Google Scholar, E6Canonica G.W. Baena-Cagnani C.E. Bousquet J. Bousquet P.J. Lockey R.F. Malling H.J. et al.Recommendations for standardization of clinical trials with Allergen Specific Immunotherapy for respiratory allergy. A statement of a World Allergy Organization (WAO) taskforce.Allergy. 2007; 62: 317-324Crossref PubMed Scopus (389) Google Scholar Rescue medication to be used in the grass pollen season was provided by the clinic and scored as follows:•1 tablet of antihistamine (8 mg of acrivastine) = 4 points (maximum × 3);•1 drop of antihistamine eye drops (0.5 mg/mL levocabastine) = 2 points (maximum × 3); and•1 tablet of corticosteroid (5 mg of prednisolone) = 6 points (maximum × 1). The combined daily SMS was calculated as follows: 0.5 × (Mean daily SS/18) + 0.5 × (Mean daily MS/24). Additionally, 2 assessments were made: a 10-point visual analog scale and the RQLQE7Juniper E.F. Guyatt G.H. Development and testing of a new measure of health status for clinical trials in rhinoconjunctivitis.Clin Exp Allergy. 1991; 21: 77-83Crossref PubMed Scopus (737) Google Scholar with 28 items in 7 domains. SPTs and intradermal tests were performed in duplicate before and after treatment. Soluprick Phleum pratense and disposable prick lancets (ALK-Abelló) were used for SPTs; saline and histamine, 10 mg/mL, were used as negative and positive controls, respectively. Intradermal tests were performed with 0.01 mL of 5000 SQ-U/mL Aquagen Phleum pratense. Readings were made after 20 minutes (immediate reaction) and 24 hours (late-phase reaction). Blood samples for determination of allergen-specific IgE and IgG4 levels were drawn before the start of treatment, after 3 and 6 treatments, and after the posttreatment season. Timothy grass–specific IgE and IgG4 antibody levels were determined by using CAP (Pharmacia), as specified by the manufacturer. The histamine release test was performed before and after treatment by using the histamine release glass-fiber technique with 10-fold titrations of grass pollen extract (RefLab, Copenhagen, Denmark).E8Skov P.S. Norn S. A simplified method for measuring basophil histamine release and blocking antibodies in hay fever patients. Basophil histamine content and cell preservation.Acta Allergol. 1977; 32: 170-182Crossref PubMed Scopus (94) Google Scholar, E9Skov P.S. Mosbech H. Norn S. Weeke B. Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Results compared with conventional leukocyte histamine release assay.Allergy. 1985; 40: 213-218Crossref PubMed Scopus (107) Google Scholar All patients reacted positively to grass pollen extract, but only 8 patients changed their cellular sensitivity (ie, reacting to a different threshold dose from before to after treatment), and thus we found nonsignificant changes both within and between treatment groups (results not shown). Intracellular T-cell cytokines after allergen or tetanus toxoid stimulation of PBMCs were identified. PBMCs were purified from 20 mL of whole blood by using Lymphoprep from Medinor (Oslo, Norway) and stained with carboxyfluorescein succinimidyl ester (CFSE) from Invitrogen (Carlsbad, Calif), according to the method of Parish et al.E10Parish C.R. Glidden M.H. Quah B.J. Warren H.S. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.Curr Protoc Immunol. 2009; (Chapter 4:Unit 4)PubMed Google Scholar The cells were cultured at 2 × 106 cells per mL in 6- or 12-well flat-bottom plates (Nunc, Roskilde, Denmark) in RPMI 1640 medium (Sigma, Saint Louis, Mo) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mmol/L l-glutamine (Invitrogen), 50 μmol/L β-mercaptoethanol (Sigma), and 5% (vol/vol) human AB serum from (Sigma). The PBMCs were cultured for 7 days with either medium control, 10 μg/mL tetanus toxoid from SSI (Copenhagen, Denmark), or 2 μg/mL Phl p 1 and Phl p 5 kindly provided by ALK-Abelló. At day 7, the cultures were harvested and fixed and permeabilized with IntraPrep from Beckman Coulter (Fullerton, Calif), according to the manufacturer's protocol, and stained for CD4–PE-cy5 or PE-cy7 and IL-10–PE-cy7 from BioLegend (San Diego, Calif); IFN-γ–PE, IL-4–PE-cy7, or FoxP3-PE from eBioscience (San Diego, Calif); and CD25-ECD from Beckman Coulter and analyzed by using a Beckman Coulter FC500 MPL flow cytometer and FlowJo from Treestar software (Ashland, Ore).E11Blom L. Poulsen L.K. IL-1 family members IL-18 and IL-33 upregulate the inflammatory potential of differentiated human Th1 and Th2 cultures.J Immunol. 2012; 189: 4331-4337Crossref PubMed Scopus (54) Google Scholar, E12Blom L. Poulsen B.C. Jensen B.M. Hansen A. Poulsen L.K. IL-33 induces IL-9 production in human CD4+ T cells and basophils.PLoS One. 2011; 6: e21695Crossref PubMed Scopus (76) Google Scholar Production of cytokines and expression of FoxP3 from the allergen-specific cells were determined from the CD4+CFSElow population. Safety data were analyzed separately as local or systemic allergic side effects and other adverse events. Side effects occurring within 1 hour of injection were documented by the investigator. Systemic reactions were graded according to the 2006 European Academy of Allergy and Clinical Immunology Position Paper.E1Alvarez-Cuesta E. Bousquet J. Canonica G.W. Durham S.R. Malling H.J. Valovirta E. Standards for practical allergen-specific immunotherapy.Allergy. 2006; 61: 1-20Crossref PubMed Scopus (399) Google Scholar The determination of sample size needed for this study was calculated, given that 13 patients per group were required to detect a 25% relative effect of 3 allergen injections compared with the placebo group with a 5% risk of type 1 or 2 error. Fifteen patients were assigned to each group to account for eventual dropouts, resulting in a total of 45 patients. Safety analysis followed the intention-to-treat format and included all subjects who received at least 1 injection. Primary end point analysis followed the efficacy population, being intention to threat with missing data according to recommendations.E13Bousquet J. Schunemann H.J. Bousquet P.J. Bachert C. Canonica G.W. Casale T.B. et al.How to design and evaluate randomized controlled trials in immunotherapy for allergic rhinitis: an ARIA-GA(2) LEN statement.Allergy. 2011; 66: 765-774Crossref PubMed Scopus (80) Google Scholar SMSs were normalized, averaged over the grass pollen season, and analyzed by means of ANOVA with Tukey correction. Additionally, all treated patients were compared with the placebo group by using a t test. The mean visual analog scale and RQLQ (domain and overall) scores were analyzed by using ANOVA with the Tukey correction. P values of less than .05 were considered statistically significant. GraphPad Prism 6.00 software (GraphPad Software, San Diego, Calif) was used. The sponsor of the study had no role in any part of the study. L.K.P., H.-J.M., and M.W. had full access to data collected for the trial and final responsibility for the decision to submit for publication.
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