Chronic pulmonary aspergillosis (CPA) is often poorly responsive to antifungal treatment; secondary infections increase morbidity/mortality, particularly in progressive cases. Interferon gamma (IFNγ) has been implicated in not only Aspergillus control but also bacterial clearance. Clinical notes of patients with CPA treated with IFNγ (2011–2018) were retrospectively hand-searched. In patients treated for >12 months (n=20), the frequency of acute exacerbation reduced from 3.1 to 1.4 episodes/year (p=0.006) in the 12 months after treatment initiation compared with the 12 months before. A significant reduction in the frequency of hospital admissions/year was also observed (0.8 to 0.3, p=0.04). These findings support further prospective studies

Denning, David W

Döffinger, Rainer

Harris, Chris

Hayes, Gemma

Kosmidis, Chris

Monk, Edward JM

English

Apollo (Cambridge)

This article has been accepted for publication in THORAX following peer review. 1 The definitive copyedited, typeset version is available online at 10.1136/thoraxjnl-2019-213606 2  3 TITLE PAGE:  4 Interferon gamma replacement as salvage therapy in chronic pulmonary aspergillosis; effects 5 on frequency of acute exacerbation and all-cause hospital admission. 6  7 Edward J.M. Monk1, Chris Harris1,2, Rainer Döffinger3, Gemma Hayes4, David W. 8 Denning1,2, Chris Kosmidis1,2 9  10 1 National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, 11 United Kingdom 12 2 Division of Infection, Immunity and Respiratory Medicine, Faculty of Biology, Medicine 13 and Health, Manchester Academic Health Science Centre, The University of Manchester, 14 Manchester, United Kingdom 15 3 Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital, 16 Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom 17 4 Derriford Hospital, University Hospitals Plymouth NHS Trust, Plymouth, United Kingdom 18  19 Correspondence:  20 Dr Chris Kosmidis, 21 National Aspergillosis Centre,  22 Manchester University NHS Foundation Trust,  23 Manchester,  24 United Kingdom 25 Email: chris.kosmidis@manchester.ac.uk 26  27 Abstract word count: 28 101 29  30 Main text word count (excluding acknowledgements, competing interests, funding: 31 1372 32  33  34  35 Key words: 36 Aspergillus Lung Disease, Immunodeficiency, Respiratory Infection  37 ABSTRACT: 38 Chronic pulmonary aspergillosis (CPA) is often poorly responsive to antifungal treatment; 39 secondary infections increase morbidity/mortality, particularly in progressive cases.  40 Interferon gamma (IFNγ) has been implicated in not only Aspergillus control but also 41 bacterial clearance.  Clinical notes of patients with CPA treated with IFNγ (2011-2018) were 42 retrospectively hand-searched.  In patients treated for >12 months (n=20), the frequency of 43 acute exacerbation reduced from 3.1 to 1.4 episodes/year (p=0.006) in the 12 months after 44 treatment initiation compared to the 12 months before.  A significant reduction in the 45 frequency of hospital admissions/year was also observed (0.8 to 0.3, p=0.04).  These findings 46 support further prospective studies. 47  48  49  50  51  52  53  54  55  56  57  58  59  60  61  62  63  64  65  66  67  68  69  70  71 MAIN TEXT: 72  73 Background: 74 Chronic pulmonary aspergillosis (CPA) is characterised by persistent Aspergillus infection, 75 usually complicating pre-existing lung disease such as COPD or bronchiectasis.  Although 76 patients are typically without immunocompromise, impaired interferon gamma (IFNγ) and/or 77 interleukin 12 (IL-12) production have been reported.1  IFNγ prevents conidial germination, 78 enhances alveolar macrophage killing capacity and manipulates Th1 CD4 cell/NK cell influx 79 through chemokine signalling.2-4  As such, IFNγ supplementation has been explored as a 80 salvage therapy for severe CPA in patients with impaired IFNγ production.5,6  IFNγ 81 immunotherapy may have additional benefits as CPA is often complicated by recurrent 82 bacterial superinfection; in chronic granulomatous disease, therapeutic IFNγ reduces bacterial 83 infections by up to 70%.7 84  85 This study reports the impact of subcutaneous IFNγ supplementation on the frequency of 86 acute exacerbation and all-cause hospital admission when administered as salvage therapy to 87 patients with severe CPA, refractory to antifungals, and proven impairment of IFNγ 88 production. 89  90 Methods: 91 For this descriptive study, clinic letters and electronic hospital records were retrospectively 92 hand-searched for all patients prescribed IFNγ salvage therapy for CPA at the National 93 Aspergillosis Centre, Manchester, UK between January 2011 and September 2018 (46 94 patients).  Subcutaneous IFNγ was self-administered at a dose of 50µg three times weekly 95 and prescribed according to clinical judgement when antifungal therapy appeared to be 96 failing due to a combination of clinical, microbiological and radiological parameters in 97 patients with impaired production of IFNγ or IL-12 after in vitro cytokine induction.   98  99 For the induction of IFNγ, whole blood samples from patients and healthy controls were 100 diluted 1:5 (Roswell Park Memorial Institute medium) into 96 well F plates (Corning) and 101 activated with lipopolysaccharide (List Biochemicals, 1µg/ml) or phytohemagglutinin 102 (Sigma, 10µg/ml), alone or in co-stimulation with IL-12 (Immunotools, 5µg/ml), and 103 incubated at 37°C/5% CO2.  For the induction of IL-12, whole blood was stimulated as above 104 with lipopolysaccharide, alone or in combination with IFNγ (Immukin, Boehringer, 2x104 105 IU/ml).  Supernatants were taken after 24 hours and cytokine levels measured by standard 106 ELISA (IFNγ: Pelikine, Sanquin, NL) or multiplexed particle-based flow cytometry (IL-12: 107 R+D Systems Fluorokinemap), according to the manufacturer’s recommendations. 108  109 Patients were excluded from analysis if they started IFNγ therapy within 12 months of the 110 patient search (n=5), were lost to follow-up (n=4) or started IFNγ prior to attending clinic 111 (n=1).  The frequency of exacerbation, defined as an acute respiratory decline clinically 112 requiring a course of antibiotics, and all-cause hospital admission were compared between 113 the 12 months pre-/post- IFNγ initiation, stratified by duration of IFNγ therapy (Wilcoxon 114 matched-pairs signed-rank test).  Incidence rates of death were compared on/off IFNγ for the 115 36 patients meeting inclusion criteria, starting from the day of IFNγ initiation through to 116 September 2018 for a maximum follow-up of 60 months. 117  118 Results: 119 Of the 36 patients meeting the inclusion criteria, 20 received IFNγ for >12 months, eight 120 stopped treatment due to side effects (all receiving IFNγ for <6 months, mean duration 2.5 121 months) and eight patients died within 12 months of initiating treatment (mean IFNγ duration 122 4.6 months, mean survival 7.5 months).  Demographic characteristics of these 36 patients are 123 shown in Table 1.   124  125 Death rate was statistically similar whilst receiving IFNγ therapy (0.16/year) compared to 126 after stopping IFNγ (0.12/year) (p=0.6), with a median follow-up of 33.5 months 127 (interquartile range 13-45 months).  The 28 patients alive at 12 months were included in 128 further analysis.  129  130 Twenty-two patients (79%) were on concomitant antifungal therapy and eight (29%) were  131 taking azithromycin long-term whilst receiving IFNγ.  Two patients stopped antifungals as 132 they transitioned to IFNγ and azithromycin was started and stopped for one/two  133 patients respectively in the 12 months preceding IFNγ initiation.  All remaining patients  134 had the same on/off antifungal and azithromycin status throughout the 24 months of 135 comparison (12 months pre-/post- IFNγ initiation), though most patients on antifungals 136 changed their regimen at some point due to adverse effects, azole resistance and/or clinical 137 failure (Figure 1). 138  139 In patients treated for >12 months (n=20), there was a significantly lower number of acute 140 exacerbations and hospital admissions in the year following IFNγ initiation compared to the 141 year prior (Table 2).  No significant changes were seen in patients treated with IFNγ for <6 142 months for both outcomes (n=8).  Patients receiving IFNγ for >12 months (versus <6 months) 143 were observed to have a greater difference between pre-/post- IFNγ initiation frequencies of 144 acute exacerbation and hospital admission, although not significantly so for exacerbation.  145 Figure 1 demonstrates the number of acute exacerbations and hospital admissions in the 12 146 months pre-/post- IFNγ therapy initiation on an individual basis.   147  148 Of the 10 patients that remained on the same antifungal treatment throughout the 24 month 149 observation period, eight received IFNγ for >12 months (negative outcomes 150 lower/equal/higher in 6/1/1 patients) and two received IFNγ for <6 months (negative 151 outcomes lower/equal/higher in 0/1/1 patients). 152  153 Discussion: 154 In this retrospective, descriptive study, patients with CPA refractory to antifungals alone 155 appeared to have lower frequencies of acute exacerbation and all-cause hospital admission 156 after the introduction of IFNγ therapy.  These effects were observed when IFNγ was given 157 for >12 months, but not for durations <6 months.  Our observation is promising as acute 158 exacerbations and hospital admissions are a substantial cause of morbidity and mortality in 159 this patient population.  160  161 Almost all patients in our study had impaired IFNγ production and those that did not had 162 reduced levels of IL-12, a cytokine that stimulates IFNγ production.  It is not clear if this 163 represents a primary immune defect or a consequence of chronic pulmonary disease or 164 infection.  The apparent beneficial effect of IFNγ suggests this is clinically relevant and could 165 be mediated by its antibacterial, antiviral or antifungal action.  Due to the chronicity of CPA 166 and the number of patients treated, it was not possible to document a meaningful effect of 167 IFNγ on CPA’s clinical course; there was no significant effect on Aspergillus serology, extent 168 of CPA on imaging, microbiological eradication of Aspergillus or significant change in 169 profile of possible pathogenic organisms from sputum culture (data not shown).  The 170 frequency of adverse events and treatment discontinuation was high in this population of 171 severely ill patients.  172  173 Long-term antifungals are the treatment of choice for CPA; most of our patients were on 174 azoles.  In addition, long-term macrolide treatment is often used for its anti-inflammatory 175 properties and to prevent bacterial exacerbations in patients with chronic lung disease; 176 several of our patients were taking azithromycin during the observation period.8,9  177 Admittedly, the concomitant use of antifungal and antibacterial treatment, with changes in 178 regimen occurring in the majority of patients during the 24 month observation, may confound 179 the results of this study.  Only a minority of patients, however,  had changes in their overall 180 on/off treatment status during this time. 181  182 The patients included in this study were highly heterogeneous and treatment was  183 individualised.  Given this heterogeneity, using patients as their own comparator was 184 considered to be the fairest way to present this retrospective data but is not without its flaws.  185 Having failed to respond to antifungal therapy they had poor prognosis from the offset.  186 There is potential for introduction for bias if patients with a poorer physiological reserve 187 responded less favourably to IFNγ therapy, self-selecting into the <6 month treatment group.  188 Patients lost to follow-up may also introduce further bias. 189  190 Patients that died within 12 months of therapy initiation were not included in the final 191 analysis, arguably introducing bias by excluding the most physiologically vulnerable or 192 patients with the most severe CPA.  It is important to note, however, that all patients had 193 declining prognostic indicators prior to starting IFNγ.  Inclusion of patients that died during 194 the study period would itself have introduced its own bias; the methodology of a self-195 controlled case series was considered but deemed inappropriate due to death being a common 196 outcome and the number of patients available.10  There was no evidence of IFNγ therapy 197 being detrimental to health; side effects leading to withdrawal were all mild and there was no 198 difference in death rates between patients on/off IFNγ.  199  200 In summary, the frequency of acute exacerbation and hospital admission were lower after the 201 introduction of IFNγ replacement therapy in patients with severe, refractory CPA.  202 Prospective data are needed to further evaluate the role of IFNγ as adjunctive therapy.  In 203 addition to control of fungal burden, IFNγ may improve CPA morbidity/mortality by 204 reducing the frequency and severity of bacterial or viral superinfection.  These findings could 205 apply to patients with other chronic lung diseases. 206  207 Table 1.  Baseline characteristics of participants at INFγ testing Characteristic Total (n = 36) n (%) Age group, years     <40   3 (8)  40-49   1 (3)  50-59   19 (53)  60-69   10 (28)  ≥70   3 (8)  Age, years, median [IQR]  56 [51 - 65.5]* Female sex   18 (50)* Smoking status     Active   8 (22)  Ex-smoker   9 (25)  Never   19 (53) Underlying pulmonary comorbidity†   ABPA   5 (14)  Asthma   6 (17)  Bronchiectasis  13 (36)  COPD   8 (22)  Fibrosis (secondary to connective tissue disease) 5 (14)  Malignancy   2 (6)  NTM infection (active/previously treated) 6 (17)  Non-COPD bullous lung disease 3 (8)  Pneumothorax (previous)  6 (17)  Sarcoidosis   2 (6)  TB (previously treated)  5 (14) IFNγ = interferon gamma, IQR = interquartile range, ABPA = allergic bronchopulmonary aspergillosis, COPD = chronic obstructive pulmonary disease, NTM = non-tuberculous mycobacterium, TB = tuberculosis *Demographics stratified by 12 month survival/exclusion:  Alive (n = 28), median age 56.5 [IQR 50.5-65] years, sex 13/28 (46%) female  Dead (n = 8), median age 55.5 [53-75 IQR] years, sex 5/8 (63%) female  †Not mutually exclusive: patients could have more than one underlying pulmonary comorbidity  208  209  210  211  212  213  214  215  216  217  218 Table 2.  Number of acute exacerbations and hospital admissions pre-/post- IFNγ initiation Outcome and population Mean events over 12 months (standard deviation) P value Pre- IFNγ initiation Post- IFNγ initiation Acute exacerbations (clinically requiring antibiotics)a     IFNγ duration      >12 months (n = 20) 3.1 (2.4) 1.4 (1.5) 0.006   <6 months (n = 8) 2.8 (1.8) 2.6 (1.8) 0.7 Hospital admissionsa     IFNγ duration      >12 months (n = 20) 0.8 (0.9) 0.3 (0.6) 0.04   <6 months (n = 8) 0.6 (0.7) 1.0 (1.1) 0.5 Comparison between >12 and <6 month IFNγ durationb    Acute exacerbations - - 0.06   Hospital Admission - - 0.02 a Wilcoxon matched-pairs signed rank test, b t-test IFNγ = interferon gamma  219  220  221  222  223  224  225  226  227  228  229  230  231  232  233  234  235  236  237  238  239 Acknowledgements: 240 We would like to thank our patients at the National Aspergillus Centre, Manchester, UK for 241 their continued engagement and support. 242  243 Funding: 244 No external funding was received. 245  246 Contributions to authorship: 247 CK, conceived the project; RD coordinated and interpreted immunodeficiency testing; CH 248 and CK performed the patient search; EJMM and CK performed data collection; EJMM and 249 CK analysed and interpreted the data; EJMM and CK drafted the manuscript; CH, RD, GH, 250 and DWD critically revised the manuscript for intellectual content.  All authors read and 251 approved the final manuscript. 252  253 Potential conflicts of interest:  254 Professor Denning and family hold Founder shares in F2G Ltd, a University of Manchester 255 spin-out antifungal discovery company.  He acts or has recently acted as a consultant to 256 Scynexis, Cidara, Pulmatrix, Zambon, iCo Therapeutics, Roivant and Fujifilm.  In the last 257 three years, he has been paid for talks on behalf of Dynamiker, Hikma, Gilead, Merck, Mylan 258 and Pfizer.  He is a longstanding member of the Infectious Disease Society of America 259 Aspergillosis Guidelines group, the European Society for Clinical Microbiology and 260 Infectious Diseases Aspergillosis Guidelines group and the British Society for Medical 261 Mycology Standards of Care committee.  EJMM, CH, RD, GH and CK have no conflicts of 262 interest to declare. 263  264  265  266  267  268  269  270  271  272  273 References: 274 1. Döffinger R, Harris C, Lear S, et al. Reduced gamma interferon (gIFN) 275 production in chronic pulmonary aspergillosis (CPA). Abstract 96, 5th Advances 276 Against Aspergillus Conference, 2012. Available at: 277 https://www.aspergillus.org.uk/content/reduced-gamma-interferon-gifn-278 production-chronic-pulmonary-aspergillosis-cpa [accessed May 2019]. 279 2. Bouzani M, Ok M, McCormick A, et al. Human NK cells display important 280 antifungal activity against Aspergillus fumigatus, which is directly mediated by 281 IFN-γ release. J Immunol. 2011;Aug 1;187(3):1369-76 doi: 282 10.4049/jimmunol.1003593 [published Online First: 22 June 2011]. 283 3. Park SJ, Hughes MA, Burdick M, et al. Early NK cell-derived IFN-{gamma} is 284 essential to host defense in neutropenic invasive aspergillosis. J 285 Immunol. 2009;Apr 1;182(7):4306-12 doi:10.4049/jimmunol.0803462. 286 4. Pak-Wittel MA, Yang L, Sojka DK, et al. Interferon-γ mediates chemokine-287 dependent recruitment of natural killer cells during viral infection. Proc Natl Acad 288 Sci U S A. 2013;Jan 2;110(1):E50–E59 doi: 10.1073/pnas.1220456110 [published 289 Online First: 17 December 2012]. 290 5. Denning DW, Cadranel J, Beigelman-Aubry C, et al. Chronic pulmonary 291 aspergillosis: rationale and clinical guidelines for diagnosis and management. Eur 292 Respir J. 2016;Jan;47(1):45-68 doi: 10.1183/13993003.00583-2015. 293 6. Kosmidis C. and Muldoon EG. Challenges in the management of chronic 294 pulmonary aspergillosis. Med Mycol. 2017;Jan 1;55(1):63-68 doi: 295 10.1093/mmy/myw119 [published Online First: 2 December 2016]. 296 7.  Errante PR, Frazao JB and Condino-Neto A. The use of interferon-gamma 297 therapy in chronic granulomatous disease. Recent Pat Antiinfect Drug 298 Discov. 2008;Nov;3(3):225-30 doi: 10.2174/157489108786242378. 299 8. Li W, Qin Z, Gao J, et al. Azithromycin or erythromycin? Macrolides for non-300 cystic fibrosis bronchiectasis in adults: A systematic review and adjusted indirect 301 treatment comparison.  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BMJ. 2016;Sep 308 12;354:i4515 doi: 10.1136/bmj.i4515. 309  310  311  312  313  314  315  316  317  318  319  320  321  322  323  324  325  326  327  328  329  330  331  332  333  334  335  336  337  338  339  340 Figure legend: 341 Figure 1.   342 Individual and mean frequencies of acute exacerbation and hospital admission in the 12 343 months pre-/post- IFNγ initiation, with illustration of concomitant antifungal/azithromycin 344 treatment regimen, stratified by IFNγ treatment duration.  SD = standard deviation.  345 

Interferon gamma replacement as salvage therapy in chronic pulmonary aspergillosis: effects on frequency of acute exacerbation and all-cause hospital admission

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Interferon gamma replacement as salvage therapy in chronic pulmonary aspergillosis: effects on frequency of acute exacerbation and all-cause hospital admission

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