Efficacy and Safety of Interferon-Gamma in Chronic Granulomatous Disease: a Systematic Review and Meta-analysis

Chronic granulomatous disease (CGD) is a primary immunodeficiency with increased susceptibility to several bacteria, fungi, and mycobacteria, caused by defective or null superoxide production by the NADPH oxidase enzymatic complex. Accepted treatment consists mainly of antimicrobial prophylaxis. The role of human recombinant subcutaneous interferon-gamma (IFNγ) is less clear since the available evidence on its efficacy derives mainly from a single clinical trial that has been challenged. We aimed to assess the efficacy and safety of IFNγ as an added treatment for CGD when compared to antimicrobial prophylaxis alone. A literature search was conducted using MeSH terms “Chronic granulomatous disease” AND (“interferon gamma” OR “interferon-gamma”), as well as antibiotics, placebo, no therapy, clinical trial, and trial, on MEDLINE, EMBASE, LILACS, WHOs, CENTRAL, KOREAMED, The Cochrane Library, clinicaltrials.gov, and abstracts from meetings, from 1976 to July 2022. We included clinical trials (CT) and prospective follow-up studies and registered the number of serious infections (requiring hospitalization and IV antibiotics) and deaths, adverse events, and autoimmune complications, in patients treated for CGD with antimicrobial prophylaxis plus IFN-γ, versus antimicrobial prophylaxis alone. We assessed the quality of the studies using risk of bias and STROBE. We performed a meta-analysis by calculating both Peto’s odds ratio (OR) and risk reduction (RR) through the Mantel–Haenszel method with a fixed-effect model, using Review Manager 5.4, and we reported the number needed to treat (NNT). We identified 54 matches from databases and 4 from other sources. We excluded 12 duplicates, 7 titles, and 9 abstracts for relevance, after which we had 30 eligible studies. Twenty-four were then excluded after reading the full text. Six papers were included: one randomized CT and 5 follow-up studies. In total, 324 patients with Chronic granulomatous disease were followed for 319 months under treatment with antibiotic prophylaxis plus interferon-gamma or placebo (or antibiotic prophylaxis alone), reported between the years 1991 and 2016. Three of the studies included a control group, allowing for the aggregate analysis of efficacy (prevention of serious infections). The aggregate OR was 0.49, with a 95% confidence interval of 0.19 to 1.23. The risk ratio for serious infection was 0.56 (95%CI 0.35–0.90) under IFN-γ. The meta-analysis thus favors interferon-gamma for a risk reduction of serious infection. The results from this meta-analysis support the use of IFN-γ in the treatment of patients with CGD. However, we found insufficient clinical evidence and believe more clinical trials are needed to better assess the efficacy and long-term safety of IFN-γ.


Introduction
Chronic granulomatous disease (CGD) is a primary immunodeficiency with increased susceptibility to several bacteria, fungi, and mycobacteria, caused by defective or null superoxide production by the NADPH oxidase enzymatic complex [1,2]. Granulomata (50%) and inflammatory disease are also common (69%) in CGD patients in the absence of detectable infection [3]. Accepted treatment consists mainly of antimicrobial prophylaxis [4]. The role of human recombinant subcutaneous interferon-gamma (IFN-γ) is less clear, as available evidence on its efficacy derives mainly from a single clinical trial that has been challenged.
When first described, CGD was named fatal granulomatosis of childhood, as affected patients usually died before adolescence. After oral antibacterial prophylaxis was introduced, the survival of CGD patients greatly increased [5]. Standard treatment usually consists of trimethoprim/sulfamethoxazole and itraconazole. Hematopoietic stem cell transplantation is advised in patients with severe and recurrent infections. Gene therapy is not yet widely available [6,7]. To date, the long-term survival rate for CGD patients remains low, at 50-55% through the fourth decade of life [8].
In the 1980s, before the genetic etiology of CGD was known, IFN-γ was introduced as a therapeutic agent. The proposed mechanism of action was not yet clear, since in vitro, ex vivo, and mice studies had shown inconclusive or contradictory results [9][10][11][12][13][14][15]. Later, IFN-γ was proven to increase the splicing efficiency of CYBB, the gene most commonly affected (60-80% of patients) in CGD [16]; it is thus thought to be beneficial to at least CGD patients who bear splice-site mutations in CYBB. Recently [17], a study in EBV-B cells from patients with CGD found an upregulation of relevant genes involved in inflammation, cell adhesion, toll-like receptor signaling, antigen transport, oxidative killing, nitric oxide synthase, and autophagolysosomes, after in vitro exposure to IFN-γ for 48 h. Another in vivo study in neutrophils from adult volunteers [18] also found altered expression of 2775 genes that lasted 24-36 h, including upregulation of genes coding for NADPH oxidase, a regulator of circulating polymorphonuclear numbers, and other genes involved in innate and adaptive immunity. Lysates from neutrophils showed a spike in nitric oxide following the administration of IFN-γ. However, IFN-γ is not defective in CGD; serum baseline levels are usually high, as are TNF-α, IL-6, IL-1β, and other pro-inflammatory cytokines [19][20][21].
Clinically, there is at least one well-designed large trial from 1991, a randomized placebo-controlled study that showed considerable risk reduction (67%) of serious infections in 128 patients with CGD from 15 centers in 4 countries, who were followed during 12 months under baseline prophylactic treatment plus IFN-γ or placebo [22]. However, that report from the International CGD Study Group was questioned by expert clinicians in Zurich and France, who were surprised by the high rate of infections per year registered during the clinical trial [23]. Other than that, most available literature supporting the use of IFN-γ in CGD consists of case reports, case series, follow-up, and beforeafter studies, with variable (mostly favorable) conclusions [24][25][26][27][28].
Long-term treatment with IFN-γ is expensive and is not exempt of adverse effects. This is probably why in Europe only 33% of patients receive it chronically [29]. In contrast, in the USA, nearly 75% of all CGD patients are under chronic IFN-γ treatment [30]. In Mexico, thanks to a generous recurrent donation from the pharmaceutical industry, 4 out of 5 patients receive IFN-γ, while its cost is prohibitive for the rest of Latin America [31]. Intriguingly, we see many more CGD patients with autoimmune manifestations in Mexico [32] and the USA (about 25%) [33], than in Europe (6-10%) [3,29].

Objective
We aimed to assess the efficacy and safety of IFN-γ as an added treatment for CGD, when compared to antimicrobial prophylaxis alone.

Design and Registry
Systematic review and meta-analysis, registered online as PROSPERO 2015 CRD42015026992. Institutional review board approval number 2015/054.

Study Selection
A literature search was conducted using MeSH terms "Chronic granulomatous disease" AND ("interferon gamma" OR "interferon-gamma"), as well as antibiotics, placebo, no therapy, clinical trial, and trial, on MEDLINE, EMBASE, LILACS, WHOs, CENTRAL, KOREAMED, The Cochrane Library, clinicaltrials.gov, and abstracts from meetings, from 1976 to July 2022, with no language restriction. We included clinical trials (CT) and prospective followup studies and registered the number of serious infections (requiring hospitalization and IV antibiotics), deaths, adverse events, and autoimmune complications, in patients treated for CGD with antimicrobial prophylaxis plus IFNγ, versus antimicrobial prophylaxis alone. We assessed the quality of the studies using risk of bias and STROBE.

Data Extraction
After filtering out for duplicates and titles, 39 abstracts were read, and studies meeting the inclusion criteria were identified. Three reviewers (SLR, YGB, DRL) independently carried out data extraction and quality assessment, with discrepancies sorted out by consensus. We used Review Manager 5.4's standardized form to extract relevant data from each included study, including design, diagnostic criteria, population, inclusion criteria, and treatment, for all intervention and control groups.

Statistical Analysis
A relative risk (RR) was calculated using the Mantel-Haenszel method to synthesize the results from included studies [34]. The relative difference (RD) and the number needed to treat (NNT) were also reported. We used fixed-effect model, as heterogeneity among studies was ruled out based on the I 2 value. The results were summarized in a frequency table and forest plot. The estimators of relative risk (RR), risk reduction (RD), and number needed to treat (NNT) are reported with their 95% confidence interval (CI). We also calculated Peto's logarithmic odds ratio.
All data were calculated using Review Manager 5.4 (Cochrane Collaboration, Oxford, England).

Results
We identified 54 matches from databases and 4 from other sources (see Fig. 1). We excluded 12 duplicates, 7 titles, and 9 abstracts, after which we had 30 eligible studies. Twentyfour were excluded after reading the full text (5 were mathematical studies, 5 were in vitro or ex vivo, 3 were reviews, 2 were letters to the editor, 2 did not report results, 2 were single case reports, 1 was a case series, 2 were retrospective studies, and 2 were cross-sectional studies). Six papers were included: one randomized clinical trial and 5 follow-up studies. In total, 324 patients with chronic granulomatous disease were followed for 319 months under treatment with antibiotic prophylaxis plus interferon-gamma or placebo (or antibiotic prophylaxis alone), reported between the years 1991 and 2016. (See Fig. 1.) Regarding genetic etiology, 58% of patients had X-linked recessive CYBB mutations (gp91 phox deficiency), 9% had autosomal recessive CYBA variants (p22 phox deficiency), 3% of patients had no identified mutation, and in 30%, the genetic etiology was not mentioned. In all six studies [11,22,[35][36][37][38], the same dose and route were used; three concluded IFNG is effective, and two reported severe adverse effects. Due to considerable heterogeneity, subgroup stratification was not feasible, and we could not evaluate differences in mortality or safety.
Severe or long-lasting side effects attributed to IFNG included abnormal liver function tests (one patient), headaches with joint stiffness (one patient), chills with fever (one patient) [36], skin rash (one), constitutional symptoms (two patients), and worsening granulomatous colitis (one patient) [22]. Most commonly, patients receiving IFNG complain of fever, chills, headache, and local erythema, which are usually mild, transient, or responsive to acetaminophen.
Three of the studies (Kang, Martire, and the CGD Study Group) [11,35,39] included a control group, allowing for the aggregate analysis of efficacy (prevention of serious infections) in 178 patients (128 + 35 + 15). Methodological quality assessment by STROBE scored a low risk of bias for all three reports. With 128 patients followed for 12 months, the CGD Study Group clinical trial from 1991 carried around 80% of the weight (see forest plot) (See Tables 1  and 2).

Discussion
We found one randomized clinical trial and 5 follow-up studies, all of them of sufficient quality and a low risk of bias, that evaluated the treatment of CGD patients with IFN-γ. Together, they account for 324 patients followed during 319 months under antibiotic prophylaxis with or without IFN-γ. Only 3 of the studies included a control group; a meta-analysis of their aggregate outcomes shows a protective relative risk of 0.56 with an acceptable confidence interval. We therefore must conclude there is sufficient evidence to support the use of IFN-γ to prevent serious infections in patients with CGD. As for adverse effects and longterm safety, we could not find enough data to perform a meta-analysis.  The power of the aggregate statistical analysis is restricted by the relatively small number of patients included. The safety and efficacy of IFN-γ in the treatment of CGD are generally accepted, based on the clinical trial from 1991 and a few other cohort studies. However, the findings from the group in Sao Paulo suggest that the cytokine might only be effective in a subgroup of patients with splice-site mutations in CYBB. The study by Kang (2015) seems to support this differential effect, as they could not find a reduction of infections in their patients with CYBA variants. This is not the first meta-analysis on the subject: in 2016, Loffredo et al. [40] reviewed the two studies by Gallin et al.
(International CGD Study Group) and Martire et al. (Italian multicenter study), 163 patients in total, and found an absolute risk reduction of 31%, with a number needed to treat of 3, as well as a reduction in pulmonary infections (RR 0.43, 95%CI 0.19-0.96, p = 0.04). However, they did not include the 15 autosomal recessive patients followed by Kang et al. in Korea (2015) [11], who found a non-significant difference in the incidence of severe infections.
Only one of the available studies is an RCT, so we had to resort to prospective studies, to assess at least one of the stated outcomes (serious infections). We believe more clinical studies are needed to better assess the efficacy and long-term safety of IFN-γ in CGD, including subgroup analyses for inheritance (X-linked versus autosomal recessive) and null versus residual superoxide production. Also, a well-designed follow-up study might be able to evaluate the long-term risk of autoimmune complications in CGD patients treated chronically.
As patients now have more longevity, a long-term, more detailed assessment of inflammatory and autoimmune complications of chronic IFN-γ treatment is needed. For the clinicians whose patients continue to die during adolescence due to invasive pulmonary aspergillosis, especially in Latin America and other regions where resources are scant, we need to be able to tell which patients benefit from the chronic use of IFN-γ and how significant the risk for complications.