The Efficacy, Safety, and Recurrence Rate of Diphenylcyclopropenone Topical Immunotherapy for Alopecia Areata: A Systemic Review and Meta-Analysis

Background and Objective . Diphenylcyclopropenone topical immunotherapy is widely used in the treatment of alopecia areata. However, previous studies have shown signifcant diferences in its efcacy. We conducted this systemic review and meta-analysis to investigate the efcacy, safety, and recurrence rate of diphenylcyclopropenone topical immunotherapy for alopecia areata. Methods . Literatures related to diphenylcyclopropenone topical immunotherapy for alopecia areata between January 1st, 2002, and July 2nd, 2022, were searched in the following databases: PubMed, Embase, Cochrane Library, Sinomed, WanFang Data, and Chinese Medical Journal Network. Results . Tis meta-analysis included a total of 40 moderate to high quality studies involving 3,002 patients. Te overall rate of any hair regrowth was 69%, and the overall rate of complete hair regrowth was 23%. Te rate of any hair regrowth in patients with alopecia totalis or alopecia universalis was 42%, and the rate of any hair regrowth in patients with other types of alopecia areata was 75%. Common side efects were mild contact dermatitis (36%), severe contact dermatitis (31%), regional lymphadenopathy (22%), hyperpigmentation (22%), and hypopigmentation (7%). Te recurrence rate was 37%. Conclusion . Diphenylcyclopropenone topical immunotherapy is an efective treatment for various types of alopecia areata, and most of its common side efects are acceptable.


Introduction
Alopecia areata (AA) is a common cause of hair loss, characterized by the sudden appearance of well-defned patches of hair loss [1].With a prevalence of approximately 2% in the general population, AA has a signifcant impact on the quality of life and increases the risk of psychopathology [2].74% of the AA patients sufer from mental disorders at least once during their lifetime [3].Despite extensive research, the pathogenesis of AA remains poorly understood.However, current understanding suggests that it is an autoimmune disease resulting from the breakdown of immune privilege of the hair follicle [4].Tis complex process involves abnormal presentation of hair follicle antigens, activation of CD8 + NKG2D + T cells, overexpression of infammatory cytokines (such as IFN-c and IL-15), and other contributing factors [1,2,4].Given the close link between AA pathogenesis and autoimmune infammation, therapeutic interventions for AA have focused primarily on modulating the immune microenvironment.Tese treatment options include topical or intralesional administration of corticosteroids as well as the systemic application of corticosteroids or other immunosuppressive agents [5].Recent studies have shown remarkable efcacy of JAK inhibitors, including baricitinib and tofacitinib, in the treatment of moderate to severe AA [6,7].Tese results underscore the importance of JAK inhibitors in the treatment of AA by targeting the immune and infammatory pathways.However, there are still many cases of severe or treatment-resistant alopecia areata, and effective long-term management of these cases remains a signifcant challenge.
Topical immunotherapy is a commonly used therapy for severe or recurrent AA.Te application of topical sensitizers such as diphenylcyclopropenone (DPCP) to the lesions of AA patients can repeatedly induce contact dermatitis and then, a certain remission rate can be achieved after a period of maintenance therapy [8].Although there are potential side efects such as severe eczema and lymphadenopathy [5], they are generally more tolerable than the side efects associated with systemic immunosuppressants.Te mechanism underlying the efcacy of topical immunotherapy remains unclear, but potential explanations include antigen competition, modulation of perifollicular lymphocyte subsets, and reduction of proinfammatory T1 and T17 cytokine levels [8].In addition to DPCP, other sensitizers used in topical immunotherapy include squaric acid dibutyl ester (SADBE) and dinitrochlorobenzene (DNCB) [8].Te latter is no longer used due to its teratogenicity and carcinogenicity.DPCP is the most commonly used sensitizer because it is more stable in acetone solution and is less expensive than SADBE.
Tere has been a surge of new, high-quality clinical studies investigating DPCP topical immunotherapy for AA.However, these studies have yielded heterogeneous results, reporting varying hair regrowth rates, ranging from as low as 34.4% (21/61) [9] to as high as 92.3% (36/39) [10].To provide a comprehensive and up-to-date assessment of DPCP as a therapeutic option for AA, we conducted a meta-analysis based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) process.Our study reviewed relevant studies conducted over the past 20 years and quantitatively evaluated the efcacy, safety, and relapse rate of DPCP in the treatment of AA.

Search Strategy.
A comprehensive search for English and Chinese literature related to DPCP in the treatment of AA was conducted in the following databases: PubMed, Embase, Cochrane Library, Sinomed, WanFang Data, and Chinese Medical Journal Network.Te articles included in the search were published between January 1st, 2002, and July 2nd, 2022.Te search strategies used in each database are shown in Tables 1 and 2.

Study Selection.
Te literature obtained was summarized in Endnote X9.3.2.Study selection and quality assessment were performed independently by two authors (Z.J.P and J.Y.Q) according to the title and abstract.When the information provided by the title and abstract was insufcient, the full text was referred to.When the two authors had diferent opinions, consensus was reached after consultation with the third author (G.X.D).

Diagnostic Criteria for AA.
Te diagnosis of AA and the diferentiation of AA types were based on clinical manifestations and medical history.

Exclusion Criteria. Te exclusion criteria were as follows:
(1) Te type of research: studies with only in vitro or in vivo research or reviews; (2) the subject of research: irrelevant to DPCP topical immunotherapy for AA; (3) the intervention: sensitizers other than DPCP were used, or other drugs were used concomitantly, or the DPCP monotherapy group was not set as a separate group, or data on the DPCP monotherapy group were not provided; (4) the research method: questionnaires were used as the main measure; (5) the response of the eyebrow area to DPCP in patients with AA was the only outcome observed; (6) the number of patients in the DPCP treatment group was less than 20 (this is not required for subgroup analysis); (7) the focus was on sensitization efects rather than efcacy; (8) detailed outcomes such as efcacy were not reported; (9) the literature was written in language other than Chinese and English; and (10) clinical studies not completed.

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Te PRISMA fow diagram is shown in Figure 1.
2.3.Quality Assessment.Te methodological index for nonrandomized studies (MINORS) was used to assess the quality of the included studies.For studies that did not apply to MINORS entries, their quality was assessed based on the following factors: the type of research design (randomized controlled trials, prospective clinical trials, and retrospective analysis), whether the study reported the proportion of areata totalis (AT) and areata universalis (AU) patients, whether the study reported the criteria for assessing hair growth outcomes, whether the study reported adverse effects, and whether the study reported the defnition of recurrence.

Data Extraction.
Te following items of data were extracted from each study: study year, author, title, type of study design (e.g., prospective or retrospective), study setting, region, mean age, sex ratio, disease duration of the study population, protocol of DPCP application, evaluation criteria for hair regrowth, rate of any hair regrowth (any improvement from a baseline), rate of complete hair regrowth (100% terminal hair growth was achieved), prevalence of adverse efect, and recurrence rate.Te data collection process was completed using WPS Ofce 4.6.2.Te data were collected independently by two researchers (Z.J.P and J.Y.Q) and compared for consistency.

Data Synthesis.
Te Shapiro-Wilk normality test was performed on the data before synthesis to confrm that they conformed to a normal distribution.Because most of the included studies were retrospective analyses or single-arm clinical trials (without a control group), only the regrowth rate was used as an outcome measure.Because the defnitions and stratifcation criteria for hair regrowth outcomes varied widely among the studies, we classifed hair regrowth outcomes as either any regrowth or complete regrowth.Te rate of each outcome was then synthesized.If the information provided in the article was insufcient to determine what level of regrowth a group belonged to, the group was categorized as lower.In addition, the recurrence rate and the incidence of side efects were synthesized.Only the side efects reported by 10 or more studies were included.
To help identify factors that may have an impact on the response to the treatment, subgroup analysis was performed according to the type of AA in studies that reported the proportion of AT and AU patients and their hair regrowth outcome.We also performed subgroup analyses according to age, disease duration, nail involvement, history of atopic disease, family history, and SALT (Severity of Alopecia Tool [11]) score in the relevant studies where data were available.Further subgroup analysis was performed by study language and region.After synthesis, all data were displayed using forest plots.Because the I 2 test indicated high heterogeneity among study results, random efects models were used for the meta-analysis.Sensitivity analysis was used to assess sources of heterogeneity after pooling efects.Egger's regression test was used to assess publication bias.All data analyses were performed using R 4.2.1 and the R package "meta."2), and the overall complete regrowth rate was 23%, 95% CI (0.16, 0.30) (Figure 3).Sensitivity analyses were performed, and all estimates difered by <3% from the original analyses.Egger's regression test indicated that publication bias was not signifcant (p � 0.7341).

Study Characteristics and the
As for the results of the subgroup analysis, only patients in the AT/AU group had a worse response to DPCP treatment.Also, when grouped by other factors such as age and disease duration, there was no signifcant diference in patient outcomes.Te rate of any hair regrowth in the AT/ AU group was 42%, 95% CI (0.33, 0.51), while the rate of any hair regrowth in patients with other AA types was 75%, 95% CI (0.67, 0.83), and there was a signifcant diference between the two groups (p < 0.01) (Figure 4).Te rate of any 4 Dermatologic Terapy hair regrowth was 48%, 95% CI (0.35, 0.62) in patients aged <18 years and 59%, 95% CI (0.51, 0.67) in patients aged ≥18 years, with no signifcant diference observed between the two groups (p � 0.19) (Supplementary Figure 1).Similarly, in patients with disease duration ≥10 years, the rate of any hair regrowth was 48%, 95% CI (0.29, 0.67), while in patients with disease duration <10 years, the rate of any hair regrowth was 69%, 95% CI (0.60, 0.79), with no signifcant diference between the two groups (p � 0.06) (Supplementary Figure 2).Regarding nail involvement, the rate of any hair regrowth in patients with nail involvement was 40%, 95% CI (0.18, 0.63), and the rate of any hair regrowth in patients without nail involvement was 67%, 95% CI (0.51, 0.82), with no signifcant diference between the two groups (p � 0.06) (Supplementary Figure 3).Furthermore, there was no signifcant diference in the rate of any hair regrowth between patients with a history of atopic disease (55%, 95% CI (0.44, 0.66)) and those without (59%, 95% CI (0.49, 0.70)) (p � 0.58) (Supplementary Figure 4).Similarly, in patients with a family history of AA, the rate of any hair regrowth was 52%, 95% CI (0.29, 0.75), and in those without a family history of AA, the rate of any hair regrowth was 55%, 95% CI (0.47, 0.63), with no signifcant diference between the two groups (p � 0.80) (Supplementary Figure 5).Te any hair regrowth rate was 64%, 95% CI (0.40, 0.89) in patients with SALT <50 and 49%, 95% CI [0.40, 0.57] in patients with SALT ≥50, with no signifcant diference between the two groups (p � 0.24) (Supplementary Figure 6).In addition, subgroup analysis was performed for all studies grouped by region and language, and there was no signifcant diference between regions and languages (Supplementary Figures 7-10).

Recurrence Rate.
Te recurrence rate for AA treated with DPCP topical immunotherapy was 37%, 95% CI [0.23, 0.50] (Figure 10).Egger's test indicated a signifcant publication bias (p � 0.0008), and funnel plots showed that the studies tended to report higher recurrence rates.

Discussion
4.1.Efcacy.Tis meta-analysis included the largest number of patients to our knowledge.Te results showed that for all the AA patients, the rate of any hair regrowth was about 69%, while the complete regrowth rate was about 23%.Te rate of any hair regrowth was about 42% in AT/AU and 75% in other AA types.In comparison, the SALT 50 (defned as a decrease in the SALT score of at least 50% from the baseline) remission rate at week 16 was 29.9% in AA patients with SALT ≧50 who received baricitinib at a dose of 4 mg/ day [12].Considering that the typical course of DPCP topical immunotherapy spans 16 weeks, our assessment indicates a sufciently high response rate.However, the existing evidence is limited in directly comparing the efcacy of DPCP with other treatments.Terefore, further research and larger-scale studies are needed to optimize the treatment regimens for AA.
Our results are in general agreement with the fndings of Lee et al. in 2017, which reported a 63.8% any hair regrowth rate and 30.7% complete regrowth rate in all alopecia areata (AA) patients, as well as 75.5% and 51.6% any hair regrowth rate in patchy AA and alopecia totalis/alopecia universalis (AT/AU) patients, respectively [13].Te meta-analysis conducted by Lee et al. included literature in English and Korean, whereas our study encompassed literature in English and Chinese.Tis further validates the results of our subgroup analysis that the DPCP treatment of AA has similar efcacy across studies in diferent regions and languages.
Moreover, subgroup analyses did not confrm statistical diferences in treatment response between patients of different age, disease duration, or SALT scores or between patients with or without family history, history of atopic disease, or nail involvement.However, the current conclusions still lack robustness as only a limited number of studies have reported patient outcomes based on specifc subgroups of the abovementioned factors.
Despite the limitations, our study provides evidence that DPCP is a feasible treatment option with demonstrated efcacy for a diverse population of patients with alopecia areata.Tis fnding is particularly signifcant for certain specifc patient groups, such as pediatric patients.Treatment options for children with AA are extremely limited.Some studies recommend only topical glucocorticoids [5].Indeed, topical steroids may not always be efective and it is in such  Dermatologic Terapy not be reported as an adverse efect in some studies.Due to the signifcant publication bias, estimates of the incidence of adverse efects may not be accurate.In addition to the adverse efects listed above, DPCP may cause some rare but serious side efects such as erythema multiforme major [14] and discoid lupus erythematosus [15] in some cases.

Recurrence Rate.
AA can be a chronic relapsing disease.Tere is currently no treatment that can reduce the recurrence rate of AA.Te recurrence rate of AA after DPCP treatment was approximately 37%.Few studies have reported the recurrence rate in patients with AT or AU.
Terefore, subgroup analysis of these patients was not performed in this study.We recommend that future studies report the recurrence rate separately by the type of AA to determine whether the type of AA is associated with recurrence after treatment.

Mechanisms of Topical
Immunotherapy.Tis study provided more sufcient evidence for the efcacy of DPCP in the treatment of AA.Tere are several hypotheses about the mechanisms of DPCP topical immunotherapy in the treatment of AA.From the perspective of skin lesions, antigen-presenting cells (APCs), CD4 + T cells, and CD8 +

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T cells (among which CD8 + NKG2D + T cells may play a more important role) may infltrate around the anagen hair follicles in active AA lesions.Tese cells recognize melanocyteand keratinocyte-associated antigens and drive anagen hair follicles (especially anagen III/IV phase) into telogen [2].DPCP can bind to endogenous epidermal proteins to form a complete antigen and induce allergic contact dermatitis in the epidermis, thereby interfering with the presentation of hair follicle antigens by APCs [8].DPCP can also stimulate the infltration of CD4 + and CD8 + T cells in the epidermis and induce the activation of suppressor Tcells (such as Treg), thereby regulating the local infammation of hair follicles [8].
From a systemic perspective, serum T1 cytokines (such as IFN-c and IL-12) and T17 cytokines (such as T17A and T23) are elevated in patients with AA, which may provide a systemic infammatory background and thus increase the susceptibility of hair follicles to autoimmune infammation [16].Gong et al. found that after DPCP treatment, serum levels of the T1 cytokines IFN-c and IL-12 decreased signifcantly in AA patients with a good response, while serum levels of the T2 cytokines IL-4 and IL-10 increased dramatically.Terefore, it is speculated that DPCP treatment may inhibit the pathogenic efect of T1-type responses on hair follicles by regulating the balance between T1-type immune responses and T2-type immune responses [17].

Protocol of DPCP Topical Immunotherapy.
For the DPCP treatment protocol for AA, most studies in this metaanalysis were similar.First, the patients' scalp was sensitized with 2% solution of DPCP in acetone.Te sensitization area was 2 cm * 2 cm∼4 cm * 4 cm.Te treatment started 2 weeks later.Te application frequency was once a week to once every 2 weeks.Te initial concentration was 0.001%.Te concentration was then gradually increased until the patients developed allergic contact dermatitis.Te concentration was then maintained.Light exposure and shampooing were avoided for 48 hours after application.However, there were diferences in the maintenance treatment after that.Some studies simply discontinued the drug administration once the desired efect was achieved.Others gradually extended the dosing interval.As a result, the length of treatment varies from as little as 2 months to as long as 15 years.On average, however, it typically spans about 4-6 months.In addition, some studies suggested a stepwise approach to DPCP application, recommending its initial use on one side of the scalp, and proceeding to treat the other side only after observing positive efects on the frst side.In most studies, DPCP was applied by physicians or nurses, but in two studies, it was applied by patients themselves at home, and these two studies found similar efcacy to hospitalbased studies [18,19].

Limitations
Due to language limitations, the literature search was conducted in English and Chinese databases only.
Most of the studies of DPCP in the treatment of AA were single-arm trials or retrospective analyses.Tere were only a few controlled trials, and the drugs used in the controlled groups were diferent.Terefore, the only outcome measure used was the regrowth rate.Tis may reduce the level of evidence and increase the heterogeneity.
Due to the diferent criteria used to assess hair regrowth in diferent studies, it was not possible to stratify the hair regrowth rate more precisely.In addition, because of the diferent focus of data collection in diferent studies, subgroup analysis of the incidence of adverse efects and recurrence rates in diferent types of AA could not be performed.

Conclusion
DPCP topical immunotherapy is an efective treatment for various types of AA, and most of its common side efects are acceptable.Te response of diferent patients to DPCP topical immunotherapy is quite diferent, and the reason needs to be further clarifed by conducting basic research on the pathogenesis of AA.

Figure 4 :
Figure 4: Any regrowth rate (subgroup analysis according to the type of alopecia areata).
Quality Assessment.A total of 40 moderate to high quality studies were included, involving 3,002 patients.Among them, 19 were retrospective analyses, 16 were single-arm clinical trials, 2 were controlled clinical trials, and 3 were randomized controlled trials.Regarding populations, 2 studies were conducted in African, 20 in Asian, 14 in European, and 4 in North American populations.Te proportion of patients with AT or AU was reported in 31 studies.All studies reported the rate of any hair regrowth.Tere were 16 studies reporting the complete regrowth rate and 15 studies reporting the recurrence rate, 7 of which reported the defnition of recurrence.However, no studies reported the duration of follow-up.Tere were 18 studies that reported the rate of any hair regrowth in the AT/ AU group, including 471 patients with AT/AU and 526 patients with other AA types.Seven studies reported any hair regrowth rates in diferent age groups, 5 studies reported any hair regrowth rates in diferent disease duration groups, 4 studies reported any hair regrowth rates in groups with and without nail involvement, 7 studies reported any hair regrowth rates in groups with and without family history, 8 studies reported any hair regrowth rates in groups with and without history of atopic disease, and 6 studies reported any hair regrowth rates in diferent SALT score groups.Regarding the incidence of side efects, mild contact dermatitis (mild to moderate pruritus and erythema) was reported in 15 studies, severe contact dermatitis (vesicles, erosions, and systemic symptoms) in 16 studies, hyperpigmentation in 12 studies, hypopigmentation in 10 studies, and regional lymphadenopathy in 17 studies.Twenty-seven studies evaluated hair growth outcomes using SALT or other quantitative criteria.Qualitative criteria (e.g., stratifcation of results into no response, scattered pigmented terminal hair, terminal hair growth but still patchy alopecia, and complete terminal hair growth) were used in 13 studies.
Figure 5: Incidence of mild contact dermatitis.