In bullous pemphigoid (BP), prolonged treatment with low-dose methotrexate (MTX) may represent an effective strategy to maintain an initial clinical remission achieved by short-duration superpotent topical steroids (STS).).

To evaluate the efficacy and safety of MTX in a real-life setting, a retrospective analysis was conducted in a reference centre for all BP patients treated between 2015 and 2020 with STS and MTX, either in monotherapy (mMTX) or in combination with oral steroids (MTX+OS). The primary endpoints were BP relapse rate and time to relapse during treatment for patients achieving initial disease control (IDC). Secondary endpoints included IDC achievement rate, relapse rate and time to relapse after MTX discontinuation, adverse effect (AE) and mortality rates, number of patients permanently discontinuing MTX owing to AE, number of patients alive and still in complete remission (CR) at the latest updates, and whether they were off treatment (cure rate).

The BP relapse rate during treatment after achievement of IDC was significantly higher in patients treated with mMTX (32.2%) than with MTX+OS (0%) (p = 0.03); mean time to relapse was 10.3 months. The BP relapse rate after MTX discontinuation and the cure rate did not differ significantly between the two groups. One patient (receiving mMTX) died during follow-up and 27.8% of patients experienced at least one AE during the study. The cure rate in patients achieving IDC was 45.2% with mMTX vs. 30% with MTX+OS (p = 0.15).

Our study highlights the value of low-dose mMTX for treating BP in a real-life setting, both for achievement of initial response and for long-term control, with a good overall safety profile and no significant additional mortality risk in elderly subjects. However, relapses appear to have been less frequent during treatment when MTX was initially combined with OS.

# Introduction

Bullous pemphigoid (BP), the most frequent auto-immune bullous disease (AIBD), predominantly affects elderly patients above 70 with an estimated incidence of 21.7 new cases/million/year in France and an overall poor prognosis compared to age- and gender-matched general population, owing to advanced age, multiple associated comorbidities, most notably neurological conditions, and adverse effects (AE) of treatments [1–4]. Although oral corticosteroids (OS) are still widely used, topical superpotent corticosteroids (STS) proved efficient in two major randomized control trials (RCT) and are currently a standard treatment in BP [1–7]. However, proper use of STS is often difficult to maintain in the long term owing to onerous and complex nursing care, and long-term cutaneous and systemic AE may also occur. This may result in poor treatment compliance or treatment discontinuation, resulting in a high risk of middle- or long-term relapse [8]. To overcome these difficulties, prolonged treatment with low-dose methotrexate (MTX) could be an effective strategy to maintain an initial clinical remission achieved with short-term STS. The use of MTX in BP was first described in 1994 by Paul et al. in 8 patients, and subsequent small-scale open series supported the efficacy of low-dose MTX in BP with no increase in mortality or serious AEs and enabled a steroid-sparing regimen [9–16]. More recently, a multicentre prospective phase 3 non-inferiority randomized controlled trial conducted by the French Taskforce for AIBD showed a favourable benefit/risk ratio for 2-step combination therapy consisting of initial STS/low-dose MTX followed by maintenance treatment with low-dose MTX alone, compared to prolonged STS in BP patients [17]. However, included patients were in good general condition and highly selected using stringent inclusion criteria and they were therefore unlikely to be representative of the general BP population. To evaluate the efficacy and safety of MTX in a real-life setting, a retrospective analysis of all patients treated for BP between 2015 and 2020 with MTX, either in monotherapy (mMTX) or in combination with oral steroids (MTX+OS), was conducted in a single tertiary reference centre. Clinical efficacy and safety data were compared with those obtained from the MTX/STS arm of the above-mentioned RCT.

# Patients and methods

## Patients and collected data

All patients treated with mMTX or MTX+OS for at least 3 months between 2015 and 2020 in the Dermatology Department of the University of Montpellier (France) were retrospectively selected and considered for inclusion. BP was diagnosed using the usual criteria: clinical, histological (subepidermal blister with dermal infiltrate by neutrophils and/or eosinophils) and immunological (positive direct immunofluorescence (DIF) with IgG and/or C3 deposits at the dermal-epidermal junction and/or the presence of circulating anti-BP230 and/or of anti-BP180 autoantibodies detected by Western blot (WB) and/or by enzyme-linked immunosorbent assay (ELISA) [18].

Digital medical records for all eligible patients were reviewed and the following data were collected: socio-demographic data, disease profile (clinical, histological and immunological data), MTX treatment data (dosage and associated treatments, i.e. STS and OS; treatment duration; treatment discontinuation during follow-up with underlying reasons; treatment status at most recent update), initial clinical response, patients’ outcome after initial response (relapse, time to relapse, clinical status at most recent update), follow-up duration and AEs.Patients with unavailable data or incomplete files were excluded.

## Endpoints

The primary endpoints were BP relapse rate and time to relapse expressed in days during follow-up for patients achieving initial disease control (IDC). IDC was defined as the absence of new lesions and of pruritic symptoms, and the start of healing of established lesions [19].

Secondary endpoints were: IDC achievement rate; specific relapse rate and time to relapse after MTX discontinuation for patients achieving IDC; AE rate with CTCAE (Common Terminology Criteria for Adverse Events) severity grade [20] and relationship with treatment; mortality rate; rate of patients who permanently discontinued MTX owing to AE; rate of patients alive and still in complete remission (CR) at most recent update(= cure rate), whether or not treatment ongoing.

## Statistical analysis

Results were expressed as percentages and absolute numbers (median and interquartile values or range (IQR); or mean value and standard error or range). Two patient groups (treated with mMTX or MTX+OS) were defined and compared. As to the primary endpoint, BP relapse rates during follow-up for patients achieving IDC were calculated for both groups and compared using the Kaplan-Meier method; time to relapse was also compared (mean and standard error). Regarding secondary endpoints, IDC achievement rates, AE rates and rates of patients permanently discontinuing MTX owing to AEs were compared between the two groups using Fisher’s exact test. Mortality rate, relapse rate after MTX discontinuation, and cure rate at the most recent update for patients achieving IDC were calculated for the two groups and compared using the Kaplan-Meier method. Finally, selected data were compared to corresponding data obtained in the MTX/STS arm of the phase 3 non-inferiority randomized controlled trial.

# Results

## Patient characteristics (Table 1)

In all, 36 of the 64 patients initially screened met the inclusion criteria and were therefore included in the final analysis: 21 women (58.3%) and 15 men (41.7%) (sex ratio 0.7) with a mean age at diagnosis of 81 years (range: 64–95). The clinical presentation was consistent with the typical profile in the majority of patients, but 3 patients exhibited a prurigo-like pattern with positive DIF and ELISA. The histopathological pattern was consistent with that of PB with subepidermal blistering in 34 patients (94.4%), while an eczematous pattern was identified in 2 patients (5.6%) with no blisters and a prurigo-like form. DIF was consistent with BP in all patients with IgG and/or C3 deposits at the dermal-epidermal junction in all patients but one, in whom ELISA revealed significant levels of anti-BP180 auto-antibodies. Similarly, the presence of anti-BP180 autoantibodies in the circulation was confirmed by ELISA in all but one of the patients in whom this assay was conducted. Clinically, 30 patients (83.3%) had a multi-bullous disease (presenting with more than 10 new blisters daily at several anatomical sites), while the remaining 6 patients had a pauci-bullous disease (fewer than 10 new bullae daily), including the 3 patients with a prurigo-like pattern.

Neurological comorbidities were identified in 50% of patients. Eleven patients (61.1%) exhibited dementia, three (16.7%) displayed symptoms of depression or anxiety disorders, and one (5.6%) presented with a Parkinsonian disorder and three additional conditions, namely paraplegia and spinal cord compression with spasticity or hemiplegia. The mean glomerular filtration rate at the time of MTX introduction was 68 mL/min (range: 36–93).

## Treatments (Table S1, supplemental material available on-line)

All patients were initially treated with STS in ointment form (propionate clobetasol). MTX was subsequently introduced after several days or weeks of whole-body STS, usually following completion of a pre-therapeutic work-up, for various reasons, namely: first-line systematic treatment in 7 patients (19.4%), unsatisfactory IDC in 19 patients (52.8%), as an OS-sparing strategy in 8 patients (22.2%), and for other reasons in 2 patients (5.6%) (simplification of treatment or pre-inclusion in a study). Of the 36 patients included in the study, 19 (52.8%) received methotrexate as monotherapy, while 17 (47.2%) received methotrexate in combination with osimertinib. The dosage of oral prednisone was titrated from 0.5 to 1 mg per kilogram per day, contingent on the initial severity of the disease. Once disease control had been achieved, progressive dosage tapering was initiated over a period ranging from 1 to 9 months, dependent on the initial dose. The median time from diagnosis to MTX initiation was 25.1 days (IQR: 11.3–63.1 days). The median weekly dosage of MTX was identical in both groups, at 10 mg (range: 7.5–15 mg). The majority of patients on a combined treatment regimen received OS prior to MTX (16/17 patients, 94.1%). The mean time between implementation of these treatments was 39 days (range: 1–420 days). The median overall duration of MTX treatment was 13.6 months (Q1-Q3 = 7.0–18 months).

Following the achievement of IDC, MTX was permanently discontinued during the follow-up period in 14 patients (6 patients with mMTX vs. 8 with MTX+OS). This was primarily due to prolonged CR (10 patients, 5 patients in each group) and less frequently in order to limit AEs (4 patients, all in prolonged CR as well). MTX was discontinued prematurely in two patients due to an initial lack of response. In patients undergoing combined treatment, OS were discontinued several weeks prior to MTX withdrawal.

## Clinical response and patient outcomes (Fig.1 and Table 2)

Median follow-up after MTX initiation was 14.7 months (Q1-Q3 = 7–26.6 months); 7/18 (38.8%) patients on mMTX and 4/16 (25%) on MTX+OS were lost to follow-up at 12 months. Regarding endpoints, considering patients lost to follow-up, the 12-month relapse rate during the treatment period following IDC achievement, as estimated using the Kaplan-Meier method, was significantly higher in patients on mMTX (32.2%) vs. MTX+OS (0%) (log-rank test p = 0.03). The mean time to relapse was 10.3 months (±0.7). Subsequently, all four patients who experienced a relapse were successfully managed with an increase in the MTX dose (in three patients) or with a course of re-treatment with steroids (in one patient).

The overall IDC rate was 94.4% (34/36 patients) and was comparable between the two subsets (94.7% with mMTX vs. 94.1% with MTX+OS, Fisher’s exact test, p = 1, non-significant (NS)). As stated previously, MTX was discontinued due to prolonged CR (associated or not with AE) in 6 and 8 patients treated with mMTX and MTX+OS respectively. After MTX discontinuation, 2/6 patients treated with mMTX relapsed vs. 3/8 patients in the MTX+OS subset. Seven months after MTX discontinuation, 3/6 patients (50%) treated with mMTX vs. 5/8 (62.5%) with MTX+OS were lost to follow-up. However, owing to the number of patients lost to follow-up, the 7-month rate of BP relapse following MTX discontinuation, as calculated using the Kaplan-Meier method, was 66.7% for mMTX vs. 42.9% for MTX+OS (log-rank test, p = 0.81, NS). Relapse appeared to manifest earlier in patients on mMTX (10.7 ± 4.3 months) compared to those on MTX+OS after initial OS (4.5 ± 0.6 months), but with no significant difference between the two subsets. Recurrences were managed in all cases by resumption of transient STS ointments in one patient initially treated with mMTX and by reintroduction of MTX in all other patients, including one treated with MTX+OS and off treatment who also required renewed treatment with systemic steroids.

The most recent data indicate that among the 36 patients under observation, MTX was still being used for 23 patients. (63.9%), including 21 patients still in CR. MTX had been permanently discontinued for prolonged CR, with or without AE, in 11 patients (30.1%), 6 of whom had been on mMTX and 5 on MTX+OS.

Once IDC was achieved, the cure rate, as calculated using the Kaplan-Meier method, was 45.2% in patients on mMTX and 30% in those on MTX+OS (log-rank test p = 0.15, NS).

## Safety data (Table S2 
                        supplemental material available on-line; Table 3)

A comprehensive safety analysis was conducted on the 36 patients included in the study. A total of 10 patients (27.8%) experienced at least one AE: 3/19 (15.8%) patients receiving mMTX vs. 7/17 (41.2%) receiving MTX+OS (Fisher’s exact test p = 0.14, NS). The most frequently reported AEs included infections (urinary and lung infections, endocarditis, viral hepatitis, colitis) and haematological toxicity (anaemia, thrombocytopenia, neutropenia). Three patients exhibited AE grades of 3 or higher: one on mMTX (1/19, 5.3%), and two on MTX+OS (2/17, 11.7%). All three AEs were deemed to be potentially or probably related to MTX.

As previously stated, the incidence of AE, irrespective of grade, led to the permanent cessation of MTX in four patients (11.1%), of whom one was receiving mMTX (1/19; 5.3%) and three were taking MTX+OS (3/17; 17.7%). Fisher’s exact test yielded a p-value of 0.33, indicating no statistically significant difference between the groups. Conversely, a transient interruption before reintroduction was observed in three patients. MTX dosage was permanently reduced without discontinuation in one patient. A single patient (on mMTX) died during the follow-up period (1/36, 2.8% overall; 1/19, 5.3% in the mMTX subset). This death was deemed to be potentially or probably associated with MTX, as evidenced by general status deterioration, cytopenia, and diarrhoea. The estimated mortality rate using the Kaplan-Meier method was 11.1% for mMTX and 0% for MTX+OS (log-rank test p = 0.46, NS).

# Discussion

Despite the encouraging results of various studies concerning the use of MTX in BP, MTX is only recommended as a second-line treatment in severe diseases, or in instances where systemic steroids are contraindicated or ineffective, and alternative therapeutic options must be considered. Examples of such instances include poorly controlled diabetes mellitus, severe heart failure, severe osteoporosis, or glaucoma. The combination of this medication with systemic steroids is a common practice, particularly for the purpose of accelerating the tapering of steroids. Nevertheless, the efficacy of this approach remains unconfirmed [7,21–23]. The objective of our retrospective study was not to define an optimal therapeutic strategy for BP; rather, it was to describe the use of MTX in real-life clinical practice in this indication by comparing two different therapeutic strategies (mMTX vs. MTX+OS), in addition to STS.

In our study, the IDC rate with mMTX following initial use of STS was 94.7% but it was slightly lower (87%) in the recent RCT [17]. In the present real-life survey, the relapse rate during treatment was 32.2% with MTX alone, which is consistent with the corresponding data from the RCT data (37.9%) and comparable to the results observed in a previous open multicentre series [11]. The incidence of BP relapse following the discontinuation of MTX for any reason was 66.7% in our study, with a median time to relapse of 10 ± 4 months. This figure is higher than that reported in another retrospective study [12]. However, the relapse rate observed in our survey following the cessation of methotrexate therapy may be an overestimation, and its interpretation is challenging due to the low incidence of the event and the number of patients who were lost to follow-up. In general, the relapse rate during MTX treatment or after MTX discontinuation is consistent with the findings presented in the updated report by Wang et al. The BP relapse rate ranged from 27.87% to 53% following disease remission, with the majority of relapses occurring within six months of disease control [24]. As in several previous studies, our survey thus substantiates the clinical efficacy of MTX in BP in a “real-life” setting, even when used in monotherapy, both for achievement of initial control and for maintenance of clinical remission obtained by initial STS.

Interestingly, combined MTX/OS did not appear to confer any discernible clinical advantage, with no significant difference being noted in IDC rate or relapse rate after treatment discontinuation. However, systemic steroid use is still of potential value in severe diseases during the initial period to achieve better and faster clinical control, but with no clearly demonstrated advantage vs. STS and at the expense of additional AEs.

The present study, conducted in a non-strictly selected population, demonstrates that MTX maintains an overall favourable safety profile, particularly regarding mortality (5.3%) and the incidence of grade 3/4 AE. Notably, only a small proportion of patients (5.3%) permanently discontinued MTX due to AE, a finding that is consistent with previous literature [10,11,13–16]. These results were also relatively consistent with (and slightly superior to) corresponding data obtained in the recent RCT. which demonstrated a mortality rate of 9.5% in the MTX arm and a discontinuation rate of 20.3% due to AEs among MTX-treated patients. However, it seems likely that these safety data were underestimated due to the considerable number of patients lost to follow-up. Furthermore, the incidence of adverse events (AEs) associated with mMTX was 15.8%, all of which were anticipated and rated as mild to moderate with only 5.3% graded as severe. In comparison, the AE incidence with MTX+OS was 41.2%, with 11.8% graded as severe. These findings indicate that mMTX may be a more tolerable option than the combined treatment. This result is consistent with those of a previous study that reported an SAE incidence of 13.2% in patients treated with MTX and methylprednisolone [25].

In this real-life survey, several bias and limitations must be acknowledged. The retrospective and descriptive design of this study resulted in the loss of data and likely introduced evaluation bias due to the subjectivity of the investigators. Additionally, the diminished statistical efficacy observed in the study may be attributable to the relatively limited number of patients incorporated into the analysis, particularly given the single-centre nature of the survey. Some patients were in fact only seen for initial BP management and MTX initiation, with subsequent follow-up occurring in private practice.Consequently, obtaining additional outcome data may be challenging for some patients, particularly regarding subsequent relapses.

In conclusion, despite the methodological issues, the present study provides evidence that low-dose mMTX is an effective treatment for BP in a real-life setting, particularly regarding achievement of initial response, but above all for long-term control, after stopping or reducing steroids. Furthermore, the study demonstrated overall fair safety and no significant additional mortality in elderly patients, even those with moderately impaired renal clearance [14]. These data are consistent with those obtained in a recent RCT involving a highly selected patient population. Furthermore, MTX offers a number of practical advantages, particularly in elderly patients, including simplicity of use and low cost [26,27]. Nevertheless, the use of low-dose MTX must be approached with caution in patients with an eGFR of less than 45 ml/min, who exhibit a markedly elevated risk of toxicity, which is predominantly manifested as myelosuppression, sepsis, or hepatotoxic effects [28].