Study about evaluation of efficacy of methotrexate in localized scleroderma using ultrasonography

Abstract Background The treatment and curative effect evaluation of localized scleroderma (LS) still perplexes many clinical workers. Purpose To investigate the efficiacy of methotrexate in the treatment of LS by the evaluation of ultrasonography. Methods A prospective study enrolled 10 patients treated with MTX for at least 6 months was conducted. Treatment outcome was evaluated by a clinical score and 15‐MHz ultrasonography. Safety assessment included the monitoring of adverse drug reactions and clinical laboratory examinations. Results Eight of the 10 patients achieved clinical remission only with MTX. One patient was relieved after MTX combined with corticosteroids, while another one does not improve after the treatment of mycophenolate mofetil and corticosteroids. The effective rate of MTX is 80%. Nine patients were significantly improved with a decrease of the Localized Scleroderma Cutaneous Assessment Tool (the mean score of the LoSCAT cutaneous activity dropped from 5.2 to 1.0, p < 0.001, the mean score of the LS cutaneous damage dropped from 4.3 to 2.3, p = 0.002). The average difference of thickness between skin lesions and normal skin evaluated by ultrasonography decreased from 0.13 cm to 0.04 cm (p = 0.009) in eight patients. No serious adverse reactions occurred. Conclusion Methotrexate is a safe and effective treatment for patients with LS. Ultrasonography can be considered as an efficient assessment tool for evaluation LS.

medicine to improve vascular microcirculation is the first choice for most doctors, such asasiaticoside tablets, Danggui Sini decoction and so on these traditional Chinese medicine treatments rely on experience without sufficient experimental evidence. Meanwhile, accurate assessment tools for evaluation of severity and efficacy of scleroderma are not available. Durometer, thermography, magnetic resonancehas, and other physical measurement tools have been used for the evaluation of scleroderma. The efficiency of these tools is uncertain. [8][9][10][11][12][13] Ultrasonography, as a cheap and convenient detection tool, has been used to evaluate the treatment of a variety of sclerosing skin diseases. In particular, the change of lesion thickness measured by ultrasonography can well reflect the therapeutic effciacy. Thus, we conducted a study to: (1) explore the efficacy of MTX in the treatment of LS in China; (2) evaluate the efficiency of ultrasonography in monitoring the thickness of skin lesions in LS patients.

Patients
From May 2020 to October 2021, a single-center nonrandomized prospective cohort study was conducted on patients with LS in the Shandong Provincial Hospital for Skin Diseases. A total of ten patients were included in the study. The flow chart has been provided in Figure 1. If the patient has adverse reactions in the course of treatment, the drug dose may be adjusted.

The LS cutaneous assessment tool (LoSCAT)
Patients are evaluated by the same dermatologist at every follow-up visit. LoSCAT, 12 which consists of an activity index LoSAI and a damage index LoSDI as well as ultrasonography, was used as measures' tools.
Additionally, we have collected clinical photos of patients at every visit, to facilitate the assessment of the improvement in the skin lesions.

Ultrasonographic evaluation
Ultrasonography was performed on the representative lesions of every patient to assess dermal thickness with a digital 15-MHz ultrasonography scanner by two radiologists. For generalized lesions, we select one of the most serious lesions as the representative to measure, and a single lesion does not need to be selected. The penetration depth of this instrument is greater than or equal to 30 mm. At the same time, the normal skin on the contralateral side or around the lesion was also measured as control. In order to reduce the measurement error and control variables as much as possible, we controlled the air conditioning temperature in the ultrasonic room so that the ambient temperature was kept between 20 and 23 • C. And, the patient rests for at least 5 min before the examination. The examiner pressed the patient's skin vertically and evaluated the thickness, echo intensity and blood flow signal of the patient's epidermis, dermis, and subcutaneous tissue, comparing it with the surrounding or contralateral normal skin. Then the difference between normal skin and affected skin was calculated.
According to the ultrasonography results before and after treatment, we defined the results as follows: (1) 9 For some insurmountable reasons, the frequency of ultrasonographic examination was not measured once a month in 10 patients, but each patient was measured at least twice during the experiment.

Statistical analysis
Data of clinical scores and difference of thickness between lesions and normal skin evaluated by ultrasonography were given as the standard deviation of mean plus minus. Normal distribution of collected data was analyzed by the Shapiro-Wilk test, while the association between LoSCAT and skin thickness measured by ultrasonography was tested by the Spearman correlation coefficient. Measurement data before and after treatment were examined by paired-samples T test. p < 0.05 was considered to be significant. Statistical analysis was performed by SPSS.

Patient
Ten patients were included in this cohort, and the characteristics of patients are showed in Table 1 and

Treatment
Ten patients were treated according to the treatment protocol as shown in Figure 1. All patients received MTX in the study.

LoSCAT
The LoSCAT scores of the nine patients were decreased except patient number 9, whose condition was not observed to improved. The average LOSAI scores of the nine patients before treatment was 5.2, which dropped to 1.0 after treatment (p < 0.01). The maximum improvement rate was 100%, and the minimum improvement rate was 40%.

Adverse events
Four patients had mild gastrointestinal reactions. Three patients were gradually tolerated, and another patient needed to reduce the dose of F I G U R E 2 Changes of LoSCAT score and lesion thickness in patient 5.
MTX (from 10 mg/week to7.5 mg/week after 2 months). One patient had a slight increase in transient transaminase, and was treated with compound glycyrrhizin tablets.

DISCUSSION
This study was conducted to evaluate the efficacy of MTX in the treatment of LS by using clinical score and ultrasonography. A total of 10 patients were recruited. Clinical and ultrasonographic evaluation revealed that nine patients markedly improved during 6-month follow up, and even linear morphea patients were found to have hair regrowth (Figures 3 and 4), showing high efficacy.

F I G U R E 3 (A and B)
Localized morphea lesions on the groin. The hardness, pigmentation, and atrophy of the skin lesions were significantly improved during the 6-month treatment period. (C and D) Changes of skin thickness (the part indicated by the red bracket) before and after treatment under ultrasonography monitoring (skin thickness decreased from 0.21 cm to 0.18 cm).
As early as 1998, Seyger et al. 12  have also been proven effective in a randomized controlled study. 14 For some stubborn lesions, mycophenolate mofetil and hydroxychloroquine have also been tried and verified by a few cohort studies. 11,15 A prospective comparative effectiveness study of three different methotrexate-based regimens for juvenile LS found there was no significant difference in efficacy between MTX alone group and MTX combined with corticosteroids treatment group. The total effective rate was 75%. 16 The researchers also found that patients with treat- assessing the efficacy of MTX in the treatment of children with LS. 8 However, due to the complexity and subjectivity of these tools, they were not widely used.
Ultrasonography has also been used to assess the therapeutic efficacy in LS. [20][21][22] Skin thickening can represent either the earlier edema stage or the later fibrosis stage. The thinning of the skin thickness represents a period of atrophy. Therefore, the change of thickness of LS lesions can be used to detect the efficacy of drugs. In our study, we found that there were significant differences between the thickness of the skin lesions before and after treatment. M. Arisi 23 found no statistical significance in skin thickness evaluated by ultrasonography in LS patients who received ultraviolet A1 phototherapy. This might be due to the 50 MHz parameter he used, which had less penetrating power. In addition, M. Arisi considered that phototherapy might only change the order of collagen fibers without reducing its total number.
In our study, the average improvement rate of skin thickness measured by ultrasonography in eight patients after 6 months of treatment was 69%, which was lower than the improvement rate of LoSAI. However, the dermis thickness assessed by ultrasonography has been changed earlier than the clinical scores, indicating that ultrasonographic assessment was more sensitive than the scores, which may aid the doctor to grasp the situation more quickly and take the appropriate treatment program. There was also no correlation between the dermis thickness measured by 14-MHz ultrasonography and LoSCAT. 24 Although the changes in the echogenicity and hyperaemia of the skin lesions were hard to obtain and compared in our study, after summing up previous studies, Suzanne C. Li 20 found that the echogenicity of the skin lesions in the early stage had different degrees of reduction, and blood flow signals were strong, which indicates dermal edema. At the same time, echo enhancement represents the hardening of dermal collagen fibers. LoSCAT was dependent on clinicians whereas ultrasonographic assessment was more objective. Thus, combination clinical score and ultrasonography would be more comprehensive.
The mechanism of MTX in treating LS remains unclear. It is generally believed that the inflammatory pathway included a variety of cytokines and chemokines are involved in the pathogenesis of LS, which is mainly caused by the Th1 cells in the early stage of the disease and the Th2 cell subsets in the late stage of LS, resulting in fibrosis. 25 Methotrexate plays an anti-inflammatory role by increasing the concentration of adenosine extra and intracellular, and it is assumed that by adenosine inhibits the oxidative burst of neutrophils and mononucleocytes, prevents the chemotaxis of leukocytes, and inhibits the secretion of multiple cytokines by monocytes and macrophages. Finally MTX interferes with inflammatory pathways in LS. 26 There are some limitations in our study. Firstly, only 10 patients were involved in our study. Secondly, according to the limited number of patients, various age groups were not separate for discussion.
In addition, the follow-up was only conducted for 6 months. It was suggested that the time of maintenance therapy should be continued for at least 2 years. After stopping treatment, these patients were advised to monitor the condition for at least 5 years. 27

CONCLUSION
In conclusion, most patients included in our study showed excellent response to MTX without any major adverse effects. Ultrasonography could be used for monitoring the improvement of dermis thickness.