Patients

All patients with early-stage MF were recruited from the outpatient department of Peking Union Medical College Hospital, Peking Union Medical College, and Chinese Academy of Medical Sciences from June 2022 to November 2022.

Of the 55 patients with MF recruited in this study, 13 completed the entire follow-up process, including before treatment and at 1, 3, 6 months after treatment. These 13 patients were defined as group A. The other 42 patients were defined as group B, whose follow-up record (urine collection) in this study was only once, including 7 patients before treatment, 9 patients at 3 months after treatment, 5 patients at 6 months after treatment, and 21 patients at 1 year after treatment.

The definitive diagnosis of early-stage MF in this study was based on standard guidelines proposed by the International Society for Cutaneous Lymphomas and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (ISCL-EORTC) [13]. Clinical staging was made using TNMB system, which included assessment of skin (T), lymph nodes (N), visceral (M), and blood (B) involvement. According to the clinical stage, stage IA, IB and IA was defined as early MF [14]. The patients’ age, sex, course of disease, skin lesion locations, staging, relevant laboratory tests, and anamnesis were recorded in detail. The patients were informed about all aspects of the study, including the study design and possible adverse effects of interferon (IFN) α-2a administration and phototherapy, and signed a detailed informed consent form prior to study initiation. This prospective study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Medical Ethics Committee affiliated with the Peking Union Medical College Hospital, Peking Union Medical College, and Chinese Academy of Medical Sciences (file number: I-22PJ131).

The inclusion criteria were as follows: early-stage MF that met the ISCL-EORTC diagnostic criteria and was confirmed by histopathology in our hospital; ≥ 18 years old; male or female; could commit to ≥ 6 months of follow-up; have not received any systemic treatment, (e.g., retinoic acid, interferon, glucocorticoids, methotrexate etc.) within the past 2 months; have not received various phototherapies (e.g., UVA, UVB, or PUVA) within the past month.

Patients were excluded if they: had other malignant tumors; had severe infection (e.g., upper respiratory tract infection, active tuberculosis, urinary tract infection, HIV infection, or other types of infection); had liver or kidney dysfunction; had diabetes; had blood diseases; had previously been allergic to IFN or phototherapy; had contraindications to phototherapy (e.g., systemic lupus erythematosus); were pregnant or breast-feeding; were unwilling or unable to attend follow-up visits.

In addition, a total of 125 control subjects were recruited. They included healthy individuals (30 cases), or patients with moderate-to-severe atopic dermatitis (EASI >15 or SCORAD >40, 30 cases), moderate-to-severe plaque psoriasis (PASI >10 or BSA >10%, 28 cases), generalized vitiligo (> 10% BSA involvement, 20 cases), or basal cell carcinoma (17 cases, all of which were confirmed by skin biopsy). All the control subjects met the diagnostic criteria and had no history of diabetes, acute or chronic hepatic or renal dysfunction, severe infection, electrolyte disorder, or malignant tumors.

Preparation for treatment

All patients underwent laboratory testing (e.g., complete blood count, liver and kidney function, blood glucose, blood lipids), electrocardiography, superficial lymph node (including neck, subclavian, axilla, and groin) ultrasound examination, along with clinical evaluation of MF; photographs were taken if the patient provided written or verbal consent.

Medications and instrument

The following medications were used in this study: recombinant human IFN α-2a injection (3 million IU) (Shenyang Sunshine Pharmaceuticals Co., Ltd., Shenyang, China), halometasone/triclosan cream (Pro Farma AG, Baar, Canton of Zug, Switzerland; Famar SA, Athens, Greece). Phototherapy was administered using a UV phototherapy instrument (Waldmann, Villingen-Schwenningen, Germany). RIGOL L-3000 High Performance Liquid Chromatography System (Beijing Puyuan Jingdian Technology Co., Ltd., Beijing, China) and Multi-functional enzyme-linked immunosorbent assay (Shanghai Delang Medical Equipment Co., Ltd., Shanghai, China) was used for LC-MS/MS and ELISA respectively. Company details of the reagents used in liquid chromatography-tandem mass spectrometry were shown in Table 1.

Table 1 Company details of the reagents used in liquid chromatography-tandem mass spectrometryTherapeutic protocol

Phototherapy options varied based on whether the patient’s skin lesions manifested as patches or plaques. NB-UVB combined with UVA was used to treat patients with skin lesions presenting as patchy or thin plaques. If the skin lesions did not improve significantly after 1 month of treatment, the patient was switched to UVA1 phototherapy. Meanwhile, patients with skin lesions presenting as thick plaques were immediately treated with UVA1. The starting dose, dose escalation, and frequency of phototherapy were as follows: NB-UVB, starting dose of 0.3 J/cm2, once every other day, three times a week, with a weekly dose increase of 0.2 J/cm2 and a maximum dose of 2.8 J/cm2; UVA, starting dose of 2 J/cm2, once every other day, three times a week, with a weekly dose increase of 0.5 J/cm2 and a maximum dose of 8–8.5 J/cm2; UVA1, starting dose of 20 mJ/cm2 or 40 mJ/cm2 (based on the thickness of the skin lesions) once a day, five times a week, for 3 consecutive months. If the skin lesions improved significantly, the patient was switched to NB-UVB, with a starting dose of 0.3 J/cm2, once every other day, three times a week, an increase of 0.2 J/cm2 per week, and a maximum dose of 2.8 J/cm2.

In addition, all patients received a subcutaneous injection of recombinant human IFN α-2a (3 × 106 IU) once every other day for 3 consecutive months. From the 4th month of treatment, IFN α-2a was stopped but phototherapy was continued.

Halometasone/triclosan cream was applied externally to the lesions twice a day. To avoid adverse reactions such as skin atrophy caused by long-term use, the halometasone/triclosan cream was applied for 2 weeks and stopped for 1 week over 3–6 consecutive months. If the skin lesions had not completely subsided by the end of the 6-month treatment period, halometasone/triclosan cream application could be continued for another month.

Treatment monitoring

Clinical evaluation of therapeutic efficacy was based on the percentage change of skin lesions relative to the total body surface area (TBSA). The BSA of patches or plaques in 12 body areas was measured using the area of patch or plaque in hand units (which was set at 1% per unit). BSA evaluation was performed by the same member of clinical staff during the full follow-up period.

The modified severity weighted assessment tool (mSWAT) was used to monitor the skin tumor load in Mycosis Fungoides/Sezary Syndrome. The percentage of TBSA for each lesion type was multiplied by a number (patch = 1, plaque = 2, tumor = 4), then summed to obtain the mSWAT score. mSWAT scores were performed by the same member of clinical staff during the entire follow-up course.

The clinical efficacy evaluation criteria were defined as follows: complete remission (CR, 100% clearance of the skin lesion), partial response (PR, a decrease of ≥ 50% in mSWAT score compared to baseline), stable disease (SD, a decrease of < 50% to an increase of < 25% in mSWAT score compared to baseline), and progressive disease (PD, an increase of ≥ 25% in mSWAT score compared to baseline, or an increase of ≥ 50% in the sum of pathological positive lymph node maximum diameter products compared to baseline). Overall response rate (ORR) was defined as the combination of CR and PR. The CR/PR had to be confirmed through repeated evaluation after ≥ 4 weeks.

Laboratory testing was performed prior to treatment and every 2 months thereafter. Adverse events during treatment were reported by the patients and observers. Patients who completed the 6-month follow-up visit were included in the efficacy analysis.

Urine collection and storage

Of the 20 patients with MF recruited in this study, 13 completed the full treatment course. These 13 (13/20) patients were defined as group A. The midstream morning urine samples were collected from group A patients before treatment and at 1, 3, 6 months after treatment.

Urine had also been collected before treatment from seven (7/20) patients with MF (defined as group B) at their initial visit; however, these patients were later lost to follow-up. The other 35 patients of group B had also received the IFN α-2a and phototherapy combination treatment in our department for 3 months (9 patients), 6 months (5 patients), or 1 year (21 patients). The midstream morning urine samples of these were collected once during a follow-up visit. Since these patients had initiated treatment before the start of this study, their before treatment urine sample could not be collected.

The midstream morning urine samples were also collected from the control subjects (i.e., healthy individuals, and patients with atopic dermatitis, psoriasis, vitiligo, or basal cell carcinoma). Urine collection from menstruating women was avoided. Urine samples were kept at room temperature for less than 4 h. Cell debris was removed by low-speed centrifugation at 2000 × g for 10 min at room temperature; the supernatants were then stored at − 80 °C until use.

Protein preparation and digestion

The urine samples were treated with 300 µL 8 M urea (containing a lysis solution to protease inhibitor ratio of 50:1), before being sonicated (using 1 s on, 2 s off pulses), for a total of 120 s. The samples were centrifuged at 14,000 × g at 4 °C for 20 min, and the protein concentration and purity within the supernatant were assessed by SDS-PAGE.

A bovine serum albumin protein standard solution was prepared according to the manufacturer’s instructions using a Bradford protein quantification kit, with a concentration gradient ranging from 0 to 0.5 µg/µL. Different ratios of bovine serum albumin standard and sample to be tested were added to a 96-well plate; the total volume in the well was made up to 20 µL. Each experiment was performed in triplicate. 180 µL of the G250 staining solution was quickly added to the 20 µL sample and the mixture incubated at room temperature for 5 min, before absorbance was determined at 595 nm. The protein concentration within each sample was calculated according to the standard curve. 10 µg of each protein sample was loaded onto a 12% SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 to reveal the protein bands.

100 µg of protein sample was incubated with 10 mM DTT at 37 °C for 1 h, before being returned to room temperature. The sample was then diluted with ammonium bicarbonate (until pH 8 was reached) and incubated with trypsin (at a 50:1 protein to trypsin ratio) at 37 °C overnight. The next day, 50 µl 0.1% formic acid (FA) was added to terminate the reaction. The samples were desalted using a C18 desalting column, which was pre-treated with 100% acetonitrile and then 0.1% FA. After loading the sample onto the column, 0.1% FA was used to wash the column to remove any impurities. Finally, 70% acetonitrile was used to elute the flowthrough, which was subsequently freeze-dried.

LC‑MS/MS

The mobile phases liquid A (100% MS water, 0.1% FA) and liquid B (80% acetonitrile, 0.1% FA) were prepared. The lyophilized powder was dissolved in 10 µL of solution A and centrifuged at 14,000 × g at 4°C for 20 min. 1 µg of supernatant was injected into the sample chamber for liquid quality detection. The Q Exactive HF-X mass spectrometer and Nanospray Flex™ (NSI) ion source were used to set the ion spray voltage to 2.4 kV and the ion transport tube temperature to 275 °C. The data-dependent acquisition mode was adopted for mass spectrum generation, and the full scanning range of mass spectrum was 350–1550 m/z. The primary MS resolution was set to 120,000 (200 m/z), the AGC was 3 × 106, and the maximum injection time was 80 ms. Parent ions with the TOP 40 ionic strengths in the full scan were selected to be splintered by high-energy impact cracking (HCD) method; a secondary MS detection was then carried out. The secondary MS resolution was set to 15,000 (200 m/z), the AGC was 5 × 104, and the maximum injection time was 45 ms. The peptide fragmentation collision energy was set to 27% and the raw MS data (.raw) were generated.

Sequence database search and data analysis

The MS data were analyzed using Proteome Discoverer 2.4 software. The parameters were set as follows: enzyme = trypsin, maximum missing cleavage = 2, static modification = carbamidomethyl (C), dynamic modification = M Oxidation (15.995 Da); Acetyl (Protein N-terminal), precursor ion mass tolerance = ± 15 ppm, and fragment ion mass tolerance = ± 0.02 Da.

Bioinformatics analyses

A protein was defined as differentially expressed if it underwent an expression fold-change of ≥ 1.5 or ≤ 1.5 in MF patients versus healthy control subjects and the P-value was < 0.05. The differentially expressed proteins were classified using principal component analysis (PCA). Next, the differentially expressed proteins were subjected to gene ontology (GO) analysis and matched against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Cluster of Orthologous Groups (COG) of proteins.

ELISA

All urine samples were thawed, centrifuged, and aliquoted according to the instructions supplied with the ELISA kits obtained from Wuhan Huamei Biological Engineering Co., LTD; Wuhan Cloud-clone Technology Co., LTD; Abcam Co.; Wuxi OriGene Technologies Co., LTD. Then, 100 µl of a biotin-conjugated antibody (1:100 dilution) and 100 µl of horseradish peroxidase (HRP)-avidin (1:100 dilution) were added to 100 µl of each sample. After the TMB substrate solution was added to each well, the reaction was terminated with 50 µl of stop solution. The optical density (OD) values were read at 450 nm, and the protein concentrations were automatically calculated according to the standard curve.

Statistical analysis

Data were analyzed using SPSS Version 27.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as the mean ± standard deviation (SD). Analysis of variance was used to compare repeated measurement data. A P-value of < 0.05 was used to indicate statistically significant differences. The receiver operating characteristic (ROC) curve method was used to determine the predictive ability of protein markers in the diagnosis of MF.