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Application of urine proteomics in the diagnosis and treatment effectiveness monitoring of early-stage Mycosis Fungoides

Clinical Proteomics volume 21, Article number: 53 (2024) Cite this article

Abstract

Background

Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma. As the early clinical manifestations of MF are non-specific (e.g., erythema or plaques), it is often misdiagnosed as inflammatory skin conditions (e.g., atopic dermatitis, psoriasis, and pityriasis rosea), resulting in delayed treatment. As there are no effective biological markers for the early detection and management of MF, the aim of the present study was to perform a proteomic analysis of urine samples (as a non-invasive protein source) to identify reliable MF biomarkers.

Methods

Thirteen patients with early-stage MF were administered a subcutaneous injection of interferon α-2a in combination with phototherapy for 6 months. The urine proteome of patients with early-stage MF before and after treatment was compared against that of healthy controls by liquid chromatography-tandem mass spectrometry. The differentially expressed proteins were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Clusters of Orthologous Groups analyses. For validation, the levels of the selected proteins were evaluated by enzyme-linked immunosorbent assay (ELISA).

Results

We identified 41 differentially expressed proteins (11 overexpressed and 30 underexpressed) between untreated MF patients and healthy control subjects. The proteins were mainly enriched in focal adhesion, endocytosis, and the PI3K-Akt, phospholipase D, MAPK, and calcium signaling pathways. The ELISA results confirmed that the urine levels of Serpin B5, epidermal growth factor (EGF), and Ras homologous gene family member A (RhoA) of untreated MF patients were significantly lower than those of healthy controls. After 6 months of treatment, however, there was no significant difference in the urine levels of Serpin B5, EGF, and RhoA between MF patients and healthy control subjects. The area under the receiver operating characteristic curve values for Serpin B5, EGF, and RhoA were 0.817, 0.900, and 0.933, respectively.

Conclusions

This study showed that urine proteomics represents a valuable tool for the study of MF, as well as identified potential new biomarkers (Serpin B5, EGF, and RhoA), which could be used in its diagnosis and management.

Background

Cutaneous T cell lymphoma (CTCL), with accounts for 65–92% of all cutaneous lymphomas, is a subtype of primary skin lymphoma caused by the abnormal clonal proliferation of cutaneous T lymphocytes [1]. Mycosis fungoides (MF) is the most common type of CTCL, accounting for ~ 54% of all cases, with an estimated incidence of 5/1,000,000 individuals per year; it is characterized by the malignant proliferation of CD4 + T lymphocytes [2].

The natural course of MF involves three stages: the patchy stage, the plaque stage, and the tumor stage [3]. Due to the heterogeneity of its clinical manifestations, early-stage MF is also known as the “universal imitator” [4, 5]. MF causes skin lesions, which can manifest as non-specific inflammatory erythema or plaques, or dry desquamation (with or without pruritus). Thus, MF is difficult to diagnose incipiently and can be misdiagnosed as skin conditions such as atopic dermatitis or psoriasis, leading to delayed treatment. At present, advanced MF cannot be effectively treated and has a poor prognosis, with a median survival time of less than 5 years [6].

To improve patient treatment and prognosis, MF should be diagnosed early. A skin biopsy is the gold standard for detecting skin lesion changes; however, it is an invasive procedure, which is not tolerated on a regular basis by most patients. At present, there are no objective and effective biological markers for the early detection and efficacy evaluation of MF. Therefore, there is an urgent need for the discovery of specific biomarkers that can be used to diagnose MF early and inform its clinical management.

Proteomics is concerned with the characterization of the total protein content of cells, tissues, or organisms. As such, it helps analyze the structure and function of proteins, both under physiological conditions and disease states. Technologies based on proteomics have been exploited to discover diagnostic biomarkers, identify vaccine candidates, understand pathogenic mechanisms, assess changes in protein expression patterns in response to treatment, and elucidate complex signaling pathways [7]. Mass spectrometry (MS) is one of the most commonly used proteomics methods. Highly sensitive, MS-based proteomics enables the identification, characterization, and quantification of proteins within complex biological samples [8]. To date, MS analysis has been predominantly applied in cancer research, whereby established bioinformatics models can be used to identify and characterize malignant tumors.

Proteomics can also be applied to the analysis of bodily fluids such as urine [9, 10]. As an important bodily fluid, urine represents a rich source of disease biomarkers. Urine is produced throughout the day and can be collected in large quantities non-invasively [10, 11]. Thus, urine proteomic testing is much more likely to appeal to patients than invasive tissue sampling techniques, improving compliance. Moreover, urine contains a plethora of proteomic information, which is indicative of changes occurring in the body at a given time. The protein composition of urine is also less complex and more stable than that of blood. Therefore, urine is an ideal source of disease markers. Liquid chromatography tandem MS (LC-MS/MS) is a noninvasive detection method used in urine proteomics, which provides qualitative and quantitative assessments of the proteome and offers clues relating to how certain biological processes are implicated in disease development [12].

In the current study, we aimed to identify the biological significance of proteins differentially expressed between patients with early-stage MF and healthy individuals using LC-MS/MS. We expected that some of the differentially expressed proteins may be implicated in biological processes involved in MF development, which may inform the prognosis and treatment of patients with this condition.

Methods

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 spectrometry
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Therapeutic 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.

Results

Patient characteristics

Among the 13 MF patients in group A (IA stage = 3 patients; IB stage = 8 patients; IIA stage = 2 patients), there were seven males and six females, aged (20–72) years, with an average age of (39.38 ± 16.01) years. Among the 42 MF patients in group B (IA stage = 4 patients; IB stage = 29 patients; IIA stage = 9 patients), there were 22 males and 20 females, aged (18–68) years, with an average age of (39.55 ± 12.58) years. Within group B, urine samples were collected from 7 patients before treatment, from 9 patients after 3 months of treatment, from 5 patients after 6 months of treatment, and from 21 patients after 1 year of treatment.

Among the control subjects, there were 30 patients with atopic dermatitis (18 males and 12 females, aged 18–79 years, with an average age of 45.50 ± 17.50 years), 28 patient with psoriasis (22 males and 6 females, aged 20–76 years, with an average age of 41.93 ± 16.27 years), 20 patients with vitiligo (12 males and 8 females, aged 19–64 years, with an average age of 37.55 ± 14.77 years), 17 patients with basal cell carcinoma (8 males and 9 females, aged 36–86 years, with an average age of 63.53 ± 14.98 years), and 30 healthy individuals (17 males and 13 females, aged 25–65 years, with an average age of 37.83 ± 10.15 years). There was no statistical difference in the sex ratio among the groups (P = 0.378). The average age of the basal cell carcinoma patients was significantly higher than that of the other groups (P < 0.001).

Routine urine examination in our hospital detected no significant abnormalities within the urine samples of any of the participants.

Clinical efficacy evaluation

After 1 month of treatment, all of the 13 patients in group A achieved SD, and none progressed. At the point, there were no significant differences in the lesion BSA and mSWAT score compared with before treatment (P = 0.182, 0.128 respectively). After 3 months of treatment, all 13 patients achieved PR, and none progressed; this time, their lesion BSA and mSWAT score significantly decreased compared with before treatment (P < 0.001). After 6 months of treatment, the lesion BSA and mSWAT score were significantly lower than those before treatment (P < 0.001) and at 3 months of treatment (P < 0.05). Overall, 11 patients achieved a CR and 2 patients achieved a PR, yielding an ORR of 100%. The clinical improvement before and after treatment is shown in Fig. 1(a-d), and the changes of BSA and mSWAT score were shown in Tables 2 and 3.

Fig. 1
figure 1

Clinical improvement experienced by a patient with MF as a result of treatment

Changes of skin lesions on the abdomen before and after treatment. Before treatment (a), and after 1 (b), 3 (c), and 6 (d) months of treatment

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Table 2 The improvement of BSA score in early-stage MF before and after treatment
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Table 3 The improvement of mSWAT score in early-stage MF before and after treatment
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Integrated proteomic information

A total of 971 non-redundant proteins were detected in the urine of patients with MF. We found that 41 of these proteins (11 overexpressed and 30 underexpressed; Table S4) were differentially expressed in MF patients versus healthy control subjects. We next subjected these 41 proteins to hierarchical clustering analysis and generated a heatmap showing protein profile differences between patients with MF and healthy control subjects (Fig. 2).

Fig. 2
figure 2

Cluster analysis of the 41 differentially expressed proteins. The hierarchical clustering results are presented as a tree heatmap, with the ordinate representing significantly differentially expressed proteins and the abscissa representing sample information. Significantly differentially overexpressed (red) and underexpressed (green) proteins are shown. A1–A6 represent patients with MF, and B1–B10 represent healthy control subjects

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GO functional analysis

We next performed a GO analysis to enrich and cluster the 41 differentially expressed proteins. The detailed information relating to the molecular functions, cellular components, and biological processes related to the differentially expressed proteins is shown in Table S1. We found that most of the proteins were involved in the phospholipase D signaling pathway, endocytosis, and the mitogen active protein kinase (MAPK) signaling pathway (Fig. 3).

Fig. 3
figure 3

GO enrichment analysis of 41 differentially expressed proteins. The main biological functions of the differentially expressed proteins were determined through the GO significance analysis. A P-value ≤ 0.05 was used as the threshold of statistical significance; GO terms that met this condition were defined as GO terms in which the differentially expressed proteins were significantly enriched

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KEGG pathway analysis

We next used KEGG analysis to search for pathways in which the 41 differentially expressed proteins were enriched (Table S2). The KEGG function classification analysis revealed that the 41 identified proteins were significantly enriched in focal adhesion, endocytosis, regulation of the actin cytoskeleton, the phospholipase D signaling pathway, the phosphatidylinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, the MAPK signaling pathway, and the calcium signaling pathway (Fig. 4).

Fig. 4
figure 4

The Pathway significance enrichment analysis method is the same as GO functional enrichment analysis; however, it uses KEGG Pathways as a unit and applies hypergeometric tests to identify pathways in which the differentially expressed proteins are significantly enriched compared with all the identified protein backgrounds. Using this method enables, the most important biochemical metabolic pathways and signal transduction pathways associated with the differentially expressed proteins can be identified

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COG functional analysis

COG functional analysis showed that the 41 differentially expressed proteins were significantly related to post-translational modification, protein turnover, chaperones, amino acids transport and metabolism, carbohydrate transport and metabolism, and lipid transport and metabolism (Fig. 5 and Table S3).

Fig. 5
figure 5

COG function classification. The COG database is used for the orthologous classification of gene products. The results of the COG classification of differentially expressed proteins in different subgroups are shown

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Sensitivity analysis of the differentially expressed proteins

The results of sensitivity analysis of the differentially expressed proteins are shown in Table S4. The area under the ROC curve (AUC) values of all 41 different proteins were ≥ 0.7, indicating their potential diagnostic value. Among them, there were seven proteins with an AUC value of 0.7–0.8, 28 proteins with an AUC value of 0.8–0.9, and six proteins with an AUC value of ≥ 0.9. Immunoglobulin heavy constant gamma 1, tenascin, and Ras homologous gene family member A (RhoA) had the highest potential predictive power, with AUC values (sensitivity, specificity) of 0.983 (0.900, 1.000) (Fig. 6a), 0.967 (1.000, 0.833) (Fig. 6b), and 0.933 (0.800, 1.000) (Fig. 6c), respectively.

Fig. 6
figure 6

The area under the receiver operating characteristic curve (AUC) values, sensitivity, and specificity for immunoglobulin heavy constant gamma 1 (a), tenascin (b), transforming protein RhoA (c), Serpin B5 (d), and EGF (e) are shown

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ELISA evaluation

To validate the results of the proteomic analysis, RhoA, programmed cell death 1 ligand 2 (PDCD1LG2), serine protease inhibitors B5 (Serpin B5), tenascin-C, lysosomal protective protein (also called cathepsin A, CTSA), epidermal growth factor (EGF), growth arrest-specific protein 1 (GAS1) were chosen for further study. The urine levels of these proteins were subsequently measured in the validation cohort of 55 patients with MF (13 patients in group A and 42 patients in group B) and 127 control subjects (30 patients with atopic dermatitis, 30 patients with psoriasis, 20 patients with vitiligo, 17 patients with basal cell carcinoma, and 30 healthy individuals), were measured by ELISA (Fig. 7).

Fig. 7
figure 7

ELISA evaluation of Serpin B5 (a), EGF (b), RhoA (c), PDCD1LG2 (d), tenascin-C (e), CTSA (f), and GAS1 (g) levels in the urine of patients with MF and healthy control subjects (i.e., patients with atopic dermatitis, psoriasis, vitiligo, and basal cell carcinoma, as well as healthy individuals). P***< 0.001 vs. healthy control subjects

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The levels of Serpin B5, EGF, and RhoA in the urine of MF patients in group A, were significantly lower before treatment compared with healthy controls (P < 0.001) (Fig. 7a, b and c). After 1 month of treatment, the urine levels of these proteins in MF patients were still significantly lower than those in healthy controls (P < 0.001). After 3 months of treatment, the urine levels of Serpin B5 and EGF of MF patients were significantly lower than those of healthy controls (P < 0.001); however, the urine levels of RhoA were now not significantly different between MF patients and healthy controls. After 6 months of treatment, the urine levels of Serpin B5, EGF, and RhoA of MF patients were no longer significantly different to those of healthy controls. We found that the AUC values (sensitivity, specificity) for Serpin B5, EGF, and RhoA were 0.817 (0.800, 0.833) (Fig. 6d), 0.900 (0.900, 0.833) (Fig. 6e), and 0.933 (0.800, 1.000) (Fig. 6c), respectively.

The levels of Serpin B5, EGF, and RhoA in the urine of MF patients in group B were also significantly lower than those of healthy controls (P < 0.001) (Fig. 7a, b and c). After 3 months of treatment, the urine levels of Serpin B5 and EGF in the urine of MF patients were still significantly lower than those of healthy controls (P < 0.001), while there was no significant difference in the urine levels of RhoA between MF patients and healthy control subjects. After 6 months and over a year of treatment, there was no significant difference in the urine levels of Serpin B5, EGF, and RhoA between MF patients and healthy control subjects. The results were consistent with those of MF patients in group A before treatment and at 3 and 6 months after treatment.

There were no significant differences in the urine levels of Serpin B5, EGF, and RhoA of patients with atopic dermatitis, psoriasis, vitiligo, or basal cell carcinoma before treatment and those of healthy control subjects (Fig. 7a, b and c).

In addition, the urine levels of PDCD1LG2, tenascin-C, CTSA, and GAS1 of MF patients (both group A and group B) before treatment were not significantly different from those of healthy controls (Fig. 7d, e, f and g).

Discussion

The popularization of high-throughput and high-precision MS technology has made proteomic analysis possible. Proteomics can reveal key protein-related information, which can contribute to the search for clinically applicable biomarkers and therapeutic targets [15]. Proteomics can give answers not only about the number and abundance of proteins but also about their involvement in metabolic pathways, interactions, post-translational modifications, synthesis, and degradation [15]. Biomarkers have numerous clinical applications. For instance, they can be used as tools for the risk stratification, screening, and early detection of cancer. In addition, they can be used to inform diagnosis, prognosis, and response to therapy, as well as aid patient surveillance and monitoring [16, 17]. However, the proteomic analysis of MF samples, and especially urine, has seldom been performed. In this study, MS technology was used to conduct a proteomic analysis of urine samples from patients with early-stage MF to find reliable biomarkers for the early diagnosis and efficacy monitoring of MF.

The pathogenesis of MF implicates multiple signaling pathways. Many products of the genes differentially expressed in MF are associated with PI3K, RAS, cell cycle/apoptosis, and MAPK signaling pathways [18]. The PI3K/AKT/mTOR axis is overactivated in various cancers, including CTCL [19, 20]. The activation of this axis can significantly promote the proliferation, survival, and migration of malignant T lymphocytes, as well as alter their microenvironment, thereby promoting the occurrence and development of T cell lymphoma [21]. PI3K can promote the continuous progression of MF toward the tumor phase of MF by interacting with the RAS pathway [18]. Studies have shown that MAPK signaling pathway dysregulation is associated with the occurrence and development of various types of lymphoma, including T cell lymphoma, chronic lymphocytic leukemia, diffuse large B cell lymphoma, and Burkitt’s lymphoma [22,23,24,25]. Moreover, the calcium signaling pathway is involved in the occurrence and development of T cell lymphoma and diffuse large B cell lymphoma [26,27,28].

In this study, we performed a proteomic analysis of the urine of MF patients before and after treatment. We identified 41 proteins which were differentially expressed between MF patients and healthy control subjects. Crucially, these proteins were enriched in various signaling pathway related to the pathogenesis of CTCL, including the calcium, MAPK, NF-κB, PI3K-Akt, phospholipase D, and RAS signaling pathways. Thus, we confirmed that the above pathways were also involved in the pathogenesis of MF.

To validate our proteomic analysis results, we evaluated changes in the urine concentrations of the three proteins (namely Serpin B5, EGF, and RhoA) most underexpressed in patients with MF versus healthy control subjects by ELISA. The findings were consistent with the MS results, suggesting that urine Serpin B5, EGF, and RhoA could represent potential biomarkers for MF. If applied clinically, these markers may help to distinguish MF from inflammatory diseases such as atopic dermatitis and psoriasis, which often obscure the MF diagnosis.

Hypopigmented MF is an uncommon variant of MF, characterized by varying sizes of hypopigmented or depigmented patches, which may be clinically mistaken for tinea versicolor, vitiligo, or pityriasis alba [29, 30]. Thus, in this study, patients with vitiligo and basal cell carcinoma (a common skin tumor) were included within the control group. The ELISA results showed that there were statistically significant differences in the urine levels of Serpin B5, EGF, and RhoA in MF patients before treatment compared with those of healthy subjects. Meanwhile, there were no statistically significant differences in the urine levels of Serpin B5, EGF, and RhoA of vitiligo and basal cell carcinoma patients before treatment compared with those of healthy subjects. These results further confirm the potential of urine Serpin B5, EGF, and RhoA to become specific biomarkers for MF.

In this study, early-stage MF patients were administered IFN α-2a by subcutaneous injection alongside phototherapy. The changes in urine Serpin B5, EGF, and RhoA levels of MF patients before and after treatment were consistent with the changes in their clinical scores. The AUC values of Serpin B5, EGF, and RhoA were 0.817, 0.900, and 0.933, respectively, indicating that these proteins have the potential to act as biomarkers for predicting and monitoring the efficacy of existing MF therapies.

Serpin B5, also known as the mammary serine protease inhibitor (Maspin), is a non-inhibitory member of the serine protease inhibitor superfamily. It can inhibit cancer cell invasion and adhesion, increase the sensitivity of cancer cells to apoptosis, and inhibit tumor angiogenesis [31, 32]. The expression level of Serpin B5 varies among various types of cancers. For instance, while its expression is reduced in breast cancer, prostate cancer, and gastric cancer, it is increased in pancreatic cancer, colorectal cancer, and thyroid cancer. These variations in Serpin B5 expression may be attributed to differences in its cell localization or its modification in different types of cancer cells [33]. The expression of Serpin B5 is also elevated in the epidermis of patients with plaque psoriasis [34] and vitiligo [35]. In this study, the expression of Serpin B5 was significantly lower in the urine of patients with MF patients but not in those with psoriasis or vitiligo than in healthy control subjects.

Studies show that Serpin B5 may be a potential target for cancer therapy [36, 37] as well as a prognostic marker for some cancers [38, 39]. This study found that the expression level of Serpin B5 was negatively correlated with lesion BSA and mSWAT score. As clinical skin lesions improved, the expression level of Serpin B5 gradually increased to that of the healthy control subjects. Therefore, Serpin B5 can also be used as a biomarker for monitoring the efficacy of MF therapy and the prognosis of MF patients.

EGF, as an endocrine growth factor, plays an important role in the regulation of cell DNA synthesis, proliferation, differentiation, migration, and angiogenesis by binding to its receptor (EGFR) on the cell membrane [40, 41]. EGF signaling is closely related to the occurrence and development of tumors, as evidenced by the fact that EGF is overexpressed in various cancers, including lung, breast, colorectal, and prostate cancers [42,43,44,45]. Courville et al. [46] found that the expression of EGF, EGFR, and TGF-α in the skin of patients with CTCL (including 28 patients with CD30+ large T cell lymphoma and 148 patients with the MF) was significantly higher than that in the normal skin of control subjects. Although T lymphocytes express EGF and TGF-α, these cytokines are not detected in patients with cutaneous B cell lymphoma or normal lymph nodes. In addition, the expression of EGFR in the skin of patients with CTCL who had pseudoepitheliomatous hyperplasia was stronger than that in CTCL patients without pseudoepitheliomatous hyperplasia. These findings suggest that EGF is likely involved in the development of epidermal hyperplasia in patients with CTCL. In this study, the urine EGF level of MF patients was lower than that of healthy control subjects. However, as the patients’ skin lesions healed as a result of treatment, EGF gradually returned to normal levels, indicating that EGF has the potential to act as a biomarker for the early diagnosis and efficacy monitoring of MF.

RhoA, as an intracellular signal transduction factor, is involved in the regulation of multiple signal transduction pathways, including cytoskeletal structure remodeling, gene expression, vesicle transport, cell proliferation, cell morphology, cell migration, and cell polarization [47]. Mutant forms of RhoA have been implicated in the occurrence and development of colorectal cancer, hepatocellular carcinoma, breast cancer, lung cancer, and other solid tumors [48,49,50,51]. Moreover, RhoA affects the biological behavior of T lymphocytes, including the regulation of T lymphocyte polarization and migration, the acceleration of T lymphocyte proliferation following TCR engagement, and the enhancement of AP-1 transcriptional activity after T lymphocyte activation [52,53,54]. RhoA mutation is involved in the pathogenesis of various types of lymphoma, including peripheral T cell lymphoma, vascular immunoblastic T cell lymphoma, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular T cell lymphoma, and adult T cell leukemia/lymphoma [55,56,57,58,59]. A study showed that a lower expression of the RhoA gene was found in the psoriasis patients compared with the controls to the 15th month of adalimumab treatment, and next a decrease to levels close to those in the healthy group [60]. The activation of RhoA was upregulated in the infiltrated inflammatory cells, fibroblasts and vasculatures in the dermis and epidermis of atopic dermatitis [61]. Insufficient RhoA activation contributes to defective tight junction formation in PLCδ1-knocked down normal human epidermal keratinocytes [62]. In the present study, we found that urine RhoA levels were lower in untreated MF patients than in healthy control subjects. After treatment, however, and as the patients’ skin lesions improved, the urine RhoA levels also gradually returned to normal. These findings indicate that urine RhoA has the potential to be used as a biomarker for MF. However, the role of RhoA in MF pathogenesis remains to be further explored.

This study also showed that the differentially expressed proteins identified via the proteomic screening of MF patient urine samples included PDCD1LG2, tenascin-C, GAS1, and CTSA. A protumorigenic microenvironment is established through the action of immune regulatory proteins in the CD28 superfamily, such as PD1 and its ligands, PD-L1/2 [63]. PD‐L1 was predominantly expressed on histiocytes/macrophages in CTCL, but focal expression on CTCL cells was seen. High expression of PD‐L1 was associated with advanced‐stage disease and with the appearance of large‐cell transformation, a histopathological feature associated with a poor prognosis [63]. Tenascin-C is preferentially expressed in malignant tissues, is spatially and temporally related to tumor neovascularization and may exert anti-adhesive and immunosuppressive activities [64, 65]. Gritti G et al. [66] used an immunohistochemistry approach using the anti-tenascin-C monoclonal antibody Tenatumomab in 75 systemic T-cell non-Hodgkin lymphoma, and 25 primary cutaneous T-cell non-Hodgkin lymphoma, and found that 93% of the cases were tenascin-C positive and 59% of systemic diseases were characterized by a predominant involvement (> 50%). However, the ELISA results confirmed that there was no significant difference in the urine PDCD1LG2, tenascin-C, GAS1, and CTSA levels of MF patients before treatment and those of healthy control subjects. The discrepancy between the ELISA and MS results may be related to the small sample size used in this study. Future multi-center, large-scale trials will be conducted to confirm our preliminary findings.

Conclusions

Urine Serpin B5, EGF, and RhoA levels have the potential to become biomarkers for diagnosing early-stage MF, as well as predicting and monitoring the efficacy of early-stage MF therapies.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

MF:

Mycosis fungoides

ELISA:

Enzyme-linked immunosorbent assay

Serpin:

Serine protease inhibitors

EGF:

Epidermal growth factor

RhoA:

Ras homologous gene family member A

CTCL:

Cutaneous T cell lymphoma

MS:

Mass spectrometry

LC-MS/MS:

Liquid chromatography tandem/Mass spectrometry

ISCL-EORTC:

International Society for Cutaneous Lymphomas and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer

IFN:

Interferon

TBSA:

Total body surface area

mSWAT:

Modified severity weighted assessment tool

CR:

Complete remission

PR:

Partial response

SD:

Stable disease

PD:

Progressive disease

ORR:

Overall response rate

PCA:

Principal component analysis

GO:

Gene ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

COG:

Cluster of Orthologous Groups

ROC:

Receiver operating characteristic

MAPK:

Mitogen active protein kinase

PI3K-Akt:

Phosphatidylinositide 3-kinase-protein kinase

PDCD1LG2:

Programmed cell death 1 ligand 2

CTSA:

Cathepsin A

GAS1:

Growth arrest-specific protein 1

Maspin:

Mammary serine protease inhibitor

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Acknowledgements

We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript, Clinical Biobank (ISO 20387), Peking Union Medical College Hospital, Chinese Academy of Medical Sciences for storage samples of our study.

Funding

This research was funded by National High Level Hospital Clinical Research Funding (2022-PUMCH-B-092) and Beijing Municipal Natural Science Foundation (Z210017) to Tao Wang, National High Level Hospital Clinical Research Funding, 2022-PUMCH-A-162 to Lu Yang.

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Authors and Affiliations

  1. Department of Dermatology, Peking Union Medical College Hospital, State Key Laboratory of Complex Severe and Rare Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, National Clinical Research Center for Dermatologic and Immunologic Diseases, Dongcheng District, Beijing, 100730, China

    Hongbin Song, Zhonghui Hu, Shiyu Zhang, Lu Yang, Jindi Feng, Lu Lu, Yuehua Liu & Tao Wang

  2. Department of Dermatology, People’s Hospital of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan, China

    Hongbin Song

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  1. Hongbin Song
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  2. Zhonghui Hu
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  4. Lu Yang
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  6. Lu Lu
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  8. Tao Wang
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Contributions

Conceptualization, Tao Wang, Yuehua Liu and Hongbin Song; investigation, Yuehua Liu and Hongbin Song; data curation, Hongbin Song, Zhonghui Hu, Shiyu Zhang, Lu Yang, Jindi Feng, and Lu Lu; writing—original draft preparation, Hongbin Song; writing—review and editing, Tao Wang and Yuehua Liu; supervision, Tao Wang and Yuehua Liu. All authors have read and agreed to the published version of the manuscript. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yuehua Liu or Tao Wang.

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Ethics approval and consent to participate

The study was approved by the Ethics Committee of Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences (approval nos. I-22PJ131). All methods were performed in accordance with the guidelines of the Institutional Review Board of Peking Union Medical College Hospital and the Declaration of Helsinki.

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All patients involved in this study have given their consent for publication.

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The authors declare no competing interests.

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Song, H., Hu, Z., Zhang, S. et al. Application of urine proteomics in the diagnosis and treatment effectiveness monitoring of early-stage Mycosis Fungoides. Clin Proteom 21, 53 (2024). https://doi.org/10.1186/s12014-024-09503-7

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Keywords

Clinical Proteomics

ISSN: 1559-0275