Markers related to curative effect of mucous membrane type melanoma and application

Abstract

The invention relates to the technical field of diagnostic markers, in particular to a marker related to the curative effect of mucous membrane type melanoma and application. The invention provides a therapeutic effect related marker CD4 < + > / CD8 < + > of mucous membrane type melanoma, and provides application of the therapeutic effect related marker CD4 < + > / CD8 < + > in non-disease diagnosis and treatment. Researches show that the score of CD4 + / CD8 + T cells activated by patients in an effective group of mucosal melanoma treated by the PD-1 / PD-L1 monoclonal antibody combined with the VEGFR inhibitor is obviously higher than that in an ineffective group, and the higher score is related to a longer survival outcome. The activated CD4 < + > / CD8 < + > T cells are scored as the curative effect marker of the mucosal melanoma PD-1 / PD-L1 monoclonal antibody combined with the VEGFR inhibitor, and the application prospect is good.

Description

Technical Field

[0001] This invention relates to the field of diagnostic biomarker technology, and in particular to a biomarker related to the treatment efficacy of mucosal melanoma and its application. Background Technology

[0002] Mucosal melanoma (MM) is a rare and clinically challenging subtype of melanoma that originates from melanocytes in mucosal tissues. Due to its insidious onset and aggressive nature, many patients are diagnosed at a locally advanced stage or with distant metastases. Compared to cutaneous melanoma (CM), mucosal melanoma is not a typical UV-driven tumor; its genomic alteration lineage, tumor antigenicity, and spatial organization of the tumor immune microenvironment differ significantly. These biological differences collectively result in limited overall benefit from existing systemic therapies for mucosal melanoma, greater heterogeneity in treatment outcomes, and a still unsatisfactory long-term prognosis.

[0003] Immune checkpoint inhibitors (ICIs) have significantly reshaped the treatment landscape for cutaneous melanoma, but objective response rates and duration of response remain generally low in mucosal melanoma. More importantly, reliable and precise treatment biomarkers for clinical stratification and decision-making are still lacking. Commonly used indicators such as programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB) have inconsistent predictive efficacy in mucosal melanoma, and precise biomarkers that can reliably play a role in treatment decisions, perioperative stratification, and recurrence risk assessment are still lacking. Therefore, establishing mechanistic biomarkers that reflect treatment-related immune status and have cross-cohort validated characteristics is a key requirement for improving the treatment efficacy of mucosal melanoma.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a biomarker related to the treatment efficacy of mucosal melanoma and its application.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] First, this invention provides CD4 + / CD8 + Applications of melanoma efficacy markers for non-disease diagnostic and treatment purposes.

[0008] Preferably, the melanoma is a mucosal melanoma.

[0009] Preferably, the application for non-disease diagnosis and treatment purposes includes: detecting activated CD4 + / CD8 + T-cell scoring reagents and / or methods are used to prepare products for identifying or predicting the efficacy of melanoma treatment.

[0010] Preferably, activated CD4 + / CD8 + T-cell scores are associated with treatment response in patients with mucosal melanoma receiving a combination of PD-1 / PD-L1 monoclonal antibody and VEGFR inhibitor therapy.

[0011] Preferably, activated CD4 + / CD8 + A high T-cell score corresponds to a high response rate in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

[0012] Preferably, activated CD4 + / CD8 + Low T-cell scores correspond to low response rates in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

[0013] Preferably, activated CD4 + / CD8 + T-cell characteristics are associated with survival in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

[0014] Preferably, activated CD4 + / CD8 + Patients with higher T-cell scores, corresponding to mucosal melanoma, have longer survival rates when treated with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors.

[0015] Preferably, activated CD4 + / CD8 + Low T-cell scores are associated with shorter survival rates in patients with mucosal melanoma treated with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors.

[0016] Preferably, mucosal melanoma patients are divided into groups with activated CD4+ according to an optimal cut-off of 0.45. + / CD8 + High and low T cell score groups; cut-off ≥0.45 corresponds to activated CD4+. + / CD8 +The high T-cell score group, with a cut-off <0.45 corresponding to activated CD4+ + / CD8 + Low T cell score group; activated CD4 + / CD8 + The high T-cell score group corresponds to a higher treatment response and survival in patients with mucosal melanoma who received PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

[0017] Beneficial effects: This invention provides CD4, a biomarker related to the treatment efficacy of mucosal melanoma. + / CD8 + This invention provides its applications for non-disease diagnosis and treatment purposes. The study demonstrates that activated CD4+ is present in patients with mucosal melanoma who responded to a treatment regimen combining PD-1 / PD-L1 monoclonal antibodies and VEGFR inhibitors. + / CD8 + The T-cell score was significantly higher in the control group than in the nullipopulation group, and the higher score was associated with longer survival. This will activate CD4+. + / CD8 + T-cell scores have promising applications as a biomarker for the efficacy of PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors in mucosal melanoma. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0019] Figure 1 This is the experimental result of transcriptomics analysis in Example 1, revealing the dynamic changes in immunity in mucosal melanoma before and after combined treatment. Specifically, a: a heatmap shows the immune cell infiltration patterns in different ROIs, assessed by ssGSEA, before and after treatment in the effective and ineffective groups. b: Box plots show the CD4 activation between the effective / ineffective groups and before / after treatment. + T cells, activated CD8 + The infiltration score of immune cells such as T cells is used. Among them, Pre-NR = Pre-Non-responder, before treatment in the ineffective group; Post-NR = Post-Non-responder, after treatment in the ineffective group; Pre-R = Pre-Responder, before treatment in the effective group; Post-R = Post-Responder, after treatment in the effective group.

[0020] Figure 2 CD4 activated in Example 1 + / CD8 +Experimental results showing the correlation between T-cell scores and cohort survival (n = 18). Among them, a: Kaplan-Meier survival curves for relapse-free survival (RFS). b: Kaplan-Meier survival curves for distant metastasis-free survival (DMFS). c: Multivariate Cox model for relapse-free survival. d: Multivariate Cox model for distant metastasis-free survival.

[0021] Figure 3 CD4 activated in Example 1 + / CD8 + Experimental results on the correlation between T cell scores and survival in the validation cohort (n = 18). Where: a: Progression-free survival (PFS) Kaplan-Meier survival curve; b: Overall survival (OS) Kaplan-Meier survival curve.

[0022] Figure 4 The CD4 activated as confirmed by multiple immunohistochemistry in Example 1 + / CD8 + Experimental results relating T-cell activity to the treatment response of mucosal melanoma to PD-1 / PD-L1 monoclonal antibody combined with a VEGFR inhibitor. In this figure, a: representative images of paired pre- and post-treatment samples, showing activated CD4. + T cells (ICOS) + CD4 + ), activated CD8 + T cells (GZMB) + CD8 + ) and proliferating melanoma cells (Ki-67) + SOX10 + (b: H-score, c: positive cell frequency, and d: positive cell density) Quantification of marker expression and cell phenotype was performed. e: Comparison of nearest neighbor cell distances between the indicated cell types within the tumor core region across groups. f: Comparison of nearest neighbor cell distances between the effective and ineffective groups within the tumor core region after treatment. Detailed Implementation

[0023] Given the limitations of single-agent immunotherapy in mucosal melanoma, combination therapy strategies have a clear biological rationale. Angiogenesis signaling pathways, particularly the vascular endothelial growth factor / VEGFR pathway, not only drive tumor growth and metastasis but also restrict immune cell entry into the tumor through abnormal vascular structure and an immunosuppressive microenvironment, thereby weakening the effect of immunotherapy. Inhibiting VEGF / VEGFR-mediated vascular abnormalities and promoting improved vascular function can enhance immune cell infiltration, alleviate local immunosuppression, and improve the effectiveness of immunotherapy. Therefore, the combination of immunotherapy and anti-angiogenic therapy has shown some anti-tumor activity in mucosal melanoma, but further investigation is needed to identify the potential beneficiary population.

[0024] To advance the establishment of translatable biomarkers in real-world clinical settings, this study evaluated a clinical cohort of patients with resectable mucosal melanoma undergoing perioperative immunotherapy combined with anti-angiogenic therapy. The tumor immune microenvironment was systematically analyzed using paired tumor samples before and after treatment. It is important to emphasize that this therapeutic window provides unique conditions for biomarker screening: direct comparison of tissues before and after treatment allows for clearer identification of key immune status changes associated with treatment response and the correlation of candidate biomarkers with clinical outcomes. Furthermore, external validation of candidate biomarkers in an independent cohort of advanced mucosal melanoma enhances their robustness and generalizability.

[0025] At the mechanistic level, T cells are central to the anti-tumor effects mediated by immunotherapy, but their role depends not only on the level of infiltration but also on their functional state, spatial organization, and interaction with tumor cells. Cytotoxic CD8 + T cells play a direct effector role in killing tumors, while effective and durable anti-tumor immunity usually requires activated CD4+. + T cells provide support, including promoting the establishment and maintenance of effector responses and the formation of immune memory; conversely, if T cells are predominantly in a state of functional exhaustion or immunosuppression, the true benefit of combination therapy may be limited. Therefore, this study is the first to discover CD4 + T cells and CD8 + The relationship between T cell activation characteristics and their spatial interactions and the clinical efficacy, recurrence risk, and survival outcomes of PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors in mucosal melanoma provides interpretable immune biomarkers for individualized stratification of mucosal melanoma.

[0026] Mucosal melanoma is a rare and highly aggressive subtype of melanoma, with a significantly lower response rate to PD-1 / PD-L1 monoclonal antibodies and targeted therapy compared to cutaneous and acral melanomas. Treatment options are limited, and molecular markers that effectively predict efficacy and prognosis are lacking. This invention is based on a phase II clinical trial of neoadjuvant PD-1 / PD-L1 monoclonal antibody combined with a VEGFR inhibitor (pembrolizumab + lenvatinib) for resectable mucosal melanoma. Transcriptome sequencing was performed on tumor tissues before and after treatment with the PD-1 / PD-L1 monoclonal antibody and VEGFR inhibitor combination. ssGSEA analysis and multiple immunohistochemical staining revealed activated CD4+. + / CD8 + T-cell scores were correlated with treatment response, and patients with higher scores had significantly longer recurrence-free survival and distant metastasis-free survival than those with lower scores. This invention was further validated in another cohort of patients with advanced mucosal melanoma treated with a combination of PD-1 / PD-L1 monoclonal antibody and a VEGFR inhibitor (atezolizumab + bevacizumab). Similarly, transcriptome sequencing combined with ssGSEA analysis confirmed that activated CD4+... + / CD8 + T-cell characteristics were associated with better treatment outcomes, and patients with higher scores had significantly longer progression-free survival and overall survival than those with lower scores. Overall, activated CD4+... + / CD8 + T-cell scores can serve as a predictive indicator of the efficacy of PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy for mucosal melanoma, helping to screen patients who respond to treatment and achieve precision diagnosis and treatment of mucosal melanoma.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0030] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0031] In the following embodiments: The cohort received lenvatinib orally 20 mg daily and pembrolizumab intravenously 200 mg every three weeks for two cycles during a 6-week neoadjuvant therapy period. Patients with resectable lesions underwent radical surgery within 1–4 weeks after completion of neoadjuvant therapy. Postoperatively, patients meeting the inclusion criteria began adjuvant pembrolizumab monotherapy (200 mg intravenously every three weeks) within 6 weeks, for a maximum extension of 15 cycles. Pre-treatment biopsy specimens and post-treatment surgical specimens were collected and formalin-embedded. Surgical specimens were centrally reviewed to assess pathological response, including: pCR (pathological complete response), defined as no surviving tumor cells in the resected specimen; near-pCR, defined as ≤10% surviving tumor cells; pPR (pathological partial response), defined as 11%–50% surviving tumor cells; and pNR (pathological non-response), defined as >50% surviving tumor cells in the tumor bed. Patients were divided into two groups based on their response level: the effective group (pCR, near-pCR, or pPR) and the ineffective group (pNR).

[0032] This validation cohort used data from patients with unresectable or metastatic mucosal melanoma treated with atezolizumab and bevacizumab. Atezolizumab was administered intravenously every 3 weeks at a fixed dose of 1200 mg. Bevacizumab was administered intravenously at a dose of 7.5 mg / kg every 3 weeks. Pretreatment biopsy or surgical samples were collected from patients and fixed in formalin. Imaging assessments were performed according to the Solid Tumor Response Assessment Criteria (v1.1), and the assessment results included: complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Patients were divided into two groups based on the degree of response: an effective group (CR or PR) and an ineffective group (SD or PD).

[0033] Example 1 The experimental procedure for this embodiment is as follows: (1) Discovery cohort digital transcriptome detection: 4 μm thick sections from formalin-fixed paraffin-embedded (FFPE) samples were dewaxed and subjected to antigen retrieval in Tris-EDTA buffer. RNA-binding proteins were then removed using the GeoMx DSP RNA Slide Prep Kit (NanoString), and the sections were fixed in 16% formaldehyde for 5 minutes at room temperature. Probes targeting 18,000 RNA molecules (GeoMxPanel and Seq Code kit, NanoString) were hybridized overnight. These probes are linked to UV-cleavable DNA oligonucleotides and contain unique molecular identifiers (UMIs), enabling precise spatial expression profiling. Sections were further stained with morphological markers: S100 (NBP2-54426, Novus, 1:400), Pmel17 (NBP2-34638, Novus, 1:400), CD45 (13917, Cell Signaling, 1:100), and Syto 13 (S7575, Thermo, 1:1000) for nuclear recognition, followed by incubation with fluorescent secondary antibody. After scanning the sections, regions of interest (ROIs) were selected on GeoMx Digital SpatialProfiler (NanoString). ROI selection was based on histological and immunofluorescence signals, strictly limited to on-tissue regions within the tumor and stromal compartments as labeled by the pathologist, with preference given to tumor-internal regions. DNA oligonucleotides from the ROIs were collected and sequenced using an Illumina NovaSeq 6000 platform. The raw digital count conversion (DCC) files were normalized using ERCC RNA exogenous spike-in controls to reduce systematic bias and correct for ROI area differences. ROI inclusion criteria were a surface area of ​​at least 1.6 × 10⁻⁶. 4 μm² and cell nuclei of at least 200. ROIs with normalization positive factors exceeding 0.3–3.0 were excluded. Normalized data were further normalized to Q3 (third quartile), logarithmically transformed, and optionally centered at the median. Data processing and analysis were performed using DSP analysis software and R (version 4.5.1). Immune cell infiltration levels, at CD45 + Within each ROI (immune cell) region, the ssGSEA method in the GSVA package was used to derive the characteristic scores of 24 immune cells in each ROI.

[0034] (2) Validation cohort digital transcriptome analysis: Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue. FFPE sections were dewaxed and rehydrated, followed by protease digestion and cross-linking reversal, and RNA purification was performed according to the kit instructions. The extracted RNA was quantified and quality-assessed before being used for library construction. Tumor RNA was reverse transcribed into cDNA using Ribo-SPIA (NuGEN) technology, and mRNA libraries were constructed, sequenced, and subjected to FASTQ data quality control. Raw sequencing data was filtered to remove low-quality reads and reads containing adapter sequences. The cleaned reads were aligned to the hg19 reference genome using HISAT. Transcript assembly was then performed using StringTie (v1.2.3), and gene expression profiles were generated from the RNA-seq data. Immune cell infiltration levels were assessed using the ssGSEA method in the GSVA package, and 24 immune cell types associated with immune responses were scored.

[0035] (3) Multiple immunohistochemical detection of activated CD4 + / CD8 + T cells: FFPE sections (4 μm) underwent routine dewaxing / rehydration and heat-induced antigen retrieval (EDTA buffer, pH 9.0; Panovue). Subsequently, iterative multiple staining based on TSA was used to detect GZMB (CST46890, Cell Signaling, 1:200), CD4 (ZA-0418, Zsbio, 1:200), Ki-67 (CST9027, Cell Signaling, 1:300), ICOS (CST89601S, Cell Signaling, 1:150), SOX10 (ZA-0624, Zsbio, ready-to-use), and CD8A (CST70306, Cell Signaling, 1:200). Primary antibodies were incubated sequentially in a pre-optimized order, with HRP-conjugated secondary antibodies and TSA signal amplification. Heat-mediated stripping was used between each round of staining to remove antibodies, and cell nuclei were counterstained with DAPI. Whole-section images were acquired using an Olympus VS200 scanner. Tumor and stromal areas were reviewed and annotated by a pathologist, excluding areas unsuitable for analysis. Single-cell segmentation and positive determination were performed in QuPath (v0.5.1), using channel-specific intensity thresholds for marker-positive identification. Cell frequency and density were quantified based on the number of phenotypic positive cells, the total number of cells, and the corresponding analysis area; the nearest neighbor distance between cells was calculated using Euclidean distance based on two-dimensional cell coordinates derived from QuPath.

[0036] The experimental results of this embodiment are as follows: (1) Discovery cohort: patient distribution and baseline characteristics: Between September 2021 and April 2023, a total of 26 patients were enrolled in the study, including 11 from the vulvovaginal region, 8 from the anorectal region, 5 from the head, neck, nasal, and oral regions, and 2 from the esophagus (Table 1). Of the 26 enrolled patients, 21 underwent surgery, and 5 did not undergo resection as planned. Among these, 4 patients refused surgery after tumor shrinkage and symptom relief. Of the 21 patients who underwent surgery, 2 achieved pathological complete response (pCR), 2 achieved near-complete response (near-pCR), and 4 achieved pathological partial response (pPR). The median recurrence-free survival (RFS) was 14.8 months (95% CI, 11.4–18.2 months), and the median distant metastasis-free survival (DMFS) was 26.8 months (95% CI, 19.2–34.4 months). There was no significant difference in median RFS and DMFS between the response group (pCR + near-pCR + pPR) and the ineffective group.

[0037] Table 1. Patient Baseline Characteristics

[0038] (2) Discovery cohort: Transcriptome sequencing identifies activated CD4 + / CD8 + T-cell score is associated with response to PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor in mucosal melanoma: This embodiment uses digital transcriptomics analysis to study the dynamic changes in the immune microenvironment before and after treatment with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors (pembrolizumab + lenvatinib). Before treatment, 11 regions of interest (ROIs) in the effective group and 30 ROIs in the ineffective group were analyzed. After treatment, 31 ROIs in the effective group and 44 ROIs in the ineffective group were sequenced. Immune cell phenotypes were quantified using single-sample gene set enrichment analysis (ssGSEA). Figure 1 Figure a) shows the comparisons between different efficacy groups and time points. Before treatment, the ineffective group exhibited a more "cold" immune phenotype, characterized by significantly lower immune scores and reduced infiltration of key anti-tumor immune cell populations, including activated CD4+. + T cells, activated CD8 + Lower levels of T cell, activated dendritic cell, and plasmacytic dendritic cell markers were also observed, consistent with the unfavorable antigen presentation and T cell activation environment prior to treatment. Post-treatment, T cell infiltration increased in the ineffective group compared to pre-treatment levels, while the effective group showed a further modest increase after treatment. Among all assessed immune populations, activated CD4+... + and CD8 +T cells showed the most significant pre-treatment differences and dynamic treatment-related increases. Figure 1 (Figure b in the middle)

[0039] (3) Discovery queue: active CD4 + / CD8 + T-cell score is associated with survival in mucosal melanoma patients receiving PD-1 / PD-L1 monoclonal antibodies in combination with VEGFR inhibitors: Activation CD4 obtained based on ssGSEA algorithm + T cells, activate CD8 + T cells, calculate the average activated CD4. + / CD8 + T-cell score. Patients were classified as having activated CD4 cells based on an optimal cutoff of 0.45. + / CD8 + T-cell high score group (≥0.45) and low score group (<0.45). Univariate log-rank analysis showed that the high score group had significantly higher recurrence-free survival (RFS, p = 0.042). Figure 2 Figure a) and survival without distant migration (DMFS, p = 0.041, Figure 2 The scores in the middle (Figure b) were significantly higher than those in the low-score group. Multivariate Cox regression analysis, incorporating factors such as gender, age, and metastasis, showed that under multivariate conditions, activated CD4... + / CD8 + The T-cell score remained at relapse-free survival (HR = 0.25, 95% CI 0.068–0.89, p = 0.033). Figure 2 (Figure c) and survival without distant metastasis (HR = 0.19, 95% CI 0.041–0.86, p = 0.031, Figure 2 Independent prognostic factors (Figure d).

[0040] (4) Validation cohort: patient distribution and baseline characteristics: Twenty-seven patients were enrolled in a clinical study between 2019 and 2020 for the treatment of advanced mucosal melanoma with a combination of PD-1 / PD-L1 monoclonal antibody and a VEGFR inhibitor (atezolizumab + bevacizumab). These patients included: 15 from the head and neck, nasal and oral regions; 6 from the vulva and vaginal regions; and 6 from the anorectal region (Table 2). Of the 27 patients, 5 achieved a response (CR+PR) and 12 did not respond to treatment.

[0041] Table 2. Patient Baseline Characteristics

[0042] (5) Validation cohort: Transcriptome sequencing to validate CD4 + / CD8 + T-cell scores are associated with treatment response and survival in mucosal melanoma treated with PD-1 / PD-L1 monoclonal antibodies in combination with VEGFR inhibitors: In this embodiment, transcriptome sequencing was performed on samples of advanced mucosal melanoma treated with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors (atezolizumab + bevacizumab). The ssGSEA algorithm was also used to calculate activated CD4+. + T cells, activate CD8 + T cell score, averaged to obtain activated CD4 + / CD8 + T cell score. Results showed that the effective group activated CD4. + / CD8 + The T-cell score was significantly higher in the control group than in the ineffective group (p = 0.032). Patients were also categorized into activated CD4+ groups based on a cut-off of 0.45. + / CD8 + T cell high score group and low score group. Univariate log-rank analysis showed that progression-free survival (PFS) in the high score group was significantly lower (p = 0.027). Figure 3 Figure a) and total survival (OS, p = 0.035, Figure 3 The scores in the middle (Figure b) were significantly higher than those in the low-scoring group.

[0043] (6) Multiple immunohistochemical studies confirmed activated CD4 + / CD8 + T cells are associated with the treatment response of mucosal melanoma to PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor: To further provide histological validation of the immunological features identified by transcriptome analysis, this embodiment performed multiplex immunohistochemical staining using a combination of ICOS / CD4 / GZMB / CD8 / Ki-67 / SOX10 antibodies. Activated CD4 + T cells are defined as ICOS + CD4 + Cells, activated CD8 + T cells are defined as GZMB. + CD8 + Cells, proliferating melanoma cells are defined as Ki-67. + SOX10 + cell. Figure 4 Figure a shows the multiplex immunohistochemical staining results before and after treatment in one representative effective patient and one representative ineffective patient. After treatment, the H scores of ICOS, CD4, GZMB, and CD8 in the effective group were significantly higher than those in the ineffective group. Figure 4 (Figure b) ICOS of the effective group+ CD4 + and GZMB + CD8 + Cell frequency ( Figure 4 (middle c diagram) and density ( Figure 4 The values ​​in the middle (d-figure) were significantly higher than those in the invalid group, while Ki-67 + SOX10 + Cell counts were significantly lower in the post-treatment group than in the effective group. Spatial proximity analysis based on nearest neighbor distance (NNC) showed that, within the tumor region, activated CD4+ cells were significantly lower in the post-treatment samples. + and CD8 + The NNCs between T cells were shorter, and the proximity between these T cell subsets and proliferating melanoma cells was also shorter; this pattern was more pronounced in the effective group. Figure 4 (Figures e and f). Overall, these data indicate that PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor treatment caused changes in the abundance and spatial organization of activated T cell subsets, and that the effective group had activated CD4+. + / CD8 + T cells were more abundant and the cells were more closely spaced.

[0044] As can be seen from the experimental results provided in this embodiment, activated CD4+ was observed in patients with mucosal melanoma who responded to treatment with PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor. + / CD8 + The T-cell score was significantly higher in the control group than in the nullipopulation group, and the higher score was associated with longer survival. Therefore, activated CD4+ + / CD8 + T-cell scores can serve as a biomarker for the efficacy of PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors in mucosal melanoma.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. CD4 + / CD8 + Application of biomarkers for efficacy in mucosal melanoma for non-disease diagnostic and therapeutic purposes.

2. The application according to claim 1, characterized in that, The applications for non-disease diagnosis and treatment purposes include: detecting CD4. + / CD8 + The reagents and / or methods are used to prepare products for identifying or predicting the efficacy of melanoma treatment.

3. The application according to claim 1, characterized in that, Activated CD4 + / CD8 + T-cell scores are associated with treatment response in patients with mucosal melanoma receiving a combination of PD-1 / PD-L1 monoclonal antibody and VEGFR inhibitor therapy.

4. The application according to claim 3, characterized in that, Activated CD4 + / CD8 + A high T-cell score corresponds to a high response rate in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

5. The application according to claim 3, characterized in that, Activated CD4 + / CD8 + Low T-cell scores correspond to low response rates in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

6. The application according to claim 1, characterized in that, Activated CD4 + / CD8 + T-cell scores are associated with survival in patients with mucosal melanoma receiving PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

7. The application according to claim 6, characterized in that, Activated CD4 + / CD8 + Patients with high T-cell scores, corresponding to mucosal melanoma, have longer survival rates when treated with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors.

8. The application according to claim 6, characterized in that, Activated CD4 + / CD8 + Patients with low T-cell scores, corresponding to mucosal melanoma, have shorter survival rates when treated with PD-1 / PD-L1 monoclonal antibodies combined with VEGFR inhibitors.

9. The application according to claim 3 or 6, characterized in that, Based on the optimal cut-off of 0.45, patients with mucosal melanoma were divided into those with activated CD4+. + / CD8 + High and low T cell score groups; cut-off ≥0.45 corresponds to activated CD4+. + / CD8 + The high T-cell score group, with a cut-off <0.45 corresponding to activated CD4+ + / CD8 + Low T cell score group; activated CD4 + / CD8 + The high T-cell score group corresponds to a higher treatment response and survival in patients with mucosal melanoma who received PD-1 / PD-L1 monoclonal antibody combined with VEGFR inhibitor therapy.

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