Application of CCR6 as evaluation marker for Langerhans cell tissue cell hyperplasia of children

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of CCR6 as a marker for evaluating children Langerhans cell tissue cell hyperplasia. According to the invention, RNA-scope, immunohistochemical and sequencing technologies are used for examining pathological tissue specimens of LCH child patients, and it is observed that significant correlation exists between increase of CCR6 expression in pathological tissues and LCH risk grading, that is to say, the risk degree of child patients with high CCR6 expression quantity is relatively high, and the relapse possibility of diseases is greater. Therefore, CCR6 expression can be used as an independent prediction index of LCH clinical case risks. Meanwhile, it is proved that CCR6 expression and CCR7 expression have positive correlation, that is, relatively high expression of CCR7 exists in a sample with high CCR6 expression, and low expression of CCR7 also exists in a sample with low CCR6 expression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of CCR6 as a marker for assessing Langerhans cell histiocytosis in children. Background Technology

[0002] Langerhans cell histiocytosis (LCH) is a rare histiocytic disease whose classification as either an inflammatory or neoplastic disease has been controversial. It wasn't until the discovery of the BRAF V600E mutation in 2010 that LCH was defined as a myeloid neoplastic lesion. In China, the annual incidence of LCH is 0.5–5.4 cases per 100,000 people, with a higher prevalence in males. Although LCH can occur in all age groups, it is more commonly diagnosed in children, and the incidence is relatively low. LCH is further classified into single-system (SS-LCH) and multi-system (MS-LCH). Furthermore, based on the extent of organ involvement, LCH can be classified as single-organ or multi-organ. MS-LCH commonly occurs in the skin, bone, lungs, and pituitary gland. MS-LCH carries a higher treatment risk, while SS-LCH (such as bone or skin lesions in children) often resolves spontaneously. Severe cases of LCH can lead to life-threatening systemic multi-organ or multi-system involvement. A staging system for LCH is still lacking. LCH patients are divided into low-risk and high-risk groups based on the presence or absence of high-risk organ involvement. The low-risk group includes patients with partial organ involvement, typically involving the skin, bones, lymph nodes, and thymus; the high-risk group includes patients with high-risk organ involvement, such as the liver, spleen, blood, and central nervous system (CNS).

[0003] LCH presents with a wide range of clinical manifestations due to its overlap with common histological features. It can manifest as a self-limiting rash or bone destruction, or lead to life-threatening multi-organ damage. Therefore, once LCH is suspected, a comprehensive assessment of clinical presentation, histopathology, and other relevant factors is necessary for diagnosis. Approximately 50% of children develop a rash on the head and neck early in the course of the disease—a common symptom of inflammatory neoplastic diseases originating in the medulla. In addition, the liver is typically characterized by sclerosing cholangitis, and spleen involvement is common. Central nervous system lesions associated with LCH can be divided into two main categories: one is caused by Langerhans cell infiltration, usually manifesting as pituitary infiltration; the other is neurodegenerative lesions, including ataxia and dysphagia. Although LCH presents with a range of clinical symptoms and imaging findings, the most reliable method for diagnosis is pathological examination, including microscopic observation using hematoxylin and eosin (HE) staining and immunophenotyping, with positive CD207 (Langerin) or CD1a antigen tests serving as diagnostic criteria. In addition, electron microscopy can be used to observe the presence of Birbeck granules in diseased cells to diagnose the disease.

[0004] The human chemokine receptor 6 (CCR6) gene is located at chromosome 6Q27, consists of four exons, and contains a total of 1235 bases. CCR6 encodes a 42.5 kDa protein composed of 374 amino acids. As the receptor for CCL20, CCR6 is present on the surface of various immune cells, including B lymphocytes, T lymphocytes, plasma cells, natural killer cells, dendritic cells, and neutrophils. CCR6 is highly expressed on pro-inflammatory cells and Treg cells, playing a crucial role in the migration and localization of immune cells.

[0005] In recent years, molecular pathology has made rapid progress, greatly improving the diagnosis and treatment of various diseases. The discovery of the BRAF V600E gene mutation not only clarified the etiology of LCH but also paved the way for exploring the signaling pathways associated with the disease. Previous studies have shown that abnormal activation of the MAPK pathway is closely related to the occurrence of LCH, affecting approximately 85% of pediatric patients. Specifically, the BRAF V600E mutation has been observed in about 60% of pediatric LCH cases. BRAF V600E mutations manifest at different stages of cell development, occurring at different stages of hematopoietic cell development, indirectly affecting clinical presentation and subtyping. If the mutation occurs at the bone marrow stem cell stage, the clinical presentation is usually classified as low-risk across multiple systems. If the mutation occurs only at the Langerhans cell stage, the clinical presentation is considered low-risk across a single system. However, no studies have shown a clear correlation between BRAF V600E mutation and patient risk. Summary of the Invention

[0006] This invention provides the application of CCR6 as a biomarker.

[0007] Applications according to specific embodiments of the present invention include the use of CCR6 as a biomarker in the preparation of a kit for detecting Langerhans cell histiocytosis in children.

[0008] CCR6: Chemokine receptor 6, CCR7: Chemokine receptor 7.

[0009] This invention provides a detection kit for Langerhans cell histiocytosis in children, the kit comprising reagents for detecting CCR6 expression levels.

[0010] The purpose of the detection kit is to detect the expression level of CCR6 in the test cells of the subject; and to compare the detected value with a reference value, where a significantly higher CCR6 level indicates a poor prognosis for the subject. The reference value is the expression level of CCR6 in normal cells, where CCR6 expression is localized in the cytoplasm of LCH cells. The normal cells are of the same strain as the test cells and are not cancerous; the test cells are known or suspected to contain tumor cells. Alternatively, the reference value can be selected based on the expression level of an internal reference gene.

[0011] In this invention, the term "expression level" generally refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., gene coding and / or epigenetics) is transformed into structures present and functioning in the cell. Therefore, as used in this invention, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotide fragments, translated polypeptide fragments, or polynucleotide and / or polypeptide modified fragments (e.g., post-translational modifications of a polypeptide) should also be considered expressed, regardless of whether they originate from transcripts generated through alternative splicing or degraded transcripts, or from post-translational processing of polypeptides (e.g., through proteolysis).

[0012] This invention also provides the use of CCR6 as a target in the development, screening, or preparation of medicaments for the prevention and / or treatment of Langerhans cell histiocytosis in children.

[0013] According to the specific embodiments of the present invention, the drug can reduce the expression of CCR6.

[0014] According to a specific embodiment of the present invention, a medicament for the prevention and / or treatment of Langerhans cell histiocytosis in children, said medicament is capable of reducing the expression of CCR6.

[0015] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0016] The above-mentioned drugs can be prepared into oral preparations or injections; the oral preparations include, but are not limited to, capsules, tablets, granules, and oral liquids; the injections include, but are not limited to, sterile powders for injection, aqueous injection solutions, and intravenous infusions of sodium chloride or glucose.

[0017] In the aforementioned drugs, the oral formulation includes additives, which are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0018] This invention demonstrates that CCR6 expression and CCR7 expression are positively correlated, that is, samples with high CCR6 expression also have relatively high CCR7 expression, and samples with low CCR6 expression also have low CCR7 expression.

[0019] Based on the above findings, this invention provides the application of CCR6 and CCR7 as combined biomarkers in the preparation of a kit for detecting Langerhans cell histiocytosis in children.

[0020] This invention provides a detection kit for Langerhans cell histiocytosis in children, the kit comprising reagents for detecting the expression levels of CCR6 and CCR7.

[0021] The purpose of the detection kit is to detect the expression of CCR6 in the test cells of the subject; and to compare the detected values ​​with reference values. If CCR6 and CCR7 are significantly higher than the reference values, it indicates a poor prognosis for the subject. The reference value level is the expression level of CCR6 and CCR7 in normal cells, where CCR6 expression is located in the cytoplasm of LCH cells and CCR7 expression is located in the cytoplasm of lymphocytes surrounding LCH cells. The normal cells are of the same strain as the test cells and are not cancerous; the test cells are known or suspected to contain tumor cells. Alternatively, the reference value level can be selected based on the expression level of an internal reference gene.

[0022] The beneficial effects of this invention are: This invention used RNA-scope, immunohistochemistry, and sequencing technologies to examine pathological specimens from pediatric patients with LCH. A significant correlation was observed between elevated CCR6 expression in pathological tissues and LCH risk stratification; specifically, children with higher CCR6 expression levels had a relatively higher risk and a greater likelihood of disease recurrence. Therefore, CCR6 expression can serve as an independent predictor of clinical LCH risk. Furthermore, CCR6-positive tumors exhibited enhanced recruitment of lymphocytes expressing high levels of CCR7. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart of the research subject selection and database construction process.

[0025] Figure 2 This image displays typical pathological morphology and electron microscopic structure of pediatric LCH, among which... A. Representative image of HE staining of pathological tissue from LCH patients; B. Representative image of CD1a and Langerin immunohistochemical staining in pathological tissues of LCH patients; C. Typical structural diagram of pathological tissue from LCH patients under electron microscopy.

[0026] Figure 3 A heatmap showing the detection and expression correlation of CCR6 and CCR7 in the skin tissue of children with LCH; A. Representative staining results of RNAscope-positive and immunohistochemical-positive Langerhans cell CCR6 and peripheral lymphocyte CCR7 in skin tissue of LCH patients; B. Statistical heatmap of the correlation between transcriptional and protein levels of CCR6 expression in Langerhans cells and CCR7 expression in peripheral lymphocytes in the pathological tissues of 48 children with LCH; C. Statistical heatmap of the correlation between the expression of CCR6 in Langerhans cells and the expression of CCR7 in peripheral lymphocytes in the pathological tissues of 48 children with LCH. Detailed Implementation

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

[0028] This invention investigated 110 children with lymphocytic leukemia (LCH), including 63 males and 47 females (male-to-female ratio 13:11). The age ranged from 2 months to 12 years. 75 cases involved a single system, and 35 cases involved multiple systems. Analysis of CCR6 expression levels in pathological tissues of LCH patients and clinical information showed a significant correlation between high CCR6 expression in pathological tissues and LCH risk stratification. Analysis of CCR6 and CCR7 staining results revealed high transcriptional and protein expression of CCR7 in lymphocytes in pathological tissues with high CCR6 expression.

[0029] Therefore, this invention proposes that CCR6 can serve as an independent predictor of risk classification for clinical cases of LCH. Based on the correlation between CCR6 and CCR7 expression, CCR6-positive tumors enhance the recruitment of lymphocytes expressing high levels of CCR7.

[0030] Example 1 1.1 Case selection and tissue processing We obtained tissue specimens and clinical information from 110 Chinese pediatric patients diagnosed with LCH (63 males, 47 females; median age 3.7 [range 0.4–14.3] years) who visited Beijing Children's Hospital and the National Center for Children's Health, affiliated with Capital Medical University, between 2020 and 2023. All pathological tissue specimens were collected and used with the prior consent of their guardians.

[0031] Diagnostic criteria and risk stratification for LCH: The diagnostic steps for LCH are as follows: (i) Preliminary diagnosis: Based on clinical presentation and imaging examination, LCH is suspected clinically.

[0032] (ii) Obtaining pathological tissue: Based on the location and size of the lesion, select the surgical method that yields the most readily available and representative tissue, such as skin biopsy or trephine, scraping or puncture of bone lesions, and resection or puncture of organs or systemic lesions.

[0033] (iii) Pathological examination: The tissue is embedded in paraffin and stained with hematoxylin and eosin (HE) and immunohistochemically. (iv) Histological evaluation: A diagnosis of LCH is confirmed if the histological morphology shows Langerhans cell myxoma foci, CD1a and Langerin positive tumor cells; otherwise, LCH is ruled out, and suspected cases are re-examined by biopsy. The risk of missed diagnosis and misdiagnosis is particularly high when LCH involves lymph nodes. A diagnosis can be made if partial destruction of the lymph node structure is observed, especially if the lesion involves the lymph sinuses.

[0034] In cases of atypical lesions, it is necessary to differentiate them from reactive hyperplasia, in which Langerhans cells are distributed in punctate or patchy patterns in LCH, often accompanied by scattered or eosinophilic infiltration. In reactive hyperplasia, lymph node structures are usually in contact with scattered Langerhans cells. This should be considered in conjunction with clinical evidence of multifocal, multisystem, and organ involvement to facilitate the diagnosis of LCH.

[0035] Risk stratification for LCH involves two steps. A positive BRAF V600E gene mutation result is crucial for guiding targeted therapy in children with symptoms or rapidly progressing disease. A comprehensive assessment is then conducted using abdominal ultrasound, liver function tests, lung function tests, central nervous system imaging, lung imaging, bone imaging, bone marrow aspiration, and other relevant examinations.

[0036] Based on the extent of involvement at diagnosis, patients are classified as SS-LCH and MS-LCH. SS-LCH refers to involvement of only one organ or system, while MS-LCH refers to involvement of two or more organs or systems, including the liver, spleen, and hematopoietic system. Central nervous system risk lesions refer to bone lesions in the mastoid process, sphenoid bone, orbit, skull, or temporal bone, as well as involvement of the oral cavity, ear, or eye, which indicate an increased risk of developing neurodegenerative central nervous system-LCH.

[0037] Based on the year of diagnosis and baseline age, 206 children diagnosed with LCH were initially included in the study group. Further screening was conducted based on the completeness of their clinical data and the adequacy of corresponding pathological tissue specimens, resulting in 175 children and their pathological specimens, which were then included in the study population database. In further sample processing and research, 61 cases were excluded due to unacceptable tissue and experimental data, and 4 cases were lost to follow-up, ultimately resulting in 110 children with valid pathological testing data. The corresponding flowchart is shown below. Figure 1 The main baseline characteristics of the enrolled children are shown in Table 1.

[0038] Table 1. Basic information, risk stratification, and organ involvement rate of the study subjects.

[0039] Childhood leukopenic purpura (LCH) is known for its diverse clinical features, unique pathological structures, immunophenotype, and ultrastructural characteristics. In the pathological diagnosis of LCH, initial diagnosis is made by HE staining. Typical LCH skin tissue, after HE staining, reveals abundant, lightly stained cytoplasm under a light microscope. Cell nuclei are kidney-shaped, coffee-bean-shaped, and have nuclear grooves. The background often shows infiltration of inflammatory cells such as lymphocytes, eosinophils, and multinucleated giant cells. Figure 2 A).

[0040] Immunohistochemical staining of pathological tissues from all children included in the study showed that Langerin (CD207) and CD1a were often double-positive. Figure 2 B).

[0041] 1.2 Transmission Electron Microscopy Formalin-fixed tissue samples were collected from all children with LCH and sent to a transmission electron microscopy laboratory (Beijing, China). Samples were fixed with 4% glutaraldehyde fixative, washed twice in PBS, and then fixed a second time with 2% osmium tetroxide fixative and 2% PB buffer. The tissues were then washed with distilled water, dehydrated using a series of ethanol fractions, filtered, and embedded in Eponate 12 epoxy resin formulation (Ted Pella, Reading, California). The samples were then sectioned, mounted on a 150-mesh copper grid, and stained with saturated uranyl acetate and lead citrate solution. Finally, the sections were examined using a Zeiss 10C and a Zeiss 906E transmission electron microscope (Zeiss ElectronMicroscopy, Thornwood, NY, USA).

[0042] Electron microscopy first clarifies the tissue morphology through semi-thin sections, revealing abundant cytoplasm with multiple lobe-like and filamentous projections, and the formation of nuclear grooves and sacs. Further tissue localization is followed by ultrathin sections to locate Birbeck granules within diseased cells for definitive diagnosis. Under electron microscopy, Birbeck granules are typically rod-shaped or tennis racket-shaped. A typical rod-shaped granule is a straight or curved rod-shaped membranous granule approximately 40 nm wide. Its longitudinal section shows two unit membranes on each side, while the center has a five-membrane longitudinal septum, collectively forming a 6-7 nm zipper-like transverse septum. Figure 2 C).

[0043] LCH was finally diagnosed based on histological morphology combined with immunohistochemical detection of CD1a and Langerin (CD207) in pathological tissue or electron microscopy showing the presence of Birbeck granules in the lesion cells.

[0044] 1.3 Immunohistochemical staining 1. Dewaxing and hydration of sections are the same as for HE staining; 2. Repair: After hydration, the sections were equilibrated with ddH2O for 1 min, and then placed in pH 9.0 EDTA repair solution (or pH 6.0 citric acid repair solution) at 100°C for 20 min for high-temperature repair. Afterward, they were allowed to cool naturally to room temperature and washed with ddH2O for 5 min × 3 times. (In this invention, immunohistochemical staining of CCR6 was repaired using pH 9.0 EDTA repair solution, and immunohistochemical staining of CCR7 was repaired using pH 6.0 citric acid repair solution). 3. Peroxidase blocking: Incubate at room temperature for 8 min; 4. Primary antibody incubation: Incubate at room temperature for 30 min; 5. Blocking after primary antibody administration: Incubate at room temperature for 8 min; 6. Blocking of anti-rabbit polymers: Incubate at room temperature for 8 min; 7. DAB color development: Mix DAB Part 1 and DAB Part 2 at a ratio of 1:50 and incubate at room temperature for 4 min; 8. Hematoxylin staining of nuclei: incubate at room temperature for 9 min; 9. After rinsing with Bond wash solution for several minutes, wash with ddH2O; 10. The dehydration and mounting procedure is the same as for HE staining; 11. Slide reading and grading: Two pathologists independently evaluated the immunohistochemically stained slides. CCR6 expression was located in the cytoplasm of LCH cells, and CCR7 expression was located in the cytoplasm of lymphocytes surrounding LCH cells.

[0045] Immunohistochemical staining was scored using the IRS (Immunoreactive Score), which combines staining intensity with the percentage of positive cells. Staining intensity was graded into four levels: 0 points: no positive staining (negative); 1 point: pale yellow (weakly positive); 2 points: brownish-yellow (positive); 3 points: brownish-red (strongly positive). The percentage of positive cells was divided into four ranges: 0 points: 0-5% of cells were positive; 1 point: 6-25% of cells were positive; 2 points: 26-50% of cells were positive; 3 points: 51-75% of cells were positive; 4 points: >75% of cells were positive.

[0046] Scoring formula: IRS = staining intensity × percentage of positive cells, with a score range of 0-12. The staining intensity of CCR6 and CCR7 is divided into three levels (low, med, and high) according to the pathology slide reading rules and the IRS immunohistochemical staining scoring standard: low (0-4 points), med (5-8 points), and high (9-12 points). Two pathologists independently and blindly evaluate the target protein expression. Negative and positive controls are included in each batch.

[0047] 1.4 RNAscope 1. Tissue pretreatment: After routine dewaxing, the fixed pathological tissue sections on the slides were treated with the RNAscope pretreatment kit, followed by heating and protease digestion to expose the target RNA; 2. Hybridization: Twenty pairs of Z-shaped target probes designed for CCR6 and CCR7 were hybridized with the target RNA in an in-furnace hybridization chamber; 3. Signal amplification: After hybridization, elution and signal amplification were performed sequentially, followed by DAB staining and hematoxylin contrast staining; 4. Visual signal formation: Under the observation of a transparent optical microscope or a multispectral imaging system, each target RNA molecule is presented as a point signal; 5. Slide reading and grading: The results were interpreted according to the mRNA expression level from low to high in 5 grades (0-4 points): 0 points: negative or <1 point / 10 cells; 1 point: 1-3 points / cell; 2 points: 4-9 points / cell, with no or very few clustered signals; 3 points: 10-15 points / cell and clustered signals <10%; 4 points: >15 points / cell and clustered signals >10%.

[0048] The staining intensity of CCR6 and CCR7 was graded into three levels—low, med, and hi—according to pathological slide reading rules: low (0-1 points), med (2 points), and hi (3-4 points). Two pathologists independently and blindly evaluated the expression of the target mRNA. Negative and positive controls were included in each batch.

[0049] RNAscope detection and immunohistochemical staining of CCR6 and CCR7 in pathological tissue sections from some LCH patients revealed that the transcriptional levels of CCR6 and CCR7 in the tissues were positively correlated with their protein expression levels (P < 0.001). Figure 3 A, B).

[0050] A comparative analysis of the CCR6 and CCR7 protein levels in the same sample was conducted, and the results are shown in the table below. Figure 3 C, Table 2.

[0051] Table 2. Statistical analysis of the correlation between clinicopathological information and CCR6 and CCR7 proteins in children with LCH.

[0052] ** P <0.01.

[0053] The results showed that CCR6 expression and CCR7 expression were positively correlated, meaning that samples with high CCR6 expression also had relatively high CCR7 expression, while samples with low CCR6 expression also had low CCR7 expression.

[0054] Table 3. Statistical analysis of the correlation between clinicopathological information and CCR6 protein levels in children with LCH.

[0055] Statistical analysis combining clinicopathological indicators yielded the following results: Table 4. Statistical analysis of the correlation between CCR6 protein and disease progression in pathological tissues of children with LCH.

[0056] As shown in Table 4, the risk level of children gradually increases with the increase of CCR6 protein expression. That is, CCR6 protein expression level is positively correlated with the risk group of children and is positively correlated with disease progression or recurrence. In other words, children with higher CCR6 expression levels have a relatively higher risk and a greater likelihood of disease recurrence.

[0057] This invention used RNAscope to detect CCR6 RNA levels in tissues. Furthermore, DNA extracted from these tissues and MAPK signaling analysis revealed that 80% of patients had BRAF mutations. BRAF V600E mutations were associated with an increased risk. Importantly, the proportion of children with BRAF V600E mutations gradually increased with increasing CCR6 expression, indicating a positive correlation between high CCR6 expression and BRAF V600E mutation.

[0058] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of CCR6 as a biomarker in the preparation of a kit for detecting Langerhans cell histiocytosis in children.

2. A kit for detecting Langerhans cell histiocytosis in children, characterized in that, The kit includes reagents for detecting CCR6 expression levels.

3. Application of CCR6 as a target in the preparation of drugs for the treatment of Langerhans cell histiocytosis in children.

4. The application according to claim 3, characterized in that, The drug can reduce CCR6 expression.

5. A medicine for the prevention and / or treatment of Langerhans cell histiocytosis in children, characterized in that, The drug can reduce the expression of CCR6.

6. The drug according to claim 5, characterized in that, The drug contains a pharmaceutically acceptable carrier.

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