KR20220072481A - Biomarker composition comprising CXCL12 in plasma membrane of cancer cells for predicting prognosis of rectal adenocarcinoma - Google Patents

Abstract

It relates to an information providing method, biomarker composition or kit for diagnosing or predicting rectal adenocarcinoma, including CXCL12, and as a result of analyzing CXCL12 protein expression in tumor tissues and normal tissues of rectal adenocarcinoma using immunohistochemical staining, normal CXCL12 protein of the present invention can be used as a biomarker for rectal adenocarcinoma by confirming that the CXCL12 protein is significantly increased in the cancer cell membrane in the tumor tissue compared to the tissue and the CXCL12 protein expression is increased in the cancer cell plasm of the rectal adenocarcinoma, and such cancer cell membrane The analysis of the expression rate of CXCL12 can predict the diagnosis or prognosis of rectal adenocarcinoma, and is expected to have high utility value in the development of prophylactic or therapeutic agents for rectal adenocarcinoma.

Description

Biomarker composition comprising CXCL12 in plasma membrane of cancer cells for predicting prognosis of rectal adenocarcinoma

The present invention relates to a composition for diagnosing or predicting recurrence prognosis of a rectal adenocarcinoma patient, and to an information providing method, composition or kit capable of predicting the prognosis of rectal adenocarcinoma recurrence, including cancer cell membrane CXCL12 (CXC chemokine ligand 12).

Rectal cancer, along with colorectal cancer, is one of the five major causes of cancer incidence and cancer mortality in Korea. The most common type of rectal cancer is adenocarcinoma. Treatments for diagnosed cancer generally include surgery, chemotherapy, and radiation therapy, but each method has many limitations. In addition, cancer has a very high probability of recurrence even after being treated, and the sensitivity to anticancer drugs varies greatly depending on the individual.

The current standard of care for locally advanced rectal cancer (LARC), which is defined as stage II (T3-4, negative peripheral lymph node metastasis) or stage III (positive peripheral lymph node metastasis) disease, is neoadjuvant chemotherapy, nCRT) and total rectal resection. Although nCRT has been shown to be effective in preserving sphincter, reducing local pelvic infiltration, and downgrading postoperative staging, the efficacy of this strategy has been stagnant due to frequent relapses in LARC patients. Early Tumor-Node-Metastasis (TNM) staging is one of the important factors predicting the prognosis of rectal cancer patients. However, in LARC patients receiving nCRT treatment, the importance of early stage before nCRT treatment as a prognostic predictor decreases due to changes in cancer cells after nCRT treatment. To overcome the problem of inaccurate prediction, various biomarkers for their ability to predict response to preoperative treatment are being studied. However, there are limitations to the study of markers for predicting prognosis in preoperative rectal cancer biopsy tissue. The reason is that endoscopic biopsy obtains insufficient tissue to allow immunohistochemical staining, may not contain normal mucosal tissue for comparison, and changes in cancer cells after nCRT treatment are unknown.

CXCL12 (CXC chemokine ligand 12) is an important chemokine in angiogenesis, apoptosis, proliferation, survival, differentiation and metastasis and is induced by hypoxia and growth arrest. Hypoxia also induces the expression of CXCL12 receptors CXCR4 (CXC chemokine recpetor 4) and CXCR7 to increase the migration, adhesion and survival of hypoxic cells. Imbalance of the CXCL12/CXCR4/CXCR7 axis is associated with antitumor immunotherapeutic effects and cancer metastasis. Therefore, the CXCL12/CXCR4/CXCR7 axis is recognized as a potential target for cancer therapy. Various studies have reported that CXCL12 can be regulated by several factors such as HIF-1α, G-CSF, SMAD, and fibroblast activation protein-α (FAPα). In a previous study, FAPα protein expressed in Carcinoma associated fibroblast (CAF) was reported as a factor regulating CXCL12 induction. In pancreatic adenocarcinoma, FAPα depletion of stromal cells or administration of AMD3100, a CXCR4 inhibitor, increased anti-tumor immunity and decreased cancer cells. This can be seen as a result showing that CXCL12 contributes to cancer growth by suppressing the anti-tumor immune response (Feig C, et al., 2013. PubMed PMID: 24277834).

Accordingly, the present inventors analyzed the expression level of CXCL12 protein in cancer cells and non-neoplastic epithelial cells of LARC surgical tissue using the immunohistochemical staining method in the process of researching biomarkers for predicting LARC prognosis after nCRT treatment. After nCRT treatment, it was confirmed that the expression of CXCL12 protein in the plasma membrane of the surviving cancer cells, especially the cancer cells in the most deeply infiltrated region, was significantly increased compared to the surrounding non-neoplastic epithelial cells. By confirming that, the present invention was able to be completed.

Korea Patent No. 1751806 relates to a marker composition for predicting the prognosis of cancer patients and anticancer drug susceptibility, and includes various biomarkers for predicting the prognosis of colorectal cancer. It contains CXCL12 as one of the indicators of colorectal cancer.

However, a composition capable of predicting the recurrence prognosis of locally advanced rectal cancer using the simultaneous measurement of CXCL12 expression and CXCL12, CXCR4 and FAPα in the cell membrane of rectal adenocarcinoma cells after nCRT treatment of the present invention has not yet been disclosed.

Korean Patent Publication No. 2018-0103539 Korea Registered Patent No. 19999371

It is an object of the present invention to provide an information providing method, biomarker composition or kit for diagnosing or predicting rectal adenocarcinoma, including CXCL12.

In order to solve the above problems, the present invention provides a first step of measuring the expression level of CXCL12 (CXC chemokine ligand 12) protein expressed in the plasma membrane of tumor cells isolated from rectal adenocarcinoma patients; Step 2 of comparing the CXCL12 protein expression level in the cell membrane with a normal control sample; And when the CXCL12 protein expression level in the cell membrane of the rectal adenocarcinoma cells of the patient is higher than that of a normal control sample, a third step of determining that the patient is rectal adenocarcinoma; It provides a method of providing information necessary for diagnosing or prognosis of rectal adenocarcinoma comprising a.

The CXCL12 protein expression level is measured through an antigen-antibody reaction, preferably western blot, immunohistochemial stain, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), It relates to at least one method selected from the group consisting of radioimmunodiffusion, immunoprecipitation assay, complement fixation assay, and protein chip.

The rectal adenocarcinoma may be locally advanced rectal cancer, and neoadjuvant chemoradiotherapy is present.

As another example, the present invention provides a biomarker composition for diagnosing or predicting rectal adenocarcinoma comprising CXCL12 as an active ingredient.

The CXCL12 cell membrane expression is increased in tumor cells of rectal adenocarcinoma compared to normal tissues.

In another embodiment, the present invention provides a kit for diagnosing or predicting a prognosis of rectal adenocarcinoma comprising an antibody that specifically binds to CXCL12.

The antibody may be a monoclonal antibody or a polyclonal antibody.

The present invention relates to an information providing method, biomarker composition or kit for diagnosing or predicting rectal adenocarcinoma, including CXCL12, and analyzing CXCL12 protein expression in cancer cells of rectal adenocarcinoma and non-tumor epithelial cells using immunohistochemical staining method As a result, the cell membrane expression of CXCL12 protein was significantly increased in cancer cells compared to non-tumor epithelial cells, and the cell membrane expression of CXCL12 protein was significantly increased in adenocarcinoma cancer cells after nCRT treatment than in rectal adenocarcinoma cancer cells that did not receive nCRT treatment. As it increased, it was confirmed that the cytoplasmic CXCL12 protein expression of rectal adenocarcinoma cancer cells increased.

Accordingly, CXCL12 of the present invention can be used as a biomarker for rectal adenocarcinoma, and it is possible to diagnose or predict the prognosis of rectal adenocarcinoma through the analysis of the expression rate of the cancer cell membrane CXCL12, and it is also useful in the development of a preventive or therapeutic agent for rectal adenocarcinoma. expected to be high.

1 shows the relationship between CXCL12 mRNA expression and the precancerous pathway in locally advanced rectal cancer (LARC) according to an embodiment of the present invention. 1A shows that CXCL12 mRNA expression level is an important prognostic factor for overall survival in GSE133057 (p=0.047; log-rank test). Figure 1b Correlation of CXCL12 expression and immune infiltration level in LARC. Figure 1C shows a gene set enrichment analysis (GSEA) correlation between CXCL12 expression and genes involved in epithelial mesenchymal metastasis and angiogenesis.
2 shows the changes in CXCL12, CXCR4 and FAPα expression after nCRT in LARC patients according to an embodiment of the present invention. 2A and 2B are box plots of CXCL12, CXCR4 and FAPα expression in tumor and irradiated tumor tissue (using data sets GSE15781 and GSE94104). 2C is a box plot of CXCL12, CXCR4 and FAPα expression in normal and irradiated normal adjacent tissues (using data set GSE15781). *p<0.05, **p<0.01, ns: meaningless. Mann-Whitney U test.
3 shows a representative image showing immunohistochemical staining of CXCL12, CXCR4 and FAPα according to an embodiment of the present invention. Rectal adenocarcinoma (A, B, C), rectal adenoma with high-grade dysplasia (D, E, F), and non-neoplastic rectal epithelium (G, H, I) after nCRT treatment. In cancer cells of rectal adenocarcinoma after nCRT treatment, the expression of CXCL12, CXCR4, and FAPα was higher than in the other two groups (original magnification 400×, scale bar = 100 μm).
4 shows LARC (LARC-nCRT, n=43) after nCRT treatment, LARC without nCRT treatment (LARC-no nCRT, n=60) and high-grade dysplasia of rectal adenoma (HD, n = 16) shows the results of comparison of expression of CXCL12, CXCR4 and FAPα. The levels of all three proteins were higher in LARC-nCRT than in LARC-nCRT or HD (p <0.001, respectively). (A) CXCL12 expression, (B) CXCR4 expression and (C) FAPα expression.
5 shows the Kaplan-Meier survival curve results of disease-free survival in LARC 43 cases after nCRT treatment according to cell membrane expression of CXCL12 and CXCR4 in tumor cells according to an embodiment of the present invention. (A) CXCL12, (B) CXCR4 and (C) FAPα.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted.

In order to achieve the object of the present invention, the present invention provides a first step of measuring the expression level of CXCL12 (CXC chemokine ligand 12) protein expressed in the plasma membrane of tumor cells isolated from a patient with rectal adenocarcinoma; Step 2 of comparing the CXCL12 protein expression level in the cell membrane with a normal control sample; And when the CXCL12 protein expression level in the cell membrane of the rectal adenocarcinoma cells of the patient is higher than that of a normal control sample, a third step of determining that the patient is rectal adenocarcinoma; It provides a method of providing information necessary for diagnosing or prognosis of rectal adenocarcinoma comprising a.

In the present invention, "for predicting prognosis" is for checking the progress of a disease, survival, or cure after cancer treatment.

As used herein, “prediction” is used herein to refer to the likelihood that a subject patient will respond favorably or unfavorably to a drug or set of drugs. In one embodiment, the prediction relates to the extent of this response. For example, prediction relates to whether and/or the probability that a patient will survive cancer recurrence-free after treatment, e.g., after treatment with certain therapeutic agents and/or surgical removal of the primary tumor and/or chemotherapy for a certain period of time . The predictions of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment modality for cancer patients. The prediction of the present invention is whether a patient will respond favorably to a therapeutic treatment, e.g., a given therapeutic treatment, e.g., administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc., or whether long-term survival of the patient is possible after the therapeutic treatment. It is a useful tool for predicting whether

In the present invention, “measurement of protein expression level” refers to a process of confirming the presence and expression level of proteins expressed from cancer-related genes in a biological sample in order to predict cancer prognosis or anticancer drug sensitivity. This is done by measuring the amount of protein. Analysis methods for this include western blot, immunohistochemial stain, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, and immunoprecipitation assay. ), a complement fixation assay, and at least one method selected from the group consisting of a protein chip, but is not limited thereto. In the present invention, the agent for measuring the protein expression level is preferably immunohistochemical staining, but is not limited thereto.

The rectal adenocarcinoma may be locally advanced rectal cancer, and neoadjuvant chemoradiotherapy is present, but is not limited thereto.

As another example of the present invention, the present invention provides a biomarker composition for diagnosing or predicting rectal adenocarcinoma comprising CXCL12 as an active ingredient.

In the present invention, "biomarker (biomarker)" refers to a marker that can distinguish normal or pathological conditions, predict a therapeutic response, and can be objectively measured. Genes such as DNA and RNA, proteins, Although lipids, metabolites, and the like, and changes in patterns thereof are used, the present invention is not limited thereto.

In the present invention, "stage" is a concept for classifying the progress of cancer, and is usually classified into 4 stages from stage 1 to stage 4, and the number increases as the degree of cancer progresses.

The CXCL12 cell membrane expression is increased in tumor cells of rectal adenocarcinoma compared to normal tissues, but is not limited thereto.

As another example of the present invention, the present invention provides a kit for diagnosing or predicting a prognosis of rectal adenocarcinoma comprising an antibody that specifically binds to CXCL12.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

In the present invention, for immunological detection of the antibody, a substrate, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like may be included. As the substrate, a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a glass slide glass, etc. may be used, and peroxidase (peroxidase) may be used as the chromogenic enzyme. ), alkaline phosphatase, etc. may be used. In addition, FITC, RITC, Cy3, Cy5, etc. may be used as a fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD as a color development substrate (o-phenylenedianim), TMB (tetramethyl benzidine), etc. may be used, but is not limited thereto.

The kit of the present invention may include an aptamer, a peptide, etc. instead of an antibody.

In the present invention, "aptamer" is a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure by itself and having high affinity and specificity to bind to a target molecule. As a kind of polynucleotide, an aptamer can specifically bind to an antigenic substance in the same way as an antibody, but has higher stability than a protein, has a simple structure, and is composed of a polynucleotide that is easy to synthesize. can be used

In the present invention, the "peptide" has the advantage of high binding strength to the target material, and does not undergo denaturation even during heat/chemical treatment. In addition, since the molecular size is small, it can be used as a fusion protein by attaching it to other proteins. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example 1> Patient and tissue samples

The present invention was carried out with the approval of the Chungnam National University Hospital Institution Review Committee (CNUH 2019-10-041-002). Formalin-fixed paraffin-embedded (FFPE) tissue samples were used for immunohistochemical staining. The biological specimens and data used in the present invention were provided by Chungnam National University Hospital Biobank, a member of the Korean Biobank Network. Because the present invention was based on immunohistochemical staining using FFPE tissue, the informed consent requirement for a retrospective comparative study was waived. In the present invention, representative FFPE whole tissue samples from 43 cases of LARC treated with nCRT, LARC from 60 cases not treated with nCRT and 16 cases of high-grade dysplastic colorectal adenoma were used for immunohistochemical staining (IHC). Forty-three LARC patients underwent medial rectal resection after preoperative radiotherapy and concomitant capecitabine treatment. After nCRT, 43 cases of LARC were in the middle or low rectum, and 60 LARCs that were not treated with nCRT were in the upper rectum. All 103 cases of LARC were moderately differentiated adenocarcinoma, and the marginal section was negative.

Patient inclusion criteria were isolation of FFPE tumor tissue from LARC patients who underwent surgery for rectal adenocarcinoma, endoscopic submucosal resection for colorectal adenocarcinoma, and follow-up clinical data sufficiently detailed. Exclusion criteria were as follows. (1) the patient had a previous history of other cancers; (2) the patient had previously undergone therapeutic resection for colonic tumor lesions; (3) the patient has received molecularly targeted therapy; (4) Surgical and curative resection of LARC was positive for perisection or pathologic stage 4 tumor (pT4); (5) patient had distant metastases at initial diagnosis; or (6) the patient achieved a complete pathological response after nCRT.

A modified Ryan system for pathologic tumor, nodule and metastasis (pTNM) stage, histological grade for LARC, and tumor regression score (R score) was determined from surgically resected tissue and was determined by the American Joint Committee on Cancer (AJCC). Based on the 8th revision of the staging system. Disease-free survival (DFS) was determined as the time interval between the date of first surgical resection and the date of LARC recurrence or metastasis. LARC recurrence or metastasis was determined via imaging and/or histological analysis. Overall survival (OS) was defined as the time from initial surgical resection to the date of death from all causes. OS or DFS time without confirmation of death, recurrence or metastasis was recorded as of the last known date of the patient.

<Example 2> Radiation therapy and cancer reactivity evaluation

Radiation therapy was performed according to the conventional method. Briefly, radiation was delivered via 6 and 10 MV photons using three-field techniques (posterior and bilateral) in most patients. Treatment was planned via computerized dosimetry and a dose of 1.8 Gy per fraction was prescribed to cover the planned target volume. Radiation therapy was administered 5 days a week, once a day at 1.8 Gy/d. Pelvic radiotherapy consisted of 45 Gy in 25 fractions over 5 weeks, after which an additional dose of 5.4 Gy was administered in 3 fractions to the primary tumor. Preoperative chemotherapy was administered concurrently with radiation therapy. Approximately 6 weeks after completion of nCRT, patients underwent final surgery. Pathological evaluation of surgical specimens, including primary tumors and resected nodes, was performed by a professional pathologist. The complete absence of residual tumor cells in the primary tumor was designated as a pathological complete response.

<Example 3> Bioinformatics analysis

TIMER is a comprehensive resource for the systematic analysis of immune invasion across various cancer types (https://cistrome.shinyapps.io/timer/). TIMER applies a previously published statistical deconvolution method to infer abundant tumor infiltrating immune cells (TIICs) from gene expression profiles. In the present invention, the correlation between CXCL12, CXCR4 and FAPα expression and abundant immune cell infiltration was analyzed.

Analysis of the LARC data set was performed essentially as previously described. Initial data can be downloaded as GSE15781, GSE94104 and GSE133057. CXCL12, CXCR4 and FAPα expression patterns were derived from GSE15781 and GSE94104 to compare the pre-nCRT and post-nCRT LARC patient groups. RNA data were analyzed using Wilcoxon-Mann-Whitney test and Benjamini-Hochberg FDR correction (P<0.05, q<0.25) to compare the expression of univariate differences in each data set. Pathway analysis was performed on the IBD data set (GSE133057) using GSEA.

<Example 4> Immunohistochemical staining analysis

Immunohistochemical staining of FFPE tissues was performed by conventional methods. A target recovery solution, pH 9 (S2368, Dako, Glostrup, Denmark) was used for antigen regeneration. Tissue sections were obtained from mouse monoclonal anti-human CXCL12 (1:300, MA5-23759, Invitrogen, Rockford, IL, USA), rabbit monoclonal anti-human CXCR4 (1:100, ab124824, Abcam, Cambridge, UK) and rabbit polyclonal. Anti-human FAPα (1:20, AHP1322, BIO-RAD, Hercules, CA, USA) was reacted at 37° C. for 30 minutes.

Plasma membrane levels of CXCL12 and CXCR4 and cytoplasmic levels of FAPα in high-grade dysplastic epithelial cells of invasive anterior or adenoma of LARC cancer cells were measured using the modified DAKO HercepTest ^TM Interpretation Manual-Breast Cancer Row version (staining was 0, 1, 1). rated at 2 or 3) [HercepTest™, Interpretation Manual Breast Cancer (online: https://www.agilent.com/cs/library/usermanuals/public/28630_herceptest_interpretation_manual-breast_ihc_row.pdf)]. A staining score of 2 or 3 was considered positive expression of CXCL12. The staining scores for CXCR4 were as follows: 0, no expression; 1 point, cytoplasmic expression only; 2 points, less than 10% of the invasive front of tumor cells with perceptible cell membrane staining; and 3 points, cell membrane staining in 10% and over 10% of the invasive front of tumor cells. Scores 2 and 3 were considered positive expression. The staining score of FAPα was determined by cytoplasmic intensity: 0, no expression; 1 point, weak; 2 points, moderate; And 3 points, strong. Staining scores 2 and 3 were considered positive expression of FAPα. Results were separately investigated and scored by a clinician unaware of the patient's clinicopathological details. Discrepancies in scores were discussed to obtain consensus.

<Example 5> Statistical analysis

Correlation of the expression of CXCL12, CXCR4 and FAPα with clinicopathological parameters was assessed using Pearson's chi-square test. The three protein expression differences between adenoma, LARC with nCRT treatment and LARC without nCRT treatment were assessed using Pearson's chi-square test. Correlation between the three protein expression profiles was assessed using Spearman's correlation. Postoperative OS and DFS were determined using log-rank tests and univariate and multivariate Cox regression analysis. Statistical significance was set at p<0.05 (SPSS v.26; SPSS Inc., Chicago, IL, USA).

<Test Example 1> CXCL12 expression is associated with poor prognosis in LARC, immune cell infiltration, epithelial-mesenchymal metastasis, and angiogenesis.

To investigate the correlation between CXCL12 mRNA and OS in pretreated LARC biopsy tissues from LARC patients, we analyzed a publicly available transcriptome data set (GSE133057). Survival analysis showed that those with high CXCL12 expression had shorter overall survival than those with low CXCL12 expression (p=0.047, FIG. 1A ). However, CXCR4 and FAPα mRNA expression in pre-treatment biopsy tissues did not correlate with overall survival of LARC patients. To further corroborate the prognostic role of CXCL12, the correlation between CXCL12 and LARC infiltration into immune cells was identified using the TIMER (Tumor IMmune Estimation Resource) database. Among the various immune cells, CD4+ T cells, macrophages and dendritic cells had the strongest correlation with CXCL12 in LARC (Fig. 1b). In addition, gene set enrichment analysis (GSEA) was performed using the LARC data set with traces of the CXCL12 gene associated with good or bad survival. In the group with high CXCL12 expression, epithelial-mesenchymal transition and enrichment of angiogenic traces were observed (Fig. 1c).

<Test Example 2> Radiation-induced changes in CXCL12 and CXCR4 expression in LARC and surrounding normal tissues

It has been reported that radiation damages tumor blood vessels to induce tumor hypoxia and ultimately induces CXCL12 in patient-derived tumor xenografts. Publicly available transcriptome data sets (GSE15781 and GSE94104) were analyzed to assess changes in CXCL12, CXCR4 and FAPα expression between pre-treatment and post-nCRT surgical specimens (Figure 2). After nCRT treatment, increased mRNA expression of CXCL12, CXCR4 and FAPα was observed in residual cancer tissues ( FIGS. 2A and 2B ). However, there was no significant difference in the expression of CXCL12, CXCR4 and FAPα in normal tissues when the pre-treatment and post-treatment were compared (Fig. 2c).

<Test Example 3> Relationship between expression of CXCL12, CXCR4 and FAPα and clinical pathological characteristics

The clinicopathological characteristics of 103 LARC patients found to be associated with the expression of CXCL12, CXCR4 and FAPα are presented in Table 1. Positive and negative classification of CXCL12 or CXCR4 was determined based on expression in the cell membrane. Positive and negative classification of FAPα was determined based on cytoplasmic expression (FIG. 3). Non-neoplastic epithelial cells and non-invasive high-grade dysplastic cells showed almost no cell membrane staining of CXCL12 or CXCR4. Cytoplasmic levels of FAPα were significantly increased in invasive LARC cells and tumor microenvironmental immune cells of adenomas, whereas adjacent non-tumor epithelial cells were negative.

- Correlation of CXCL12, CXCR4 and FAPα expression with clinicopathological factors in 103 patients with LARC -

variable No. CXCL12 CXCR4 FAPα (-) (+) p * (-) (+) p * (-) (+) p * Gender N = 71 N = 32 0.504 N = 79 N = 24 0.079 N = 59 N = 44 0.620 Male 66 47 19 47 19 39 27 Female 37 24 13 32 5 20 17 Age (years) 0.021 0.364 0.011 ≤65 68 52 16 54 14 45 23 >65 35 19 16 25 10 14 21 nCRT <0.001 <0.001 <0.001 No 60 57 3 60 0 45 15 Yes 43 14 29 19 24 14 29 (y)pT 0.716 0.202 0.565 2 36 24 12 25 11 22 14 3 67 47 20 54 13 37 30 (y)pN 0.064 0.300 0.120 0 65 49 16 52 13 41 24 1-2 38 22 16 27 11 18 20 (y)pTNM Stage 0.064 0.300 0.120 II 65 49 16 52 13 41 24 III 38 22 16 27 11 18 20

* Pearson's chi-square test; nCRT, neoadjuvant chemoradiotherapy; (y)pTNM stage, (neoadjuvant)pathologic stage in American Joint Committee on Cancer (AJCC) cancer staging system ( ^8th edition).

Cell membrane CXCL12 levels in 103 cases of LARC were positive in LARC with lymph node metastasis compared to LARC without lymph node metastasis (p=0.064). Cell membrane CXCL12 expression in LARC 43 cases after nCRT was positive at R score 2-3 compared to R score 1 (p = 0.055; Fisher's exact test). In addition, CXCL12, CXCR4, and FAPα positivity rates were higher in LARC after nCRT treatment than in LARC without nCRT treatment (p<0.001, respectively) (Table 1). The positivity rates for CXCL12, CXCR4 and FAPα were stronger in LARC cases after nCRT treatment (p<0.001, respectively) than in 16 high-grade dysplastic adenoma cases or LARC cases not receiving nCRT treatment (p<0.001, respectively) (Figure 4). There was a significant correlation between membrane CXCL12 expression levels and CXCR4 and FAPα levels in LARC cancer cells after nCRT treatment (Spearman's Rho: p=0.001 and p<0.003).

<Test Example 4> The positive CXCL12 cancer cell membrane expression is predicted to shorten the disease-free survival period in 43 cases of LARC after nCRT treatment.

At a follow-up period with a median of 56 months, 9/43 patients died from unspecified causes, 9 became ill, and 25 were alive without evidence of disease. Patients with high CXCL12 expression in residual viable cancer cell membranes had worse DFS at high significance and significance thresholds in the univariate long-term rank test and multivariate Cox regression analysis (p=0.027 and p=0.064, respectively) (Table 2 and Table 3). ). The Kaplan-Meier curve also showed that high CXCL12 expression was strongly associated with DFS exacerbation (p = 0.006) (Fig. 5). Higher CXCR4 membrane levels were associated with shorter DFS duration at the significance threshold in the univariate long-term rank test and Kaplan-Meier curves (p=0.078 and p=0.065, respectively) (Table 2, Figure 5).

- Univariate analysis of overall survival and disease-free survival in LARC 43 cases using nCRT -

prognostic factor overall survival Disease-free survival HR (95% CI) P* HR (95% CI) P* CXCL12 expression 0.304 0.027 negative (n=14) 1 (reference) 1 (reference) positive (n=29) 2.308 (0.468-11.381) 9.955 (1.305-75.953) CXCR4 expression 0.357 0.078 negative (n=19) 1 (reference) 1 (reference) positive (n=24) 1.924 (0.478-7.754) 2.768 (0.891-8.599) FAPα expression 0.928 0.165 negative (n=14) 1 (reference) 1 (reference) positive (n=29) 1.066 (0.266-4.263) 2.437 (0.692-8.580) Age at operation 0.772 0.710 ≤65 (n=25) 1 (reference) 1 (reference) >65 (n=18) 1.215 (0.326-4.533) 1.208 (0.446-3.275) Sex 0.973 0.655 Male (n=29) 1 (reference) 1 (reference) Female (n=14) 0.976 (0.244-3.905) 1.261 (0.456-3.481) R score 0.593 0.235 1 (n=10) 1 (reference) 1 (reference) 2-3 (n=33) 0.681 (0.166-2.787) 2.499 (0.551-11.331) ypTNM stage 0.229 0.093 II (n=27) 1 (reference) 1 (reference) III (n=16) 2.243 (0.602-8.361) 2.336 (0.869-6.284)

* Multivariate Cox Regression; HR, hazard ratio; CI, confidence interval; CXCL12 expression, cell membrane expression in cancer cells; CXCR4 expression, cell membrane expression in cancer cells; FAPα expression, cytoplasmic expression in cancer cells; R score, modified Ryan scheme for tumor regression score in American Joint Committee on Cancer (AJCC) cancer staging system ( ^8th edition).

- Multivariate analysis of overall survival and disease-free survival in LARC 43 cases with nCRT -

prognostic factor overall survival Disease-free survival HR (95% CI) P* HR (95% CI) P* CXCL12 expression 0.413 0.064 negative (n=14) 1 (reference) 1 (reference) positive (n=29) 2.611 (0.262-26.029) 8.830 (0.884-88.231) CXCR4 expression 0.156 0.543 negative (n=19) 1 (reference) 1 (reference) positive (n=24) 3.818 (0.600-24.313) 1.619 (0.343-7.637) FAPα expression 0.269 0.765 negative (n=14) 1 (reference) 1 (reference) positive (n=29) 0.331 (0.046-2.354) 1.290 (0.243-6.849) Age at operation 0.347 0.668 ≤65 (n=25) 1 (reference) 1 (reference) >65 (n=18) 2.290 (0.407-12.898) 0.786 (0.261-2.368) Sex 0.808 0.853 Male (n=29) 1 (reference) 1 (reference) Female (n=14) 1.247 (0.209-7.443) 0.898 (0.290-2.785) R score 0.070 0.809 1 (n=10) 1 (reference) 1 (reference) 2-3 (n=33) 0.185 (0.030-1.150) 0.808 (0.144-4.530) ypTNM stage 0.188 0.651 II (n=27) 1 (reference) 1 (reference) III (n=16) 3.062 (0.578-16.215) 1.299 (0.418-4.037)

* Multivariate Cox Regression; HR, hazard ratio; CI, confidence interval; R score, modified Ryan scheme for tumor regression score in American Joint Committee on Cancer (AJCC) cancer staging system ( ^8th edition).

Claims (9)

Step 1 of measuring the expression level of CXCL12 (CXC chemokine ligand 12) protein expressed in the plasma membrane of tumor cells isolated from a patient with rectal adenocarcinoma;
Step 2 of comparing the CXCL12 protein expression level in the cell membrane with a normal control sample; and
Step 3 of determining that the patient is rectal adenocarcinoma when the CXCL12 protein expression level in the cell membrane of the rectal adenocarcinoma cells is higher than that of a normal control sample;
A method of providing information necessary for diagnosis or prognosis of rectal adenocarcinoma comprising The method of claim 1, wherein the CXCL12 protein expression level is measured through an antigen-antibody reaction. The method of claim 2, wherein the antigen-antibody reaction is performed by western blot, immunohistochemial stain, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), radioimmunodiffusion, Immunoprecipitation assay (immunoprecipitation assay), complement fixation assay (complement fixation assay), characterized in that at least one method selected from the group consisting of a protein chip (protein chip) to provide information necessary for diagnosing or predicting prognosis of rectal adenocarcinoma Way The method of claim 1, wherein the rectal adenocarcinoma is a locally advanced rectal cancer. The method of claim 1, wherein the rectal adenocarcinoma has prior chemoradiotherapy (neoadjuvant chemoradiotherapy). Biomarker composition for diagnosing or predicting rectal adenocarcinoma comprising CXCL12 as an active ingredient [Claim 7] The biomarker composition for diagnosing or predicting prognosis of rectal adenocarcinoma according to claim 6, wherein the CXCL12 cell membrane expression is increased in tumor cells of rectal adenocarcinoma compared to normal tissue. A kit for diagnosing or predicting prognosis of rectal adenocarcinoma comprising an antibody that specifically binds to CXCL12 of claim 6 or 7 The kit for diagnosing or prognosing rectal adenocarcinoma according to claim 8, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

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