KR20210042836A - Biomarkers for prediction of response to neoadjuvant chemoradiation therapy in rectal cancer - Google Patents

Abstract

The present invention relates to a biomarker for predicting a response to chemoradiotherapy, which is selected from a group consisting of human cathelicidin antimicrobial peptide (CAMP), lysophosphatidylcholine acyltransferase 2 (LPCAT2), and aldo-keto reductase family 1, member B1 (AKR1B1). More specifically, the present invention relates to a composition for predicting a response to chemoradiotherapy, a prediction kits, and an information providing method for prediction. According to the present invention, since the biomarker of the present invention is used, the presence or absence of a response to the chemoradiotherapy in a rectal cancer patient can be predicted before surgery, thereby providing great help in determining a treatment plan for the rectal cancer patient.

Description

Biomarkers for prediction of response to neoadjuvant chemoradiation therapy in rectal cancer

The present invention relates to a biomarker for predicting response to anticancer radiation therapy selected from the group consisting of CAMP (human cathelicidin antimicrobial peptide), LPCAT2 (Lysophosphatidylcholine acyltransferase 2) and AKR1B1 (Aldo-keto reductase family 1, member B1). , Specifically, a composition for predicting response to anticancer radiation therapy; Prediction kit; It relates to a method of providing information for prediction.

According to IARC (International Agency for Research on Cancer) data, the incidence of gastric cancer among Koreans is the world's first, colon cancer is second, and liver cancer is 10th. The socio-economic burden due to the treatment of cancer patients is increasing.

Colorectal cancer (CRC) is the third most common malignant tumor in the world. Almost 940,000 patients with colon cancer each year, and 1.5 million patients die of colon cancer every year. According to Korea's national cancer registration project report in 2012, colon cancer (colon cancer, rectal cancer) is the third most common (28,919 people, 12.9%) after thyroid cancer and stomach cancer. In males, it is the second most common cancer in 17,419 (15.5%) after gastric cancer, and in females, it is a high frequency cancer that occurs in 11,514 (10.3%), which is the second most common after thyroid gland and breast cancer. Of the patients diagnosed with colorectal cancer, 50 to 55% are rectal cancer patients, and the improvement of the treatment performance of rectal cancer is thought to have a greater impact on the health and health promotion of Korea as a whole more than any other cancer tumors in the present as well as in the future.

In addition, rectal cancer refers to a malignant tumor composed of cancer cells in the rectum. The large intestine is largely divided into colon and rectum. Depending on where the cancer occurs, cancer in the colon is called colon cancer, and cancer in the rectum is called rectal cancer, which is collectively referred to as colon cancer or colorectal cancer. The rectum is the last part of the large intestine, and its length is about 15 cm, and can be divided into upper, middle, and lower rectum. It goes down from the front of the sacrum along the middle and ends in the anus. The rectum is a pipe-shaped tube and is divided into four layers: the mucous layer, the submucosa layer, the muscle layer, and the serous layer from the inside. Most rectal cancers are adenocarcinomas that occur in the mucous membrane of the intestine. In addition, there are carcinoid tumors, lymphomas, sarcomas, squamous cell carcinomas, and metastatic lesions of other cancers.

Currently, it is recommended to perform chemotherapy before surgery as the standard treatment for rectal cancer, so about 50% of all rectal cancer patients undergo chemotherapy and then proceed with surgery. However, the degree of response to radiation therapy varies greatly from person to person. If the response to radiation therapy is good, there is a possibility of choosing a surgical method due to the treatment, and it is known that cancer treatment results are improved, but if the response is not good, not only will you receive unnecessary radiation treatment, but you will also be waiting for surgery after treatment. In the process of, cancer progresses and treatment is delayed. In addition, 50 to 60% of patients treated with poor response or disease progression after radiation therapy.

For the efficient treatment of rectal cancer, research on biomarkers and diagnostic methods for predicting the response to radiation therapy in rectal cancer patients is ongoing, but the development and commercialization of markers and diagnostic kits with excellent predictive specificity and efficiency are urgently needed.

Under this background, the present inventors have made intensive research efforts to develop a method for predicting the presence or absence of a response to radiation therapy in a rectal cancer patient, and as a result, three biomarkers capable of predicting the presence or absence of a reaction were selected, and the present invention was completed.

One object of the present invention is a formulation capable of detecting the expression of one or more proteins selected from the group consisting of CAMP (human cathelicidin antimicrobial peptide), LPCAT2 (Lysophosphatidylcholine acyltransferase 2) and AKR1B1 (Aldo-keto reductase family 1, member B1). It is to provide a composition for predicting whether a response to the anticancer radiation treatment comprising a.

Another object of the present invention is to provide a kit for predicting response to anticancer radiation therapy using the composition.

Another object of the present invention is to provide a method of providing information for predicting response to anticancer radiotherapy, comprising the step of detecting protein expression using the composition from a sample isolated from an individual.

One embodiment of the present invention for achieving the above object is one or more proteins selected from the group consisting of CAMP (human cathelicidin antimicrobial peptide), LPCAT2 (Lysophosphatidylcholine acyltransferase 2) and AKR1B1 (Aldo-keto reductase family 1, member B1). It provides a marker for predicting whether the response to the anti-cancer radiotherapy, including.

In a specific embodiment of the present invention, proteomics analysis was performed to discover protein diagnostic biomarkers to confirm the efficacy of anticancer radiotherapy before treatment. Rectal cancer tissues were obtained through colonoscopy before chemotherapy, and after surgery, tissues were obtained in the operating room to extract proteins, and then trypsin was cut to obtain LC-MS/MS data, and peptides showing reliability were identified using the SEQUEST algorithm. And, through Mass Profile Professional (MPP) software, a protein with a p-value of 0.05 or less and a fold change of 2 times or more was identified (Table 1).

In addition, six biomarker candidate groups were derived using a statistical method and a relative quantitative analysis method, and based on this, the final three biomarkers were derived through final verification.

The term "human cathelicidin antimicrobial peptide (CAMP) of the present invention is a precursor of LL-37 and is known as a direct target of vitamin D receptors, but there is no known use for predicting the response of anticancer radiation therapy in the present invention. , Was first identified by the inventors.

The term "LPCAT2 (Lysophosphatidylcholine acyltransferase 2)" of the present invention is known to be involved in the activities of acyltransferase and acetyltransferase. On the other hand, there is no known use of the present invention for predicting the response of anticancer radiation therapy, and was first identified by the present inventors.

The term "AKR1B1 (Aldo-keto reductase family 1, member B1)" of the present invention is also known as an aldose reductase, and is a NADPH-dependent reducing enzyme that promotes the reduction of various aldehydes and ketones to the corresponding alcohols. On the other hand, there is no known use of the present invention for predicting the response of anticancer radiation therapy, and was first identified by the present inventors.

The anticancer radiotherapy may be performed before a patient's surgery in cancer treatment, but is not limited thereto.

The cancer may be rectal cancer, and specifically, may be locally advanced rectal cancer, but is not limited thereto.

The term "anti-cancer radiation therapy" of the present invention is one of the three major cancer treatments along with surgery and anti-cancer drug treatment, and by irradiating high-energy radiation using a radiation-generating device or radioactive isotope, the symptoms of an individual with cancer It refers to any action that improves or is beneficially altered. In addition, the term "response to anti-cancer radiotherapy" refers to any aspect in which cancer cells are lost in a tissue or symptoms of an individual with cancer are improved or beneficially altered.

The term "prediction" of the present invention is to determine the susceptibility of an individual to a specific disease or disorder, to determine whether an individual currently has a specific disease or disorder, or to determine the prognosis of an individual suffering from a specific disease or disorder. determining prognosis, or therametrics (eg, monitoring the condition of an individual to provide information about treatment efficacy).

The term "prediction" may be used with the same meaning as "diagnosis".

The predictive composition may be judged to comply with anticancer radiotherapy when CAMP or LPCAT2 has a higher protein expression level than that of the normal control group, and when it is low, it can be determined to be refractory to the anticancer radiotherapy, and AKR1B1 expresses protein compared to the normal control group. If the level is low, it may be judged to comply with anticancer radiation therapy, and if it is high, it may be judged to be refractory to anticancer radiation therapy.

In a specific embodiment of the present invention, six biomarker candidate groups were finally selected through a statistical method. Specifically, the higher the expression of CAMP, CAD, LPCAT2 and STAT3 in the individual, the lower the expression of ANXA13 and AKR1B1, the more anticancer radiotherapy. It was confirmed that it conforms to (Fig. 3). Based on this, after final verification, it was confirmed that CAMP, LPCAT2, and AKR1B1 among the biomarker candidate groups showed reliable results (FIG. 6), and were selected as the final biomarkers.

One aspect of the present invention provides a composition for predicting response to anticancer radiotherapy, comprising an agent capable of detecting the expression of one or more proteins selected from the group consisting of CAMP, LPCAT2, and AKR1B1.

At this time, descriptions of the terms "CAMP", "LPCAT2", "AKR1B1", "response to anticancer radiation therapy", "prediction", and the like are as described above.

Another aspect of the present invention provides a kit for predicting response to anticancer radiotherapy using the composition.

At this time, descriptions of the terms "CAMP", "LPCAT2", "AKR1B1", "response to anticancer radiation therapy", "prediction", and the like are as described above.

The kit of the present invention refers to a kit including an agent capable of detecting the expression of the protein, and the response to anticancer radiation therapy can be predicted by using the kit of the present invention. The kit for predicting response to anticancer radiotherapy of the present invention may include not only the protein, but also one or more other constituent compositions, solutions, or devices suitable for the analysis method. In addition, the kit of the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, and the like for immunological detection of the antibody. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polyester resin, and a slide glass made of glass, and the color developing enzyme is peroxidase, alkaline Phosphatase (Alkaline Phosphatase) may be used, fluorescent material may be used, such as FITC, RITC, etc., the color developing substrate solution ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) )) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

Another aspect of the present invention provides a method of providing information for predicting response to anticancer radiotherapy, comprising the step of detecting protein expression using the composition from a sample isolated from an individual.

In this case, the descriptions of the terms "response to anticancer radiation therapy", "prediction", and the like are as described above.

The term "sample" of the present invention includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine that are derived from the individual and can predict the response to anticancer radiation therapy, and in particular It may be a blood sample isolated from an individual, but is not limited thereto.

The information providing method of the present invention is a method for predicting whether or not a response to anticancer radiotherapy has occurred, and to determine the degree of up-regulation or down-regulation of the proteins, detection of a protein in a sample isolated from an individual and measuring the expression thereof It is an information providing method including the step of.

The information providing method may further include comparing the expression level of the protein with the expression level of the protein in a sample isolated from a normal control.

In the above information provision method, when CAMP or LPCAT2 has a higher protein expression level compared to the normal control, it can be determined to comply with anticancer radiotherapy, and when it is low, it can be determined to be refractory to the anticancer radiotherapy, and AKR1B1 expresses protein compared to the normal control. When the level is low, it can be judged to comply with anticancer radiotherapy, and when it is high, it can be judged to be refractory to anticancer radiotherapy.

By using the biomarker of the present invention, the presence or absence of a reaction to anticancer radiation therapy in a rectal cancer patient can be predicted before surgery, which can greatly help in determining a treatment policy for a rectal cancer patient.

1 is a schematic diagram showing a sample group for discovering diagnostic biomarkers.
2 is a schematic diagram showing an experimental process for analyzing a protein using a mass spectrometer.
3 is a graph showing the results of parallel recation monitoring (PRM) of a biomarker protein capable of predicting the response to anticancer radiotherapy through identification.
4 is a graph showing the cell survival rates of the rectal cancer cell line SNU-503 and the radiation resistant rectal cancer cell line SNU-503R80Gy according to the radiation dose.
5 is a graph showing the cell survival rates of the rectal cancer cell line SNU-503 and the radiation-resistant rectal cancer cell line SNU-503R80Gy by time after irradiation.
6 is a result of confirming the expression level of each protein after irradiation with respect to the rectal cancer cell line SNU-503 and the radiation-resistant rectal cancer cell line SNU-503R80Gy.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: Peptide identification

Peptides were identified through proteomic analysis to discover protein diagnostic biomarkers to confirm the efficacy of anticancer radiotherapy before treatment.

More specifically, a rectal cancer tissue was obtained through colonoscopy from a rectal cancer patient before anticancer radiotherapy, and tissue was obtained in an operating room after surgery. Acquired tissues were classified into pre-treatment (CR-S) and post-treatment (CR-T) of the completely affected CR group, and pre-treatment (Std3-S) and post-treatment (Std-T) of stage 3 without response. Was done (Fig. 1). Thereafter, after extracting protein from the tissue with an efficiency of 10% or more through the Pulverization and Focused Sonication method from the groups, trypsin digestion through the Filter Aided Sample Preparation (FASP) Digestion method is performed, and then LC-MS through Hybrid Orbitrap MS. /MS data was obtained and the experiment was repeated 3 times for non-labeled quantitative analysis. Then, a peptide showing reliability was identified using the SEQUEST algorithm (Table 1). Abundance, retention time, and m/z information for each protein are entered into Agilent's Mass Profile Professional (MPP) software, and the p-value is 0.05 or less and the fold change is 1.5 times or more different through t-test and ANOVA statistical analysis. Obtained was confirmed the function and relationship of each protein (Fig. 2).

As a result, as shown in Table 1, a protein having peptide information showing high reliability was identified.

Example 2: KEGG pathway analysis

After obtaining significant protein information representing a p-value of 0.05 or less and a fold change of 1.5 times or more, functions and associations of each protein were confirmed through KEGG pathway, GO Ontology, and protein network analysis.

More specifically, Wnt signaling is an important mediator for tissue homeostasis and repair, and is a delivery system that often appears during tumor development. Almost all colorectal cancers exhibit overactivation of the Wnt pathway. In addition, JAK-STAT signaling is closely related to immune function and cell growth. The combination of IGF1/2 and IGF1R/2R or multiple growth factors bind to IGF1R to select the JAK-STAT pathway, a lower delivery system, to inhibit tumor growth and cell death. IGF2 mRNA and protein are overexpressed in about 1/3 of colorectal cancers, and IGF1R protein is expressed in more than 90% of colorectal cancers.

Example 3: Biomarker candidate selection

Example 3-1: Selection through statistical method

Finally, six biomarkers were selected through a statistical method.

More specifically, the proteins identified in Example 1 were subjected to a statistical method to determine a protein corresponding to FDR 1% and a protein having at least two unique peptides, CR-S: 1,481, CR-T: 1,418, Std3-S:2,064 and Std3-T:1,853 were selected. Subsequently, the total was quantified based on the quantification values of the pre-treatment group, CR-S, the post-treatment group, the CR-T, the pre-treatment sample, the Std3-S, and the post-treatment group, Std3-T protein 1,268 proteins were selected.

Example 3-2: Selection through a relative quantitative analysis method

Among the four groups quantified above, proteins with differences in the presence or absence of treatment effects (CR-S and Std3-S) were selected through relative quantitative analysis, and a total of 59 up-regulations, 131 down-regulations, and a total of 190 proteins were selected, and among the selected protein candidate groups, six biomarker candidate proteins showing relatively quantitative changes in CR-S and Std3-S through PRM were finally selected.

As a result, as can be seen in Figure 3, it was confirmed that the higher the expression of CAMP, CAD, LPCAT2, and STAT3, the lower the expression of ANXA13 and AKR1B1, the more conforming to the anticancer radiotherapy.

Through this, six biomarker candidate proteins that can predict the presence or absence of a response to anticancer radiotherapy were finally selected.

Example 4: Validation of 6 biomarker candidates

The six biomarker candidate groups were selected through Example 3, and from this, it was intended to finally derive a biomarker meaningful for clinical usefulness in locally advanced rectal cancer. To this end, it was attempted to confirm using a rectal cancer cell line and a radiation resistant rectal cancer cell line.

Specifically, the rectal cancer cell line SNU-503 and the radiation-resistant rectal cancer cell line SNU-503R50Gy were distributed from the Korea Cell Line Bank, and the usefulness of the candidate material was evaluated.

Experiment method

4-1: Cell Counting: Counting cells

Two cultured cell lines were irradiated with 0, 2, 4, 6 Gy of radiation, and then stained with trypan blue to determine the number of living cells, respectively. Through this, it is possible to confirm the radiation sensitivity of rectal cancer cell lines and radiation resistant rectal cancer cell lines.

4-2: Cell Viability Assay: Cell viability test

After irradiation of the two cultured cell lines at 4 Gy, the survival rate of the cells was confirmed using EZ-Cytox reagent after 0, 6h, 24h, 48h, and 72h. In addition, after 48 h after irradiation of the two cultured cell lines with 0, 2, 4, 6 Gy of radiation, the cell viability was confirmed using the EZ-Cytox reagent.

4-3: Western Blot Analysis: protein expression level

The two cultured cell lines were divided into 0 Gy and 4 Gy, irradiated, and cells were harvested after 0, 6h, 24h, 48h, and 72h, and 20ug of total protein was separated on an 8-12% SDS-PAGE gel. Expression was confirmed using antibodies of the derived candidate biomarkers CAMP, CAD, LPCAT2, ANXA13, AKR1B1, and STAT3.

Experiment result

First, as a result of confirming the sensitivity to radiation for the rectal cancer cell line SNU-503 and the radiation-resistant rectal cancer cell line SNU-503R80Gy, there is a difference in the cell viability of the two cell lines according to the radiation dose and time as shown in FIGS. 4 and 5. Was confirmed. Figure 4 shows the cell survival rate according to the radiation dose, Figure 5 shows the cell survival rate over time. For example, 48 h after irradiation of 4 Gy, the SNU-503 cell line was 46.9% and the SNU503-R80Gy cell line was 86.0%, confirming that it was a suitable cell line for this experiment.

Next, as a result of confirming the expression levels of six candidate proteins by Western Blot analysis in the rectal cancer cell line SNU-503 and the radiation-resistant rectal cancer cell line SNU-503R80Gy, CAMP and LPCAT2 were highly expressed in the SNU-503 cell line. In the case of AKR1B1, it was confirmed that the expression was high in the SNU-503R80Gy cell line (FIG. 6). It was confirmed that, in the case of CAMP and LPCAT2, when the protein expression level was higher than that of the normal control group, it was conformed to anticancer radiotherapy, and when it was low, it was confirmed that it was consistent with the result of Example 3, which was judged to be refractory to anticancer radiotherapy. In addition, in the case of AKR1B1, when the protein expression level was lower than that of the normal control group, it was confirmed that it conformed to the anticancer radiotherapy, and when it was higher, it was consistent with the result of Example 3, which was judged to be refractory to the anticancer radiotherapy.

However, in the case of the remaining three biomarkers (ANXA13, CAD, STAT3), the expression level between the two cell lines was similar, or rather, the pattern appeared opposite to the result of Example 3, indicating that it is not suitable as an actual biomarker. there was.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (12)

Anticancer radiotherapy comprising an agent capable of detecting the expression of one or more proteins selected from the group consisting of CAMP (human cathelicidin antimicrobial peptide), LPCAT2 (Lysophosphatidylcholine acyltransferase 2), and AKR1B1 (Aldo-keto reductase family 1, member B1) A composition for predicting whether a reaction to.
The method of claim 1, wherein the anti-cancer radiotherapy is performed before the patient's surgery in the anti-cancer treatment, the predictive composition.
The composition of claim 1, wherein the cancer is rectal cancer.
The method of claim 3, wherein the rectal cancer is locally advanced rectal cancer.
The method of claim 1, wherein the response to the anti-cancer radiotherapy is that cancer cells are lost in the tissue, the composition for prediction.
The method of claim 1, wherein CAMP (human cathelicidin antimicrobial peptide) and LPCAT2 (Lysophosphatidylcholine acyltransferase 2) are compliant with anticancer radiotherapy when the protein expression level is higher than that of the normal control group, and when low, refractory to anticancer radiotherapy. It is determined that the composition for prediction.
The method of claim 1, wherein AKR1B1 (Aldo-keto reductase family 1, member B1) is determined to comply with anticancer radiotherapy when the protein expression level is lower than that of the normal control group, and when it is high, it is judged to be refractory to anticancer radiotherapy. That is, the composition for prediction.
A kit for predicting a response to anticancer radiotherapy, comprising the composition of any one of claims 1 to 7.
Including the step of detecting the expression of one or more proteins selected from the group consisting of CAMP (human cathelicidin antimicrobial peptide), LPCAT2 (Lysophosphatidylcholine acyltransferase 2), and AKR1B1 (Aldo-keto reductase family 1, member B1) from a sample isolated from an individual. A method of providing information for predicting whether or not to respond to anticancer radiation therapy.
The method of claim 9, further comprising comparing the expression level of the protein with the expression level of the protein in a sample isolated from a normal control.
The method of claim 9, wherein CAMP (human cathelicidin antimicrobial peptide) and LPCAT2 (Lysophosphatidylcholine acyltransferase 2) are compliant with anticancer radiotherapy when the protein expression level is higher than that of the normal control group, and when low, refractory to anticancer radiotherapy. It is determined to be, the method of providing information.
The method of claim 9, wherein AKR1B1 (Aldo-keto reductase family 1, member B1) is determined to comply with anticancer radiotherapy when the protein expression level is lower than that of the normal control group, and when it is high, it is judged to be refractory to anticancer radiotherapy. How to provide information.

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