TWI619945B - Method for predicting survival time of a human subject with a cancer - Google Patents

Abstract

The present invention discloses a method for predicting the survival time of a human subject with a cancer after performing a surgical resection of the cancer, comprising dividing a biological sample taken from the human individual into a tumor portion and a non- a portion of the tumor; each of which is detected in the tumor portion and the non-tumor portion of the lncRNA AOC4P expression level; such that the lncRNA AOC4P expression level detected in the tumor portion is detected in the non-tumor portion Comparing, 俾 obtains a ratio; and predicting the survival time of the human individual based on the obtained ratio, wherein the ratio is positively correlated with an increased survival time.

Description

Used to predict the survival time of a human with a cancer Methods

The present invention relates to a method for predicting a survival time of a human subject with a cancer after performing a surgical resection of the cancer, comprising a biological entity obtained from the human individual The sample is divided into a tumor portion and a non-tumor portion; the LncRNA AOC4P expression level in the tumor portion and the non-tumor portion is separately detected; the LncRNA AOC4P expression level detected in the tumor portion is Comparing the detected ones in the non-tumor portion, 俾 obtains a ratio; and predicting the survival time of the human individual based on the obtained ratio, wherein the ratio is positively correlated with an increased survival time.

Cancer is one of the leading causes of human death today. Although cancer formation is still not fully understood, it is believed that carcinogenesis or tumorigenesis can be attributed to when cells accumulate exogenously or Endogenous When factors cause genetic abnormalities, the signal transduction pathways in these cells may be erroneous and cause cell division to be out of control, thereby causing the cells to gradually form cancer cells. Cancer cells are able to evade apoptosis and have the ability to migrate and invade, so cancer cells proliferate and pass through the lymphatic or vascular system. Transfer metastasize to other parts of the body.

Surgical resection is one of the main treatments for cancer, but the effect of treatment is not ideal, and the survival time (including survival-free survival time or overall survival time) ] may be limited by the recurrence and/or metastasis of cancer. Evaluation of the prognosis after surgical resection is useful for the physician to determine whether to use other treatments [eg, chemotherapy and radiotherapy] and to arrange for adjuvant therapy (adjuvant jherapy). ). Therefore, researchers in the field are looking for a reliable biomarker for assessing the prognosis after surgical resection.

Long non-coding RNA (lncRNA) is an RNA that falls between 200 bp and 100 kb and cannot be translated into a protein. In recent years, in human genomes, many lncRNAs have been found to predict the prognosis of cancer patients after surgery. For example, lncRNA ZEB1-AS1, lncRNA MVIH, lncRNA PCAT-1, lncRNA NEAT, and lncRNA HOTAIR (Yuan SX et al . (2012), Hepatology , 56: 2231-2241; Wu ZH et al . (2014), Oncol . Rep ., 32:395-402; Li Y. et al . (2015), Oncotarget , 6:27641-27650; Yan TH et al . (2015), Int.J.Clin.Exp . Pathol ., 8:4126 -4131; Li T. et al . (2015), Oncogene , doi: 10.1038/onc.2015.223). Therefore, these lncRNAs have become the focus of research and research in today's medical community in the search for biomarkers for the prognosis of cancer.

Copper-containing amine oxidases (AOCs) are copper dependent enzymes that catalyze the oxidation of many different endogenous amines. Human AOC genes include the following four types: (1) AOC1 gene, which encodes a diamine oxidase (DAO), which is mainly found in the kidneys, intestines, and lungs, and its function mainly involves histamine (histamine). Metabolism; (2) AOC2 gene, which encodes a retina-specific amine oxidase (RAO), which is a membrane protein with unknown physiological functions; (3) AOC3 Gene, which encodes a semicarbazide-sensitive amine oxidase (SSAO) [also known as vascular adhesion protein-1 (VAP-1)], which is in the lungs, The aorta, liver, and ileum are highly expressed, and their functions include leukocyte adhesion and glucose uptake; and (4) the AOC4 gene (also known as the AOC4P gene). ), which is considered to be a truncated and non-functional gene [also known as a pseudogene], which is transcribed into a 2073 bp lncRNA AOC4P (its RNA sequence can be seen) NCBI registration number NR_002 773.1).

Studies have been conducted to explore the association between AOC and cancer prognosis, and these studies focus on VAP-1. In Kaplan MA et al . (2014), Oncol. Res.Treat . , 37:340-344, in order to investigate the association between serum VAP-1 levels and the prognosis of gastric cancer, Kaplan MA et al analyzed 54 Serum VAP-1 levels before surgery with 32 patients with operable gastric cancer and 32 patients with metastatic gastric cancer before receiving chemotherapy -1 position, and found that patients with operable gastric cancer have a serum VAP-1 level that is significantly lower than patients with metastatic gastric cancer. Next, all patients were divided into 2 groups according to a cut-off value estimated by the receiver operating characteristic curve, and the 2 groups of patients were treated. The overall survival time afterwards. As a result, it was found that the survival time of a patient having a serum VAP-1 level lower than the cutoff value was significantly lower than that of a patient having a serum VAP-1 level higher than the cutoff value. Therefore, serum VAP-1 levels are considered to be useful for prognostic evaluation of gastric cancer.

Although the above-mentioned literature has been reported, as far as the Applicant is aware, no literature or patents have so far revealed the association of lncRNA AOC4P with cancer. Upon investigation, the applicant unexpectedly discovered that lncRNA AOC4P can be used as a biomarker for predicting a patient with a cancer [especially hepatocellular carcinoma (HCC)] after undergoing surgical resection of the cancer. Survival time.

Summary of invention

Accordingly, the present invention provides a method for predicting a survival time of a human subject with a cancer after performing a surgical resection of the cancer, comprising dividing a biological sample taken from the human individual into a tumor portion and a non-tumor portion; separately detecting the expression level of the lncRNA AOC4P in the tumor portion and the non-tumor portion; causing the lncRNA AOC4P expression level detected in the tumor portion to be detected in the non-tumor portion Compare the measured ones, get a ratio a value; and predicting the survival time of the human individual based on the ratio obtained, wherein the ratio is positively correlated with an increased survival time.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to find a reliable biomarker for assessing the prognosis after surgical resection, Applicants analyzed the lncRNA that was down-regulated in liver tumor tissue of patients with hepatocellular carcinoma and found that 43 lncRNAs were Down regulation, in which lncRNA AOC4P is down-regulated most prominently. Then apply Human analysis of liver tumor tissues of 108 patients and corresponding lncRNA AOC4P expression levels in adjacent normal liver tissues revealed that the expression level of lncRNA AOC4P in liver tumor tissues of 68% of patients was significantly reduced. The applicant further analyzed the correlation between the ratio of the lncRNA AOC4P expression level in the liver tumor tissue of the patient relative to the corresponding adjacent normal liver tissue and the survival time after the surgical resection of the liver tumor. The analysis found that if the ratio is higher, the overall survival time of the patient is longer.

Accordingly, the present invention provides a method for predicting the survival time of a human subject with a cancer after performing a surgical resection of the cancer, comprising: dividing a biological sample taken from the human individual into a tumor portion And a non-tumor portion; separately detecting the expression level of the lncRNA AOC4P in the tumor portion and the non-tumor portion; allowing the lncRNA AOC4P expression level detected in the tumor portion to be in the non-tumor portion Comparing the detected ones to obtain a ratio; and predicting the survival time of the human individual based on the obtained ratio, wherein the ratio is positively correlated with an increased survival time.

According to the invention, the survival time is disease free survival time or overall survival time.

As used herein, the term "disease-free survival time" means the time from the date of surgical resection of a cancer to the date of death of the cancer or any cause of death.

As used herein, the term "overall survival time" means the time from the date of surgical resection of a cancer to the date of death caused by any cause.

According to the present invention, the cancer is selected from the group consisting of hepatocellular carcinoma, oral cancer, colorectal cancer, lung cancer, and breast cancer. In a preferred embodiment of the invention, the cancer is hepatocellular carcinoma.

In accordance with the present invention, the biological sample can be removed from the human subject by methods well known in the art including, but not limited to, surgical resection, biopsy, aspiration, and peritoneum. Peritoneoscopy. In a preferred embodiment of the invention, the biological sample is tissue removed by surgical resection.

In accordance with the present invention, the lncRNA AOC4P expression level can be detected by any method known to those of ordinary skill in the art for quantifying lncRNA AOC4P. Preferably, the lncRNA AOC4P expression level is detected using at least one of the following methodologies to quantify lncRNA AOC4P: hybridization and polymerase chain reaction. The setting of the operating conditions for these methodologies and the selection of reagents fall within the professional literacy and routine technology of those skilled in the art.

The invention also provides a kit for predicting the survival of a human subject with a cancer after undergoing a surgical resection of the cancer, comprising an agent for quantifying the level of expression of the lncRNA AOC4P.

The prediction of survival time is useful in deciding whether a more conservative or more thorough treatment should be used, or whether different treatments should be combined, and therefore the method according to the invention is expected to be useful for determining a patient with cancer. The course of treatment. As used herein, the term "treatment procedure" means the treatment that is taken after the diagnosis of the cancer by the patient and the span of time at which the patient is treated.

Other features and effects of the present invention will be apparent from the following description of the drawings, wherein: Figure 1 shows the relative performance levels of lncRNA AOC4P in 108 patients with hepatocellular carcinoma; and Figure 2 shows The Kaplan-Meier curve of the overall survival time of the high relative level group and the low relative level group within 160 months after the surgical resection.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example

Experimental individuals:

The subjects involved in the study were 108 patients with hepatocellular carcinoma (HCC) who underwent surgery for liver tumors at Chang Gung Memorial Hospital between 2000 and 2012. Surgical resection, while their liver tumor tissue and adjacent normal liver tissue were obtained and stored in the Tissue Bank of Linkou Chang Gung Memorial Hospital. The clinical information of these patients (such as gender, age, duration of disease, pathological features, and whether there is recurrence or death of hepatocellular carcinoma within 160 months after the surgical resection) are recorded in their medical records. In addition, the study was approved by the ethics committee of Chang Gung Memorial Hospital and all patients who participated in the study were informed of their consent.

General experimental method:

1. Quantitative real-time polymerase chain reaction (qRT-PCR):

In the examples below, total RNA from each tissue sample was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To remove any contaminating genomic or plastid DNA, the RNA samples were processed in a DNase without RQ1 RNase (Promega, Madison, WI, USA) according to the manufacturer's protocol. 2 μg of these treated RNA samples were quantified by real-time polymerase chain reaction using a TaqMan non-coding RNA assay (Applied Biosystems) to detect long non-coding RNA (long non-coding RNA, lncRNA) expression; GAPDH was used as an internal control group.

2. Statistical analysis:

In the following examples, all statistical analyses were performed using SPSS 16.0 and Excel 2007 software. All statistical tests are two-sided, and the threshold for significance is set at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Example 1. Screening and Identification of Down-regulated lncRNAs in Hepatocellular Carcinoma Tissues

A, lncRNA screening and identification:

In order to confirm that lncRNA is involved in the progression of hepatocellular carcinoma, total RNA is isolated from liver tumor tissues of three patients and corresponding adjacent normal liver tissues, and then the isolated total RNA is used by using Ambion ^® WT exhibit a kit ^{(Ambion ® WT expression Kit) (} Applied Biosystems) and according to the manufacturer's protocol and is used to generate cDNA microarray analysis ^{(microarray analysis) (Affymetrix GeneChip ®} Human Gene 2.0). A fold-change greater than 2 is set as a threshold for gene expression differences. Using this threshold, the applicant detected 41 upregulated and 43 down-regulated lncRNAs in these liver tumor tissues compared to the corresponding adjacent normal liver tissue.

To validate these findings, Applicants selected 10 lncRNAs (including AOC4P) that showed a fold change greater than 4 and their performance was in accordance with item 1 "Quantitative Real-Time Polymerase Chain Reaction" in the "General Experimental Methods" above. The method was analyzed in liver tumor tissues of 15 patients and corresponding adjacent normal liver tissues [lncRNA AOC4P is expressed by using the AOC4P expression assay kit (Thermo Fisher Scientific, Cat) .No.4426961) was analyzed]. The experimental results showed that lncRNA AOC4P was down-regulated in all liver tumor tissues.

B. Analysis of the expression level of lncRNA AOC4P in liver tumor tissues and normal liver tissues:

To investigate the biological significance of lncRNA AOC4P downregulation for hepatocellular carcinoma (biological Significance), the applicant analyzed the liver tumor tissue of the 108 patients and the corresponding lncRNA AOC4P in the adjacent normal liver tissue according to the method described in Item 1 "Quantitative Instant Polymerase Chain Reaction" of the "General Experimental Methods" above. The performance level was calculated, and the ratio of the lncRNA AOC4P expression level in the liver tumor tissue of each patient to the corresponding adjacent normal liver tissue (hereinafter referred to as the relative performance level of lncRNA AOC4P) was calculated.

Figure 1 shows the relative performance levels of lncRNA AOC4P in 108 patients with hepatocellular carcinoma. As can be seen from Figure 1, the performance level of lncRNA AOC4P in liver tumor tissues was significantly reduced to 10% in 68% of patients (73/108) ( p < 0.0001) compared with normal liver tissue. Up to 90%. Applicants believe that the relative performance level of lncRNA AOC4P may be related to the survival time of patients after surgical resection of liver tumors.

Example 2. Correlation between the relative performance level of lncRNA AOC4P and survival time after surgical resection of liver tumors

A. Preliminary analysis:

First, the patients were divided into the following two groups according to the medical records of 108 patients: (1) survival group (n=44), these patients were alive within 60 months after the surgical resection. And (2) the death group (n=46), who died within 60 months of the surgical resection. The remaining 18 patients were not included in the group because they lost track before they died. The phase of lncRNA AOC4P in these two groups of patients A preliminary comparison of performance levels was performed, and it was found that the relative performance level of lncRNA AOC4P in the survival group was generally higher than that in the death group, and the relative performance level of lncRNA AOC4P was generally positive with increased survival time. Correlation.

Next, you can use Youden's index analysis (Youden WJ (1950), Cancer, 3:32-35) to determine the best relative position of lncRNA AOC4P for distinguishing the two groups of patients. The optimal cut-off value. The analysis found that the optimal cutoff value was 0.86.

B. Kaplan-Meier survival analysis:

To investigate changes in survival over time after surgical resection, the applicant divided the 108 patients into the following two groups based on the results of the analysis in item A above: (1) High relative level group (n= 41), the relative performance level of lncRNA AOC4P in these patients was greater than or equal to 0.86; and (2) the low relative level group (n=67), and the relative performance level of lncRNA AOC4P in these patients was less than 0.86. Next, Kaplan EL and Meier P. (1958), Journal of the American Statistical Association, 53:457-481 was used to examine the two groups of patients within 160 months after the surgical resection. The overall survival time and the Breslow test were used for statistical analysis to assess the difference between the two groups. The results obtained are shown in Figure 2.

As can be seen from Figure 2, the high relative level group has a longer overall survival time compared to the low relative level group, in particular, the overall survival time of approximately 50% of patients is greater than 73 months.

In addition, the analysis results of the Bresrov test are summarized in Table 1 below. As can be seen from Table 1, the average overall survival time of the high relative level group is significantly higher than that of the low relative level group.

Based on the above experimental results, the applicant believes that lncRNA AOC4P can be used as a biomarker for predicting the survival time of a patient with HCC after undergoing surgical resection of a liver tumor.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Claims (4)

A method for predicting survival time of a human subject with a cancer after performing a surgical resection of the cancer, comprising: dividing a biological sample taken from the human individual into a tumor portion and a non-tumor portion Individually detecting the level of expression of the lncRNA AOC4P in the tumor portion and the non-tumor portion; causing the level of the lncRNA AOC4P detected in the tumor portion to be detected in the non-tumor portion Comparing, 俾 to obtain a ratio; and predicting the survival time of the human individual based on the ratio obtained, wherein the ratio is positively correlated with an increased survival time; wherein the cancer is hepatocellular carcinoma. The method of claim 1, wherein the survival time is disease-free survival time. The method of claim 1, wherein the survival time is an overall survival time. The method of claim 1, wherein the 1ncRNA AOC4P expression level is detected by quantifying lncRNA AOC4P using at least one of the following methodologies: hybridization and polymerase chain reaction.