A PLEC mutant gene and its application in gastric neuroendocrine carcinoma

Abstract

The present invention provides for the first time a specific human PLEC mutant gene, which is formed by the g.1377-1380delATGT mutation of the wild-type PLEC gene, and its specific sequence is shown in SEQ ID NO: 1. The gene mutation can be used as a molecular marker for evaluating the efficacy of G-NEC chemotherapy. In addition, the present invention also provides a PLEC mutant gene detection kit and detection method based on digital PCR, including digital PCR primers as shown in SEQ ID NO: 3-4 sequences and digital PCR probes as shown in SEQ ID NO: 7-8, which can achieve sensitive, specific, efficient, comprehensive, rapid and accurate mutation detection, provide effective support for the application of PLEC mutant genes in the evaluation of the efficacy of G-NEC chemotherapy, and will help to accurately evaluate the efficacy of G-NEC chemotherapy and monitor the condition in order to guide the formulation and timely adjustment of treatment plans.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection and application, and specifically relates to PLEC mutant gene, PLEC mutant gene detection and its application in evaluating the chemotherapy efficacy of gastric neuroendocrine carcinoma. Background Art

[0002] Neuroendocrine neoplasms (NENs) are a group of tumors that originate from peptidergic neurons and neuroendocrine cells. They can occur in many organs and tissues throughout the body, but are more common in the gastrointestinal tract and pancreas. Gastroenteropancreatic NENs (GEP-NENs) are the most common type of NENs, accounting for as high as 65%-75%. Although the overall incidence of neuroendocrine tumors is low, epidemiological surveys have shown that the incidence of neuroendocrine tumors has increased by about 5 times in the past 30 years. Neuroendocrine carcinoma (NEC) does not manifest itself in the early stages, but it is highly heterogeneous, poorly differentiated, and highly malignant. When diagnosed, it is mostly local progression or distant metastasis, so the prognosis is poor. Data show that the median survival of patients in the local advanced stage is 16 months, while the median survival of NEC patients with distant metastasis is only 5 months.

[0003] Chemotherapy is an important part of the comprehensive treatment of GEP-NEN and the main treatment for advanced patients. Taking G-NEC patients as an example, the survival of metastatic G-NEC patients receiving chemotherapy varies greatly (from 7 months to 19 months), but there is a substantial improvement compared with patients who only receive best supportive care (1 month). At present, there is no standard unified chemotherapy regimen for G-NEC in clinical practice. Cisplatin combined with etoposide regimen (ie, EP regimen) is the most widely used chemotherapy regimen for G-NEC, with a remission rate of up to 30% and a median survival of about 1 year. At present, the clinical evaluation of the efficacy of G-NEC chemotherapy mainly relies on conventional imaging methods and serological indicators. However, imaging methods often have a certain lag and sometimes cannot accurately reflect the molecular nature of the cancer lesions; and serum markers are generally not specific, and their monitoring has limited value for the prognosis and diagnosis and treatment guidance of G-NEC.

[0004] The occurrence and development of cancers, including G-NEC, involve temporal mutations of multiple genes. It is a complex disease with multiple genes, multiple pathways and multiple stages. As a result, the occurrence and development mechanisms of most cancers have not been fully analyzed. Different molecular characteristics not only determine the growth, progression, invasion and metastasis of cancer, but also determine its response to specific treatment methods (including chemotherapy), that is, the prognosis of patients. Since molecular characteristics change earlier than cell tissue and imaging morphology, they have good timeliness and predictive value. Therefore, finding sensitive and specific tumor molecular markers has positive clinical significance for early evaluation of chemotherapy efficacy, monitoring disease progression and timely replacement of drug resistance regimens. At present, there is a lack of accurate and effective molecular markers for G-NEC chemotherapy efficacy, and previous G-NEC cases and samples are a good entry point. There are a large number of previous G-NEC cases with rich samples, and their treatment information and clinical outcomes are known; in addition, with the application of second-generation DNA sequencing technology, long-term preserved clinical samples, especially pathological paraffin blocks, can also be well analyzed. Therefore, conducting reasonable grouping analysis based on previous cases will help to quickly screen important gene mutations related to the occurrence and development of cancer, and then find specific and effective clinical molecular markers. Summary of the invention

[0005] In view of the lack of specific and effective molecular markers for evaluating the efficacy of chemotherapy for gastric neuroendocrine carcinoma in the clinic, the present invention provides a specific mutation of the PLEC gene and a detection method thereof, and applies it to the evaluation of the efficacy of chemotherapy for gastric neuroendocrine carcinoma. These can provide a basis and foundation for evaluating the efficacy of chemotherapy for gastric neuroendocrine carcinoma, monitoring disease progression, adjusting treatment plans, and analyzing the occurrence and development mechanism.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] The first object of the present invention is to provide a human PLEC mutant gene with a g.1377-1380delATGT mutation, wherein the mutant gene is formed by the deletion of ATGT at the 1377-1380 position of the wild-type PLEC gene, and the wild-type PLEC gene sequence within the range of 143915153 to 143976745 of NCBI accession number NC_000008.11 is used as a reference sequence, and the gene fragment sequence where the g.1377-1380delATGT mutation is located is shown in SEQ ID NO: 1. The g.1377-1380delATGT mutation of the PLEC gene in cancer cells of patients with gastric neuroendocrine carcinoma can be used as a molecular marker for evaluating the efficacy of chemotherapy.

[0008] The second object of the present invention is to provide a human PLEC mutant gene with a g.38110-38115delATCAAG mutation, wherein the mutant gene is formed by the deletion of ATCAAG at the 38110-38115 position of the wild-type PLEC gene, and the wild-type PLEC gene sequence within the range of 143915153 to 143976745 of NCBI accession number NC_000008.11 is used as a reference sequence, and the gene fragment sequence where the g.38110-38115delATCAAG mutation is located is shown in SEQ ID NO: 2. The g.38110-38115delATCAAG mutation of the PLEC gene in cancer cells of patients with gastric neuroendocrine carcinoma can be used as a molecular marker for evaluating the efficacy of chemotherapy.

[0009] The third object of the present invention is to provide a detection kit for detecting a human PLEC mutant gene with a g.1377-1380delATGT mutation, the kit comprising a digital PCR primer pair as shown in SEQ ID NO: 3-4, a digital PCR probe targeting the g.1377-1380delATGT mutation site as shown in SEQ ID NO: 8, and a corresponding digital PCR probe targeting the wild-type site as shown in SEQ ID NO: 7. The 5'-end nucleotide of the probe shown in SEQ ID NO: 7 is modified with a fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group; similarly, the 5'-end nucleotide of the probe shown in SEQ ID NO: 8 is modified with another fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group.

[0010] The fourth object of the present invention is to provide a detection kit for detecting a human PLEC mutant gene with a g.38110-38115delATCAAG mutation, the kit comprising a digital PCR primer pair as shown in SEQ ID NO: 5-6, a digital PCR probe targeting the g.38110-38115delATCAAG mutation site as shown in SEQ ID NO: 9, and a corresponding digital PCR probe targeting the wild-type site as shown in SEQ ID NO: 10. The 5'-end nucleotide of the probe shown in SEQ ID NO: 9 is modified with a fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group; similarly, the 5'-end nucleotide of the probe shown in SEQ ID NO: 10 is modified with another fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group.

[0011] Objective 5 of the present invention is to provide a detection method for detecting the human PLEC gene fragment, targeting the g.1377-1380delATGT mutation, using a digital PCR primer pair as shown in SEQ ID NO: 3-4, a digital PCR probe targeting the g.1377-1380delATGT mutation site as shown in SEQ ID NO: 8, and a corresponding digital PCR probe targeting the wild-type site as shown in SEQ ID NO: 7, and the corresponding detection conditions are: pre-denaturation at 96°C for 6 minutes; denaturation at 94°C for 15 seconds, annealing and extension at 56°C for 30 seconds, and 40 cycles; final reaction at 98°C for 10 minutes. For the g.38110-38115delATCAAG mutation, a digital PCR primer pair as shown in SEQ ID NO: 5-6, a digital PCR probe targeting the g.38110-38115delATCAAG mutation site as shown in SEQ ID NO: 10, and a corresponding digital PCR probe targeting the wild-type site as shown in SEQ ID NO: 9 were used. The corresponding detection conditions were: pre-denaturation at 96°C for 6 min; denaturation at 94°C for 15 s, annealing and extension at 57°C for 30 s, and 40 cycles; final reaction at 98°C for 10 min.

[0012] The two PLEC mutant genes of g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation provided by the present invention can be used as molecular markers for evaluating the efficacy of G-NEC chemotherapy, and can also provide a basis and foundation for the analysis and research and development of the mechanism of action of G-NEC chemotherapy drugs. In addition, the present invention also provides a PLEC mutant gene detection kit and detection method based on digital PCR, which can be applied to the detection of samples with low content and severe fragmentation such as ctDNA and FFPE genomes, to achieve sensitive, specific, efficient, comprehensive, rapid and accurate mutation detection, and provide effective support for the application of PLEC mutant genes in the evaluation of the efficacy of G-NEC chemotherapy, which will help to accurately evaluate the efficacy of G-NEC chemotherapy and monitor the disease in order to guide the formulation and timely adjustment of treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Clustering map of high-frequency gene mutations in 23 G-NEC patients.

[0014] Figure 2 Sanger sequencing peak diagram of the PLEC mutant gene at the g.1377-1380delATGT mutation site. The arrow in the figure indicates the site where the mutation occurs, and the underlined bold sequence ATGT is the mutation region.

[0015] Figure 3Sanger sequencing peak diagram of the PLEC mutant gene at the g.38110-38115delATCAAG mutation site. The arrow in the figure indicates the site where the mutation occurs, and the underlined bold sequence ATCAAG is the mutation region.

[0016] Figure 4 Growth curves of gastric cancer cell lines under the action of EP chemotherapy drugs. The growth of gastric cancer cell lines without PLEC gene mutations in the absence of EP chemotherapy drugs was used as the benchmark, where series 1 is the relative growth curve of gastric cancer cell lines without PLEC gene mutations, series 2 is the relative growth curve of gastric cancer cell lines with PLEC mutation genes containing g.1377-1380delATGT mutations, and series 3 is the relative growth curve of gastric cancer cell lines with PLEC mutation genes containing g.38110-38115delATCAAG mutations. DETAILED DESCRIPTION

[0017] At present, there is no standard unified chemotherapy regimen for G-NEC in clinical practice. Cisplatin combined with etoposide (EP regimen) is the most widely used chemotherapy regimen for G-NEC, but its remission rate can only reach 30%. Due to the high heterogeneity and individual differences of G-NEC, different patients respond differently to EP-based chemotherapy regimens and benefit differently. In addition to patient factors, the main reason for the difference in efficacy is the difference in tumors at the molecular level, especially at the genetic level. The efficacy of chemotherapy is closely related to the molecular type of G-NEC. How to analyze the cancer gene feature spectrum of G-NEC patients and find specific molecular markers to guide the selection of chemotherapy regimens, disease progression monitoring and timely replacement of chemotherapy regimens has become very important. Previous cases are a valuable resource library with a large number of known clinical processes and outcomes. Therefore, we can try to analyze the clinical information and pathological samples of previous cases, use the second-generation DNA sequencing technology to analyze the molecular feature spectrum of the cancer lesion itself at the genomic level, and combine clinical progress and benefit information to screen specific and effective molecular markers for chemotherapy prognosis.

[0018] The present invention is further described below in conjunction with specific embodiments.

[0019] Example 1 Screening of candidate molecular markers related to G-NEC chemotherapy efficacy

[0020] The hospital's previous case data on gastrointestinal neuroendocrine carcinoma were systematically sorted out and combined with the retained samples, and 23 patients with postoperative recurrence or metastasis of gastric neuroendocrine carcinoma (confirmed by clinical pathology) were selected. Their postoperative paraffin block samples were intact and the clinical information was complete. Most of these patients were treated with EP-based chemotherapy regimens (cisplatin combined with etoposide). According to the chemotherapy efficacy evaluation results based on traditional clinical imaging combined with survival, they were divided into two groups, one group was the chemotherapy benefit group and the other group was the chemotherapy non-benefit group. The relevant information is shown in Table 1. The evaluation of chemotherapy efficacy in previous cases was mainly based on the results of imaging examinations (mainly computed tomography, CT), and then evaluated according to the solid tumor efficacy evaluation criteria (RECIST 1.1) at that time. The results of efficacy evaluation are divided into complete response (CR: all target lesions completely disappear), partial response (PR: the sum of the longest diameters of target lesions decreases by at least 30% compared with the baseline state), progressive disease (PD: the sum of the longest diameters of target lesions increases by at least 20% compared with the sum of the longest diameters of the smallest target lesions recorded after the start of treatment; or one or more new lesions appear), and stable disease (SD, between PR and PD).

[0021] There were 9 patients in the chemotherapy benefit group, including 5 male patients and 4 female patients, with an average age of 52.4 years. These patients had at least one PR and / or CR lasting more than 2 months during chemotherapy, and the survival period from recurrence and metastasis was 8-34 months (average 17.5 months); while there were 14 patients in the chemotherapy non-benefit group, including 8 male patients and 6 female patients, with an average age of 51.8 years. These patients were always SD and / or PD during chemotherapy, and the survival period from recurrence and metastasis was 3-14 months (average 8.2 months). As shown in Table 1, the male-female ratio and average age of the two groups of patients were similar, but there was a significant difference in survival, which was more than double, and their genetic profiles may be significantly different. The differences in the characteristic profiles of the two groups at the genomic level were further analyzed through cancer pathology samples to screen candidate molecular markers related to chemotherapy efficacy.

[0022] Table 1 Grouping and gene mutation information of 23 G-NEC patients

[0023]

[0024] Pathological paraffin block specimens of these 23 G-NEC patients were obtained, and at least 6 paraffin sections were cut from the specimens at the cancer site (i.e., FFPE sample sections, length, width and thickness of 1cm×1cm×10um, and the area of ​​the cancer site occupied at least half of the section area); at the same time, paired adjacent cancer tissues as far away from the cancer site as possible were selected and cut into at least 10 paraffin sections (FFPE sections were 1cm×1cm×10um in length, width and thickness, and the area of ​​normal tissue occupied at least 30% of the section area, and only the normal tissue part was taken). The QIAamp DNA FFPE Tissue Kit of Qiagen Company of Germany was used to extract genomic DNA from FFPE sections. RNase was added to completely remove RNA, and the product was tested by Qubit4.0, etc., requiring the OD 260 / 280 value to be between 1.8-2.0, the minimum concentration to be not less than 50ng / μl, and the total amount to be not less than 3ug. All the samples met the quality standards and were sent to a gene company for next-generation sequencing (NGS) analysis. Whole exome sequencing (WES) was selected. Exons are the protein coding regions of genes. All exons in the genome are called exomes. The human genome contains about 1.8×10 5 Exons, with a total of 30Mb of bases, account for 1% of the human genome. More than 90% of human disease gene mutations occur in the exon region and the non-coding region around the exons. For cancer, a disease with cumulative multi-gene mutations, the exome is the preferred region for mining specific molecular biomarkers. Therefore, sequencing and analyzing the whole exome, which only accounts for 1% of the genome, is a very cost-effective method.

[0025] For whole exome sequencing, the SureSelect Human All Exon V7 kit (Agilent) was used to construct the sequencing library, which fully covers the gene protein coding regions in major databases such as RefSeq, CCDS and GENCODE. During the library construction process, the fragmented genomic DNA was first blunt-ended, and a phosphate group was added to the 5' end and a PolyA tail was added to the 3' end. Then, the adapter sequence was added to both ends of the fragment through TA connection, and after a round of PCR amplification, it became a complete fragment library. Then, these DNA fragments were hybridized with the capture chip to obtain the enriched target fragments; then the target fragments were randomly connected to long-chain DNA fragments, and then randomly interrupted again and connected to sequencing adapters at both ends, and then PCR amplification was performed using the sequence matching the adapter as primer to obtain the sequencing library. Qubit4.0 was first used to perform preliminary quantification of the sequencing library, and then the Insert size of the library was detected, and then the effective concentration of the library was accurately quantified using qPCR to ensure the quality of the gene library. The valid library was sequenced on a HiSeq 4000 sequencer (Illumina, USA) using the PE150 (Pair-End 150) sequencing method, requiring the average coverage depth to be no less than 250× (cancer samples) and 100× (adjacent cancer samples).

[0026] The FASTQ raw data obtained by SureSelect Human All Exon V7 capture kit was used, and the software provided by the sequencer was used to remove poor quality reads and adapters. The clean reads were then aligned to GRCh38 (human genome reference sequence) using BWA software (Burrows-Wheeler Aligner). GATK (GenomeAnalysis Toolkit), VarScan251 and ANNOVAR software were used to analyze and annotate somatic DNA variations (the p value for mutation identification was 0.1, and the minimum quality score was 15). Databases such as dbSNP, 1000 Genomes and ESP6500 were then used to exclude polymorphic sites and determine mutation site information, and high-frequency mutations were selected for Sanger sequencing verification. The WES sequencing results showed (Table 1) that for the sequencing samples of 23 G-NEC patients, an average of 25.7 high-frequency gene mutations were detected (ranging from 8 to 76 genes, involving a total of 83 genes, with a mutation frequency >1%, and sequencing reads >200), and each gene may have multiple mutation types.

[0027] Systematic analysis and comparison of high-frequency gene mutations showed that no high-frequency gene mutation was found in all 9 G-NEC patient samples in the chemotherapy benefit group, nor was there a high-frequency gene mutation in all 14 G-NEC patient samples in the chemotherapy non-benefit group. Therefore, there is no high-frequency gene mutation that only exists in the samples of patients in the chemotherapy benefit group but not in the samples of patients in the chemotherapy non-benefit group, and correspondingly, there is no high-frequency gene mutation that only exists in the samples of patients in the chemotherapy non-benefit group but not in the samples of patients in the chemotherapy benefit group. This shows that the heterogeneity of G-NEC is high, and even in the same clinical category (such as the chemotherapy benefit group), there may be multiple molecular groups with certain individual differences. Therefore, relying solely on the grouping of clinical efficacy may not be able to analyze the similarities and differences of cancer foci at the molecular group level. Since high-frequency gene mutations can represent the gene characteristic spectrum of cancer samples, the molecular nature and type of cancer can be reflected based on the gene characteristic spectrum. The grouping analysis of cancer molecular groups will be more helpful to analyze the molecular basis behind the differences in clinical treatment efficacy. Therefore, based on the detected high-frequency gene mutations, cluster analysis was performed using software such as DEseq2, and the results are as follows: Figure 1 As shown. Based on the high-frequency gene mutation data, 23 G-NEC patients were clustered into three categories, among which 6 patients in the chemotherapy benefit group were clustered into category I ( Figure 1 1-6 in the figure, for the convenience of presentation, the patient numbers have been re-regularized to some extent), 3 patients in the chemotherapy benefit group ( Figure 1 7-9) and 9 patients who did not benefit from chemotherapy ( Figure 1 10-14 and 20-23) were clustered into Class II, and 5 patients who did not benefit from chemotherapy were clustered into Class III ( Figure 1 15-19 of ).

[0028] Specific analysis found that the cancer samples of the six chemotherapy benefit group patients in category I all contained PLEC gene mutations, involving two PLEC gene mutations, g.1377-1380delATGT and g.38110-38115delATCAAG (verified by Sanger sequencing, the Sanger sequencing peak of the g.1377-1380delATGT mutation is shown in Figure 2). Figure 2 As shown, the Sanger sequencing peak diagram of the g.38110-38115delATCAAG mutation is as follows Figure 3As shown in SEQ ID NO: 1, and SEQ ID NO: 2, the sequences of the PLEC mutant gene fragments with g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The PLEC mutant gene with g.1377-1380delATGT mutation is formed by the deletion of ATGT at the 1377-1380 position of the wild-type PLEC gene (the first nucleotide of the PLEC wild-type gene is the starting position, i.e., the first position), and the PLEC mutant gene with g.38110-38115delATCAAG mutation is formed by the deletion of ATCAAG at the 38110-38115 position of the wild-type PLEC gene (the first nucleotide of the PLEC wild-type gene is the starting position, i.e., the first position). The sequence of the wild-type PLEC gene is the gene fragment within the range of 143915153 to 143976745 of NCBI accession number NC_000008.11, with a total length of 61593 bp. This is used as the reference sequence of the PLEC wild-type gene. The remaining sequences of the PLEC mutant genes with g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation refer to the PLEC wild-type gene reference sequence (gene fragment within the range of 143915153 to 143976745 of NCBI accession number NC_000008.11).

[0029] The above two gene mutations (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation of PLEC gene) were not present in the cancer samples of the remaining 17 patients (including 14 patients in the chemotherapy-unbenefited group). Literature review revealed that PLEC gene mutations are closely related to the function of mitochondria. PLEC gene mutations will cause cancer cells to be under stress, such as when chemotherapy drugs are acting, and the stress amplification of cancer cell mitochondria will be inhibited, and the mitochondrial energy effect cannot be fully exerted, which will lead to the inability of cancer cells to resist and escape the killing effect of chemotherapy drugs. Therefore, it is speculated that PLEC gene mutations are potential prognostic molecular markers for chemotherapy in G-NEC patients.

[0030] Example 2 Expression of PLEC mutant gene in G-NEC cancer cells and analysis of drug resistance of gastric cancer cell lines

[0031] Furthermore, G-NEC patients who were scheduled for surgical treatment were collected, and fresh cancerous and adjacent tissues (not less than 0.1 g) were immediately obtained within 1 h after surgery. Fresh postoperative genomic DNA and RNA were extracted using conventional tissue genomic DNA and RNA extraction kits (Beijing Tiangen) according to the instructions, and RNA was reverse transcribed to obtain cDNA (Beijing Tiangen Quant cDNA first-strand synthesis kit, random primers). For the above two PLEC gene mutations (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation), the extracted genomic DNA was used as a template to design primer pairs as shown in SEQ ID NO: 3-6 for amplification (see Table 2 for specific corresponding relationships), and 2×Taq PCR Mastermix (Beijing Tiangen Company) was used to prepare the amplification system according to the instructions. The amplification conditions are shown in Table 2. The specific amplification product electrophoresis bands were detected by agarose gel electrophoresis and sent to Shanghai Biotech for Sanger sequencing. At the same time, digital PCR primers and probes were designed (the relevant primers and probes for digital PCR are shown in SEQ ID NO: 3-10, and the specific corresponding relationship is detailed in Table 2). Since PLEC gene mutation will lead to the loss of gene function, and thus fail to activate the amplification of mitochondrial copy number under stress conditions (preoperative chemotherapy and other treatments), the obtained cDNA was used as a template, and only digital PCR was used to detect the expression of normal PLEC gene, and the wild-type gene probes used were SEQ ID NO: 7 and SEQ ID NO: 9. In addition, according to the literature, the extracted genomic DNA was used as a template, and the Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara) was used to detect the copy number of mitochondrial DNA by real-time fluorescence quantitative PCR according to the method in the kit manual. In addition, in order to explore the impact of PLEC gene mutations on gene expression in cancer cells, the extracted RNA was sent to a gene company for RNA-Seq analysis. The results showed that since the g.1377-1380delATGT mutation and the g.38110-38115delATCAAG mutation both occurred at the junction of exons and introns, the RNA of the PLEC gene was detected to have undergone variable splicing in cancer cells with these two mutations, resulting in the loss of associated exons, which affected the function of the gene.

[0032] Table 2 PLEC gene mutation amplification system and conditions

[0033]

[0034] Results Among the 19 patients' cancer samples collected, 9 were found to contain the g.1377-1380delATGT mutation or g.38110-38115delATCAAG mutation of the PLEC gene, and 10 were found to not contain these two mutations of the PLEC gene. The specific information is shown in Table 3. The average expression of the wild-type PLEC gene in cancer samples with PLEC gene mutations was significantly lower than that in paired paracancerous tissue samples (the average difference was more than 20 times), and the corresponding mitochondrial DNA copy number was also significantly lower than that in paired paracancerous tissue samples (the average difference was more than 50 times). However, the expression of the wild-type PLEC gene and the mitochondrial DNA copy number in cancer samples without PLEC gene mutations were not significantly different from those in paracancerous tissues, and were significantly higher than those in cancer samples with PLEC gene mutations. It can be seen that due to the mutation of the PLEC gene in cancer cells, the expression of the functional wild-type PLEC gene is significantly reduced, which in turn leads to the inability to greatly expand the mitochondrial DNA copy number under stress. The mitochondrial copy number is at a very low level and cannot maintain and provide the energy required for cancer cell metabolism and stress response. Therefore, it is easy to be sensitive to chemotherapy drugs. Therefore, the PLEC gene mutation in G-NEC patients is an important genetic marker for the efficacy of chemotherapy, and the presence of PLEC gene mutation can indicate benefit from chemotherapy.

[0035] Table 3 Detection of PLEC gene mutation and expression in fresh postoperative tissue samples

[0036]

[0037] Further, verification was performed at the cellular level. The above analysis shows that G-NEC cancer cells containing specific mutations in the PLEC gene (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation) are sensitive to chemotherapy drugs. Therefore, cancer cell lines containing these two PLEC gene mutations were constructed for chemotherapy drug resistance testing, and EP chemotherapy drugs were selected. Since there are currently no mature commercial G-NEC cancer cell lines, the typical gastric cancer cell line SGC-7901 (purchased from Beijing Zhongyuan Company) was selected and cultured and passaged according to the conditions of the instructions. Sanger sequencing verified that the gastric cancer cell line did not contain the g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation of the PLEC gene. Beijing Saibei Biotechnology Co., Ltd. was commissioned to construct a knock-in gastric cancer cell line with PLEC gene mutations using the CRISPR / Cas9 method. The KI gastric cancer cell line with positive PLEC gene mutation and the original gastric cancer cell line (without PLEC gene-related mutation) were selected and divided into two groups. The cell concentration was adjusted to 1×10 6 / ml, according to the EP chemotherapy regimen, a certain amount of cisplatin (final concentration of 3ng / ml) and etoposide (final concentration of 5ng / ml) were added, and no chemotherapy drugs were used as the drug-free control group. The cell concentration was measured every 24h by flow cytometry (BD Company, USA). Results ( Figure 4 ) found that compared with the original gastric cancer cell line without PLEC gene (added EP chemotherapy drug) in the negative control group without chemotherapy drugs, the number of gastric cancer cell lines containing PLEC gene mutations (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation) constructed by KI decreased rapidly to near zero under the action of chemotherapy drugs, and the rate of decrease was much higher than that of the original gastric cancer cell line without PLEC gene. This indicates that the cancer cell lines containing these two PLEC gene mutations are sensitive to chemotherapy drugs, indicating that the g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation of the PLEC gene can be used as molecular markers for evaluating the efficacy of chemotherapy.

[0038] Example 3 Clinical Application Analysis

[0039] Cancer tissue samples obtained by surgery can be directly used as the object of molecular detection, but G-NEC patients who are undergoing chemotherapy cannot use cancer tissue samples for real-time and continuous genetic detection and monitoring. In the past ten years, studies have found that the blood of cancer patients contains cancer cells and nucleic acids derived from cancer cells, called circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Therefore, the molecular characteristic spectrum of the cancer can be characterized by detecting ctDNA in the blood of cancer patients to understand the development and changes of the cancer, so as to guide clinical diagnosis and treatment. This is a non-invasive detection method with the advantages of being economical, simple, flexible, easy to use, accurate and effective. The results in Examples 1 and 2 show that cancer cell lines containing PLEC gene mutations are sensitive to chemotherapy drugs. Further, by detecting ctDNA of G-NEC patients and comparing it with the traditional chemotherapy evaluation method based on impactology, the effectiveness and accuracy of the chemotherapy evaluation gene markers for G-NEC patients based on PLEC gene mutations are verified, and their clinical applications are explored and corresponding detection methods are constructed.

[0040] Ten patients with advanced G-NEC diagnosed by pathology in the hospital (numbered G1-G10) were selected. All of them were newly diagnosed and untreated patients. Chemotherapy (mainly EP regimen) was given according to the reference standard regimen of the guidelines. With the signing of the informed consent form, blood samples were collected during the treatment (each time at an interval of 1-3 months), ctDNA was extracted and used as a template to detect the copy number of PLEC wild-type gene and PLEC mutant gene (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation) fragments by digital PCR (refer to the method in Example 2, and wild-type and mutant probes SEQ ID NO: 8-10 were used at the same time). At the same time, their clinical treatment and disease assessment information were collected, and finally a comprehensive analysis was performed (the results are shown in Table 4).

[0041] (1) Blood sample collection and ctDNA extraction

[0042] 5 ml of EDTA-anticoagulated blood was collected from the patient's cubital vein (the two blood draws were between 1 and 3 months apart), plasma was separated within 1 hour, and ctDNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany).

[0043] (2) PLEC gene fragment detection

[0044] Using the extracted ctDNA as a template, digital PCR primers, probes, and conditions in Table 2 were used to detect the contents of PLEC mutant gene fragments (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation) and the corresponding wild-type gene fragments.

[0045] (3) Application of new gene markers in the evaluation of chemotherapy efficacy

[0046] The evaluation of the clinical efficacy of chemotherapy in 10 patients with advanced G-NEC and the PLEC gene mutations in the corresponding blood sample ctDNA are shown in Table 4. The data showed that PLEC gene mutation fragments were detected in the ctDNA of the 6 G-NEC patients G1-G6. Through the association analysis of the evaluation of the clinical efficacy of chemotherapy in these 6 patients and the PLEC gene mutations in the corresponding blood sample ctDNA, it was found that G-NEC patients with PLEC gene mutations all had PR during chemotherapy. PLEC gene mutations can guide the selection of chemotherapy regimens and be used to screen the beneficiary population. In addition, when the relative proportion of PLEC gene mutations (the number of copies of mutant gene fragments / the sum of the number of copies of wild gene fragments and the number of copies of mutant gene fragments) increased, the evaluation of the patient after chemotherapy was SD or PR, and the overall trend was PR; when the relative proportion of PLEC gene mutations decreased, the evaluation of the patient after chemotherapy was SD or PD, and the overall trend was PD, and the evaluation of PLEC gene mutations was earlier than the traditional imaging-based clinical evaluation method. This shows that PLEC gene mutations can be used for chemotherapy efficacy evaluation and monitoring.

[0047] Taking G1 patients as an example, the PLEC mutant gene of g.1377-1380delATGT was detected in the three blood samples before and after, and the proportion of mutant genes increased from 10.2% (first blood sample) to 25.5% (second blood sample) to 86.0% (third blood sample), that is, the PLEC mutant gene of g.1377-1380delATGT was always detected, and the mutation ratio showed an increasing trend. The corresponding clinical evaluation results of chemotherapy efficacy based on imaging showed that the synchronous evaluation during the first two blood draws was SD, and the third time was converted to PR. It can be seen that for the chemotherapy regimen adopted, G1 patients are effective and benefited, and its effectiveness is a continuous process. By detecting the PLEC mutant gene in the blood sample and the trend of the proportion change, it can be verified and predicted that the chemotherapy regimen is effective for G1 patients in the second blood sample, so as to adhere to the chemotherapy regimen.

[0048] Taking G5 patient as an example, the PLEC mutant gene of g.38110-38115delATCAAG was detected in the four blood samples before and after, and the proportion of mutant genes decreased from 85.8% (first blood sample) to 18.2% (second blood sample) and 8.6% (third blood sample) to 4.4% (fourth blood sample). That is, the PLEC mutant gene of g.38110-38115delATCAAG was always detected, and the mutation ratio showed a rapid downward trend. The corresponding clinical evaluation results of chemotherapy efficacy based on imaging showed that the synchronous evaluations conducted during the first two blood draws were PR, the third was PD, and the fourth was SD. It can be seen that for the chemotherapy regimen adopted, G5 patients are gradually failing. By detecting the PLEC mutant gene in the blood sample and the trend of the proportion change, it is possible to verify and predict that the chemotherapy regimen is failing for G5 patients in the second or third blood sample, so as to adjust the treatment plan in time.

[0049] Table 4 Evaluation of chemotherapy efficacy in 10 patients with advanced G-NEC and PLEC gene mutations in corresponding blood ctDNA

[0050]

[0051] In summary, the PLEC gene mutations (g.1377-1380delATGT mutation and g.38110-38115delATCAAG mutation) provided by the present invention are accurate, specific and effective gene markers for chemotherapy in patients with G-NEC. The molecular markers of the PLEC mutant gene can well predict the benefit of chemotherapy, i.e., the efficacy, and are more effective and accurate than conventional methods, and more importantly, can achieve early prediction.

[0052] Although the present invention is described by preferred specific embodiments, it is obvious to those skilled in the art that the present invention may be modified and varied in various ways. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection kit for PLEC mutant gene, characterized in that: The kit comprises a digital PCR primer pair as shown in SEQ ID NO: 3-4, a digital PCR probe as shown in SEQ ID NO: 8, and a digital PCR probe as shown in SEQ ID NO: 7; wherein the 5'-end nucleotide of the probe shown in SEQ ID NO: 7 is modified with a fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group; similarly, the 5'-end nucleotide of the probe shown in SEQ ID NO: 8 is modified with another fluorescent group, and the 3'-end nucleotide is modified with a paired fluorescent quenching group.

Images