CN111910007A - Use of RNF43 gene variation in predicting sensitivity of solid tumor patients to immune checkpoint inhibitor therapy - Google Patents

Abstract

The invention relates to the field of clinical molecular diagnostics, in particular, to the application of an RNF43 gene mutation in predicting the sensitivity of solid tumor patients to immune checkpoint inhibitor therapy. In the present invention, the RNF43 gene mutation is screened as a biomarker for predicting the group sensitive to immune checkpoint inhibitor therapy in solid tumor patients, and the prediction result is more accurate compared to the co-mutation of other gene combinations; The RNF43 gene variant can be used as an independent predictive risk factor in practical applications and improve the detection efficiency.

Description

RNF43 gene variant predicts response to immune checkpoint inhibitor therapy in patients with solid tumors Applications in Sensitivity

technical field

The invention relates to the field of clinical molecular diagnostics, in particular, to the application of an RNF43 gene mutation in predicting the sensitivity of solid tumor patients to immune checkpoint inhibitor therapy.

Background technique

Tumor immunotherapy is now in full swing, among which immune checkpoint inhibitors (anti-PD-1/PD-L1 inhibitors) are the "star" drugs in the field of tumor treatment in recent years, and have entered non-small cell lung cancer, melanoma and kidney cancer. First-line treatment of solid tumors such as cancer. However, we must also see that although immune checkpoint inhibitors are effective, the overall ORR is still only about 20%, so how to accurately screen the beneficiaries has become an urgent problem for clinicians to solve.

PD-L1, TMB (tumor mutational burden) and MSI (microsatellite instability) are three immunotherapy biomarkers approved by FDA or recommended by NCCN guidelines, but these three biological Each marker has advantages and disadvantages. PD-L1 is the most widely used immunotherapy biomarker, and PD-L1 IHC detection has also been approved by the FDA as a companion diagnostic for first-line Pembrolizumab. However, the results of multiple clinical trials have shown that the predictive ability of PD-L1 expression on the efficacy of immunotherapy is inconsistent, and some PD-L1-negative patients can still benefit from immunotherapy, and the duration of remission is not inferior to that of PD-L1-positive patients; TMB It is also the immunotherapy biomarker recommended by the NCCN guidelines for non-small cell lung cancer, but due to the different TMB algorithms of different companies or laboratories, it is difficult to establish a consensus on the TMB threshold; MSI has been used as a key tumor biomarker for FDA to agree to be based on MSI status, not histopathological type However, the proportion of MSI-H in tumors is too low, and clinical promotion has certain limitations. The most important point is that the existing study (including 11,348 solid tumors) found that the overlap rate of PD-L1 positive, TMB high expression and MSI-H was only 0.6%, suggesting that any biomarker alone will miss many potential immunotherapy. beneficiaries. Therefore, it is necessary to further explore immunotherapy biomarkers.

With the increasingly widespread use of next-generation sequencing in precision tumor therapy, somatic mutations in specific genes have been found to affect tumor immune function or response to immunotherapy, suggesting that specific somatic mutations may be potential predictors of immunotherapy. EGFR mutations and ALK rearrangements are potential predictors of poor prognosis in ICI immunotherapy. A retrospective analysis found that only 3.6% of these patients responded to ICI immunotherapy, compared with 23.3% of EGFR wild-type and ALK-negative or unknown patients. A meta-analysis of 5 trials including 3025 patients with advanced NSCLC treated with PD-(L)1 inhibitors found no improvement in overall survival compared with docetaxel in patients with EGFR mutations. Lung cancers with EGFR mutations or ALK rearrangements showed lower PD-L1 and CD8+ T cell infiltration, which may account for the poor response to ICI immunotherapy. In addition to alterations in EGFR and ALK, cooperative mutations in TP53 and KRAS and in the DNA damage response (DDR) pathways of homologous recombination repair and mismatch repair (HRR-MMR) or HRR and base excision repair (HRR-BER) It is considered to be a positive predictor of better efficacy of ICI immunotherapy, and synergistic mutations of KRAS and STK11 are associated with poor prognosis of ICI immunotherapy. However, these gene mutations as biomarkers still cannot cover all potential immunotherapy benefit populations. Since different anti-PD-(L)1 drugs have their own PD-(L)1 detection antibodies and platforms, there is no unified standard for PD-L1 detection; in addition, the expression of PD-L1 has the characteristics of dynamic changes, which leads to PD-(L)1 detection. -The relationship between L1 expression and immunotherapy efficacy is still somewhat controversial; on the other hand, although a large number of randomized controlled studies and large sample real-world studies have confirmed the correlation between TMB and immune efficacy, TMB still only reflects The number of tumor mutations can not indicate the state of the tumor microenvironment, and TMB detection has higher requirements on the technical platform, longer working cycle, and higher cost, which restrict its clinical application. Therefore, there remains a need in the art for methods and tools to more efficiently and accurately identify patients with solid tumors suitable for treatment with immune checkpoint inhibitors.

SUMMARY OF THE INVENTION

The present invention relates to the use of RNF43 gene variants as biomarkers to predict the sensitivity of cancer patients to immune checkpoint inhibitor therapy.

Specifically, the present invention relates to the application of a RNF43 gene mutation detection agent in the preparation of a kit for predicting the sensitivity of solid tumor patients to immune checkpoint inhibitor therapy. Indications of susceptibility to checkpoint inhibitor therapy;

The solid tumor is selected from the group consisting of osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, small bowel cancer, small cell cancer One or more of lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, urothelial cancer.

According to yet another aspect of the present invention, the present invention also relates to the use of a RNF43 gene mutation detection agent in the preparation of a kit for predicting the degree of tumor mutation burden in patients with solid tumors, wherein the presence of RNF43 gene mutation is an indicator of high tumor mutation burden sign;

The solid tumor is selected from the group consisting of osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, small bowel cancer, small cell cancer One or more of lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, urothelial cancer.

The beneficial effects of the present invention are:

In the present invention, the RNF43 gene mutation is screened as a biomarker for predicting a group sensitive to immune checkpoint inhibitor therapy in a specific solid tumor patient. Compared with co-mutation of other gene combinations, the prediction result is more accurate; and the present invention adopts The RNF43 gene variant can be used as an independent predictor of risk factors in practical applications to improve detection efficiency. This method is conducive to simplifying the test content, reducing the cost of patient testing, and speeding up the issuance of test reports. Compared with the PD-L1 immunohistochemical method, which requires manual interpretation of immunohistochemical films and TMB requires manual determination of the threshold, and the gene Detection of mutation status is more reliable.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the accompanying drawings required in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative efforts.

Figure 1 shows the proportion distribution of 10,010 solid tumor patients in an embodiment of the present invention.

Figure 2 shows the mutation frequency distribution of RNF43 in 10,010 patients with different solid tumors in an embodiment of the present invention.

Figure 3 is a comparison of the RNF43 gene mutation and the tumor mutational burden (TMB) of wild-type patients in an embodiment of the present invention.

Figure 4 shows the frequencies of different variants in solid tumor patients according to an embodiment of the present invention.

Figure 5 is the analysis of RNF43 mutation sites in one embodiment of the present invention.

FIG. 6 is a comparison of immunotherapy of RNF43 gene mutation and wild-type patients receiving immune checkpoint inhibitor according to an embodiment of the present invention.

Figure 7 is a comparison of immunotherapy with BRCA1 gene variant and wild-type patients receiving immune checkpoint inhibitors.

Figure 8 is a comparison of IRS1 gene variant and wild-type patients receiving immune checkpoint inhibitor immunotherapy.

Figure 9 is a comparison of NCOR1 gene variant and wild-type patients receiving immune checkpoint inhibitor immunotherapy.

Figure 10 is a comparison of immunotherapy with RAD54L gene variant and wild-type patients receiving immune checkpoint inhibitors.

Figure 11 is a Cox multivariate regression analysis of independent risk factors associated with the efficacy of immunotherapy using immune checkpoint inhibitors in one embodiment of the present invention.

FIG. 12 is a comparison of the survival rate of patients with RNF43 mutation in gastroesophageal tumor patients after receiving immunotherapy and the survival rate of RNF43 wild-type patients in an embodiment of the present invention.

Figure 13 is a comparison of the survival rate of melanoma patients with RNF43 mutation after receiving immunotherapy and the survival rate of RNF43 wild-type patients according to an embodiment of the present invention.

Figure 14 shows the survival rate of patients with BRAF mutations after receiving immunotherapy in patients with unidentified primary tumors, melanoma and breast cancer in the whole solid tumor patient population and the survival rate of BRAF wild-type patients in an embodiment of the present invention Survival comparison.

Figure 15 shows the survival rate of patients with BRCA2 mutations after receiving immunotherapy and the survival rate of BRCA2 wild-type patients in patients with unidentified primary tumors, melanoma and breast cancer in the whole solid tumor patient population according to an embodiment of the present invention Survival comparison.

Detailed ways

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of illustration and not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

Therefore, it is intended that this invention covers such modifications and changes as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or will be apparent from the following detailed description. It should be understood by those of ordinary skill in the art that this discussion is a description of exemplary embodiments only, and is not intended to limit the broader aspects of the invention.

The present invention relates to the application of a detection agent for RNF43 gene mutation in the preparation of a kit for predicting the sensitivity of solid tumor patients to immune checkpoint inhibitor therapy, wherein the existence of RNF43 gene mutation is the result of immune checkpoint inhibition in the solid tumor patient Indications of drug sensitivity;

The tumor suffered by the solid tumor patient is selected from osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, One or more of small bowel cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, and urothelial cancer.

According to yet another aspect of the present invention, the present invention also relates to the use of a RNF43 gene mutation detection agent in the preparation of a kit for predicting the degree of tumor mutation burden in patients with solid tumors, wherein the presence of RNF43 gene mutation is an indicator of high tumor mutation burden sign;

The tumor suffered by the solid tumor patient is selected from osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, One or more of small bowel cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, and urothelial cancer.

As used herein, the term "immune checkpoint" refers to some inhibitory signaling pathways present in the immune system. Under normal circumstances, immune checkpoints can maintain immune tolerance by regulating the intensity of autoimmune responses. However, when the body is invaded by tumors, the activation of immune checkpoints will inhibit autoimmunity and facilitate the growth and escape of tumor cells. Through the use of immune checkpoint inhibitors, the body's normal anti-tumor immune response can be restored to control and eliminate tumors.

The immune checkpoints described in the present invention include but are not limited to programmed death receptor 1 (PD1), PD-L1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4); also include some newly discovered immune checkpoints such as lymphocytes Cell activation gene 3 (LAG3), CD160, T cell immunoglobulin and mucin-3 (TIM-3), V domain immunoglobulin inhibitor of T cell activation (VISTA), adenosine A2a receptor (A2aR) and many more.

Preferred immune checkpoint inhibitors are PD1 inhibitors and/or PD-L1 inhibitors.

The PD1 inhibitor can further be selected as one of Nivolumab (OPDIVO; BMS-936558), Pembrolizumab (MK-3475), Jembrolizumab, lambrolizumab, Pidilizumab (CT-011), Toripalizumab (JS001) and Ipilimumab or more.

The PD-L1 inhibitor can further be selected as one or more of Atezolizumab (MPDL3280A), JS003, Durvalumab, Avelumab, BMS-936559, MEDI4736 and MSB0010718C.

The terms "mutation burden", "mutation burden", "mutation burden" and "mutational burden" are used interchangeably herein. In the context of tumors, mutational burden is also referred to herein as "tumor mutational burden," "tumor mutational burden," or "TMB."

In the present invention, gene variation may include point mutation and fragment mutation; point mutation may be single nucleotide polymorphism (SNP), base substitution, single base insertion or base deletion, or silent mutations (eg, synonymous mutations); fragment mutations can be insertion mutations, truncation mutations or gene rearrangement mutations.

In some embodiments, the genetic variation includes at least one of fusion/rearrangement mutations, insertion/deletions, and truncation mutations.

In some embodiments, the mutation is located at nucleotides 904-3255 of the RNF43 gene (Gene ID: 54894 NM_017763).

In some embodiments, the solid tumor is a clinical stage III and/or IV solid tumor.

Stage III: divided into stage IIIA and IIIB. Many stage IIIA and nearly all stage IIIB tumors are difficult or impossible to remove surgically. For example, the tumor involves mediastinal lymph nodes, or the tumor invades adjacent structures in the lung. In another case, due to various factors, the tumor cannot be completely removed at one time, but can only be removed in multiple times. In this case, it is difficult to completely remove the tumor.

Stage IV: Involves: Cancer has metastasized to multiple sites in the contralateral lung, or fluid builds up around the lung or around the heart, or through the bloodstream to other parts of the body. Once in the bloodstream, cancer cells can metastasize to any part of the body, but the more common sites of metastasis are the brain, bone, liver, and adrenal glands.

Since the RNF43 gene is a protein-encoding gene, it can encode the antagonistic protein RNF43 protein in the Wnt signaling pathway, so the mutation of the gene is usually expressed at the transcription level and the response level. Those skilled in the art can learn from RNA and protein Detecting its mutation at the level to indirectly reflect whether it has gene mutation, all of which can be applied to the present invention.

In some embodiments, the detection agent detects at the nucleic acid level.

The nucleic acid level (DNA or RNA level) detection reagent can be selected from a reagent known to those skilled in the art, such as a nucleic acid (usually a probe or primer) that can hybridize to the DNA or RNA and is labeled with a fluorescent label. In addition, those skilled in the art can easily imagine that the mRNA is reverse transcribed into cDNA and then the cDNA is detected. The conventional replacement of these technical means does not exceed the protection scope of the present invention.

In some embodiments, the detection agent is used to perform any of the following methods:

Polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM.

In some embodiments, the polymerase chain reaction is selected from the group consisting of restriction fragment length polymorphism, single-strand conformation polymorphism, Taqman probe, competitive allele-specific PCR, and allele-specific PCR.

In some embodiments, the biological mass spectrometry is selected from mass spectrometry-in-flight detection.

In some embodiments, the nucleic acid sequencing method is selected from the Snapshot method.

In some embodiments of the present invention, the nucleic acid sequencing method may be transcriptome sequencing or genome sequencing. In other embodiments of the invention, the nucleic acid sequencing method is high-throughput sequencing, also referred to as next-generation sequencing ("NGS"). Next-generation sequencing generates thousands to millions of sequences simultaneously in parallel sequencing processes. NGS is distinguished from "Sanger sequencing" (first generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. Sequencing platforms that can use the NGS of the present invention are commercially available, including, but not limited to, Roche/454 FLX, Illumina/SolexaGenomeAnalyzer, and Applied Biosystems SOLID system, among others. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a specific cell or tissue of a species in a certain state through the next-generation sequencing platform, which can be used to study gene expression, gene function, structure, and availability. Alternative splicing and new transcript prediction, etc.

In some embodiments, the detection agent detects at the protein level.

In some embodiments, the detection agent is used to perform any of the following methods:

Biological mass spectrometry, amino acid sequencing, electrophoresis, and detection with antibodies designed specifically for the mutation site.

The detection method using antibodies specifically designed for the mutation site may further include immunoprecipitation, co-immunoprecipitation, immunohistochemistry, ELISA, and Western Blot.

In some embodiments, the kit further includes a sample processing reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

In some embodiments, the sample is selected from at least one of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen, and saliva samples of the solid tumor patient.

As used herein, "tissue or tissue lysate" may also be used generically with the terms "lysate", "lysed sample", "tissue or cell extract", etc. to refer to a sample and/or biological sample material comprising lysed tissue or cells , i.e. where the structural integrity of a tissue or cell has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to dissolve, degrade or disrupt the cell walls and membranes of such tissue or cells. The skilled artisan is very familiar with appropriate methods for obtaining lysates. This process is encompassed by the term "lysis" and typically requires the use of sample lysis reagents and/or sample purification reagents.

In some embodiments, the tissue is cancerous tissue or paracancerous tissue.

In some embodiments, the tissue is the primary tumor.

The test sample can also be blood, serum, plasma, in some embodiments from peripheral blood.

According to yet another aspect of the present invention, the present invention also provides a method for predicting the sensitivity of patients with solid tumors to immune checkpoint inhibitor therapy, the method comprising:

The presence or absence of gene variants in the RNF43 family is measured using the assays described above.

An ideal scenario for diagnosis is a situation where a single event or process causes various diseases, eg, in infectious diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types. As the skilled artisan will appreciate, for a given multifactorial disease, diagnosis without biochemical markers is 100% specific and with 100% sensitivity. Conversely, biochemical markers (eg, RNF43 family gene variants) can be used to assess, eg, the presence or severity of a disease with some probability or predictive value. Therefore, in routine clinical diagnosis, various clinical symptoms and biological markers are usually considered comprehensively to diagnose, treat and control the underlying disease.

In some embodiments, the methods are used for prognostic assessment of patients with solid tumors following immune checkpoint inhibitor therapy.

The embodiments of the present invention will be described in detail below with reference to the examples.

Example

The research method adopted in the embodiment of the present invention is as follows:

Comprehensive Genome Analysis

FFPE tumor samples and paired peripheral whole blood control samples from Chinese solid tumor patients were studied. All patients provided written informed consent. Targeted capture next-generation sequencing (NGS) at OrigiMed involving a panel of 450 cancer-related genes. DNA was extracted from whole unstained FFPE sections and whole blood with a tumor content of not less than 20% by DNA FFPE Tissue Kit and DNA Mini Kit (QIAamp), respectively, and then quantified by dsDNA HS Assay Kit (Qubit). Libraries were constructed by fragmenting ~250 bp sonicated DNA using the KAPA Hyper Prep Kit (KAPA Biosystems), followed by PCR amplification and quantification. Hybrid capture was performed using a custom combination covering the 2.6Mb human genome targeting 450 cancer-related genes and certain frequently rearranged introns. The captured libraries were pooled, denatured, and diluted to 1.5–1.8 pM, followed by paired-end sequencing on an Illumina NextSeq 500 according to the manufacturer's protocol.

The samples used the following three sets of primer pairs to amplify the ACTIN gene for quality detection:

i) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-CACGCTCGGTGAGGATCTTC-3',

ii) 5’-CACACTGTGCCCATCTATGAGG-3’ and 5’-TCGAAGTCCAGGGGCAACATAGC-3’, and

iii) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-AAGGCTGGAAGAGCGCCTCGGG-3', which amplified fragments of 100 bp, 200 bp and 300 bp, respectively. When the three sets of primers all amplified to the target fragment, the quality of the tissue sample was judged to be qualified.

Genome Alteration Analysis

Genomic alterations, including single base substitutions (SNVs), short and long indels, copy number variations (CNVs), and gene rearrangements and fusions, were assessed. Alignment of raw reads to the human genome reference sequence (hg19) was performed using the Burrows-Wheeler Aligner, followed by PCR deduplication using Picard's MarkDuplicates algorithm. Variants with read depths less than 30x, strand bias greater than 10% or VAF < 0.5% were removed. Common single nucleotide polymorphisms (SNPs) defined as either from the dbSNP database (version 147) or with a frequency exceeding 1.5% of the Exome Sequencing Project 6500 (ESP6500) or more than 1.5% of the 1000 Genomes Project were also excluded outer.

Whether the identified mutation is true is judged by the following criteria:

(1) For point mutation:

The depth of sequencing coverage at the location of the point mutation is >500 times; the quality value of each read containing the point mutation is >40, and the quality value of each read containing the point mutation corresponding to the point mutation is >21 ; the number of reads containing the point mutation ≥ 5; the ratio of forward reads to reverse reads in all reads containing the point mutation < 1/6; and variant alleles in tumor tissue Frequency/variant allele frequency in control tissue ≥ 20;

(2) For insertion and deletion (indel):

If there are <5 consecutive identical bases in the indel, the depth of sequencing coverage at the position of the indel is >600 times; the quality value of each read containing the indel is >40; on each read containing the indel The base quality value corresponding to the indel mutation is >21; the number of reads containing the indel is greater than or equal to 5; the forward read length and the reverse read length in all the reads containing the indel The ratio is <1/6; the variant allele frequency of the tumor tissue/the variant allele frequency of the control tissue is greater than or equal to 20;

If the consecutive identical bases in the indel are ≥5 and <7, the sequencing coverage depth of the indel position is >60 times; the quality value of each read containing the indel is >40; each read containing the indel The base quality value corresponding to the indel mutation on the read is >21; the number of reads containing the indel is ≥ 5; the forward read length and reverse direction in all the reads containing the indel The read length ratio of the tumor tissue is less than 1/6; the variant allele frequency of the tumor tissue/the variant allele frequency of the control tissue is >20; and the variant allele frequency of the tumor tissue is greater than or equal to 10%;

If the indel has ≥7 consecutive identical bases, the depth of sequencing coverage at the indel position is >60 times; the quality value of each read containing the indel is >40; on each read containing the indel The base quality value corresponding to the indel mutation is >21; the number of reads containing the indel is greater than or equal to 5; the forward read length and the reverse read length in all the reads containing the indel The ratio is <1/6; the variant allele frequency of the tumor tissue/the variant allele frequency of the control tissue is >20; and the variant allele frequency of the tumor tissue is ≥20%.

(3) Amplification mutation

Refers to the type of variation in gene copy number variation. Amplifications are CNVs with increased copy number. CNV, that is, copy number variation, generally refers to the copy number duplication and deletion of large genomic fragments ranging from 1 kb to several Mb in length.

TMB calculation

In addition to routine detection of genomic alterations, TMB is also determined by NGS-based algorithms. TMB was estimated by counting somatic mutations including SNVs and indels per megabase of the coding region sequence examined. Driver mutations and known germline alterations in dbSNPs were excluded.

Immunohistochemistry

Immunohistochemical (IHC) staining procedures were performed as previously described. Briefly, deparaffinization, rehydration, and target recovery were performed, followed by incubation with monoclonal antibodies against PD-L1 (DAKO, clones 22C3 and 28-8). The slides were incubated with ready-to-use chromogenic reagents consisting of secondary antibody molecules and horseradish peroxidase (HRP) molecules coupled to the dextran polymer backbone. Subsequent enzymatic conversion with addition of a chromophore and enhancer results in the precipitation of visible reaction products at the antigenic site. The samples were then counterstained with hematoxylin.

Public database queue data acquisition

To further validate the clinical predictive role of RNF43 mutations for immune checkpoint inhibitor therapy, we downloaded a cohort of 1610 solid tumors from the tumor genomics database cBioPortal website (http://www.cbioportal.org/), including Patient clinical baseline data, immune checkpoint inhibitor treatment efficacy assessment data, and patient genomic data.

Example 1 Clinical characteristics of patients

A total of 10,010 Chinese solid tumor patients participated in this study. The distribution of the main tumor types of the patients is shown in Figure 1: among the 10010 patients, 2026 (20.24%) were non-small cell lung cancer, 1120 (11.19%) were liver cancer, 858 (8.57%) were gastric cancer, and 590 (5.89%) were esophageal cancer. , 550 cases (5.49%) of cholangiocarcinoma, 544 cases (5.43%) of soft tissue sarcoma, and 492 cases (4.92%) of pancreatic cancer (see Figure 1 for details).

The characteristics of the patients are shown in Table 1. There was no significant difference in age and sex ratio between the two groups of patients with RNF43 mutation and wild type. The median age at diagnosis of RNF43 mutation patients was 58 years old. The samples with RNF43 gene mutation were mainly collected from the primary tumor (p=0.03). The results of TMB detection in 10,100 patients showed that the median TMB of the overall population was 4.6 muts/Mb, and the TMB of patients with RNF43 gene mutation was significantly higher than that of RNF43 wild type (10.7 vs. 4.6, p<0.001). The number of pathological stages was stage III (12% vs. 25% vs. 31% vs. 24%, p<0.001).

Table 1. Clinical characteristics of patients with solid tumors

At the same time, the correlation between RNF43 mutations and specific solid tumors is also relatively specific. After verification, it was found that 118 patients with gastroesophageal tumors out of 1610 patients with solid tumors, and patients with RNF43 mutations in patients with gastroesophageal tumors received immunization The survival rate after treatment was not significantly different from that of RNF43 wild-type patients (p=0.52) (Fig. 12); among 1610 solid tumor patients, 313 patients with melanoma, and in patients with melanoma patients with RNF43 mutation The survival rate after immunotherapy was not significantly different from that of RNF43 wild-type patients (p=0.44) (Fig. 13). In addition to these tumor types, the survival rates of patients with RNF43 mutations in other tumor types in Table 1 after receiving immunotherapy were significantly different from those of RNF43 wild-type patients (p<0.05).

Example 2 Frequency of RNF43 mutation in Chinese solid tumor population and its correlation with immunotherapy biomarker TMB

Among the 10,010 solid tumor patients in the Chinese population, 310 patients carried RNF43 gene mutations, and the overall mutation rate was 3.1%. Among them, colorectal cancer (10.2%), gastric cancer (8.0%), pancreatic cancer (6.9%), small bowel cancer (7.0%) and bile duct cancer (3.6%) had higher mutation rates (see Figure 2 for details).

The TMB of RNF43 mutant patients was significantly higher than that of RNF43 wild-type patients (median TMB: 10.7 vs. 4.6, p<0.001) (Fig. 3).

The mutation frequency of the main variant forms of RNF43 gene is shown in Figure 4. The variant types include 13 cases (0.13%) fusion/rearrangement, 96 cases (0.96%) insertion/deletion, 223 cases (2.23%) truncating variant, and 13 cases (0.13%) with two or more variants.

The RNF43 mutation sites were relatively scattered, and there was no obvious hot spot mutation region (Fig. 5).

Example 3 Clinical data validation of RNF43 gene mutation as immunotherapy biomarker

To further validate the predictive value of RNF43 mutations for treatment with immune checkpoint inhibitors (ICIs), we performed external validation by downloading cohort information from public databases. Downloaded cohort data uploaded by Robert M. Samstein et al. The Robert M. Samstein cohort included 1661 (1610 patients with solid tumors in this study) receiving anti-PD-(L)1 monotherapy or anti-PD-(L) )1+ anti-CTLA-4 combination therapy for patients with solid tumor cancer. For the specific patient baseline data, please refer to Samstein RM, Lee C-H, Shoushtari AN et al. Tumor mutational loadpredicts survival after immunotherapy across multiple cancer types. NatureGenetics 2019; 51: 202-206. In the whole solid tumor patient population, 47 (4.0%) of 1171 patients after deletion of unidentified primary tumor, melanoma, and breast cancer were patients with RNF43 mutation, and the survival rate of patients with RNF43 mutation after receiving immunotherapy was significant Higher survival than RNF43 wild-type patients (p<0.0001) (Fig. 6). The present invention also selects many genes related to ICIs therapy reported in the prior art for verification, but their mutations are not related to the survival rate after receiving immunotherapy, and some of the results are shown in Figures 7-10, 14, and 15: In the whole solid tumor patient population, 39 (3.3%) of 1171 patients after deletion of unidentified primary tumor, melanoma and breast cancer were patients with BRCA1 mutation. The survival rate of patients with BRCA1 mutation after receiving immunotherapy was similar to There was no statistically significant difference in survival among BRCA1 wild-type patients (p=0.49) (Fig. 7); in the full solid tumor patient population, among 1171 patients after deletion of tumors of unknown primary, melanoma, and breast cancer, IRS1 There were 38 (3.2%) mutated patients, and the survival rate of patients with IRS1 mutation after receiving immunotherapy was not significantly different from the survival rate of IRS1 wild-type patients (p=0.092) (Figure 8); in the whole solid tumor patient population Among the 1171 patients after deletion of tumors of unknown primary, melanoma and breast cancer, 50 (4.3%) of patients with NCOR1 mutation, the survival rate of patients with NCOR1 mutation after receiving immunotherapy was compared with that of patients with NCOR1 wild type. There was no statistically significant difference in the rate (p=0.19) (Fig. 9); in the whole solid tumor patient population, 10 of the 1171 patients with unidentified primary tumor, melanoma, and breast cancer were deleted (10 patients with RAD54L mutation). 0.9%), the survival rate of patients with RAD54L mutation after receiving immunotherapy was not significantly different from that of RAD54L wild-type patients (p=0.21) (Figure 10); in the whole solid tumor patient population, the primary tumor was deleted Among 1171 patients with unidentified tumors, melanoma, and breast cancer, there was no statistically significant difference in survival after immunotherapy between patients with BRAF mutations and BRAF wild-type patients (p=1) (Fig. 14). ); in the whole solid tumor patient population, 1171 patients after deletion of tumors of unknown primary, melanoma, and breast cancer, in these patients, the survival rate of patients with BRCA2 mutations after receiving immunotherapy was compared with that of patients with BRCA2 wild type The rate difference was not statistically significant (p=0.085) (Figure 15).

The results of Cox multivariate analysis further indicated that the RNF43 gene variant was an independent predictive risk factor for the prognosis of immunotherapy (RNF43-MUT vs. RNF43-WT, HR: 0.24, 95%CI: 0.12-0.49, p<0.001) (Figure 11).

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are more specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be noted that, for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. The application of a detection agent for RNF43 gene mutation in the preparation of a kit for predicting the sensitivity of a patient with a solid tumor to an immune checkpoint inhibitor therapy, wherein the presence of the RNF43 gene mutation is an indication that the patient with a solid tumor is sensitive to an immune checkpoint inhibitor Indications for sensitivity to therapy; The tumor suffered by the solid tumor patient is selected from osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, One or more of small bowel cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, and urothelial cancer. 2. The application of a RNF43 gene mutation detection agent in the preparation of a kit for predicting the degree of tumor mutation burden in patients with solid tumors, wherein the presence of RNF43 gene mutation is an indication of high tumor mutation burden; The tumor suffered by the solid tumor patient is selected from osteosarcoma, colorectal cancer, cervical cancer, esophageal cancer, gastrointestinal neuroendocrine tumor, head and neck cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, One or more of small bowel cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer, and urothelial cancer. 3. The use according to claim 1 or 2, wherein the immune checkpoint inhibitor is a PD1 inhibitor and/or a PD-L1 inhibitor. 4. The use according to claim 1 or 2, wherein the genetic variation comprises at least one of fusion/rearrangement mutation, insertion/deletion and truncation mutation. 5. The use according to claim 1 or 2, wherein the detection agent is detected at the nucleic acid level. 6. The use according to claim 5, wherein the detection agent is used to perform any one of the following methods: Polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM. 7. The use according to claim 1 or 2, wherein the detection agent is detected at the protein level. 8. The use according to claim 7, wherein the detection agent is used to perform any one of the following methods: Biological mass spectrometry, amino acid sequencing, electrophoresis, and detection with antibodies designed specifically for the mutation site. 9. The application according to claim 1 or 2, wherein the kit further comprises a sample processing reagent, and the sample processing reagent comprises at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent. A sort of. 10. The application according to claim 9, wherein the sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen and saliva of the solid tumor patient at least one of the samples.

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