CN111910007A

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

Info

Publication number: CN111910007A
Application number: CN202011093320.8A
Authority: CN
Inventors: 张琳; 王飞; 张史钺
Original assignee: Origimed Technology Shanghai Co ltd
Current assignee: Origimed Technology Shanghai Co ltd
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2020-11-10

Abstract

The invention relates to the field of clinical molecular diagnostics, in particular to application of RNF43 gene variation in predicting sensitivity of a solid tumor patient to immune checkpoint inhibitor therapy. In the invention, the RNF43 gene variation is screened as a biomarker for predicting a population sensitive to an immune checkpoint inhibitor therapy in a solid tumor patient, and compared with the co-mutation of other gene combinations, the prediction result is more accurate; furthermore, the RNF43 gene variation adopted in the invention can be used as an independent prediction risk factor in practical application, and the detection efficiency is improved.

Description

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

Technical Field

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

Background

Tumor immunotherapy is now well developed, and among them, immune checkpoint inhibitors (anti-PD-1/PD-L1 inhibitors) are more "star" drugs in the field of tumor therapy in recent years, and have entered first-line treatment of solid tumors such as non-small cell lung cancer, melanoma and renal cancer. However, it is also seen that although the immune checkpoint inhibitor has good effect, the overall ORR is still only about 20%, so how to accurately screen the population with benefit becomes a problem that needs to be solved by the clinician urgently.

PD-L1, TMB (tumor mutational burden) and MSI (microsatellite instability) are three immunotherapeutic biomarkers (biomarker) that have been approved by FDA or recommended by NCCN guidelines, but each of these three biomarkers has advantages and disadvantages. PD-L1 is most widely used as an immunotherapy biomarker, and PD-L1 IHC detection is also approved by the FDA as a concomitant diagnosis of Pembrolizumab first-line drug administration. However, the results of multiple clinical trials show that the prediction ability of the expression of PD-L1 on the curative effect of immunotherapy is inconsistent, part of PD-L1 negative patients still can benefit from the immunotherapy, and the sustained remission time is not inferior to that of PD-L1 positive patients; TMB is also the immunotherapeutic biomarker recommended by the non-small cell lung cancer NCCN guidelines, but TMB thresholds are difficult to establish consensus given the differences in TMB algorithms by different companies or laboratories; MSI has been used as a key biomarker for tumors to allow FDA to agree to administer medication based on MSI status, rather than histopathological type, but the tumor MSI-H ratio is too low, and clinical popularization has certain limitations. The most important point is that the overlapping rate of PD-L1 positive, TMB high expression and MSI-H is only 0.6% in the existing research (including 11348 cases of solid tumors), which suggests that many potential immunotherapy benefit groups are missed by any biomar alone. Further exploration of immunotherapeutic biorarkers is required.

With the development of the second generation sequencing in the precise treatment of tumors, somatic mutations of specific genes are found to possibly influence the immune function of the tumors or the response to immunotherapy, namely, the specific somatic mutations are suggested to be potential predictors of immunotherapy. EGFR mutations and ALK rearrangements are potential predictors of poor prognosis for ICI immunotherapy. A retrospective analysis found that only 3.6% of these patients responded to ICI immunotherapy, while the response rate for EGFR wild-type and ALK-negative or unknown patients was 23.3%. Meta-analysis of 5 trials involving 3025 patients with advanced NSCLC treated with PD- (L)1 inhibitors found no improvement in overall survival compared to docetaxel in EGFR-mutated patients. EGFR-mutated or ALK-rearranged lung cancers show lower PD-L1 and CD8+ T cell infiltration, which may be responsible for poor ICI immunotherapy response. In addition to the synergistic mutations of EGFR and ALK that alter TP53 and KRAS and those in the DNA Damage Response (DDR) pathway of homologous recombination repair and mismatch repair (HRR-MMR) or HRR and base excision repair (HRR-BER) are considered to be positive predictors of better efficacy of ICI immunotherapy, whereas the synergistic mutations of KRAS and STK11 are associated with poor prognosis of ICI immunotherapy. However, these gene mutations still do not cover all potential immunotherapeutic benefit groups as biomarkers. PD-L1 test lacks uniform standards due to different anti-PD- (L)1 drugs having their own corresponding PD- (L)1 test antibodies and platforms; in addition, the expression of PD-L1 has the characteristic of dynamic change, so that the relationship between the expression of PD-L1 and the effect of immunotherapy is still controversial; on the other hand, although a large number of random control studies and large-sample real-world studies have confirmed the correlation between TMB and the immune efficacy, TMB still can only reflect the tumor mutation number, but cannot prompt the state of the tumor microenvironment, and TMB detection has high requirements on a technical platform, a long working period and high cost, which restricts clinical application thereof. Thus, there remains a need in the art for methods and tools for more efficiently and accurately identifying solid tumor patients for treatment with immune checkpoint inhibitors.

Disclosure of Invention

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

In particular, the invention relates to the use of a detection agent for an RNF43 genetic variation in the manufacture of a kit for predicting the sensitivity of a solid tumor patient to an immune checkpoint inhibitor therapy, wherein the presence of an RNF43 genetic variation is indicative of the sensitivity of said solid tumor patient to an immune checkpoint inhibitor therapy;

the solid tumor is selected from one or more 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, renal cancer, small intestine cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer and urothelial cancer.

According to a further aspect of the invention, the invention also relates to the use of a detection agent for RNF43 gene variation in the manufacture of a kit for predicting the degree of tumor mutation burden in a solid tumor patient, wherein the presence of RNF43 gene variation is indicative of high tumor mutation burden;

The invention has the beneficial effects that:

in the invention, the RNF43 gene variation is screened as a biomarker for predicting a population sensitive to an immune checkpoint inhibitor therapy in a specific solid tumor patient, and compared with the co-mutation of other gene combinations, the prediction result is more accurate; furthermore, the RNF43 gene variation adopted in the invention can be used as an independent prediction risk factor in practical application, and the detection efficiency is improved. The method is beneficial to simplifying detection content, reducing the detection cost of a patient and shortening the time for issuing a detection report, and compared with the PD-L1 immunohistochemical method which needs manual interpretation of immunohistochemical fragments and the TMB which needs manual determination of a threshold value, the detection of the gene mutation state is more reliable.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Fig. 1 is a proportion distribution of 10010 patients with solid tumors in one embodiment of the present invention.

Figure 2 is a graph of the sudden frequency distribution of RNF43 in 10010 different patients with solid tumors, according to one embodiment of the present invention.

FIG. 3 is a comparison of the variation of the RNF43 gene with the Tumor Mutational Burden (TMB) of a wild-type patient in one embodiment of the present invention.

FIG. 4 is a graph showing the frequency of variant variation in patients with solid tumors, in accordance with one embodiment of the present invention.

FIG. 5 shows the RNF43 mutation site analysis in one embodiment of the present invention.

FIG. 6 is a comparison of an RNF43 gene variation in one embodiment of the invention with wild-type patients receiving immunotherapy with immune checkpoint inhibitors.

FIG. 7 is a comparison of BRCA1 gene variation with wild-type patients receiving immunotherapy with immune checkpoint inhibitors.

Figure 8 is a comparison of IRS1 gene variation with wild type patients receiving immunotherapy with immune checkpoint inhibitors.

Figure 9 shows a comparison of NCOR1 gene variation with wild type patients receiving immunotherapy with immune checkpoint inhibitors.

Figure 10 is a comparison of RAD54L gene variation to wild type patients receiving immunotherapy with immune checkpoint inhibitors.

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

FIG. 12 is a comparison of survival of patients with a mutation in RNF43 after immunotherapy to that of patients with RNF43 wild-type in gastroesophageal tumor patients, according to one embodiment of the invention.

FIG. 13 is a comparison of the survival of melanoma patients with mutations in RNF43 compared to the survival of RNF43 wild-type patients, according to one embodiment of the invention.

Fig. 14 is a comparison of survival of patients with BRAF mutations after immunotherapy compared to survival of BRAF wild-type patients in a population of patients with solid tumors, with unknown primary foci, melanoma and breast cancer deleted in accordance with an embodiment of the present invention.

FIG. 15 is a graph of survival of patients after receiving immunotherapy with a mutation in BRCA2 in patients with deletion of tumors with unknown primary foci, melanoma, and breast cancer, compared to the survival of patients with BRCA2 wild-type in a population of patients with solid tumors, in accordance with an embodiment of the present invention.

Detailed Description

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 explanation, 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 instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The present invention relates to the use of a detector of an RNF43 gene variation in the manufacture of a kit for predicting the sensitivity of a solid tumor patient to an immune checkpoint inhibitor therapy, wherein the presence of an RNF43 gene variation is indicative of the sensitivity of said solid tumor patient to an immune checkpoint inhibitor therapy;

the tumor of the patient with solid tumor is selected from one or more 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, renal cancer, small intestine 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 pathway present in the immune system. Under normal conditions, the immune checkpoint can maintain immune tolerance by adjusting the strength of autoimmune reaction, however, when the organism is invaded by tumor, the activation of the immune checkpoint can inhibit autoimmunity, which is beneficial to the growth and escape of tumor cells. By using the immune checkpoint inhibitor, the normal anti-tumor immune response of the body can be restored, so that the tumor can be controlled and eliminated.

Immune checkpoints according to the invention include, but are not limited to, programmed death receptor 1 (PD 1), PD-L1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4); also included are some newly discovered immune checkpoints such as lymphocyte activation gene 3 (LAG 3), CD160, T cell immunoglobulin and mucin-3 (TIM-3), T cell activated V domain immunoglobulin inhibitor (VISTA), adenosine A2a receptor (A2 aR), and the like.

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

The PD1 inhibitor may further be selected from one or more of Nivolumab (OPDIVO; BMS-936558), Pembrolizumab (MK-3475), Jembrolizumab, lambrolizumab, Pidilizumab (CT-011) Terepril mab (JS 001), and Iplilimumab.

The PD-L1 inhibitor may further be selected from one or more of Atezolizumab (MPDL 3280A), JS003, Durvalumab, Avelumab, BMS-936559, MEDI4736 and MSB001071 0010718C.

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

In the present invention, the genetic variation may include point mutation (point mutation) and fragment mutation (fragment mutation); the point mutation may be a Single Nucleotide Polymorphism (SNP), a base substitution, a single base insertion or base deletion, or a silent mutation (e.g., a synonymous mutation); the fragment mutation may be an insertion mutation, a truncation mutation or a gene rearrangement mutation.

In some embodiments, the genetic variation comprises at least one of a fusion/rearrangement mutation, an insertion/deletion, and a truncation mutation.

In some embodiments, the mutation is located at nucleotide 904-3255 of the RNF43 Gene (Gene ID: 54894 NM-017763).

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

Stage III: it is divided into stages IIIA and IIIB. Many stage IIIA and almost all stage IIIB tumors are difficult or impossible to remove by surgery. For example, a tumor affects the mediastinal lymph nodes, or a tumor invades adjacent structures within the lung. There is also a case where the tumor cannot be completely excised once and can be taken out in multiple times due to various factors, which makes it difficult to completely remove the tumor.

Stage IV: the method comprises the following steps: cancer cells metastasize to multiple sites in the contralateral lung, or to fluid accumulation around the lung or around the heart, or to other sites in the body via blood flow. Once in the bloodstream, cancer cells can metastasize to any part of the body, but more commonly metastasize to the brain, bone, liver and adrenal glands.

Since the gene RNF43 encodes protein and antagonist protein RNF43 in Wnt signaling pathway, and the mutation of the gene is usually expressed at the transcription level and response level, the skilled person can detect the mutation from RNA and protein level to indirectly reflect whether the gene mutation occurs, and these can be applied to the present invention.

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

As the detection agent for a nucleic acid level (DNA or RNA level), a known agent known to those skilled in the art can be used, for example, a nucleic acid (usually a probe or primer) which can hybridize to the DNA or RNA and is labeled with a fluorescent label, and the like. And one skilled in the art would also readily envision reverse transcribing mRNA into cDNA and detecting the cDNA, and routine replacement of such techniques would not be outside the scope of the present invention.

In some embodiments, 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 method.

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 biomass spectrometry is selected from flight mass spectrometer detection.

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

In some embodiments of the invention, the nucleic acid sequencing method may be transcriptome sequencing or genome sequencing. In some further embodiments of the invention, the nucleic acid sequencing method is high throughput sequencing, also known as next generation sequencing ("NGS"). Second generation sequencing produces thousands to millions of sequences simultaneously in a parallel sequencing process. NGS is distinguished from "Sanger sequencing" (one generation sequencing), which is based on electrophoretic separation of chain termination products in a single sequencing reaction. Sequencing platforms that can be used with the NGS of the present invention are commercially available and include, but are not limited to, Roche/454 FLX, Illumina/SolexaGenomeAnalyzer, and Applied Biosystems SOLID system, among others. Transcriptome sequencing can also rapidly and comprehensively obtain almost all transcripts and gene sequences of a specific cell or tissue of a certain species in a certain state through a second-generation sequencing platform, and can be used for researching gene expression quantity, gene function, structure, alternative splicing, prediction of new transcripts and the like.

In some embodiments, the detection agent is detected at the protein level.

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

The detection method using an antibody specifically designed for the mutation site may further be immunoprecipitation, co-immunoprecipitation, immunohistochemistry, ELISA, Western Blot, or the like.

In some embodiments, the kit further comprises a sample treatment 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 in common with the terms "lysate", "lysed sample", "tissue or cell extract", and the like, to denote a sample and/or biological sample material comprising lysed tissue or cells, i.e., where the structural integrity of the tissue or cells 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 lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. The process is encompassed by the term "lysis", and typically the process requires the use of sample lysis reagents and/or sample purification reagents.

In some embodiments, the tissue is cancerous or para-cancerous.

In some embodiments, the tissue is a primary lesion.

The test sample may also be blood, serum, plasma, and in some embodiments they are from peripheral blood.

According to a further aspect of the invention, there is also provided a method for predicting the sensitivity of a solid tumor patient to immune checkpoint inhibitor therapy, the method comprising:

the presence or absence of a variation in the RNF43 family gene was measured using the detection agent as described above.

An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases, e.g. 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, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Conversely, biochemical markers (e.g., RNF43 family gene variations) can be used to assess, for example, the presence or absence or severity of a disease with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.

In some embodiments, the methods are used for prognostic evaluation of solid tumor patients following administration of immune checkpoint inhibitor therapy.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

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

comprehensive genomic analysis

FFPE tumor samples from chinese solid tumor patients and paired peripheral whole blood control samples were studied. All patients provided written informed consent. Next generation sequencing for targeted capture (NGS) at OrigiMed involves a combination comprising 450 cancer-associated genes. DNA was extracted from all unstained FFPE sections and whole blood containing not less than 20% of tumor content by DNA FFPE Tissue Kit and DNA Mini Kit (QIAamp), respectively, and then quantified by dsDNA HS determination Kit (Qubit). The 250bp sonicated DNA was fragmented using KAPA Hyper Prep Kit (KAPA Biosystems) to construct a library, which was then PCR amplified and quantified. Hybrid capture was performed using custom combinations, this group and the human genome covering 2.6Mb, targeting 450 cancer-associated genes and some frequently rearranged introns. The captured libraries were mixed, denatured and diluted to 1.5-1.8 pM, followed by paired-end sequencing on Illumina NextSeq 500 according to the manufacturer's protocol.

Wherein the samples were subjected to quality detection using the following three primer pairs for amplifying the ACTIN gene:

i) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-CACGCTCGGTGAGGATCTTC-3' of the group consisting of,

ii) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-TCGAAGTCCAGGGCAACATAGC-3', and

iii) 5'-CACACTGTGCCCATCTATGAGG-3' and 5'-AAGGCTGGAAGAGCGCCTCGGG-3', which amplify fragments of 100bp, 200bp and 300bp, respectively. And when the three groups of primers are amplified to the target fragment, judging that the tissue sample is qualified.

Genome alteration analysis

Genomic alterations, including single base Substitutions (SNVs), short and long insertion deletions, Copy Number Variations (CNVs), and gene rearrangements and fusions were evaluated. Alignment of the original reads to the human genome reference sequence (hg19) was performed using a 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 from the dbSNP database (version 147) or with frequencies exceeding 1.5% of exome sequencing project 6500 (ESP6500) or exceeding 1.5% of the 1000 genome project were also excluded.

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

(1) for point mutations:

the sequencing coverage depth of the position of the point mutation is more than 500 times; a quality value for each read comprising the point mutation of >40, and a base quality value corresponding to the point mutation on each read comprising the point mutation of > 21; the number of the reads containing the point mutation is more than or equal to 5; a ratio of reads in forward to reads in reverse of all reads comprising the point mutation < 1/6; and the frequency of the variant allele of the tumor tissue/the frequency of the variant allele of the control tissue is more than or equal to 20;

(2) for indels (indels):

if the consecutive identical bases in the indel are <5, the sequencing coverage depth of the position of the indel is >600 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; the frequency of the variant allele of the tumor tissue/the frequency of the variant allele of the control tissue is more than or equal to 20;

if the continuous identical basic groups in the insertion deletion are more than or equal to 5 and less than 7, the sequencing coverage depth of the position of the insertion deletion is more than 60 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; (ii) the variant allele frequency of the tumor tissue/variant allele frequency of a control tissue > 20; and the frequency of the variant allele of the tumor tissue is more than or equal to 10 percent;

if the continuous same basic groups in the insertion deletion are more than or equal to 7, the sequencing coverage depth of the position of the insertion deletion is more than 60 times; the quality value of each read containing the indels is > 40; (ii) a base quality value corresponding to the indel mutation on each read comprising the indel of > 21; the number of reads containing the insertion deletion is more than or equal to 5; the ratio of forward read length to reverse read length in all reads containing the indel is < 1/6; (ii) the variant allele frequency of the tumor tissue/variant allele frequency of a control tissue > 20; and the frequency of the variant allele of the tumor tissue is more than or equal to 20 percent.

(3) Amplifying mutations

Refers to the type of variation in copy number variation of a gene. Amplification is an increased copy number of CNV. CNVs, i.e. copy number variations, generally refer to copy number duplications, deletions of large genomic fragments ranging from 1kb to several Mb in length.

TMB calculation

In addition to routine detection of genomic changes, TMB is also determined by NGS-based algorithms. TMB was estimated by counting somatic mutations including SNVs and indels per megabase of coding region sequence examined. The driver mutations and known germline changes in dbSNP 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 a ready-to-use chromogenic reagent consisting of a secondary antibody molecule and a horseradish peroxidase (HRP) molecule coupled to a dextran polymer backbone. Subsequent enzymatic conversion with the addition of chromophores and enhancers 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 variants for immune checkpoint inhibitor therapy, we downloaded an entry 1610 panel of solid tumor cohorts in the tumor genomics database, cbioport website (http:// www.cbioportal.org /), including patient clinical baseline data, immune checkpoint inhibitor therapy efficacy assessment data, and patient genomic data.

Example 1 clinical characteristics of patients

A total of 10010 patients with solid chinese tumors participated in this study. The main tumor distribution of the patient is shown in figure 1: 2026 non-small cell lung cancers (20.24%), 1120 liver cancers (11.19%), 858 gastric cancers (8.57%), 590 esophageal cancers (5.89%), 550 cholangiocarcinomas (5.49%), 544 soft tissue sarcomas (5.43%), 492 pancreatic cancers (4.92%) in 10010 patients (see FIG. 1 for details).

The characteristics of the patients are shown in table 1. The age and sex ratio of the RNF43 mutant and wild-type patients in the two groups are not significantly different, and the median age of the RNF43 gene mutant patients at diagnosis is 58 years. Samples of RNF43 gene mutations were collected mainly from primary foci (p = 0.03). The results of TMB tests on 10100 patients showed that the median TMB of the whole population was 4.6 muts/Mb, and the TMB of the RNF43 gene mutant patients was significantly higher than that of the RNF43 wild type (10.7 vs. 4.6, p < 0.001). The pathological stages are more in stage III (12% vs. 25% vs. 31% vs. 24%, p < 0.001).

TABLE 1 clinical characteristics of patients with solid tumors

Meanwhile, the correlation between the RNF43 mutation and a specific solid tumor is relatively specific, and after verification, it is found that the survival rate of 118 patients with gastroesophageal tumor in 1610 solid tumor patients is statistically meaningless (p = 0.52) than that of RNF43 wild-type patients after receiving immunotherapy in patients with the RNF43 mutation in the patients with gastroesophageal tumor (fig. 12); of the 1610 solid tumor patients, 313 melanoma patients, whose survival after immunotherapy in patients with the mutation in RNF43 was not statistically different from that in patients with the wild-type RNF43 (p = 0.44) (fig. 13). In addition to these tumor species, the survival of patients with mutations in RNF43 among the other tumor species in table 1 after immunotherapy was statistically different from that of patients with RNF43 wild-type (p < 0.05).

Example 2 frequency of RNF43 mutations in the population of solid tumors in China and correlation with the immunotherapeutic biomarker TMB

Among 10010 patients with solid tumors of Chinese population, 310 patients carried the mutation of RNF43 gene, and the overall mutation rate was 3.1%. The mutation rates of colorectal cancer (10.2%), gastric cancer (8.0%), pancreatic cancer (6.9%), small intestine cancer (7.0%) and bile duct cancer (3.6%) were high (see fig. 2 for details).

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

The mutation frequencies of the major variants of RNF43 gene are shown in FIG. 4, and the variant types include 13 (0.13%) fusions/rearrangements, 96 (0.96%) insertions/deletions, 223 (2.23%) truncated variants, and 13 (0.13%) variants with two or more variants.

RNF43 mutation sites were relatively scattered with no distinct hot spot mutation regions (fig. 5).

Example 3 validation of RNF43 Gene variants as clinical data for immunotherapy biomarker

To further validate the predictive value of RNF43 mutations for Immune Checkpoint Inhibitor (ICIs) treatment, we performed external validation by downloading public database cohort information. The cohort data uploaded by Robert M.Samstein et al, incorporated 1661 (this study was incorporated into 1610 patients with solid tumors) patients with solid Tumor cancer who received anti-PD- (L)1 monotherapy or anti-PD- (L)1+ anti-CTLA-4 combination therapy, and specific patient baseline data were described in Samstein RM, Lee C-H, Shoushtari AN et al, Tumor multiple load predictions provided herein and animal immunology Genetics 2019, 51: 202- "206. Among 1171 patients with deletion of tumors of unknown primary foci, melanoma and breast cancer in the population of patients with solid tumors, 47 patients with RNF43 mutation (4.0%) and significantly higher survival rates after immunotherapy with RNF43 mutation than with RNF43 wild-type (p < 0.0001) (fig. 6). In the invention, a plurality of genes related to ICIs therapy reported in the prior art are simultaneously selected for verification, but the mutation of the genes is not related to the survival rate after receiving immunotherapy, and partial results are shown in FIGS. 7-10, 14 and 15: of 1171 patients with deletion of tumors of unknown primary foci, melanoma and breast cancer in the population of patients with solid tumors, 39 of the BRCA1 mutant patients (3.3%), the survival rate of the BRCA1 mutant after immunotherapy was statistically insignificant compared to that of the BRCA1 wild-type patient (p = 0.49) (fig. 7); among 1171 patients with deletion of tumors of unknown primary foci, melanoma and breast cancer in the population of patients with solid tumors, 38 patients with the IRS1 mutation (3.2%), and the survival rate of patients with the IRS1 mutation after immunotherapy was statistically insignificant from that of patients with the IRS1 wild type (p = 0.092) (fig. 8); of 1171 patients in the population of patients with solid tumors, with deletion of tumors of unknown primary foci, melanoma and breast cancer, 50 patients with the NCOR1 mutation (4.3%), and the survival rate of patients with the NCOR1 mutation after immunotherapy was statistically insignificant compared to that of NCOR1 wild-type patients (p = 0.19) (fig. 9); of 1171 patients with deletion of tumors of unknown primary foci, melanoma and breast cancer in the population of patients with solid tumors, 10 of the patients with RAD54L mutation (0.9%), no statistical difference in survival after immunotherapy for patients with RAD54L mutation from that of wild-type RAD54L (p = 0.21) (fig. 10); 1171 patients with deletion of tumors of unknown primary foci, melanoma and breast cancer in the population of patients with solid tumors, among which patients with BRAF mutations received no statistical difference in survival from patients with wild type BRAF (p = 1) (fig. 14); in the population of patients with solid tumors, 1171 patients after deletion of tumors with unknown primary foci, melanoma and breast cancer, the survival of patients with the BRCA2 mutation after immunotherapy was statistically insignificant compared to that of wild-type BRCA2 (p = 0.085) (fig. 15).

The Cox multifactorial analysis results further demonstrated that the RNF43 gene variation is an independent predictive risk factor for the prognosis of immunotherapy (RNF 43-MUT vs. RNF43-WT, HR: 0.24, 95% CI: 0.12-0.49, p < 0.001) (FIG. 11).

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of a detector of an RNF43 gene variation in the manufacture of a kit for predicting sensitivity of a solid tumor patient to an immune checkpoint inhibitor therapy, wherein the presence of an RNF43 gene variation is indicative of the sensitivity of the solid tumor patient to an immune checkpoint inhibitor therapy;

the tumor of the patient with solid tumor is selected from one or more 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, renal cancer, small intestine cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer and urothelial cancer.
Use of a detection agent for RNF43 genetic variation in the manufacture of a kit for predicting the degree of tumor mutation burden in a patient with a solid tumor, wherein the presence of RNF43 genetic variation is indicative of high tumor mutation burden;

the tumor of the patient with solid tumor is selected from one or more 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, renal cancer, small intestine cancer, small cell lung cancer, soft tissue sarcoma, gastric cancer, thymic tumor, thyroid cancer and urothelial cancer.
3. 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 of claim 1 or 2, wherein the genetic variation comprises at least one of a fusion/rearrangement mutation, an insertion/deletion and a truncation mutation.
5. The use of claim 1 or 2, wherein the detection agent detects at the nucleic acid level.
6. The use of 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 method.
7. The use of claim 1 or 2, wherein the detection agent is detected at the protein level.
8. The use of 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 using antibodies specifically designed for the mutation site.
9. The use of claim 1 or 2, wherein the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents and sample nucleic acid extraction reagents.
10. The use according to claim 9, wherein 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.