CN116837103A - ZFHX3 gene mutation can be used as SCLC immune therapeutic biomarker - Google Patents

Abstract

The application relates to a biomarker for immunotherapy with SCLC patients, which is a mutant ZFHX3 gene. According to the application, through sequencing of genome and transcriptome of SCLC patient samples (including tumor and paired normal samples), deep analysis finds that the SCLC patient with ZFHX3 gene mutation has higher immunity and better prognosis, and clinical test results further prove that the patient with ZFHX3 gene mutation has higher pathological remission rate (MPR), so that the SCLC patient with ZFHX3 mutation is likely to benefit from immunotherapy, the current situation that SCLC lacks an immune treatment biomarker is filled, and the method has important significance for immune treatment of the SCLC patient.

Description

ZFHX3 gene mutation can be used as SCLC immune therapeutic biomarker

Technical Field

The application relates to the field of molecular biology, in particular to a ZFHX3 gene mutation which can be used as an SCLC immunotherapeutic biomarker.

Background

Small cell lung cancer (small cell lung cancer, SCLC) is a heterogeneous neuroendocrine tumor of Kulchitsky cells originating from bronchial mucosal epithelium, and is the most invasive subtype of lung cancer, accounting for about 15% -20% of lung cancer. 98% of SCLC is attributed to smoking, and is characterized by high malignancy, high invasiveness, rapid disease progress, easy occurrence of distant metastasis in early stage, and the like, and although initial treatment is sensitive, the disease is easy to recur and transfer, drug resistance is formed, and the like, so that SCLC patients have extremely poor prognosis and survival rate of less than 7% in 5 years.

Since 1980, a plurality of clinical trial researches show that platinum-based combined etoposide can achieve higher complete remission rate and better prognosis when used for treating SCLC, and lays the foundation of the SCLC treatment scheme. However, in recent 20 years, other drugs have failed to achieve good therapeutic effects in the first-line treatment of SCLC, such as drugs for inhibiting vascular endothelial growth (bevacizumab, tharidomide, etc.), multi-target tyrosine kinase inhibitors (sunitinib), bcl-2 inhibitors (belimersen), PARP inhibitors (Veliparib, etc.), cyclin-dependent kinase 4/6 (cyclin-dependent kinase 4/6, CDK 4/6) inhibitors (trilacilib), DLL3 inhibitors (Rovalpituzumab tesirine), etc. have not achieved the expected effects in the clinical trials of SCLC.

In recent years, the advent of immunotherapy has brought new hopes for the treatment of tumor patients, and has been expected by tumor researchers. SCLC has the characteristics of strong heterogeneity, unstable genome, higher tumor mutation load and the like, so as to stimulate the organism to release more tumor-related antigens, and is possibly beneficial to promoting immune cells to kill tumor cells. It was found that the combination of an anti-apoptotic receptor-ligand 1 (PD-L1) immune drug such as Atezolizumab, durvalumab and Adebrelimab with standard chemotherapy regimens increased the overall median survival of SCLC over the extensive period, but it was noted that the median survival of SCLC only benefited by about 2 months compared to NSCLC immunotherapy. Subsequent scholars further analyzed the data in depth, finding that the survival time in a few SCLC patients can benefit by about 8 months. Therefore, deep research into the potential benefit of SCLC is of great clinical importance.

Chinese patent CN113943806a, publication date 2022.01.18, discloses a biomarker for predicting susceptibility of lung adenocarcinoma patients to immune checkpoint inhibitor therapy, comprising a mutant cell differentiation regulating gene selected from at least one of ZFHX3 and PTPRD genes, the mutant being missense mutation and/or nonsense mutation. The biomarker for predicting the sensitivity of the lung adenocarcinoma patients to the immune checkpoint inhibitor therapy has obvious correlation with TMB value, can be used as a new biomarker for predicting the sensitivity of the lung adenocarcinoma patients to the immune checkpoint inhibitor therapy, can accurately screen beneficiary populations of the immune checkpoint inhibitor therapy, and saves detection cost.

Lung cancer is largely divided into non-small cell lung cancer and small cell lung cancer, wherein non-small cell lung cancer accounts for about 85%, and mainly comprises lung squamous cell carcinoma and lung adenocarcinoma, whereas small cell lung cancer accounts for about 15%. SCLC is a heterologous neuroendocrine tumor derived from Kulchitsky cells of the bronchial mucosal epithelium, and is the most invasive subtype of lung cancer, accounting for about 15% -20% of lung cancer. 98% of SCLC is attributed to smoking, and is characterized by high malignancy, high invasiveness, rapid disease progress, easy occurrence of distant metastasis in early stage and the like, and although initial treatment is sensitive, the disease is easy to relapse and transfer, drug resistance and other characteristics are formed, so that SCLC patients have extremely poor prognosis, the survival rate of 5 years is less than 7%, and the essential difference exists between SCLC and non-small cell lung cancer.

In addition, the mode of drug treatment is also different for SCLC and non-small cell lung cancer. For treatment of non-squamous cell carcinoma driving gene negative or unknown non-small cell lung cancer patients, it is generally recommended that platinum-based combination pemetrexed, paclitaxel, gemcitabine or docetaxel be subjected to chemotherapy; for the treatment of patients with squamous cell carcinoma driving gene negative or unknown non-small cell lung cancer, platinum-based therapies in combination with paclitaxel, gemcitabine or docetaxel are generally recommended; for lung adenocarcinoma patients with genetic mutation, corresponding targeted therapies, such as drugs for targeting EGFR mutation, ALK mutation and the like, can be recommended; for SCLC, however, it is generally recommended that platinum is used in combination with etoposide or irinotecan for chemotherapy without the corresponding targeted drug for treatment.

Regarding immunotherapy, current domestic guidelines recommend only a few immune drugs for the treatment of extensive SCLC, including Atezolizumab, durvalumab, adebrelimab and Serpalimab, whereas immune drugs for non-small cell lung cancer are far more than SCLC, suggesting that there is a large difference between the two tumors. For the investigation of biomarkers for lung cancer immunotherapy, non-small cell lung cancer and SCLC are also different. For non-small cell lung cancer, no completely exact immunotherapeutic biomarker is currently available, and it is generally speculated that patients with high expression of PD-L1, TMB, tumor neoantigens may benefit from immunotherapy, and studies suggest that patients with SKT11, KEAP1, KRAS gene mutations may not benefit from immunotherapy. In addition, students use public databases to resolve potential biomarkers for non-small cell lung cancer immunotherapy, and they find that ZFHX3 mutated non-small cell lung cancer patients may benefit from immunotherapy.

While there are currently few studies on biomarkers for SCLC immunotherapy, foreign scholars divide SCLC into four subtypes ASCL1, plurod 1, POU2F3 and Inflamed according to transcriptome data, wherein Inflamed subtype can benefit from immunotherapy, but no clear biomarker is available, and no report has been made on ZFHX3 gene mutation invented herein as a biomarker for SCLC immunotherapy.

Disclosure of Invention

The object of the present application is to provide a biomarker for detecting sensitivity of SCLC patients to immunotherapy and its use, which address the deficiencies in the prior art.

In one aspect, a biomarker for detecting sensitivity of an SCLC patient to immunotherapy is provided, the biomarker being a mutant ZFHX3 gene.

In a second aspect, reagents for detecting the biomarkers described above are provided.

As a preferred example, the reagent is selected from one or more of a primer, a primer pair, a probe, an antibody and a nucleic acid chip.

In a third aspect, a diagnostic kit for SCLC immunotherapy is provided, said kit comprising the above-described reagents.

In a fourth aspect, there is provided the use of the above-described reagents in the preparation of a diagnostic reagent or kit for SCLC immunotherapy.

In a fifth aspect, there is provided the use of a biomarker as described above in the manufacture or screening of a SCLC immunotherapeutic agent.

In a sixth aspect, there is provided the use of the above-described reagents and kits for the preparation of a product for predicting or judging the effect of immunotherapy in a patient with SCLC.

In a seventh aspect, there is provided the use of a biomarker as described above as a target in the manufacture of a product for inhibiting the occurrence and/or development of SCLC.

In an eighth aspect, a method for predicting the efficacy of SCLC immunotherapy and for prognosis is provided, said method comprising detecting the biomarker as described above.

The application has the advantages that:

at present, for SCLC, platinum-based combined etoposide or irinotecan is generally recommended for chemotherapy, and no corresponding targeting drug is used for treatment. For immunotherapy of SCLC, the median survival of SCLC benefits only around 2 months, whereas the median survival of a few SCLC patients benefits up to around 8 months, but lacks the corresponding immunotherapeutic biomarkers. The application has the advantages that the deep analysis discovers that SCLC patients with ZFHX3 gene mutation have better immune infiltration and better prognosis, which indicates that SCLC patients with ZFHX3 gene mutation possibly benefit from immunotherapy, and fills the current situation that SCLC lacks an immunotherapy biomarker.

Drawings

FIG. 1 is a diagram of the results of immunophenotyping based on transcriptome sequencing. A: SCLC immunophenotyping can be classified as hot tumor, cold tumor and beside cancer; b: SCLC patient prognosis effect of immunocold tumor; c: the mutation situation of ZFHX3 gene in immunothermic tumor; immunization and TMB scoring in ZFHX3 gene mutated tissues.

FIG. 2 shows the results of genomic sequencing-based gene mutation. A: the first 6 with higher mutation frequencies are in turn: TP53 (72%), RB1 (56%), KMT2C (21%), ZFHX3 (19%), KMT2D (16%), FAT1 (12%).

FIG. 3 shows prognosis and pathway enrichment analysis of ZFHX3 gene mutant patients. A: prognosis of ZFHX3 gene mutated patients compared to unmutated patients; b: pathway enrichment analysis of ZFHX3 gene mutation samples.

FIG. 4 shows the rate of pathological remission of SCLC patients with ZFHX3 mutations versus non-mutated patients by exon sequencing analysis.

Detailed Description

The application is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the description of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available usual instruments and reagents, and can be referred to in the molecular cloning test guidelines (3 rd edition) (scientific press), microbiological tests (4 th edition) (higher education press) and manufacturer specifications of the corresponding instruments and reagents.

EXAMPLE 1 application of ZFHX3 Gene mutation as SCLC immunotherapeutic biomarker

1 test sample

All SCLC tumors and Normal Adjacent Tissues (NATs) were collected in Shanghai city pulmonary department hospital (university of Shangji, china Shanghai) from 4 months 2012 to 6 months 2019 in this study. The study was approved by the Shanghai department of Lung hospital institutional review board and informed consent was obtained for all participating patients. All patients underwent surgical excision and had not previously received chemotherapy, radiation or immunotherapy.

2 experimental procedure

2.1DNA sequencing

Genomic DNA was extracted from tumors and NATs using QIAamp Fast DNA tissue kit (QIAGEN, hilden, germany) according to the protocol of the kit. Total DNA was quantified using a Qubit 2.0Flurometer (Life Technologies, CA, USA) and NanoDrop 2000 (Thermo Fisher Scientific), and integrity was assessed using a TapeStation (Agilent Technologies, CA, USA). Genomic DNA was fragmented to an average of 180-280bp using a Covaris focused ultrasound apparatus. A WES library was then prepared and captured using a Agilent SureSelect HumanAll ExonV kit (Agilent Technologies, CA, USA) according to the kit instructions, and a DNA library with 150bp paired-end reads was sequenced on an IlluminaNovaseq 6000 platform (Illumina, CA, USA). All genomic sequencing was done by the Tianjin Norhe origin company.

2.2RNA sequencing

Total RNA was extracted and purified from fresh frozen tissues using TRIzol reagent (Invitrogen, CA, USA). UsingRNAassaykit, in->2.0Flurometer (Life Technologies, CA, USA) andthe RNA concentration and purity of all RNA analytes were detected in a spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNANano 6000Assay Kit of the Bioanalyzer 2100 System (Agilent Technologies, CA, USA). RNA integrity value (RIN)>The sample of 6.0 was a high quality sample used to construct a transcriptome library. Use->UltraTM RNA library Prep Kit for/>The (NEB, MA, USA) kit performs total RNA-seq library construction on RNA samples and adds an index code to the property sequence of each sample. Each step was quality controlled and using the Agilent Bioanalyzer 2100 system (Agilent technologygies, CA, USA) quantitates the library. The paired-end library was then sequenced on an Illumina Novaseq6000 platform (Illumina, CA, USA) to generate 150bp paired-end reads. A total of 107 paired tumor and NAT samples were used for RNA sequencing by RNA quality control. All transcriptome sequencing was done by the Tianjin Norhe source company.

2.3 analysis of Gene mutations

To detect Single Nucleotide Variations (SNV) and small insertions/deletions (INDEL), analysis was performed using Genome Analysis Toolkit (GATK, version 4.0.6.0). After excluding low quality reads, matched paired-end WES sequencing reads were aligned with human reference genome (hg 38) using BWA MEM (version 0.7.15, http:// bio-BWA. Sourceforge. Net /). The resulting BAM file was further processed with a Picard tool (version 2.9.0, http:// broadensite. Gitgub. Io/Picard /) to remove PCR duplicates. Subsequently, recalibration and INDEL recalibration were achieved using the GATK modules INDEL realignor and baserecalcifier. Cross sample contamination was assessed using the GATK module calculecotaining tool with a threshold of 5%. Snv and INDELs were detected using mutact 2 tool (cibulsky et al 2013) embedded in SCLC tumor and paired non-tumor samples GATK, followed by filtering of the short tandem repeat regions downloaded from UCSC table browser, and finally annotated with funcators in GATK.

2.4TMB analysis

For each patient we calculated the TMB score, i.e. total number of truncated mutations x 1.5+ total number of non-truncated mutations x 1.0. Truncation mutations include nonsense mutations, frameshift deletion mutations, frameshift insertion mutations, and splice site mutations, while non-truncation mutations include missense mutations, intraframe deletion mutations, intraframe insertion mutations, and uninterrupted mutations. Silent mutations are excluded from these analyses because they do not lead to amino acid changes. Considering that truncation mutations have a greater detrimental effect on gene function than non-truncation mutations, higher weights are given.

2.5 pathological remission rate analysis

The applicant subject group of the present application has registered and performed 2 clinical trials (NCT 04539977, NCT 04542369) to evaluate the efficacy of immune drugs in small cell lung cancer. Currently 12 pre-treatment small cell lung cancer samples (9 from the NCT04539977 clinical trial and 3 from the NCT04542369 clinical trial) have been collected and analyzed for the rate of pathological remission of ZFHX3 gene mutated and unmutated patients by exon sequencing.

3 statistics and analysis

3.1 immunoassay

Using RNA-seq data, the abundance of 64 different cell types in 107 SCLC tumors and paired NAT samples was calculated by xCell (https:// xCell. Ucsf. Edu /). Unsupervised clustering is then achieved using NMF R packages (version 0.23.0) to sign these units. We select k=3 from the largest coherence coefficient using 50 iterations, followed by 200 iterations of the NMF analysis.

3.2 survival analysis

The Kaplan-Meier analysis was used to explore the survival differences from ZFHX3 mutant status and immune subtypes.

4 experimental results

Based on transcriptomic data, using xCell algorithm, unsupervised cluster analysis, tumor and paracancestral tissues were divided into 3 subtypes, i.e., hot tumor, cold tumor and paracancestral tumor (fig. 1A), wherein most of the tumors were immunocold tumor, with worse prognosis (fig. 1B), whereas hot tumor was enriched for more killer immune cells, such as cd8+ T, NK, activated dendritic cells, M1 macrophages (fig. 1A). Based on genome data, we analyzed the mutation situation in SCLC tumor tissue, and we found that there were many gene mutations in SCLC, with higher mutation frequencies of TP53 (72%), RB1 (56%), KMT2C (21%), ZFHX3 (19%), KMT2D (16%), FAT1 (12%), etc. in order (fig. 2A).

Further analysis of the correlation of the gene mutation with cold and hot tumors, we found that there was a higher ZFHX3 gene mutation (p=0.0378) in hot tumors (fig. 1C). Further analysis found that tumors with ZFHX3 gene mutations possess higher immune scores and tumor mutation loads (Tumor Mutational Burden, TMB) than tumors without ZFHX3 gene mutations (fig. 1D), these data suggest that ZFHX3 gene mutations may be closely related to higher tumor immunity and may serve as molecular markers for tumor immunity. Survival analysis further suggested that patients with ZFHX3 gene mutation had a better prognosis than patients without ZFHX3 mutation (fig. 3A). Pathway enrichment analysis further demonstrates that tumors with ZFHX3 gene mutations enrich for several immune pathways, including response to interferon-gamma, antigen processing, and presentation pathways (fig. 3B), which further suggest that ZFHX3 gene mutations can be used as molecular markers for immunization of small cell cancerous tumors.

Exon sequencing analysis found that patients with ZFHX3 gene mutation had higher pathology remission rate (MPR) (fig. 4), suggesting that small cell lung cancer patients with ZFHX3 mutation might benefit from immunotherapy.

The foregoing is merely a preferred embodiment of the present application, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present application, which modifications and additions are also to be considered as within the scope of the present application.

Claims (9)

1. A biomarker for detecting sensitivity of an SCLC patient to immunotherapy, characterized in that the biomarker is a mutant ZFHX3 gene.

2. A reagent for detecting the biomarker of claim 1.

3. The reagent according to claim 2, wherein the reagent is one or more selected from the group consisting of a primer, a primer pair, a probe, an antibody and a nucleic acid chip.

4. A diagnostic kit for SCLC immunotherapy, characterized in that said kit comprises the reagent of claim 2.

5. Use of the agent of any one of claims 2-3 for the preparation of a diagnostic reagent or kit for SCLC immunotherapy.

6. Use of the biomarker of claim 1 in the manufacture or screening of a SCLC immunotherapeutic agent.

7. Use of the biomarker of claim 1 in the manufacture of a product for assessing or aiding in assessing the efficacy of an immune single drug therapy of SCLC.

8. Use of the biomarker of claim 1 as a target in the preparation of a product that inhibits the occurrence and/or development of SCLC.

9. A method for predicting the efficacy of an SCLC immunotherapy and for prognosis, said method comprising detecting the biomarker of claim 1.