Gene marker for detecting small cell mixed type lung cancer of adenocarcinoma and application of gene marker

Abstract

The invention relates to the technical field of tumor typing, and discloses a genotype marker for subpopulation typing of adenocarcinoma small cell mixed type lung cancer in small cell lung cancer patients and application of the genotype marker. The gene marker comprises EGFR, KRAS, BRAF and PIK3CA, and point mutation sites of subpopulation typing related genes of adenocarcinoma small cell mixed type lung cancer in the gene marker can be used for distinguishing whether a small cell lung cancer patient is adenocarcinoma small cell mixed type lung cancer or not, so that an important judgment basis is provided for precise treatment of adenocarcinoma small cell mixed type lung cancer. The marker can also be used for gene detection on free tumor cells or tumor DNA in biopsy tumor tissue or body fluid of a small cell lung cancer patient, so that small cell lung cancer typing is rapidly carried out, the defects of current clinical pathological typing are overcome, and the marker has a good application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of tumor classification, and in particular to gene markers for detecting adenocarcinoma, small cell mixed lung cancer and their application. Background Art

[0002] According to the 2020 global cancer statistics, lung cancer is the most common malignant tumor and cause of cancer death in my country. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). SCLC is a high-grade neuroendocrine carcinoma, accounting for about 15% of all lung cancers. It is characterized by high proliferation rate, easy metastasis and poor prognosis, with a five-year survival rate of only 7%. Several studies have confirmed that small cell lung cancer can be combined with non-small cell lung cancer components such as large cell carcinoma, adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). These mixed tumors are called combined small cell lung cancer (CSCLC), accounting for about 2-5% of patients with small cell lung cancer. Compared with pure SCLC, CSCLC shows more aggressive behavior and worse prognosis. However, because the clonal origins and molecular characteristics of the different components in the histopathology of CSCLC tumors are unclear, CSCLC is clinically regarded as pure SCLC without special treatment.

[0003] At present, the main method of clinical diagnosis and classification of lung cancer is to obtain tumor tissue samples through surgery or biopsy, and use cytology and immunohistochemistry methods. However, due to the hidden nature of small cell lung cancer and the strong invasive ability of tumor cells, it is usually in the late clinical stage when diagnosed and cannot be operated on. Most of the pathological diagnosis and classification of small cell lung cancer rely on biopsy tissue. In mixed small cell lung cancer tumor tissue, the proportion of lung adenocarcinoma tumor cell components is relatively low, and the biopsy puncture sample is small, and it may not be possible to obtain both small cell and lung adenocarcinoma components. Therefore, it is often impossible to accurately diagnose adenocarcinoma-small cell mixed lung cancer, resulting in missed detection and incorrect classification, which makes it difficult to meet the clinical demand for accurate classification of adenocarcinoma-small cell mixed lung cancer.

[0004] Therefore, in order to meet the requirements of accurate classification and precise treatment of adenocarcinoma-small cell mixed lung cancer, a simple and accurate classification method for adenocarcinoma-small cell mixed lung cancer is urgently needed. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a gene marker for performing adenocarcinoma small cell mixed lung cancer subtype typing in small cell lung cancer patients. By detecting the mutation of these genes, it is possible to accurately distinguish whether a small cell lung cancer patient has adenocarcinoma small cell mixed lung cancer.

[0006] In the first aspect, gene markers are used to perform subtype typing of adenocarcinoma-small cell mixed lung cancer in patients with small cell lung cancer, wherein the gene markers include EGFR, KRAS, BRAF, and PIK3CA.

[0007] By adopting the above technical scheme, using gene markers composed of EGFR, KRAS, BRAF, and PIK3CA, and detecting point mutation sites of specific genes, it is possible to accurately classify the adenocarcinoma-small cell mixed lung cancer subgroup in patients with small cell lung cancer, accurately distinguish whether patients with small cell lung cancer have adenocarcinoma-small cell mixed lung cancer, provide a clinical basis for formulating personalized treatment plans for patients, and improve patients' treatment effects and survival rates.

[0008] On the other hand, the present application also provides the use of a detection reagent for detecting gene markers composed of EGFR, KRAS, BRAF, and PIK3CA in the preparation of a detection tool for subtyping adenocarcinoma small cell mixed lung cancer subtypes in patients with small cell lung cancer.

[0009] Furthermore, each of the gene mutation sites includes base mutation, base deletion or base insertion.

[0010] Preferably, the mutation sites of the EGFR include but are not limited to G719A, G719C, G719S, Ex19del, L747S, D761Y, V765A, S768I, V774A, T783A, S784P, T790M, V769M, Ex20Ins, L858R, L861Q, L861R, C797S, T854A and A871E, wherein the Ex19del indicates a fragment deletion in exon 19, and the Ex20Ins indicates a fragment insertion in exon 20.

[0011] Preferably, the mutation sites of KRAS include but are not limited to G12A, G12C, G12D, G12F, G12S, G12R, G12V, G13C, G13A, G13R, G13D, G13S, G13V, Q61R, Q61H, Q61K, Q61L, Q61P and A146T.

[0012] Preferably, the mutation sites of BRAF include but are not limited to V600E, V600D, V600K, V600R, K601E, K601N, L597R, L597S, L597Q, L597V, G464E, G464V, G466A, G466R, G466E, G466V, G469V, G469A, G469E, G469V, G469R, N581S, N581I, N581T, N581Y, D594A, D594N, D594G, D594H, D594E and D596R.

[0013] Preferably, the mutation sites of PIK3CA include but are not limited to E81K, E542K, E542Q, E542A, E543K, E545K, E545G, E545A, E726K, H1047R, H1047L, H1047R, H1047Y, H1047I, N345K, C420R, P539R, Q546K, Q546P, T1025S, M1043I, M1043V, R38H and K111N.

[0014] Furthermore, the reagent is a reagent that can detect the EGFR, KRAS, BRAF, and PIK3CA gene mutation site information using fluorescent quantitative PCR method, high-throughput sequencing method, nucleic acid mass spectrometry, and DNA chip technology.

[0015] By adopting the above technical scheme, the gene sequence is obtained by sequencing each gene in the gene marker, and the detection primer pair can be combined to achieve amplification of each gene sequence in the gene marker to obtain a complete gene sequence for comparison of mutation sites.

[0016] Furthermore, the application specifically includes the following steps: (1) Select ex vivo tissues or body fluids from patients with small cell lung cancer as test samples, and process each test sample to obtain tumor cell tissue genomic DNA; (2) constructing a high-throughput sequencing library of the gene marker using the detection reagent, and performing high-throughput sequencing; (3) Perform gene mutation analysis and somatic mutation analysis on sequencing data; (4) When at least one of the gene markers EGFR, KRAS, BRAF, and PIK3CA has a somatic mutation site, the patient is identified as a subgroup of adenocarcinoma-small cell mixed lung cancer.

[0017] By adopting the above technical scheme, the test samples can be detected through mature molecular biological methods such as high-throughput sequencing, which is simple and rapid to operate. At the same time, combined with gene mutation analysis and somatic mutation analysis, including point mutations, single nucleotide variations (SNVs), insertions and deletions of small fragments, etc., it is determined whether there are somatic mutations in the genes of EGFR, KRAS, BRAF, and PIK3CA in the gene markers, thereby facilitating and quickly determining the subtype of mixed small cell lung cancer samples.

[0018] Preferably, the mutation sites of the EGFR include but are not limited to G719A, G719C, G719S, Ex19del, L747S, D761Y, V765A, S768I, V774A, T783A, S784P, T790M, V769M, Ex20Ins, L858R, L861Q, L861R, C797S, T854A and A871E, wherein the Ex19del indicates a fragment deletion in exon 19, and the Ex20Ins indicates a fragment insertion in exon 20.

[0019] Preferably, the mutation sites of KRAS include but are not limited to G12A, G12C, G12D, G12F, G12S, G12R, G12V, G13C, G13A, G13R, G13D, G13S, G13V, Q61R, Q61H, Q61K, Q61L, Q61P and A146T.

[0020] Preferably, the mutation sites of BRAF include but are not limited to V600E, V600D, V600K, V600R, K601E, K601N, L597R, L597S, L597Q, L597V, G464E, G464V, G466A, G466R, G466E, G466V, G469V, G469A, G469E, G469V, G469R, N581S, N581I, N581T, N581Y, D594A, D594N, D594G, D594H, D594E and D596R.

[0021] Preferably, the mutation sites of PIK3CA include but are not limited to E81K, E542K, E542Q, E542A, E543K, E545K, E545G, E545A, E726K, H1047R, H1047L, H1047R, H1047Y, H1047I, N345K, C420R, P539R, Q546K, Q546P, T1025S, M1043I, M1043V, R38H and K111N.

[0022] Furthermore, the body fluid includes but is not limited to blood, pleural effusion, peritoneal effusion, cerebrospinal fluid, saliva and alveolar lavage fluid.

[0023] By adopting the above technical solution, the typing of the test samples can be achieved non-invasively through liquid biopsy technology.

[0024] On the other hand, the present application also provides a system for performing adenocarcinoma small cell mixed lung cancer subtype typing in patients with small cell lung cancer, comprising the following modules: A sequencing module for testing ex vivo tissue or fluid samples from a patient population with small cell lung cancer; Data analysis module, used to analyze point mutation sites in sequencing data; The determination module is used to determine the subgroup typing of the test sample using the gene markers composed of EGFR, KRAS, BRAF, and PIK3CA as detection targets. When at least one of the genes EGFR, KRAS, BRAF, and PIK3CA in the gene markers has a mutation site, it is determined to be a mixed adenocarcinoma small cell lung cancer subgroup.

[0025] Furthermore, the sequencing module includes reagents capable of detecting the EGFR, KRAS, BRAF, and PIK3CA gene mutation site information using fluorescent quantitative PCR method, high-throughput sequencing method, nucleic acid mass spectrometry, and DNA chip technology.

[0026] Furthermore, the mutation site includes base mutation, base deletion or base insertion at the mutation site.

[0027] Preferably, the mutation sites of the EGFR include but are not limited to G719A, G719C, G719S, Ex19del, L747S, D761Y, V765A, S768I, V774A, T783A, S784P, T790M, V769M, Ex20Ins, L858R, L861Q, L861R, C797S, T854A and A871E, wherein the Ex19del indicates a fragment deletion in exon 19, and the Ex20Ins indicates a fragment insertion in exon 20.

[0028] Preferably, the mutation sites of KRAS include but are not limited to G12A, G12C, G12D, G12F, G12S, G12R, G12V, G13C, G13A, G13R, G13D, G13S, G13V, Q61R, Q61H, Q61K, Q61L, Q61P and A146T.

[0029] Preferably, the mutation sites of BRAF include but are not limited to V600E, V600D, V600K, V600R, K601E, K601N, L597R, L597S, L597Q, L597V, G464E, G464V, G466A, G466R, G466E, G466V, G469V, G469A, G469E, G469V, G469R, N581S, N581I, N581T, N581Y, D594A, D594N, D594G, D594H, D594E and D596R.

[0030] Preferably, the mutation sites of PIK3CA include but are not limited to E81K, E542K, E542Q, E542A, E543K, E545K, E545G, E545A, E726K, H1047R, H1047L, H1047R, H1047Y, H1047I, N345K, C420R, P539R, Q546K, Q546P, T1025S, M1043I, M1043V, R38H and K111N.

[0031] Compared with the prior art, the present application has the following technical effects.

[0032] 1) For the first time, the gene markers composed of EGFR, KRAS, BRAF, and PIK3CA were used as the detection objects. Through the detection of somatic mutation sites of specific genes, the accurate classification of adenocarcinoma small cell mixed lung cancer subgroups in patients with small cell lung cancer was achieved, and whether the small cell lung cancer patients had adenocarcinoma small cell mixed lung cancer was accurately distinguished. At the same time, combined with clinical screening methods and clinical manifestations, it can provide important basis for disease treatment and medication guidance for patients with small cell lung cancer, and improve the treatment effect and survival rate of patients.

[0033] 2) The typing method provided in this application uses non-invasive biopsy technology and mature high-throughput sequencing technology, which is simple, fast and efficient to achieve accurate typing of adenocarcinoma small cell mixed lung cancer subgroups. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.

[0035] Figure 1 The results of gene marker detection and analysis of patient samples in Example 1.

[0036] Figure 2 The results of gene marker detection and analysis of patient samples in Example 2.

[0037] Figure 3 This is the analysis result of the sensitivity of gene marker detection in this application. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.

[0039] Example 1 1. Sample collection: In order to verify the detection sensitivity of gene markers, 17 SCLC / ADC patients confirmed by histopathology in publicly published academic papers were collected as a public data set. The applicant collected 10 samples.

[0040] In the applicant's own cohort, WES was used to sequence and analyze the mutation spectrum of SCLC and NSCLC components in the same formalin-fixed and paraffin-embedded (FFPE) slide of CSCLC samples. In each tumor sample, the regions of SCLC and ADC components were clearly defined. SCLC and ADC components in CSCLC samples were collected on tissue samples by microdissection and whole exome sequencing (WES) was performed. Paired normal adjacent tissues for each sample were also sequenced by WES. The average WES depth of tumors was 188× (range: 64-894×) and that of normal controls was 182× (range: 78-303×). The average number of mutations in tumors was 547 (range: 85-1133).

[0041] 2. Extraction of genomic DNA: The FFPE section genomic DNA extraction kit (QIAamp DNA FFPET issue Kit, #CAT: 56404) was used, and the experiment was strictly performed according to the instructions of the kit. The concentration of genomic DNA was determined using the Qubit 4.0 Fluorometer, and the Qubit dsDNA HS (High Sensitivity) Assay Kit (#CAT: Q33232) was used in conjunction.

[0042] 3. Capture of whole exome sequencing library: 200ng of genomic DNA was collected from each sample, and the intact genomic DNA was ultrasonically broken using Covaris S2 ultrasonicator to fragment the genomic DNA. The fragmented DNA was repaired at the end using KAPA DNA Library Preparation Kit, adenylate deoxyribonucleotide "A" was added, and the library adapter was connected. The whole genome library was then amplified, and the whole exon region was captured using Sure Select Human All Exon V6. The experiment was performed in strict accordance with the instructions of the kit.

[0043] 4. High-throughput gene sequencing: Use Geneplus-2000 sequencer to complete whole exome sequencing, using a double-end, 150bp sequencing scheme.

[0044] 5. Gene mutation analysis: Whole exome sequencing data preprocessing and variation analysis were based on Sentieon-genomics. Adapter sequences and low-quality bases in the original data were deleted according to the filtering criteria, sequencing reads were aligned with the human reference genome (hg19) using BWA MEM, PCR duplications were marked and removed using LocusCollector and Dedup, and realigned and recalibrated using Sentieon-genomics Realigner, with paracancerous tissue DNA used as a control.

[0045] 6. Somatic mutation detection Single nucleotide variants (SNVs) were analyzed by Sentieon-genomics Tnscope and MuTect2 software. Small insertions and deletions (indels) were identified by the Sentieon-genomics VarCall algorithm. High-quality reads were selected based on the following criteria: Phred score ≥ 30, mapping quality score ≥ 30, and no paired-end read bias. Candidate somatic mutations were selected using the following screening strategy: (i) mutations were detected in at least 5 high-quality reads and supported by at least 10 normal reads, and the total depth of the tumor site was greater than 30×. (ii) The mutant allele had to appear in ≥ 3% of the variant allele frequency (VAF) determined by TNscope. (iii) The mutation did not appear in more than 1% of the population in the 1000 Genomes Project, dbSNP database, and (iv) the local blacklist database. All these mutations were further filtered by the "PASS" output of MuTect2. The final overlapping variants were annotated using Ensembl Variant Effect Prediction (VEP v93.3) software.

[0046] 7. Analysis of genetic markers for adenocarcinoma-small cell combined lung cancer subtype classification: Analyze the somatic mutations of EGFR, KRAS, BRAF and PIK3CA genes in each tumor sample.

[0047] The results are as follows Figure 1 As shown, it can be seen that among the 17 samples in the publicly published academic literature, 14 patients were detected to be adenocarcinoma mixed with small cell lung cancer patients through the gene marker detection of the present application, and the sensitivity of the CSCLC detector was 82% ( Figure 1 Among the 10 samples collected by the applicant, 6 patients with adenocarcinoma mixed with small cell lung cancer could be detected by the CSCLC detector ( Figure 1 At the same time, multiple samples were taken from different spatial locations in the tumor tissue of the same patient, and the results of gene mutation detection showed that different components of small cell lung cancer tumor cells and lung adenocarcinoma tumor cells from different locations carried the same gene somatic mutation ( Figure 1 C), thus proving the feasibility of the typing method provided in the present application. Even if only a small amount of a single tumor component is obtained, the present gene marker can still be used to accurately diagnose adenocarcinoma small cell mixed lung cancer.

[0048] Example 2 1. Sample collection: The references in the database (10.2147 / OTT.S159612) were read, and the free tumor DNA (ctDNA) data in the peripheral blood of 99 patients with small cell lung cancer were sorted out from the literature, of which one patient had mixed small cell lung cancer and had been excluded.

[0049] 2. Extraction of ctDNA from plasma: According to the literature description, 5 mL of peripheral blood was drawn from each patient using an EDTA anticoagulation blood collection tube, and the plasma was separated by centrifugation at 4000 rpm for 10 minutes. ctDNA was extracted from the plasma using the High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany).

[0050] 3. Gene mutation detection: High-resolution melting fluorescence quantitative PCR technology (HRM) was used to detect EGFR, KRAS, BRAF and PIK3CA gene mutations respectively.

[0051] 4. Analysis of gene markers for adenocarcinoma-small cell mixed lung cancer subtype classification: Analyze the ctDNA EGFR, KRAS, BRAF and PIK3CA gene mutations of each patient. Figure 2 As shown, among 98 small cell lung cancer samples, 15% of the patients were classified as adenocarcinoma-small cell mixed lung cancer.

[0052] Example 3 We downloaded tissue-based sequencing data from two SCLC cohorts (n=64) and identified 14% of samples that tested positive on the CSCLC detector test ( Figure 3 In addition, the CSCLC Detector was used to test the ctDNA data of 254 SCLC patients, and 14% of the samples were identified as positive ( Figure 3 These results suggest that biopsy-based diagnosis underestimates the incidence of CSCLC in patients, especially combined adenocarcinoma-small cell lung cancer.

[0053] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A gene marker for subtyping adenocarcinoma small cell mixed lung cancer subtypes in patients with small cell lung cancer, characterized in that: The gene markers include EGFR, KRAS, BRAF, and PIK3CA.

2. Use of a reagent for detecting each gene mutation site in the gene marker according to claim 1 in the preparation of a detection tool for subtyping adenocarcinoma small cell mixed lung cancer subtypes in patients with small cell lung cancer.

3. The use according to claim 2, characterized in that: The various gene mutation sites include base mutation, base deletion or base insertion.

4. The use according to claim 2, characterized in that: The reagent is a reagent capable of detecting the EGFR, KRAS, BRAF, and PIK3CA gene mutation site information by using a fluorescent quantitative PCR method, a high-throughput sequencing method, a nucleic acid mass spectrometry method, and a DNA chip technology.

5. The use according to claim 2, characterized in that: The application comprises the following steps, (1) Select ex vivo tissues or body fluids from patients with small cell lung cancer as test samples, and process each test sample to obtain tumor cell tissue genomic DNA; (2) constructing a high-throughput sequencing library of the gene marker using the detection reagent, and performing high-throughput sequencing; (3) Perform gene mutation analysis and somatic mutation analysis on sequencing data; (4) When at least one of the gene markers EGFR, KRAS, BRAF, and PIK3CA has a somatic mutation site, it is diagnosed as adenocarcinoma small cell mixed lung cancer.

6. The use according to claim 5, characterized in that: The body fluids include, but are not limited to, blood, pleural effusion, peritoneal effusion, cerebrospinal fluid, saliva and alveolar lavage fluid.

7. A system for classifying adenocarcinoma-small cell mixed lung cancer in patients with small cell lung cancer, characterized in that: Includes the following modules, A sequencing module for testing ex vivo tissue or fluid samples from a patient population with small cell lung cancer; Data analysis module, used to analyze gene mutation and somatic mutation of sequencing data; A determination module is used to determine the subgroup typing of the test sample using the gene markers EGFR, KRAS, BRAF, and PIK3CA as the comparison standard, when the gene markers If at least one of the EGFR, KRAS, BRAF, and PIK3CA genes has a somatic mutation site, it is determined to be adenocarcinoma-small cell mixed lung cancer subtype.

8. The system according to claim 7, characterized in that The sequencing module includes reagents that can detect the EGFR, KRAS, BRAF, and PIK3CA gene mutation site information by using fluorescent quantitative PCR method, high-throughput sequencing method, nucleic acid mass spectrometry, and DNA chip technology.

9. The system according to claim 7, characterized in that The mutation site includes base mutation, base deletion or base insertion at the mutation site.

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