CN114277129A - Application of SWI/SNF gene change in auxiliary diagnosis and prognosis of chordoma - Google Patents
Application of SWI/SNF gene change in auxiliary diagnosis and prognosis of chordoma Download PDFInfo
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Abstract
The invention relates to application of SWI/SNF gene change in auxiliary diagnosis and prognosis of chordoma. SWI/SNF gene alterations can be used as biomarkers for predicting chordoma patient-specific survival (CSS) and relapse-free survival (RFS). The results of the invention reveal the broad significance of SWI/SNF gene alterations as prognostic markers for chordoma.
Description
Technical Field
The invention relates to the field of chordoma auxiliary diagnosis, in particular to application of the chordoma on basis of skull to auxiliary diagnosis and prognosis.
Background
Chordoma is a rare cancerous tumor that occurs in about one million people per year. Chordoma accounts for less than 1% of tumors affecting the brain and spinal cord. Chordoma can occur anywhere in the spine, from the base of the skull to the coccyx. Chordoma grows slowly, gradually extending into the bone and surrounding soft tissue. They usually recur after treatment, and in about 40% of cases, the cancer spreads (metastasizes) to other parts of the body, such as the lungs.
About half of all chordomas occur at the bottom of the spine (sacrum), about one third at the bottom of the skull (occiput), and the remainder at the vertebrae of the cervical (neck), thoracic (upper back), or lumbar (lower back). As the chordoma grows, it applies pressure to adjacent areas of the brain or spinal cord, resulting in signs and symptoms of the disease. Spinal cord tumors anywhere in the spine can cause pain, weakness, or numbness in the back, arms, or legs. Basicranial chordoma (occipital chordoma) can lead to diplopia and headache. Chordoma (coccygeal chordoma) occurring in the coccyx can cause lumps large enough to be felt through the skin and can cause bladder or bowel function problems.
Chordoma usually occurs in adults between the ages of 40 and 70. About 5% of chordoma in children are diagnosed. For reasons of clarity, men are affected approximately twice as much as women. According to american epidemiological monitoring and end-of-care outcome (SEER) data, the incidence of chordoma varies by gender and race, but the etiology of the chordoma is poorly understood. To date, germline repeat coding of the T gene, which encodes the Brachyury protein, a transcription factor that plays an important role in embryonic development, is thought to be the major susceptibility mechanism for familial chordoma. Genetic polymorphisms common in the T gene are associated with an increased risk of familial and sporadic chordoma.
Chordoma is considered to grow slowly, but the rate of recurrence is high, especially in patients with basicranial chordoma, primarily due to incomplete tumor resection. Clinical progression of craniofacial chordoma is very different, and no verified clinical or molecular prognosis evaluation index exists at present. Treatment of cranial base tumors typically involves surgery with or without adjuvant radiation therapy. Chemotherapy or other systemic therapies are not effective enough to treat chordoma. Although several molecular targets for potential drug therapy have been identified, and some are being evaluated in clinical trials, treatment options for patients with chordoma, particularly those in the advanced stage of the tumor, remain limited.
Genomic analysis of chordoma is not a well studied. To date, the largest sequencing analysis included 104 patients with sacral chordoma, but only 11 tumors were genome-wide sequenced. The results of this study indicate that T amplification, homozygous deletion of CDKN2A, and mutations in the SWI/SNF chromatin remodeling complex gene (PBRM1, SETD2, ARID1A) and PI3K signaling pathways are the most common genomic alteration events in sacral chordoma. The study did not examine driver mutations, structural variations and mutation signatures, limited by the small number of samples. It is also unclear whether craniofacial chordoma, which is an earlier disease than sacral chordoma, is associated with similar genomic alterations. Furthermore, the correlation of these genomically altered events with clinical characteristics of tumors is largely unknown.
Therefore, there is an urgent need for better understanding of the molecular processes in chordoma to develop prognostic prediction tools and to discover new drug therapeutic targets.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to screen out SWI/SNF genomic variation susceptible to chordoma as a molecular diagnostic marker, develop a corresponding diagnostic kit and diagnose the chordoma or provide prognosis thereof.
Specifically, the invention provides the following technical scheme:
in one aspect, the invention provides the use of a product for detecting SWI/SNF gene alteration in the preparation of a reagent or kit for aiding in the diagnosis of chordoma.
In one aspect, the invention provides the use of a product that detects changes in the SWI/SNF gene in the manufacture of a reagent or kit for predicting survival of a chordoma patient.
In one aspect, the invention provides an agent or kit for aiding diagnosis of chordoma or predicting survival of chordoma patients, the agent or kit comprising an agent for detecting a mutation in the SWI/SNF gene; and/or the reagent or kit comprises a reagent for detecting structural variation of the SWI/SNF gene.
Drawings
FIG. 1 shows a genome-driven gene map of basicranial chordoma. Wherein the top bar graph shows the number of non-synonymous mutations per tumor. The upper gene panel shows non-synonymous mutations in the potential chordoma driver gene. The middle gene panel shows non-synonymous mutations in known tumor driver genes detected in this study. Wherein, SNV: single nucleotide variations; INDEL: base insertions or deletions; SV: structural variation; HOMO DEL: homozygous deletion; AMP: amplification; germline DUP: the germ line repeats. The bottom panel shows whether the tumor has recurred (non-recurrent: grey; recurrent: red), age (young to old: blue to yellow), gender (female: red; male: blue).
FIG. 2 shows SWI/SNF gene alteration profiles, wherein FIG. 2A shows that non-synonymous mutations were found in PBRM1 gene (5 patients) and SETD2 gene (2 patients). Each dot represents a single mutation, and the length of the line under the dot reflects the number of samples with that mutation. The colored squares represent the functional domains in the protein. The dotted line represents a splicing mutation in the non-coding region. FIG. 2B shows that a variation in gene structure was found in the PBRM1 gene (5 patients) and the SETD2 gene (1 patient). Different colors represent different types of structural variation.
Figure 3 shows GISTIC analysis identifying regions of focal somatic copy number variation, where figure 3A shows regions of chromosomal focal amplification and figure 3B shows regions of chromosomal deletion.
Fig. 4 shows the genomic changes associated with chordoma-specific survival (CSS) and recurrence-free survival (RFS), wherein the correlation analysis of chordoma-specific survival (top half) and recurrence-free survival (bottom half) with SWI/SNF gene status for chordoma patients is shown. P-value, risk ratio (HRs) and 95% Confidence Interval (CIs) were calculated from Cox proportional hazards models and corrected for age, gender, pre-and post-operative radiotherapy, and other influencing factors. "+" indicates that the patient carries the genomic variation.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
The term "chordoma" refers to a rare low-grade malignant primary tumor that originates from residual spinal cord tissue of the embryo.
The term "classic chordoma" refers to a chordoma which is characterized in that tumor tissues are pathologically divided into masses with different sizes, clear or unclear boundaries by fibrous tissues, tumor cells at the periphery of the masses are small in volume and are arranged in a nest shape or a strip shape, the tumor cells at the central area of the masses are obviously increased in volume, vacuoles with different sizes are arranged in cytoplasm to form typical droplet cells, and Brachyury is strongly and positively expressed in cell nuclei.
The term "chondrogenic chordoma" refers to chordoma that is pathologically characterized by more or less distinct hyaline cartilage-like regions in addition to typical chordoma, and that is also expressed strongly positive for Brachyury in the nucleus.
The term "dedifferentiated chordoma" refers to chordoma that is pathologically represented by the presence of both classic chordoma structures and sarcoma-like structures (i.e., undifferentiated spindle-shaped nuclei, resembling osteosarcoma cells) within the tumor tissue, but with well-defined components, no morphological features in the transition region between the two, and no Brachyury expressed in the nuclei of the sarcoma components.
The term "SWI/SNF" is a nucleosome remodeling complex that occurs in both eukaryotes and prokaryotes. They are a group of proteins involved in the remodeling DNA packaging mode. The human SWI/SNF complex has inhibitory effects on many human malignancies.
The SWI/SNF complex is mainly comprised of two classes: BRG 1/BRM-associated factor complex (BRG1/BRM associated factor, BAF) and polybromo-associated BAF complex (PBAF). BAF and PBAF both include three core subunits (SMARCB1, SMARCC1 and SMARCC2), with differences in auxiliary regulatory subunits, BAF mainly includes ARID1A/1B, DPF1/2/3, SS18, SMARCE1, SMARCD1/D2/D3, ACTL6A and BRD9, PBAF mainly includes ARID2, PBRM1, PHF10, SMARCE1, SMARCD1/D2/D3, ACTL6A and BRD 7. SMARCA2 and SMARCA4 contain 6 conserved domains: QLQ domain, proline-rich domain, small helicase/SANT related domain, DNA-dependent ATPase domain, Retinoblastoma (RB) binding domain (LxCxE), and Bromo domain. The Bromo domain interacts with acetylated histones and is involved in the binding and stability of the SWI/SNF complex to DNA. The LxCxE domain binds to a member of the RB tumor suppressor family, while the QLQ domain is involved in protein-protein interactions. Finally, helicase and DExDc domains separate DNA duplexes requiring ATP hydrolysis. However, the two ATP hydrolases have significant differences in function and cannot compensate each other. INI1/SMARCB 1, BAF 155/SMARCC1 and BAF170/SMARCC2 are termed "core subunits" and are essential for ATP-dependent chromatin remodeling activity of SMARCA2 or SMARCA4, primarily involved in double strand break and nucleotide excision repair. The SWI/SNF compound also has 7-10 auxiliary regulation subunits, targets specific DNA or gene locus and is responsible for specific genome targeted by different compounds. BAF complex comprises ARID1A/B, PBAF complex comprises PBRM1, ARID2, BRD7 and PHF 10. It contains specific domains (bromodomains, chromatin domains, DNA binding domains, ARIDs and Zing fingers, etc.) required for interaction with DNA or histones.
The term "gene" refers to a segment of DNA involved in the production of a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) involved in transcription/translation of the gene product and the regulation of said transcription/translation, as well as intervening sequences (introns) between the individual coding regions (exons).
The term "genome" relates to the total amount of genetic information in the chromosome of an organism or cell.
The term "mutation" refers to a permanent change to the nucleotide sequence, extrachromosomal DNA or other genetic element of the genome of an organism.
The term "structural variation" may refer to a variation in the structure of a chromosome. Structural variations can be deletions, duplications, copy number variants, insertions, inversions, and translocations. In some cases, two regions that are far apart are brought into proximity. A hybrid gene formed from two previously separated genes, which may be linked by, for example, a translocation, deletion, or inversion event, may be referred to as a "gene fusion" or "fusion gene".
The term "non-synonymous mutation" refers to a mutation, preferably a nucleotide substitution, that results in an amino acid change (e.g., amino acid substitution) in the translation product, which preferably results in the formation of a neo-epitope.
The term "single nucleotide variant/variation" (SNV) refers to the difference in nucleic acid sequence at a particular site (allele) when comparing the genome from a diseased cell (e.g., a tumor cell) to the genome of a preferably matching (corresponding) normal non-diseased cell or a reference genome.
The term "insertion" refers to the addition of one or more additional nucleotides to the DNA. Insertions in the coding region of a gene can alter splicing of the mRNA (splice site mutations), or cause a shift in the reading frame (frameshifting), both of which can significantly alter the gene product.
The term "deletion" refers to the removal of one or more nucleotides from the DNA. Like insertions, these mutations can alter the reading frame of a gene.
The term "repeat" refers to multiple copies of the same base sequence. The term "tandem repeat" refers to multiple copies of the same base sequence in the same orientation. Thus, they are, for example, copies of the tandem nucleotide sequence repeated several times in the chromosomal direction. Any of the arrays of repeating series in series may comprise multiple copies of a single element or may have at least one other element interspersed within the array or within an element of the array.
The term "inversion" refers to reversing the orientation of a chromosome segment.
The term "translocation" or "chromosomal translocation" generally refers to the exchange of equal or unequal amounts of chromosomal material between the same or different chromosomes. Typically, crossover occurs between non-homologous chromosomes.
The term "amplification reaction" refers to a process for copying nucleic acid one or more times. In embodiments, the amplification methods include, but are not limited to: polymerase chain reaction, self-sustained sequence reaction, ligase chain reaction, rapid amplification of cDNA ends, polymerase chain reaction and ligase chain reaction, Q-beta phage amplification, strand displacement amplification, or overlap-extension-splice polymerase chain reaction. In some embodiments, a single molecule nucleic acid is amplified, for example, by digital PCR.
The term "chromosomal fragmentation" refers to a genetic phenomenon that is fragmented through a specific region of a single catastrophic event genome and then spliced together.
The term "biomarker" refers to a biomolecule or fragment of a biomolecule, the change and/or detection of which may be associated with a particular physical condition or state. Throughout the present disclosure, the terms "marker" and "biomarker" are used interchangeably. For example, the biomarkers of the invention are associated with chordoma. These biomarkers include, but are not limited to, biomolecules including nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, fats, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules, and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins). The term also includes portions or fragments of biomolecules, for example, peptide fragments of proteins or polypeptides comprising at least 5 contiguous amino acid residues, at least 6 contiguous amino acid residues, at least 7 contiguous amino acid residues, at least 8 contiguous amino acid residues, or more contiguous amino acid residues.
The terms "patient" and "subject" are used interchangeably to refer to both human or other mammalian patients and subjects, and include any individual examined or treated using the methods of the invention. However, it should be understood that "patient" does not mean that symptoms are present. Suitable mammals falling within the scope of the present invention include, but are not limited to, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., corals, bears, wild cats, wild dogs, wolves, Australian wild dogs, foxes, etc.).
The term "kit" refers to any delivery system used to deliver a substance. In reaction assays, such delivery systems include systems that store, transport or deliver reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and/or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., cassettes) containing the relevant reaction reagents and/or support materials.
In one aspect, the invention provides the use of a product for detecting SWI/SNF gene alteration in the preparation of a reagent or kit for aiding in the diagnosis of chordoma.
In one aspect, the invention provides the use of a product that detects changes in the SWI/SNF gene in the manufacture of a reagent or kit for predicting survival of a chordoma patient.
In a preferred embodiment, the survival is chordoma-specific survival (CSS) and/or relapse-free survival (RFS).
In a preferred embodiment, the SWI/SNF gene alteration is a SWI/SNF gene mutation and/or a genetic structural variation.
In a preferred embodiment, the SWI/SNF gene is PBRM1 gene and/or SETD2 gene.
In a preferred embodiment, the genetic mutation is a non-synonymous mutation; preferably, the non-synonymous mutation is a frameshift mutation and/or a splicing mutation.
In a preferred embodiment, the structural variation is selected from one or more of duplication, inversion, translocation and deletion.
In a specific embodiment, the repeats are tandem repeats.
In a particular embodiment, the inversion is an intra-arm inversion and/or an inter-arm inversion.
In a specific embodiment, the translocation is an interchromosomal translocation.
In a specific embodiment, the chordoma is basicranial chordoma.
In a preferred embodiment, the product comprises reagents for detecting mutations in the SWI/SNF gene.
In a specific embodiment, the detection is by PCR technology; preferably, the PCR is digital PCR or quantitative fluorescence PCR (qpcr).
In a specific embodiment, the product comprises a PCR amplification reaction reagent mixture, a probe for SWI/SNF gene mutation; preferably, the PCR amplification reaction reagent mixture comprises TaqDNA polymerase, dNTP mixture and MgCl2The kit comprises a solution, a fluorescent quantitative PCR reaction buffer solution and deionized water; preferably, the probe is a TaqMan MGB probe, more preferably, the 5 'end of the TaqMan MGB probe is marked with a fluorescent reporter group, and the 3' end of the TaqMan MGB probe is marked with a non-fluorescent quenching group MGB; preferably, the fluorescent reporter is a FAM or HEX fluorescent reporter.
In a preferred embodiment, the product comprises reagents for detecting structural variations of the SWI/SNF gene.
In a specific embodiment, the assay is a chip-based assay.
In one aspect, the invention provides an agent or kit for aiding diagnosis of chordoma or predicting survival of chordoma patients, the agent or kit comprising an agent for detecting a mutation in the SWI/SNF gene; and/or the reagent or kit comprises a reagent for detecting structural variation of the SWI/SNF gene.
Changes in the PBRM1 gene and/or the SETD2 gene detected in SWI/SNF gene expression profiling studies indicate their potential importance for survival in chordoma patients, particularly basicranial chordoma patients. SWI/SNF gene alterations can be used as biomarkers for predicting chordoma patient-specific survival (CSS) and relapse-free survival (RFS). The results of the invention reveal the broad significance of SWI/SNF gene alterations as prognostic markers for chordoma.
Examples
The present invention will be described in more detail with reference to specific examples, which, however, are for illustrative purposes only and do not limit the present invention. The reagents and biomaterials described in the following examples are commercially available, unless otherwise specified.
Example 1 relationship of SWI/SNF Gene alteration to survival of chordoma patients
In this example, data and samples of patients with cranial base chordoma who underwent endoscopic nasal surgery in neurosurgery at the beijing Temple Hospital, affiliated with the university of capital medicine, between 10 months 2010 and 2017 and 11 months were analyzed. Primary tumor specimens, recurrent tumor specimens, and matched peripheral blood samples were collected from these patients. Their clinical pathology was recorded, including age, tumor histology type, tumor volume, Ki67 values, preoperative radiotherapy, postoperative radiotherapy, relapse and death. The study protocol was approved by the ethical committee of the beijing tiantan hospital and informed consent was obtained from all study participants.
Materials and methods
Sample collection, quality control and processing
A small portion of the chordoma specimen frozen in liquid nitrogen was embedded with optimal cutting temperature compound (OCT) and frozen with cold N-hexane, which was then cut into frozen sections 10 μm thick using a cryomicrotome. Frozen sections were fixed in cold acetone, stained with hematoxylin and eosin (H & E), and dehydrated by increasing the concentration of ethanol and xylene. Chordoma specimens with > 50% tumor cells were selected for subsequent DNA/RNA extraction.
Extraction of DNA and RNA
Genomic DNA was extracted from frozen tumor tissue specimens and patient peripheral blood samples matched thereto, respectively, using DNeasy blood & tissue kit. A total of 500ng of DNA with a large molecular mass (> 20Kb for a single band) was used to prepare the library. Tumor tissue was treated with TRIzol reagent to extract total RNA. The extracted RNA was first electrophoresed on a 1% agarose gel to check for degradation and contamination. RNA quality and quantity was assessed in a Bioanalyzer 2100 system using the RNA Nano 6000 detection kit. RNA samples with RNA integrity counts (RIN) greater than 6.8 were used to prepare transcriptome libraries and sequenced.
Library construction, sequencing and data Generation
The sequencing depth is the ratio of the total number of bases obtained by sequencing to the size of a genome, and is one of indexes for evaluating sequencing quantity, and the error rate or false positive rate caused by sequencing is reduced along with the increase of the sequencing depth.
Whole Genome Sequencing (WGS) and RNA sequencing (RNA-Seq) were performed by the Poa novo, Beijing. A whole genome sequencing library was constructed using a Truseq Nano DNA HT Sample Prep kit and whole genome sequencing was performed on an Illumina HiSeq X platform with a mean sequencing depth of 76X for tumors and 41X for paired peripheral blood. After excluding reads containing adaptor contamination and low quality/unrecognized nucleotides, sequencing data were mapped to the human genome (UCSC hg19) using Burrows-Wheeler Aligner software to obtain raw mapping results, and the results were stored in BAM format. SAMtools software, Picard software (http:// broadinstruction. githu. io/Picard /) and GATK software were used to order BAM files and base quality recalibration, repeat read deletion, local adjustment to generate the final BAM file for mutation analysis. The BAM-checker software was used to check if two BAM files were generated from the same patient.
Potential tumor-driven gene analysis
dNdScv software was used to identify tumor driver genes, while PBRM1 gene was the only significantly mutated gene identified in the patient cohort (FDR < 0.01). The PBRM1 gene belongs to the SWI/SNF chromatin remodeling complex gene, and the alteration of the SWI/SNF gene has a potential tumor-driving role in sacral chordoma, which is also considered as a potential tumor-driving event of craniofacial chordoma. Changes in the SWI/SNF gene include mutations in the PBRM1 gene and the SETD2 gene, structural variations involving both genes, and significant chromosomal fragmentation in the 3p21.3 region of chromosome, all of which have been confirmed by targeted sequencing.
Mutation validation
A plurality of nonsynonymous mutated genes including PBRM1 gene, SETD2 gene, CDKN2A/2B gene, MAP3K4 gene, BAG1 gene, ITGA6 gene, CSDE1 gene, etc. were screened in whole genome sequencing data, and these mutations were verified by Sanger sequencing.
Analysis of somatic copy number variation
Allele-specific somatic copy number variation was analyzed using FACETS software (version 0.5.6, https:// github. com/mskcc/faces). The GISTIC2.0 software was used to detect significantly mutated chromosomal regions.
Analysis of structural variation
The Meerkat algorithm was used to analyze somatic structural variation and the location of breakpoints in the genome from the re-calibrated BAM file. The parameters used have been adjusted accordingly to the sequencing depth of the tumor and blood samples.
Chromosome fragmentation analysis
Chromosome fragmentation was analyzed using ShatterSeek software (https:// github. com/parklab/ShatterSeek). Such chromosomal rearrangements were confirmed by permutation test. Chromosomal fragmentation events are identified only in regions of the chromosome that have a large number of interactions.
RNA sequencing
An amount of 3. mu.g of RNA was extracted from each sample as a starting material for RNA sample preparation. First, ribosomal RNA was removed using Epicentre Ribo-zeroTM rRNA Removal kit. Subsequently, rRNA-deplated RNA and
the UltraTM directed RNA Library Prep kit generated the sequencing Library. After adaptor ligation and library amplification, the library fragments were purified using the AMPure XP system in order to be able to select fragments of 150-200 bp in length. Strands labeled dUTP were not amplified for strand-specific sequencing. Finally, library quality was assessed on the Agilent Bioanalyzer 2100 system. After generation of the clusters, the library was sequenced on the Illumina Hiseq platform, yielding 150bp end-paired reads. Processing fastq formatRaw data, clean data was generated by removing reads containing adaptors or ploy-N and low quality reads. Gene expression was quantified using the TPM method using RSEM software, after which the values were logarithmized and statistically analyzed as log2TPM values.
Statistical analysis
The Wilcoxon scale test is used to compare the mean difference in genomic changes between different groups of patients stratified by treatment, clinical characteristics, or genomic characteristics. Multifactorial analysis is used to assess the association between multiple genomic features and patient features and to adjust for factors such as age of diagnosis, gender, pre-and post-operative radiotherapy, and the like. The Kaplan-Meier method was used to calculate the relapse-free survival and chordoma-specific survival of patients and was stratified for different genomic events. The multifactor Cox proportional hazards model is also used to test for differences in survival outcomes after adjustment for factors such as age, gender, pre-and post-operative radiotherapy, etc. for confirmation based on genomic characteristics. All statistical tests in this example are two-sided tests.
Results
Clinical characteristics of the patient
This example included 80 patients with craniofacial chordoma who had received diagnosis and treatment at the Beijing Temple Hospital. The mean age of the primary diagnosis of chordoma in these patients was 44.7 years (range: 7-79 years). Males accounted for 62.5%, most (80%) were classic chordoma, 12 of which received radiation therapy prior to surgery. After a mean follow-up period of 50 months, 59 relapses and 17 deaths all succumb to chordoma.
Whole genome sequencing analysis of DNA from 91 surgically excised chordoma specimens and their matched blood samples from 80 patients (including 11 matched primary and recurrent tumor samples) was performed.
Analysis of mutant genes
When non-synonymous mutations were analyzed, PBRM1 gene (6.25%), B2M gene (3.75%), and MAP3K4 gene (3.75%) were found to be the most common cancer driver genes for mutations in this patient cohort. Finally, only one significantly mutated gene was identified: PBRM1 gene. Five different types of PBRM1 mutations were found in total (4 INDEL +1 SNVs), each occurring in a different patient (fig. 1). Two mutations have also been found in the LYST gene, which encodes a protein that modulates protein trafficking functions in endosomes and was previously considered a potential chordoma driver gene. One of the carriers of the mutations in the two LYST genes also had germline T gene repeats. The TP53 gene mutation is common in different types of cancers, but the TP53 gene mutation was found in only one patient with chordoma.
Somatic copy number variation
Somatic copy number variation at the chromosomal level was found in most primary chordoma samples (77.5%). Consistent with previous research reports, frequent somatic copy number variation events at the chromosomal level were also observed in this example. Among them, 17 significant events were found by GISTIC analysis, including amplification of chromosomes 1q, 7p and 7q and deletion of chromosomes 1p, 3, 4, 9, 10, 13q, 14q, 18 and 22 q. Five different patient groups were assigned based on somatic copy number variation events. Group 1 (n-19) had no or only few somatic copy number variations. Group 2 (n-13) had sporadic somatic copy number variations, most of which were deletions. Group 3 (n-16) and group 4 (n-25) have a large number of somatic copy number variations, but group 4 lacks the amplification and deletion of chromosomes 4, 9 and 14. Group 5 (n-7) is characterized by enrichment of a large number of chromosomal amplifications.
GISTIC analysis of the highlighted somatic copy number variation regions identified 6 important amplifications and 12 important deletion regions (FIG. 3). The most important deletion region is 9p21.3(p ═ 5.87x 10)-13) This region contains the known cancer suppressor gene CDKN2A, while the deleted 3p21.1 region (p ═ 0.062) contains PBRM1 and SETD2, both SWI/SNF chromatin remodeling complex genes.
Driving gene profile of basicranial chordoma
In order to reveal potential drivers of basicranial chordoma, genomic alterations of the SWI/SNF gene (SWI/SNF +) were examined, and only alterations of the PBRM1 gene and SETD2 gene were found in the cohort of patients examined, including 7 patients with genetic mutations (5 with PBRM1 gene mutations, 2 with SETD2 gene mutations) and 6 patients with structural variations (5 with PBRM1 gene structural variations, 1 with SETD2 gene structural variations) (fig. 2). Wherein, figure 2A shows that non-synonymous mutations were found in PBRM1 gene (5 patients) and SETD2 gene (2 patients); a frameshift mutation in PBRM1 gene of patient 16, 34, 50, 71; splicing mutation occurred in PBRM1 gene of patient No. 06; this missense mutation occurred in the SETD2 gene of patient No. 09 and 73. FIG. 2B shows that tandem repeat structural variation (tandem _ dup) occurred in PBRM1 gene of patient No. 02; an inter-arm-to-site (inverters _ r) structural variation occurred in PBRM1 gene of patient No. 27; structural variations in intra-arm inversion (inverters _ f) and inter-arm inversion (inverters _ r) occurred in PBRM1 gene from patient No. 44; a deletion (del) in the PBRM1 gene of patient No. 76; an interchromosomal translocation (trans inter) occurred in PBRM1 gene of patient No. 32; structural variations in intraarm inversion (inverters _ f) and interarm inversion (inverters _ r) occurred in the SETD2 gene of patient No. 21. These results were verified by targeted sequencing and accounted for 16% of the chordoma samples that were sequenced for the whole genome (13 out of 80).
Chordoma with SWI/SNF gene alterations (SWI/SNF +) has a higher total number of mutations (Wilcoxon rank sum, p 0.004) and nonsynonymous mutation burden (p 0.01) and also a higher number of structural variations, in particular tandem repeats (p 0.0001) and interchromosomal translocations (p 0.02), than chordoma without SWI/SNF gene alterations (SWI/SNF-).
Basis cranii chordoma driver gene profile and clinical characteristics of patients
Of the 80 chordoma patients undergoing whole genome sequencing, 64 were classical, 14 were cartilaginous, and 2 were dedifferentiated. There were no significant differences in mutation load, total number of structural variations and tumor genomic drivers (SWI/SNF) between canonical and chondrogenic chordomas. Both patients with dedifferentiated chordoma had homozygous deletions of chromosome 9p21, as well as high mutation loads and complex structural variations. One patient was diagnosed with chordoma at 9 years of age and the tumor had recurred 6 months after surgery. While the other 9 pediatric chordoma patients (diagnosed. ltoreq.20 years) appeared to have a silent genome characterized by a low tumor mutation burden and lack of tumor drivers.
Basis cranii chordoma driver gene profile and clinical prognosis
Since it is clinically important to identify chordoma patients with malignant characteristics, it is aimed at finding genomic alterations that correlate with the prognosis of the patient. Table 1 shows the association between genomic alterations and chordoma-specific survival (CSS) (table 1A) and relapse-free survival (RFS) (table 1B). HR: a risk rate; lower and Upper: 95% confidence interval.
TABLE 1
A.CSS.
| Genomic features | HR | Lower | Upper | P |
| SWI/SNF+ | 5.32 | 1.88 | 15.05 | 0.002 |
| CDKN2A/B/C+ | 0.88 | 0.20 | 3.92 | 0.86 |
| SCNA_Group2a | 1.90 | 0.25 | 14.33 | 0.53 |
| SCNA_Group3a | 2.21 | 0.39 | 12.43 | 0.37 |
| SCNA_Group4a | 3.39 | 0.62 | 18.48 | 0.16 |
| SCNA_Group5a | 1.22 | 0.11 | 14.04 | 0.87 |
| 1p deletion | 0.94 | 0.31 | 2.83 | 0.92 |
| 3p deletion | 3.94 | 1.09 | 14.29 | 0.04 |
| 3q deletion | 2.81 | 0.89 | 8.89 | 0.08 |
| 4p deletion | 1.06 | 0.33 | 3.38 | 0.93 |
| 4q deletion | 1.04 | 0.31 | 3.42 | 0.95 |
| 9p deletion | 2.01 | 0.56 | 7.17 | 0.28 |
| 9q deletion | 2.46 | 0.80 | 7.55 | 0.12 |
| 10p deletion | 2.25 | 0.75 | 6.80 | 0.15 |
| 10q deletion | 2.28 | 0.75 | 6.92 | 0.15 |
| 13q deletion | 2.79 | 0.85 | 9.23 | 0.09 |
| 14q deletion | 3.21 | 0.93 | 11.06 | 0.06 |
| 18p deletion | 3.03 | 1.08 | 8.55 | 0.04 |
| 18q deletion | 3.08 | 1.09 | 8.75 | 0.03 |
| 22q deletion | 5.88 | 1.85 | 18.68 | 0.003 |
| 1q amplification | 1.51 | 0.45 | 5.09 | 0.50 |
| 7p amplification | 1.47 | 0.54 | 4.03 | 0.45 |
| 7q amplification | 1.87 | 0.63 | 5.61 | 0.26 |
| 9p21.3 | 1.44 | 0.42 | 4.88 | 0.56 |
| 9p11.2 | 1.33 | 0.42 | 4.16 | 0.62 |
| 9q21.11 | 1.80 | 0.61 | 5.32 | 0.29 |
| SWI/SNF+or 22q deletion* | 17.46 | 3.69 | 82.56 | 0.0003 |
B.RFS.
| Genomic features | HR | Lower | Upper | P | HR+Ki67 | pa |
| SWI/SNF+ | 3.89 | 1.96 | 7.68 | 9.6x10-5 | 3.70 | 0.01 |
| CDKN2A/B/C+ | 1.68 | 0.66 | 4.31 | 0.28 | 2.12 | 0.27 |
| SCNA_Group2 | 2.22 | 0.93 | 5.30 | 0.08 | 5.42 | 0.08 |
| SCNA_Group3 | 2.55 | 1.07 | 6.11 | 0.04 | 3.40 | 0.06 |
| SCNA_Group4 | 1.77 | 0.78 | 3.99 | 0.17 | 2.16 | 0.17 |
| SCNA_Group5 | 1.39 | 0.48 | 4.04 | 0.54 | 0.92 | 0.92 |
| 1p deletion | 1.41 | 0.77 | 2.57 | 0.26 | 2.79 | 0.03 |
| 3p deletion | 1.53 | 0.86 | 2.73 | 0.15 | 3.33 | 0.02 |
| 3q deletion | 1.84 | 1.05 | 3.24 | 0.03 | 4.18 | 0.004 |
| 4p deletion | 1.66 | 0.83 | 3.33 | 0.15 | 1.97 | 0.19 |
| 4q deletion | 1.84 | 0.89 | 3.81 | 0.10 | 1.83 | 0.26 |
| 9p deletion | 3.36 | 1.74 | 6.94 | 0.0003 | 2.97 | 0.02 |
| 9q deletion | 3.99 | 2.08 | 7.65 | 3.06x10-05 | 2.21 | 0.10 |
| 10p deletion | 1.71 | 0.97 | 3.04 | 0.07 | 2.16 | 0.07 |
| 10q deletion | 1.56 | 0.89 | 2.71 | 0.12 | 2.29 | 0.05 |
| 13q deletion | 1.08 | 0.62 | 1.88 | 0.78 | 1.22 | 0.65 |
| 14q deletion | 2.13 | 1.20 | 3.79 | 0.01 | 3.74 | 0.01 |
| 18p deletion | 2.39 | 1.36 | 4.20 | 0.002 | 1.83 | 0.13 |
| 18q deletion | 2.65 | 1.51 | 4.65 | 0.001 | 2.06 | 0.07 |
| 22q deletion | 3.74 | 1.89 | 7.38 | 0.0001 | 3.16 | 0.02 |
| 1q amplification | 1.53 | 0.81 | 2.91 | 0.19 | 1.65 | 0.29 |
| 7p amplification | 1.68 | 0.97 | 2.93 | 0.07 | 1.26 | 0.55 |
| 7q amplification | 1.49 | 0.85 | 2.59 | 0.16 | 1.01 | 0.98 |
| 9p21.3 | 2.65 | 1.42 | 4.93 | 0.002 | 3.24 | 0.01 |
| 9p11.2 | 2.54 | 1.38 | 4.67 | 0.003 | 3.79 | 0.004 |
| 9121.11 | 3.63 | 1.97 | 6.69 | 3.4x10-5 | 4.36 | 0.002 |
| SwI/SNF+or 9q21.11deletion or 22q deletion* | 4.06 | 2.22 | 7.40 | 4.96x10-6 | 3.88 | 0.007 |
Two potential tumor drivers, namely the status of the SWI/SNF gene and the CDKN2A/B gene, were first analyzed for correlation with chordoma-specific survival (CSS) and recurrence-free survival (RFS). SWI/SNF gene changes were significantly correlated with poorer prognosis, i.e., shorter chordoma-specific survival (5.32 for hazard ratio [ HR ], 1.88-15.05 for 95% confidence interval [ CI ], 0.002 for p) and relapse-free survival (3.89 for HR, 1.96-7.68 for 95% CI, 9.6x10-5) after correction for age, gender, pre-and post-operative radiation therapy, etc. (figure 4, table 1).
In contrast, CDKN2A/B gene changes were not significantly correlated with both chordoma-specific survival (HR 0.88, 95% CI 0.20-3.92, p 0.86) and relapse-free survival (HR 1.68, 95% CI 0.66-4.31, p 0.28).
When SWI/SNF gene alterations are combined with chromosome 22q deletions, their association with chordoma-specific survival becomes stronger (HR 17.46, 95% CI 3.69-82.56, p 0.0003). In contrast, the association of SWI/SNF gene alterations with chromosome 9q21.11 and 22q deletions only slightly increased the association with relapse-free survival (HR 4.06, 95% CI 2.22-7.40, p 4.96x 10-6). These genomic changes did not significantly correlate with chordoma-specific survival and relapse-free survival (changes in risk rates were not significant) after further correction of KI-67 values for tumors (table 1).
Claims (10)
1. Use of a product for detecting SWI/SNF gene alteration in the preparation of a reagent or kit for the auxiliary diagnosis of chordoma.
2. Use of a product that detects SWI/SNF gene alterations in the manufacture of a reagent or kit for predicting survival of a chordoma patient.
3. The use of claim 2, wherein the survival is chordoma-specific survival (CSS) and/or relapse-free survival (RFS).
4. The use of any one of claims 1-3, wherein the SWI/SNF gene alteration is a SWI/SNF gene mutation and/or a genetic structural variation.
5. The use of any one of claims 1-4, wherein the SWI/SNF gene is a PBRM1 gene and/or a SETD2 gene.
6. The use of claim 4 or 5, wherein the genetic mutation is a non-synonymous mutation; preferably, the non-synonymous mutation is a frameshift mutation and/or a splicing mutation.
7. The use of any one of claims 4-6, wherein the genetic structural variation is selected from one or more of a duplication, inversion, translocation, deletion;
preferably, the repeats are tandem repeats;
preferably, the inversion is intra-arm inversion and/or inter-arm inversion;
preferably, the translocation is an interchromosomal translocation;
preferably, the chordoma is basicranial chordoma.
8. The use of any one of claims 1-7, wherein the product comprises reagents for detecting mutations in the SWI/SNF gene;
preferably, the detection is by PCR technique; preferably, the PCR is digital PCR or quantitative fluorescence PCR (qpcr);
preferably, the product comprises a PCR amplification reaction reagent mixture, a probe aiming at SWI/SNF gene mutation; preferably, the PCR amplification reaction reagent mixture comprises TaqDNA polymerase, dNTP mixture and MgCl2The kit comprises a solution, a fluorescent quantitative PCR reaction buffer solution and deionized water; preferably, the probe is a TaqMan MGB probe, more preferably, the 5 'end of the TaqMan MGB probe is marked with a fluorescent reporter group, and the 3' end of the TaqMan MGB probe is marked with a non-fluorescent quenching group MGB; preferably, the fluorescent reporter is a FAM or HEX fluorescent reporter.
9. The use of any one of claims 1-8, wherein the product comprises reagents for detecting structural variation of the SWI/SNF gene; preferably, the detection is a chip-based detection.
10. An agent or kit for aiding in the diagnosis of chordoma or predicting the survival of a patient with chordoma, comprising an agent that detects a mutation in the SWI/SNF gene; and/or the reagent or kit comprises a reagent for detecting structural variation of the SWI/SNF gene;
preferably, the chordoma is basicranial chordoma;
preferably, the survival is chordoma-specific survival (CSS) and/or relapse-free survival (RFS);
preferably, the SWI/SNF gene alteration is a SWI/SNF gene mutation and/or a gene structural variation;
preferably, the SWI/SNF gene is a PBRM1 gene and/or a SETD2 gene;
preferably, the gene mutation is a non-synonymous mutation; preferably, the non-synonymous mutation is a frameshift mutation and/or a splicing mutation;
preferably, the genetic structural variation is selected from one or more of duplication, inversion, translocation, deletion; preferably, the repeats are tandem repeats; preferably, the inversion is intra-arm inversion and/or inter-arm inversion; preferably, the translocation is an interchromosomal translocation;
preferably, the product comprises a PCR amplification reaction reagent mixture, a probe aiming at SWI/SNF gene mutation; preferably, the PCR amplification reaction reagent mixture comprises TaqDNA polymerase, dNTP mixture and MgCl2The kit comprises a solution, a fluorescent quantitative PCR reaction buffer solution and deionized water; preferably, the probe is a TaqMan MGB probe, more preferably, the 5 'end of the TaqMan MGB probe is marked with a fluorescent reporter group, and the 3' end of the TaqMan MGB probe is marked with a non-fluorescent quenching group MGB; preferably, the fluorescent reporter is a FAM or HEX fluorescent reporter;
preferably, the detection is a chip-based detection.
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| Title |
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| PATRICK S. TARPEY ET AL: "The driver landscape of sporadic chordoma", 《NATURE COMMUNICATIONS》, vol. 8, no. 1 * |
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