CN116479152A - Application of microbial marker in preparation of head and neck squamous cell carcinoma detection product - Google Patents
Application of microbial marker in preparation of head and neck squamous cell carcinoma detection product Download PDFInfo
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Abstract
The invention provides an application of a microbial marker in preparing a head and neck squamous cell carcinoma detection product, which belongs to the field of microbial detection, and takes a microorganism with association with Head and Neck Squamous Cell Carcinoma (HNSCC) as a microbial marker, wherein the microbial marker comprises ciliated cellsLeptotrichia). Solves the technical problems of lacking of predictive evaluation products and life-prolonging auxiliary treatment schemes for patients in different stages of HNSCC, especially for patients in late stages, and lacking of high sensitivity and high specificity for preparing the microbial markers in the HSNCC detection products in different stages in the prior art.
Description
Technical Field
The invention belongs to the field of microorganism detection, and particularly relates to application of a microorganism marker in preparation of a head and neck squamous cell carcinoma detection product.
Background
Head and neck squamous cell carcinoma (HNSCC, abbreviated as head and neck squamous cell carcinoma) is the most common type of head and neck tumor, accounting for more than 90% of head and neck malignant tumors, and has become the sixth malignant tumor worldwide. Most patients with HNSCC are already at a locally advanced stage at the time of diagnosis due to atypical early symptoms. HNSCC causes more than 7000 ten thousand cancers worldwide each year.
The pathogenesis of HNSCC is not completely understood, and some risk factors are associated with HNSCC, such as smoking, drinking and human papillomavirus infection. Studies have been investigated in the microbiome of head and neck cancer to show that microbial variation is associated with the onset and progression of HNSCC, and that some potential oncogenic bacteria have been identified. In 2018, hayes et al report on the university of new york, U.S. medical college, determined the abundance of oral cavity microorganism constitution and specific microorganisms by bacterial 16S rRNA gene sequencing, and compared the abundance of microorganism constitution and specific microorganisms in patients and normal human bodies, it was found that the greater the content of corynebacteria and escherichia coli in the oral cavity, the lower the risk of Head and Neck Squamous Cell Carcinoma (HNSCC), suggesting that these two bacterial groups may have an effect of preventing cancer. However, reports of searching microbiome for patients with different stages of HNSCC are very limited.
Although recent studies have shown that intratumoral microbiota is a key marker for HNSCC, the relationship between the patient's microbiota and its clinical improvement in HNSCC disease progression is not clear. Lack of microbiota studies on patients with different stages of HNSCC, especially lack of theoretical basis and effective protocols for life expectancy extension in patients with advanced HNSCC.
U.S. publication No. US2017233817A1 has disclosed that 16S rRNA saliva analysis reveals microbiome biomonitors associated with human papillomaviruses and head and neck squamous cell carcinoma. Methods and compositions useful for diagnosing and treating head and neck squamous cell carcinoma are specifically disclosed. OTUs and several microflora at different classification levels were found to distinguish HNSCC from normal control samples, hpv+ and HPV-samples, pre-and post-operative treatment samples. So that appropriate diagnostic and therapeutic strategies can be employed based on the identification of microbiota in the patient's saliva. However, the application of the invention does not provide a study of the microbiota of patients at different stages and how to provide adjuvant therapy strategies for patients at advanced stages.
In addition, chinese publication No. CN113684242A discloses a head and neck cancer prognosis biomarker based on lymph node microbial flora and application thereof, and specifically discloses that a difference species exists in flora species of metastatic lymph nodes and non-metastatic lymph nodes in a head and neck cancer patient by performing high-throughput sequencing on 16S ribosomal RNA genes of the lymph node microbial flora of the head and neck cancer patient; further, through a microbial flora symbiotic relationship net, the flora correlation between the species of the metastatic lymph node and the non-metastatic lymph node of the HNC patient is found to be different at the genus level, the characteristics of the microbial groups of the metastatic lymph node and the non-metastatic lymph node of the HNC patient are found for the first time, and meanwhile, the analysis shows that the characteristics of the flora difference have good prediction effect on the survival period (total survival period and three-year survival period) of the patient with the head and neck cancer, so that the microbial flora symbiotic relationship net has good practical application value. The technical scheme of the invention mainly aims at prognosis evaluation, and does not provide research basis and assistance for disease prediction evaluation and life expectancy extension of patients in different stages of HSNCC, especially advanced patients.
Disclosure of Invention
For this reason, the invention provides the application of the microbial marker in preparing the head and neck squamous cell carcinoma detection product. Solves the technical problems of the prior art that the prediction and evaluation products and life-prolonging auxiliary treatment schemes for patients in different stages of HNSCC, especially for patients in late stages are lack, and the technical problems of the prior art that the prior art lacks the microbial markers with higher sensitivity and specificity for preparing the HSNCC detection products in different stages.
The technical scheme provided by the invention is as follows: use of a microbial marker in the preparation of a head and neck squamous cell carcinoma detection product, wherein a microorganism having relevance to head and neck squamous cell carcinoma is used as the microbial marker, and the microbial marker comprises ciliated genus #Leptotrichia）。
Preferably, the product comprises a reagent, kit, chip, high throughput sequencing platform, predictive model or assessment system for assessing the stage of development of head and neck squamous cell carcinoma.
Preferably, the stage of head and neck squamous cell carcinoma development includes early and late stages.
Preferably, the product is a reagent for detecting the microbial marker in a sample to be detected, including detecting the abundance or content of the microbial marker.
Preferably, the product is a kit, chip or high-throughput sequencing platform comprising reagents for detecting the microbial markers.
Preferably, the product is a predictive model of the stage of development of squamous cell carcinoma of the head and neck, and the input variable of the predictive model is the abundance or content of the microbial marker.
Preferably, the product is an assessment system of the stage of development of squamous cell carcinoma of the head and neck, comprising the following units:
(1) An input unit: the input unit is used for inputting the abundance data of the microbial markers obtained from the subject sample into the processing unit;
(2) And a processing unit: the processing unit is used for analyzing and processing the abundance of the microbial marker input by the input unit to obtain a prediction result of the subject;
(3) An output unit: the output unit is used for outputting the prediction result of the subject, which is obtained by the analysis and the treatment of the processing unit;
wherein the saidThe microbial markers comprise ciliated genus @Leptotrichia）。
The beneficial effects are that:
the application of the microbial marker in preparing the head and neck squamous cell carcinoma detection product provided by the invention obtains the microbial cilium genus which has relevance with the head and neck squamous cell carcinoma through screeningLeptotrichia) It was used as a microbial marker. The microbial marker can be applied to the preparation of detection products for the development stage of head and neck squamous cell carcinoma. Such as reagents, kits, chips, predictive models, evaluation systems or high throughput sequencing platforms for diagnosing the stage of squamous cell carcinoma of the head and neck. The invention clarifies the microbial differences between patients with early (T1-2) HNSCC and patients with late (T3-4) HNSCC, and provides assistance for future research on treatment schemes and strategies for prolonging the service life of patients with advanced HNSCC. New steps are put forward for advancing HNSCC research, and a solid foundation is laid for subsequent research.
The invention provides ciliated fungus genusLeptotrichia) The microbial marker for preparing and detecting the development stage of HNSCC has higher efficacy on preparing detection products of the development stage of HNSCC. The prediction model can effectively predict the tumor development period of the HNSCC patient, and has good in-situ application prospect; ciliated genus according to the study of the present inventionLeptotrichia) The relationship between the microbial markers and the clinical manifestations can assist in formulating treatment schemes and strategies for patients with advanced HNSCC, and prolong the life of patients with advanced disease.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
FIG. 1 shows the significant differences in the tumor microflora at different stages (T1-2 and T3-4) in a sample of HSNCC patients in example 1 of the present invention;
FIG. 2 shows the total survival and detection of ciliated cells in the sample of HSNCC patients in example 1 of the present inventionLeptotrichia) Is a correlation of (1);
FIG. 3 shows the various stages (T) of a sample of HSNCC patients in example 1 of the present invention1-2 and T3-4) ciliated genus [ ]Leptotrichia) Detecting rate;
FIG. 4 is a graph showing the results of the detection of FISH signals and the corresponding analysis of different phases (T1-2 and T3-4) of HPF of a sample of an HSNCC patient in example 1 of the present invention;
FIG. 5 shows ciliated strain of the different stages (T1-2 and T3-4) of the sample of HSNCC patient in example 1 of the present inventionLeptotrichia) Analysis results of relative expression levels of genomic DNA.
Detailed Description
The invention will be described in detail below with reference to the drawings in connection with embodiments. The principles and features of the present invention are described below with reference to the drawings, and it should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
Terms and noun interpretation in the present invention:
TNM staging: a classification method for determining the range of tumor lesions;
PCoA: a visualization method for researching data similarity or difference, a main coordinate analysis method;
Bray-Curtis distance matrix: comparing the composition differences of the two community microorganisms based mainly on the count statistics of OTUs;
OUT: i.e., a classification operation unit, in the analysis of the diversity of microorganisms, OTU classification is performed on all sequences according to different similarity levels, and in general, if the similarity between sequences is higher than 97% (seed level), it can be defined as one OTU, each OTU representing one species;
chao1: an index of species richness;
shannon index: shannon index, diversity index;
alpha diversity: intra-sample diversity;
beta diversity: sample-to-sample diversity;
Kaplan-Meier curve: analyzing the influence of a single factor on the survival time, and estimating the survival rate of a patient and drawing a survival curve;
single factor Cox proportional hazards model: evaluating an influence model of a single factor on survival;
fluorescence in situ hybridization: hybridization is carried out on a nucleic acid probe marked by fluorescein directly or indirectly and a nucleic acid probe marked by fluorescein and the like and a nucleic acid sequence in a sample to be detected according to the principle of base complementary pairing, and the hybridization is directly observed under a fluorescence microscope after washing;
real-time quantitative PCR: the molecular biological technology of amplifying specific DNA fragments in vitro can greatly increase trace DNA;
QIIME: amplicon analysis means;
usaearch: a sequence analysis tool;
greengenes database: database of 16S rRNA genes;
wilcoxon sign rank test: a median for paired sample differences is compared to 0; but also for single sample median and overall median comparisons;
permanva: replacing multivariate analysis of variance;
RNeasy Mini Kit: RNA extraction kit;
SYBR Green PCR Master Mix: real-time fluorescent quantitative PCR kit;
StepOne Plus Realtime PCR: a real-time fluorescent quantitative PCR system;
2-delta delta Ct method: a simple method for analyzing the relative change of gene expression in a real-time quantitative PCR experiment, namely a simple method for relative quantification;
FITC: fluorescein isothiocyanate;
DAPI: a fluorescent dye;
study samples: the study samples described in the present invention include patient samples and control samples; the patient samples included a TCGA patient cohort and a validated patient cohort.
The method adopted in the specific embodiment of the invention is as follows:
1. biometric analysis
Double-ended reads are incorporated into tags with FLASH (version 1.2.7). The original tag is filtered using the command split_lists_fastq.py in QIIME (version 1.8). After quality control, chimeric tags were deleted using userch (version 6.1). The reads that were required were clustered into an Operational Taxon (OTU) using QIIME with 97% similarity. The classification information of the OTU is obtained by a ribosome database entry classifier against Greengenes database (version 13.8).
The alpha diversity of the number of species observed was measured and the beta diversity distance matrix of the pairwise differences between the samples was measured using QIIME calculations. Wilcoxon signed rank test detects the differences in Chao1 index, shannon index and observed OTU. Principal coordinate analysis (PCoA) was performed using R software (version 4.0.2). PERMANOVA uses the vegan package in the R language to elucidate the differences in the microbial communities between groups. The Wilcoxon signed rank test was applied to identify clusters with rich inter-group variability, with a false discovery rate <0.05 considered statistically significant.
2. Quantitative RT-PCR
RNA was isolated using the RNeasy Mini Kit (QIAGEN, valencia, calif., USA) according to the manufacturer's instructions. Quantitative RT-PCR was performed using SYBRGreen PCR Master Mix (Invitrogen, themo Fisher, grand Island, NY, USA) and StepOnePlus Realtime PCR systems (Applied Biosystems, thermo Fisher, grand Island, NY, USA). The PCR cycle included 40 cycles of template DNA amplification, with primers annealed at 60 ℃. The relative levels of gene expression were calculated using the 2-delta delta Ct method. Actin acts as a housekeeping gene, often yielding similar results.
Forward primer sequence: 5'-GTTAAATGGGCTTCTTAAACCAA-3';
reverse primer sequence: 5'-AACAACTGTAAAATTGCCTT-3'.
3. Fluorescence in situ hybridization
Tumor tissues of HSNCC patients were collected for frozen sections and RNA fluorescence in situ hybridization was performed using lncRNA FISH kit (GenePharma, shanghai, china). Frozen sections were reconstituted according to the instructions, proteinase K cut, denatured, FITC-labeled ciliated nucleotide probe hybridization, DAPI staining, and observed under a fluorescence microscope.
Probe sequence: FAM-CACTTCATTCGGCCCTAATAATC-FAM.
Example 1
This example provides the microbial marker ciliated genus of squamous cell carcinoma of head and neckLeptotrichia) The method for obtaining the microbial marker comprises the following steps:
1. collection and processing of research samples
The study samples included patient samples including TCGA patient cohorts and validated patient cohorts and control samples. Wherein, the TCGA patient queue is the sequence data and clinical characteristics (http:// cancetrgenome.nih.gov/, month 2 of 2020) of HNSCC patients obtained from a cancer genome map (TCGA) database; verifying that the patient cohort surgically obtained cancerous tissue from the central region of the lesion for the patients enrolled in the study; the control sample was a paired paracancerous control tissue of patients in the TCGA patient cohort.
All HNSCC patients did not receive a radical resection of the head and neck squamous cell carcinoma and received antibiotic treatment for one month; infection with HBV, HCV, syphilis or HIV; and patients with a history of malignancy, chemotherapy, or radiation therapy.
According to 2021 National Comprehensive Cancer Network (NCCN) guidelines, TNM staging was determined for all HNSCC patients in the group. Patient cohorts were divided into early (T1-2) and late (T3-4).
The participants were prohibited from eating, smoking, and oral cleaning for at least 2 hours prior to sampling. Cancerous tissue from the central lesion area was obtained by surgery and tissue samples were placed in sterile 2mL centrifuge tubes and frozen at-80 ℃ prior to treatment.
2. Study of the diversity in alpha and beta for three groups of control, patient (T1-2), and patient (T3-4).
Using a principal coordinate analysis (PCoA) based on a Bray-Curtis distance matrix of the genus microorganism, the results showed that there was a significant difference in distribution between the control sample and the patient sample (control sample and patient sample (T1-2),P=0.035; control samples and patient samples (T3-4),P=0.004）。
alpha-diversity alignment of sequences using the OTU, chao1 and Shannon indices showed a significant decrease in microbial alpha-diversity in patient samples (T3-4) compared to control samplesP<0.01 And patient sample (T1-2) Compared with the patient sample (T3-4), the sample also has a reductionP<0.05）。
At the door classification level, the microbial flora of three groups of samples is classified by Bacteroides doorBacteroidetes) And thick-wall fungus doorFirmicutes) Mainly, the second is Fusobacterium gateFusobacteria) Proteus gateProteobacteria) The actinomycota isActinobacteria) The helicobacter gate isSpirochaetes) And the soft wall fungus door isTenericutes). At the genus classification level, the relative abundance of the first 20 genera in the three groups of samples was compared, and the results showed that the Fusobacterium species in patient sample (T1-2) and patient sample (T3-4) wereFusobacterium) The treponema pallidum belongs toTreponema) The carbon dioxide is a genus of the fibrinopsisCapnocytophaga) And the specific microorganism genus is relatively enriched.
3. Analysis of the relative abundance of a particular microbial flora of patient samples (T1-2) and patient samples (T3-4).
Comparisons were made at the one, one class, two order, two family, five genus levels by analyzing the average abundance of microbial flora between early (T1-2) and late (T3-4) tumor tissues of HNSCC. Curvularia genus of patient sample (T1-2) compared to patient sample (T3-4)Campylobacter) Genus Luo's bacteriumRothia) Ciliates genus [ ]Leptotrichia) And tanna genusTannerella) The average abundance of (c) increases significantly, see figure 1.
4. Screening of microbial flora benign related to HNSCC clinical results
The relationship between the microbial flora and the total survival (OS) of HSNCC patients was further studied by dividing the patient samples into two groups according to whether the above five microbial species were detected in the patient samples. By plotting Kaplan-Meier curve against survival distribution, it was found that ciliated genus @ could be detected in 37.7% of HNSCC patient samplesLeptotrichia) And ciliated genus @Leptotrichia) The average total survival for the positive and negative groups was 37.5 months and 20.5 months, respectively. Adopting a single factor Cox proportion risk model to display ciliated genus @, andLeptotrichia) The total survival time of the patients in the positive group is obviously longer than that of ciliated genus #Leptotrichia) Patients of negative groupP=0.0005), see fig. 2.
Furthermore, ciliated genus of patient sample (T1-2)Leptotrichia) The number of patient samples in the positive group was ciliated for patient sample (T3-4)Leptotrichia) About 1.5 times the number of patient samples in the positive group, which indicates ciliated genus @ in patient sample (T1-2) as compared to patient sample (T3-4)Leptotrichia) More generally, see fig. 3.
5. Forward promotion analysis of ciliated genus
Fluorescence In Situ Hybridization (FISH) and real-time quantitative PCR were performed on patient samples using ciliated genomic DNA as a probe pair, FISH signals being detected in tumor slides of patient samples. By calculating the signal for each High Power Field (HPF) in each sample, it was found that more FISH signals were identified in patient sample (T1-2) than in patient sample (T3-4), see FIG. 4. The use of real-time PCR allows a better quantification of the ciliated gene expression, which results show that in the validated patient cohort, the expression level of ciliated genome genes in patient sample (T1-2) is still about 7 times higher than in patient sample (T3-4)P=0.0375), see fig. 5.
This example shows the characterization of the ciliated micro-marker ciliated genus for different stages of HNSCCLeptotrichia) Ciliated bacteria of the genus Cellostachys in patients with early HSNCC compared with patients with late HSNCCLeptotrichia) The number is obviously increased, and the number is positively correlated with the total survival period, so that a powerful theoretical basis is provided for further research, and potential clinical application opportunities are provided for cancer therapy based on microbial flora, especially for prolonging the service life of patients with advanced HSNCC. And will have great potential in other detection of HSNCC related products.
Example 2
This example provides reagents for detecting the micro-biomarker ciliated genus @ in a sample to be tested in products of different stages of HSNCCLeptotrichia) Comprising detecting the abundance or content of the microbial marker.
Example 3
This example provides a kit for use in the detection of products of different stages of HSNCC, the kit comprising the reagents of example 2.
Example 4
The present embodiment provides a predictive model of HSNCC. The construction method comprises the following steps:
s1, collecting and processing a microbial specimen;
study samples were collected and processed. The specific method is the same as that of embodiment 1, and will not be described in detail in this embodiment.
S2, detecting and analyzing a microbial specimen, and screening out a microbial marker, wherein the microbial marker is ciliated bacteriaLeptotrichia）。
S3, performing model training by a machine learning method;
and (3) constructing and obtaining the prediction model of the HSNCC at different stages based on the microbial markers screened in the step (S2).
The microbial marker construction model obtained by the invention is used for analyzing different stages of the HSNCC, assisting in screening of different stages of the HSNCC, realizing the provision of an individual treatment scheme for patients in different stages, improving the life quality of the patients and providing powerful assistance for the formulation of a scheme for prolonging the service life of advanced patients.
Example 5
The embodiment provides a prediction system or device of HNCC. Comprising the following units:
(1) An input unit: the input unit is used for inputting the abundance data of the microorganism marker obtained from the sample of the subject into the processing unit, wherein the microorganism marker is ciliated genus obtained in the embodiment 1Leptotrichia）；
(2) And a processing unit: the processing unit is used for analyzing and processing the abundance of the microbial marker input by the input unit to obtain a prediction result of the subject;
(3) An output unit: the output unit is used for outputting the prediction result of the subject, which is obtained by the analysis and the treatment of the processing unit.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (7)
1. Use of a microbial marker for the preparation of a head and neck squamous cell carcinoma detection product, characterized in that a microorganism having a correlation with head and neck squamous cell carcinoma is used as a microbial marker, said microbial marker comprising ciliated genus #Leptotrichia）。
2. The use according to claim 1, wherein the product comprises a reagent, kit, chip, high throughput sequencing platform, predictive model or evaluation system for assessing the stage of development of head and neck squamous cell carcinoma.
3. The use according to claim 2, wherein the stage of development of squamous cell carcinoma of the head and neck comprises early and late stages.
4. The use according to claim 3, wherein the product is a reagent for detecting the microbial marker in a sample to be tested, comprising detecting the abundance or content of the microbial marker.
5. The use according to claim 3, wherein the product is a kit, chip or high-throughput sequencing platform comprising reagents for detecting the microbial markers.
6. The use according to claim 3, wherein the product is a predictive model of the stage of development of squamous cell carcinoma of the head and neck, the input variable of said predictive model being the abundance or content of said microbiological marker.
7. Use according to claim 3, characterized in that said product is an evaluation system of the stage of development of squamous cell carcinoma of the head and neck, characterized in that said evaluation system comprises the following units:
(1) An input unit: the input unit is used for inputting the abundance data of the microbial markers obtained from the subject sample into the processing unit;
(2) And a processing unit: the processing unit is used for analyzing and processing the abundance of the microbial marker input by the input unit to obtain a prediction result of the subject;
(3) An output unit: the output unit is used for outputting the prediction result of the subject, which is obtained by the analysis and the treatment of the processing unit;
wherein the microbial markers comprise ciliated genus [ ]Leptotrichia）。
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