CN110305965A - A method of sensibility of prediction non-small cell lung cancer (NSCLC) patient to immunotherapy - Google Patents

Abstract

The invention discloses using KMT2C and TP53 the two genes predicted as biomarker cancer (non-small cell lung cancer) patient to immunotherapy, such as using immunologic test point inhibitor immunotherapy sensibility method；The reagent of biomarker described in specific detection is also disclosed in preparation prediction cancer patient (non-small cell lung cancer) to immunotherapy, for example, using immunologic test point inhibitor immunotherapy sensibility kit in purposes.In the present invention, TP53 and KMT2C gene mutation state is considered by combination, group that can be sensitive to ICI in Accurate Prediction NSCLC patient avoids blindness medication, improves the economic performance of ICI treatment.

Description

A kind of prediction non-small cell lung cancer (NSCLC) patient is to the sensibility of immunotherapy Method

Technical field

The present invention relates to biomedicine fields.Specifically, the present invention relates to one kind for predicting non-small cell lung cancer (NSCLC) patient is to immunotherapy, such as the method for sensibility of immunotherapy using immunologic test point inhibitor, kit And system.More particularly it relates to which KMT2C gene and TP53 gene is used to predict that cancer is suffered from as biomarker Person is to immunotherapy, such as the sensibility of the immunotherapy using immunologic test point inhibitor.

Background technique

Lung cancer is divided into two major classes type: Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC) by clinic.This differentiation It is considerable, because being completely different to the therapeutic scheme of the lung cancer of both types.Identify two kinds of partings, is desirable By bronchoscope, aspiration biopsy or postoperative acquisition pathology, just can determine that by Pathologis diagnosis.Non-small cell lung cancer It is the general name, including squamous carcinoma, gland cancer, large cell carcinoma etc. of a really major class disease for Small Cell Lung Cancer.Face now Bed is less and less to use this appellation, more directly with titles such as squamous carcinoma, gland cancer.Wherein squamous carcinoma is most commonly seen, accounts for about 50%, the speed of growth is slower, and the course of disease is longer, relatively sensitive to chemicotherapy.Gland cancer women is relatively common, general growth compared with Slowly, but sometimes hematogenous metastasis occurs for early stage i.e. hair mouth.Non-small cell lung cancer can pass through operation more than its early stage and obtain root It controls, less recurrence.The non-small cell lung cancer of Locally Advanced mostly uses multi-disciplinary means to treat, need thoracic surgery, radiotherapy department, The doctor of internal medicine formulates the therapeutic scheme for being suitable for patient after discussing.Some patientss can also be cured.Majority is shifted Property lung cancer, based on systemic therapy.The therapeutic effect of part of patient is preferable, and patient can be made to obtain long term survival, for example, For ALK positive lung cancer, the survival of patients time for now passing through after treating about 50% is more than 4 years.Small Cell Lung Cancer accounts for about 20% Left and right, belongs to neuroendocrine carcinoma, is a kind of very high tumour of grade malignancy often with cryptorrhea or carcinoid syndrome, more Positioned at lung central portion, growth is rapidly, relatively early to shift.It is how very sensitive for initial chemotherapy or radiotherapy, but most of trouble There is drug resistance quickly in person, once drug resistance, successive treatment is more difficult.Small Cell Lung Cancer is divided into Limited-stage and extensive phase, Limited-stage are small Cell lung cancer emphasizes to start combined radiotherapy as early as possible after chemotherapy；The extensive phase mostly after end of chemotherapy some patientss can row chest put It treats.

And immunotherapy of tumors has developed like a raging fire, wherein immunologic test point inhibitor (anti-PD-1/PD-L1 suppression Preparation) be even more therapeutic field of tumor in recent years " star " drug, entered non-small cell lung cancer first-line treatment.But we also want Although seeing that immunologic test point inhibitor effect is pretty good, entirety ORR still only has 20% or so, obtains so how precisely to screen Beneficial crowd becomes clinician's problem in the urgent need to address.

PD-L1, TMB(Tumor mutations load, tumor mutational burden) and MSI(microsatellite instability, Microsatellite instability) it is three immunization therapy biological markers for obtaining FDA approval or the recommendation of NCCN guide Object (biomarker), but these three biomarkers respectively have advantage and disadvantage.PD-L1 is as immunization therapy biomarker using most To be extensive, PD-L1 IHC detection is also approved the adjoint diagnosis as Pembrolizumab fiest-tire medication by FDA.But it is multiple Clinical test results show PD-L1 expression it is not consistent to the predictive ability of immunization therapy curative effect, part PD-L1 negative patient according to Can so benefit from immunization therapy, and the lasting remission time and be no less than PD-L1 positive patient；TMB is equally non-small cell lung cancer The immunization therapy biomarker that NCCN guide is recommended, but the difference in view of different company or laboratory for TMB algorithm, TMB Threshold value is difficult to set up common recognition；MSI has been used as tumour key biomarker that FDA is allowed to agree to based on MSI state, rather than organizes Histological type carries out medication treatment, but MSI-H ratio is too low in tumour, and clinical expansion has certain limitation.Most important one Point is exactly existing research (being included in 11348 solid tumors) discovery, and PD-L1 is positive, TMB high is expressed and MSI-H three's Duplication Only 0.6%, many potential immunization therapies benefit crowds can all be omitted by prompting any biomarker to be applied alone.So needing further Explore immunization therapy biomarker.

As development of the two generations sequencing in tumour is precisely treated is increasingly extensive, the somatic mutation of specific gene is found Tumour immunity function or the response to immunization therapy may be influenced, that is, prompting specific somatic mutation may be that potential be immunized is controlled Treat predictive factor.EGFR mutation and ALK rearrangement are the potential predictive factors of ICI immunization therapy poor prognosis.One retrospective point Analysis discovery, only 3.6% pair of ICI immunization therapy has a reaction in these patients, and EGFR wild type and ALK is negative or unknown patient Reactivity be 23.3%.To include using PD- (L) 1 inhibitor for treating 3025 advanced NSCLC patients 5 tests progress Meta-analysis discovery, EGFR mutation patient in, compared with docetaxel, overall survival does not improve.EGFR mutation Or the lung cancer that ALK is reset shows lower PD-L1 and CD8⁺T cell infiltration, this may be the original that ICI immunization therapy reacts bad Cause.In addition to the collaboration mutation of the change of EGFR and ALK, TP53 and KRAS and homologous recombination repair and mispairing reparation (HRR- MMR) or the collaboration mutation in the DNA damage of HRR and base excision repair (HRR-BER) reaction (DDR) access is considered as ICI The preferable forward prediction factor of immunization therapy curative effect, and the collaboration of KRAS and STK11 mutation has with ICI immunization therapy poor prognosis It closes.But the above gene mutation cannot still cover all potential immunization therapies as biomarker and benefit crowd, ability There is still a need for more efficient, more accurately identification is suitable for the side of the Patients with Non-small-cell Lung of immunologic test point inhibitor for treating in domain Method and tool.

PD-L1 and TMB has been considered as selecting 2 that treat sensitive NSCLC patient to ICI the most frequently used prediction lifes Object marker.However, PD-L1 detection has corresponding PD- (L) 1 to detect antibody peace due to different anti-1 drugs of PD- (L) Platform causes to lack unified standard；In addition the expression of PD-L1 has the characteristics that dynamic change, and PD-L1 expression is caused to be controlled with immune Relationship between therapeutic effect still has some disputes；On the other hand, although a large amount of randomized control studies and large sample real world are ground Study carefully the correlation having confirmed that between TMB and immunotherapeutic, but TMB can only still reflect Tumor mutations quantity, and cannot mention Show the state of tumor microenvironment, and TMB detection is more demanding to technology platform, the duty cycle is longer, and higher cost all restricts it Clinical application.Therefore, it is badly in need of a kind of prediction technique that can efficiently to immunotherapy sensibility.

Summary of the invention

To solve the above-mentioned problems, the present invention provides KMT2C genes and TP53 gene to predict as biomarker Cancer patient to immunotherapy, such as using immunologic test point inhibitor immunotherapy sensibility method and kit, And device and system.

KMT2C gene order of the present invention is of the present invention as shown in GenBank accession number NG_033948.1 TP53 gene order is as shown in GenBank accession number NG_017013.2.

In a first aspect, the present invention provides specific detection TP53 gene mutation and/or expression, and/or specifically Property detection KMT2C gene mutation/or expression reagent preparation for predicting cancer patient to the sensibility of immunotherapy Kit or device in purposes.

In some embodiments, the immunotherapy is the immunotherapy using immunologic test point inhibitor.

In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

In some embodiments, the reagent such as gene order-checking reagent, gene-specific primer or probe, gene The specific antibody etc. of expression product.

In second aspect, the present invention provides one kind for predicting that cancer patient to immunotherapy, such as uses immune inspection The method for making an inventory of the sensibility of the immunotherapy of inhibitor, the method includes

Step a) assesses the TP53 gene mutation and/or expression in the tumor tissues of the patient；With

Step b) assesses the KMT2C gene mutation and/or expression in the tumor tissues of the patient；With

Step c) is based on assessment result prediction cancer patient a), b) to immunotherapy, such as uses immunologic test point inhibitor Immunotherapy sensibility.

In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

As used herein, term " immunologic test point " refers to some inhibition signal paths present in immune system.Machine Under normal circumstances, immunologic test point can maintain immune tolerance by adjusting the intensity of autoimmune response to body, however machine For body when by tumor invasion, the activation of immunologic test point can inhibit autoimmunity, be conducive to the growth and escape of tumour cell. By using immunologic test point inhibitor, the normal anti tumor immune response of body can be restored, thus control and removing tumour. A variety of immunologic test point inhibitor that can be used for oncotherapy known in the art.For example, immunologic test point of the present invention inhibits Agent includes but is not limited to PD1 inhibitor or PD-L1 inhibitor, such as the domestic auspicious Puli's monoclonal antibody of spy, Xin Dili monoclonal antibody, card Rayleigh Pearl monoclonal antibody and pyridine aldoxime methyliodide (PAM) monoclonal antibody receive Wu Dankang, Aunar pearl monoclonal antibody, Avelumab and Durvalumab.

In some embodiments of various aspects of the present invention, the cancer is non-small cell lung cancer (NSCLC).

In some embodiments of various aspects of the present invention, by comparing the sequencing number of tumor tissues and control tissue According to, such as TP53 gene mutation described in gene order-checking data and/or sequencing of extron group data assessment and KMT2C gene be prominent Become.

In some embodiments of various aspects of the present invention, the control tissue is the normal tissue from the object (nonneoplastic tissue), such as blood tissues.

In some embodiments of various aspects of the present invention, the sequencing is high-flux sequence, the sequencing of also referred to as two generations ("NGS").The sequencing of two generations generates thousands of to millions of sequences simultaneously in parallel sequencing procedure.NGS is different from " Sanger Sequencing " (generation sequencing), the latter is the electrophoretic separation based on the chain termination product in single sequencing reaction.It can be with of the invention The microarray dataset of NGS is commercialization available, including but not limited to Roche/454 FLX, Illumina/Solexa Genome Analyzer and Applied Biosystems SOLID system etc..

Sequencing of extron group is that height is carried out after genomic exon regional DNA is captured and is enriched with using sequence capturing technology The genome analytical method of flux sequencing.Since it has to common and rare variation high sensitivity, the base to 2% is only needed Most of disease correlation variation of exon region can be found because group is sequenced.The sequencing can be full-length genome survey Sequence, can also be with the sequencing in portion gene or region in covering gene group.

In some embodiments, assessment TP53 gene mutation includes determining that its code area whether there is mutation, such as move Code mutation.In some embodiments, the mutation is located at the position 285-864 nucleotide, the 954-1074 nucleosides of TP53 gene In acid.In some preferred embodiments, assessment TP53 gene mutation includes determining that its code area whether there is to make TP53 albumen Truncated mutation.

In some embodiments, TP53 base is assessed by transcript profile sequencing, polymerase chain reaction (PCR) or immunoassays Because of expression.The various methods that can detecte specific gene expression known in the art, these can be applied to the present invention.

For example, transcript profile sequencing is quickly comprehensively to obtain a certain species specific cells or tissue by two generation microarray datasets Almost all of transcript and gene order under a certain state, can be used for studying gene expression amount, gene function, knot Structure, alternative splicing and the prediction of new transcript etc..In addition, can be surveyed by PCR such as reverse transcription PCR by designing suitable primer The transcriptional expression for determining TP53 gene is horizontal.Further, it is also possible to for example be exempted from using TP53 protein specific antibody by immunoassays The protein expression level of the methods of epidemic disease group (IHC), ELISA measurement TP53 gene.

In some embodiments, after determining that the presence of the gene coding region TP53 makes the mutation of TP53 protein truncation, assessment TP53 gene expression, such as the protein expression level of TP53 gene.

In some embodiments, assessment KMT2C gene mutation includes assessing its code area with the presence or absence of mutation.

In some embodiments, the KMT2C gene mutation is to lead to the mutation of high Tumor mutations load (TMB).It is swollen Tumor mutational load (TMB) is defined as the somatic cell gene code error being detected in every megabase, base replacement, gene The sum of insertion or missing errors.TMB is generally indicated with the total quantity of mutation or the mutation quantity of every 1Mb (1 megabasse) (muts/Mb)。

In some embodiments, the mutation is located at the position the 3999-7788 nucleotide of KMT2C gene, 9375-13071 In nucleotide.In some preferred embodiments, assessment KMT2C gene mutation includes determining that its code area whether there is to make The mutation of KMT2C protein truncation.

In some embodiments, if i) TP53 gene has mutation and/or TP53 gene expression missing, and ii) There is mutation and/or KMT2C gene expression missing in KMT2C gene, then predict that the patient is sensitive to the immunization therapy.

In some embodiments, if i) mutation is not present in TP53 gene and/or TP53 gene expression is complete, and ii) There is mutation and/or KMT2C gene expression missing in KMT2C gene, then predict that the patient is insensitive to the immunization therapy.

In some embodiments, if i) mutation is not present in TP53 gene and/or TP53 gene expression is complete, and ii) Mutation is not present in KMT2C gene and/or KMT2C gene expression is complete, then predicts that the patient is insensitive to the immunization therapy.

In some embodiments, the method includes obtaining the tumor tissues from the cancer patient.

In some embodiments, the method also includes carrying out quality testing to acquired tumor tissues.

In some embodiments, by carrying out institute from the nucleic acid amplification ACTIN gene for extracting from the tumor tissues State quality testing.In some embodiments, using following three groups of primer pair amplifies ACTIN gene:

I) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '-CACGCTCGGTGAGGATCTTC-3 ' (SEQ ID NO:2),

Ii) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '- TCGAAGTCCAGGGCAACATAGC-3 ' (SEQ ID NO:3), and

Iii) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '- AAGGCTGGAAGAGCGCCTCGGG-3 ' (SEQ ID NO:4) expands the segment of 100bp, 200bp and 300bp respectively. Determine that tumor tissues are up-to-standard when three groups of primers, which expand, arrives target fragment.

Quality testing is carried out to tumor tissues through the above steps, up-to-standard tissue is subjected to subsequent sequencing and is commented Estimate step, be avoided that because tissue sample it is of poor quality caused by make a variation information missing inspection, so that it is sensitive to improve immunologic test point inhibitor Property prediction accuracy.

In some embodiments, when passing through the gene mutation of high-flux sequence data assessment, only assessment meets with subscript Quasi- mutation:

(1) for point mutation:

Sequencing overburden depth > 500 time of the point mutation position；Each read mass value > 40 comprising the point mutation, packet Base corresponding with point mutation mass value > 21 in each read containing the point mutation；It include the read of the point mutation Item number >=5；It include read positive in all reads of the point mutation and reversed read ratio < 1/6；And tumor group Variation gene frequency/control tissue variation gene frequency >=20 knitted;

(2) for insertion and deletion (indel):

If consecutive identical base<5 in insertion and deletion, sequencing overburden depth>600 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency >=20 knitted;

If consecutive identical base>=5 and<7 in insertion and deletion, sequencing overburden depth>60 of the insertion and deletion position It is secondary；Each read mass value > 40 comprising the insertion and deletion；It is prominent with the insertion and deletion in each read comprising the insertion and deletion Covert corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the insertion and deletion Long and reversed long ratio < 1/6 of reading of positive reading in all reads；Variation gene frequency/control of the tumor tissues Variation gene frequency > 20 of tissue；And variation gene frequency >=10% of the tumor tissues；

If consecutive identical base >=7 in insertion and deletion, sequencing overburden depth > 60 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency > 20 knitted；And variation gene frequency >=20% of the tumor tissues.

It will be understood by those skilled in the art that all or part of function of above method step can be by way of hardware It realizes, can also be realized by way of computer program.When function all or part of in above method step passes through computer When the mode of program is realized, which be can be stored in a computer readable storage medium, and storage medium may include: read-only Memory, random access memory, disk, CD, hard disk etc. execute the program by computer to realize above-mentioned function.For example, will Program is stored in the memory of equipment, and when executing program in memory by processor, above-mentioned all or part can be realized Function.In addition, the program can also when function all or part of in above embodiment is realized by way of computer program To be stored in the storage mediums such as server, another computer, disk, CD, flash disk or mobile hard disk, by downloading or again System is saved in the memory of local device, or carries out version updating to the system of local device, is deposited when by processor execution When program in reservoir, all or part of function in above embodiment can be realized.

In the third aspect, the present invention provides a kind of for predicting that cancer patient to immunotherapy, such as uses immunologic test Point inhibitor immunotherapy sensibility device, the cancer is non-small cell lung cancer (NSCLC), described device include with Lower three modules:

Evaluation module I), assess TP53 gene mutation and/or the expression in the tumor tissues of the patient；

Evaluation module II), assess KMT2C gene mutation and/or the expression in the tumor tissues of the patient；With

Prediction module III), be based on evaluation module I), II) assessment result prediction cancer patient to immunotherapy, such as make With the sensibility of the immunotherapy of immunologic test point inhibitor.

In some embodiments, evaluation module I), II) by comparing tumor tissues and control tissue sequencing data, Such as TP53 gene mutation described in gene order-checking data and/or sequencing of extron group data assessment and KMT2C gene mutation.

In some embodiments, evaluation module II) determine that the gene coding region TP53 is prominent with the presence or absence of mutation, such as frameshit Become.In some embodiments, the mutation is in the position the 285-864 nucleotide, 954-1074 nucleotide of TP53 gene. In some preferred embodiments, evaluation module II) it determines that the gene coding region TP53 whether there is and makes the prominent of TP53 protein truncation Become.

In some embodiments, evaluation module II) determine the gene coding region KMT2C with the presence or absence of mutation.In some realities It applies in scheme, the KMT2C gene mutation is to lead to the mutation of high Tumor mutations load (TMB).It is described in some embodiments Mutation is in the position the 3999-7788 nucleotide, 9375-13071 nucleotide of KMT2C gene.

In some embodiments, evaluation module I), II) only assessment meet the mutation of following standard:

(1) for point mutation:

Sequencing overburden depth > 500 time of the point mutation position；Each read mass value > 40 comprising the point mutation, packet Base corresponding with point mutation mass value > 21 in each read containing the point mutation；It include the read of the point mutation Item number >=5；It include read positive in all reads of the point mutation and reversed read ratio < 1/6；And tumor group Variation gene frequency/control tissue variation gene frequency >=20 knitted;

(2) for insertion and deletion (indel):

If consecutive identical base<5 in insertion and deletion, sequencing overburden depth>600 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency >=20 knitted;

If consecutive identical base>=5 and<7 in insertion and deletion, sequencing overburden depth>60 of the insertion and deletion position It is secondary；Each read mass value > 40 comprising the insertion and deletion；It is prominent with the insertion and deletion in each read comprising the insertion and deletion Covert corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the insertion and deletion Long and reversed long ratio < 1/6 of reading of positive reading in all reads；Variation gene frequency/control of the tumor tissues Variation gene frequency > 20 of tissue；And variation gene frequency >=10% of the tumor tissues；

If consecutive identical base >=7 in insertion and deletion, sequencing overburden depth > 60 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency > 20 knitted；And variation gene frequency >=20% of the tumor tissues.

In some embodiments, if i) TP53 gene has mutation and/or TP53 gene expression missing, and ii) There is mutation and/or KMT2C gene expression missing in KMT2C gene, then predict that the patient is sensitive to the immunization therapy.

In some embodiments, if i) mutation is not present in TP53 gene and/or TP53 gene expression is complete, and ii) There is mutation and/or KMT2C gene expression missing in KMT2C gene, then predict that the patient is insensitive to the immunization therapy.

In some embodiments, if i) mutation is not present in TP53 gene and/or TP53 gene expression is complete, and ii) Mutation is not present in KMT2C gene and/or KMT2C gene expression is complete, then predicts that the patient is insensitive to the immunization therapy.

In fourth aspect, the present invention also provides one kind for predicting that cancer patient to immunotherapy, such as uses immune inspection The device of the sensibility of the immunotherapy of inhibitor is made an inventory of, the cancer is non-small cell lung cancer (NSCLC), described device packet It includes: memory, for storing program；Processor, for the program by executing the storage of above-mentioned memory to realize the present invention the The all or part of the steps of the method for two aspects.

At the 5th aspect, the present invention also provides a kind of computer readable storage mediums comprising program, the program can be by Processor executes all or part of the steps to realize the method for second aspect of the present invention.

Compared with prior art, the present invention has following technical effect that

1. currently, PD-L1 and TMB is used only as suitable treatment in immunologic test point inhibitor (ICI) treatment of NSCLC patient Biomarker.However, objective response rate still only has about 20% in the NSCLC patient selected by PD-L1 or TMB Left and right, while the NSCLC patient of some PD-L1 feminine genders expression is also reported as having response to ICI.In the present invention, pass through combination Consider TP53 and KMT2C gene mutation and/or expression status, group that can be sensitive to ICI in Accurate Prediction NSCLC patient, Blindness medication is avoided, the economic performance of ICI treatment is improved.

2. filtering out TP53 and KMT2C gene in the present invention to be mutated altogether as group sensitive to ICI in prediction NSCLC patient The biomarker of body, for the total mutation of other assortments of genes, prediction result is more accurate；And used in the present invention TP53 and KMT2C gene is mutated in practical applications altogether can be used as independent prediction risk factors, improve detection efficiency.

The mutation status of two genes of 3.TP53 and KMT2C discloses patient to immunotherapy, such as uses immunologic test point The sensibility of the immunotherapy of inhibitor is conducive to simplify detection content, reduces patient's testing cost, accelerates examining report and provides Time, and compared to PD-L1 ImmunohistochemistryMethods Methods need artificial interpretation immunohistochemistry piece and the artificial threshold value of TMB needs this For two o'clock, the detection of gene mutation state is more reliable.

Detailed description of the invention

Fig. 1 KMT2C is mutated compared with wild type patient tumors mutational load (TMB)；

Fig. 2 KMT2C mutation is compared with wild type patient tumors PD-L1 expression；

Fig. 3 KMT2C is compared with the Tumor mutations load (TMB) of TP53 difference mutation status；

Fig. 4 KMT2C is compared with the PD-L1 of TP53 difference mutation status expression；

Fig. 5 KMT2C mutation is compared with the curative effect that wild type patient receives using the immunotherapy of immunologic test point inhibitor；

The patient of Fig. 6 KMT2C and TP53 difference mutation status receives the curative effect of the immunotherapy using immunologic test point inhibitor Compare；

Fig. 7 KMT2C and TP53 is total to mutation combination, Cox multinomial logistic regression and the immune treatment using immunologic test point inhibitor The relevant independent risk factor of method curative effect；

The patient of Fig. 8 KRAS and TP53 difference mutation status receives the curative effect of the immunotherapy using immunologic test point inhibitor Compare；

Fig. 9 KRAS and TP53 is total to mutation combination, Cox multinomial logistic regression and the immune treatment using immunologic test point inhibitor The relevant independent risk factor of method curative effect；

The patient of Figure 10 PTPRD and TP53 difference mutation status receives the treatment of the immunotherapy using immunologic test point inhibitor Effect compares；

Figure 11 PTPRD and TP53 is total to mutation combination, Cox multinomial logistic regression and being immunized using immunologic test point inhibitor The relevant independent risk factor of therapy curative effect；

The patient of Figure 12 HGF and TP53 difference mutation status receives the curative effect of the immunotherapy using immunologic test point inhibitor Compare；

Figure 13 HGF and TP53 is total to mutation combination, Cox multinomial logistic regression and the immune treatment using immunologic test point inhibitor The relevant independent risk factor of method curative effect.

Specific embodiment

The present invention is described in detail with embodiment with reference to the accompanying drawing.

It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment is briefly described, it should be apparent that, be described below in embodiment be some embodiments of the present invention, for For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these embodiments His embodiment.

Embodiment 1

Genome analysis

It has studied from the Chinese FFPE tumor sample of non-small cell lung cancer (NSCLC) patient and the periphery whole blood control sample of pairing Product.All patients are provided which Written informed consent.The next-generation sequencing (NGS) of targeted capture is carried out in OrigiMed, is related to Combination comprising 450 cancer related genes.Pass through DNA FFPE Tissue Kit and DNA Mini kit (QIAamp) point Whole not from tumour content not less than 20% is unstained in FFPE slice and whole blood and extracts DNA, is then measured with dsDNA HS Kit (Qubit) is quantitative.About 250bp is ultrasonically treated using KAPA Hyper Prep Kit (KAPA Biosystems) DNA fragmentationization constructs library, then carries out PCR amplification and quantifies.Hybrid capture, the group and covering are carried out using self-defined combination The human genome of 2.6Mb, targets 450 cancer related genes and certain intrones often reset.By the text after capture Library mixing is denaturalized and is diluted to 1.5-1.8pM, then carries out on Illumina NextSeq 500 according to the scheme of manufacturer Paired end sequencing.

Wherein sample carries out quality testing using following three groups of primer pair amplifies ACTIN genes:

I) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '-CACGCTCGGTGAGGATCTTC-3 ' (SEQ ID NO:2),

Ii) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '- TCGAAGTCCAGGGCAACATAGC-3 ' (SEQ ID NO:3), and

Iii) 5 '-CACACTGTGCCCATCTATGAGG-3 ' (SEQ ID NO:1) and 5 '- AAGGCTGGAAGAGCGCCTCGGG-3 ' (SEQ ID NO:4) expands the segment of 100bp, 200bp and 300bp respectively. When three groups of primers, which expand, arrives target fragment, decision set tissue samples are up-to-standard.

Genome changes analysis

Genome change is had evaluated, including single base replaces (SNV), short and long insertion and deletion, copy number variation (CNV) and gene It resets and merges.Original read and human genome reference sequences (hg19) are carried out using Burrows-Wheeler Aligner Comparison, then using Picard MarkDuplicates algorithm carry out PCR duplicate removal.It reads depth and is less than 30x, chain Preference (strand bias) is greater than 10% or the variant of VAF < 0.5% is removed.It is defined as from dbSNP database (version 147) Or frequency be more than sequencing of extron group project 6500 (ESP6500) 1.5% or it is more than 1000 genome plans 1.5% common single nucleotide polymorphism (SNP) is also excluded from outside.

Judge whether identified mutation is true by following standard:

(1) for point mutation:

Sequencing overburden depth > 500 time of the point mutation position；Each read mass value > 40 comprising the point mutation, packet Base corresponding with point mutation mass value > 21 in each read containing the point mutation；It include the read of the point mutation Item number >=5；It include read positive in all reads of the point mutation and reversed read ratio < 1/6；And tumor group Variation gene frequency/control tissue variation gene frequency >=20 knitted;

(2) for insertion and deletion (indel):

If consecutive identical base<5 in insertion and deletion, sequencing overburden depth>600 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency >=20 knitted;

If consecutive identical base>=5 and<7 in insertion and deletion, sequencing overburden depth>60 of the insertion and deletion position It is secondary；Each read mass value > 40 comprising the insertion and deletion；It is prominent with the insertion and deletion in each read comprising the insertion and deletion Covert corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the insertion and deletion Long and reversed long ratio < 1/6 of reading of positive reading in all reads；Variation gene frequency/control of the tumor tissues Variation gene frequency > 20 of tissue；And variation gene frequency >=10% of the tumor tissues；

If consecutive identical base >=7 in insertion and deletion, sequencing overburden depth > 60 time of the insertion and deletion position； Each read mass value > 40 comprising the insertion and deletion；It is mutated in each read comprising the insertion and deletion with the insertion and deletion Corresponding base mass value > 21；It include item number >=5 of the read of the insertion and deletion；It include the institute of the insertion and deletion Long ratio < 1/6 of reading for having reading positive in read long and reversed；Variation gene frequency/control group of the tumor tissues Variation gene frequency > 20 knitted；And variation gene frequency >=20% of the tumor tissues.

TMB is calculated

Other than the conventional detection that genome changes, TMB is determined also by the algorithm based on NGS.It is prominent by counting body cell Become to estimate TMB, the somatic mutation includes the SNV and insertion and deletion of every megabasse of checked coding region sequence.Row In addition to the driving gene mutation and the change of known germline in dbSNP.

Embodiment 2

A total of 637 non-small cell lung cancers (NSCLC) patient takes part in this research.The feature of patient is as shown in table 1.Mostly Number patients are male (355/637,55.7%), and the median age when diagnosis is 60 years old (IQR, 53-67 year old).The most common tissue Type is non-squamous cell carcinoma (N=553,86.8%), including gland cancer (N=537) and other non-squamous cell carcinomas (N=16).Its In I phase 211 (33.1%), II phase 64 (10%), III phase 138 (21.7%), IV phase 224 (35.2%).To 637 patients Carry out PD-L1 and TMB detection.The expression of PD-L1 and TMB are as shown in table 1 according to the distribution situation of Demographics.Weak sun Property (PD-L1 TPS scoring=1-49%) and strong positive PD-L1 expression (PD-L1 TPS scores >=be 50%) it is respectively 16.5% He 10%.We to PD-L1 express (as classified variable assess, cutoff value be 1% and 50%) with the Clinical symptoms of non-small cell lung cancer Between relationship carried out single factor analysis (table 1).There was no significant difference for average age between TPS<1%, 1-49% and>=50% (P>0.05).PD-L1 expression in male (P=0.002) and squamous cell carcinoma (P < 0.001) is higher.In the TMB of whole crowd Digit is 4.6 muts/Mb (IQR, 2.3-10).The tumour of TMB >=10muts/Mb accounts for 29.4%.We have found that age and TMB Be positively correlated (p < 0.001).In addition, male patient's group (p < 0.001) and squamous cell carcinoma patients group (p < 0.001) TMB value is higher.

Embodiment 3

The mutated gene of front three is TP53 (55.9%), EGFR (51.3%) and KRAS in 637 NSCLC patient's queues (12.6%).There are 32 patients to carry KMT2C gene mutation in 637 NSCLC patients, accounts for 5%.In 32 KMT2C mutation patients There are 24 patients while carrying TP53 gene mutation, accounts for 75%.The TMB of KMT2C mutation patient is significantly higher than KMT2C wild type Patient (middle position TMB:13.1 vs. 4.6, p < 0.001) (Fig. 1).PD-L1 positive expression rate (the PD-L1 of KMT2C mutation patient TPS >=1%) it is higher than KMT2C wild type patient (34.4% vs. 26.1%, p > 0.05) (Fig. 2).

In 637 NSCLC patient's queues, 42.9% (273/637) is TP53-WT/KMT2C-WT patient, 1.3% (8/ 637) it is TP53-WT/KMT2C-MUT patient, 52.1%(332/637) it is TP53-MUT/KMT2C-WT patient, 3.7%(24/ It 637) is TP53-MUT/KMT2C-MUT patient.TP53-WT/KMT2C-MUT patient tumors TMB is significantly higher than TP53-WT/ KMT2C-WT patient (middle position TMB:10.8 vs. 3.1, p=0.007)；TP53-MUT/KMT2C-MUT patient tumors TMB is significant Higher than TP53-MUT/KMT2C-WT patient (middle position TMB:15.2 vs. 6.6, p=0.002)；TP53-MUT/KMT2C-MUT suffers from Person's tumour TMB is significantly higher than TP53-WT/KMT2C-WT patient (middle position TMB:15.2 vs. 3.1, p < 0.001) (Fig. 3). The PD-L1 positive expression rate (PD-L1 TPS >=1%) of TP53-WT/KMT2C-MUT patient tumors is higher than TP53-WT/KMT2C-WT Patient (25% vs. 16.1%, p > 0.05).PD-L1 positive expression rate (the PD- of TP53-MUT/KMT2C-MUT patient tumors L1 TPS >=1%) it is higher than TP53-MUT/KMT2C-WT patient (37.5% vs. 34.4%, p > 0.05) (Fig. 4).

As it can be seen that clinical laboratory data shows TP53 and KMT2C gene mutation and/or expression status, it being capable of Accurate Prediction The economic performance of ICI treatment improves in the group sensitive to ICI in NSCLC patient.

Embodiment 4

It is mutated the predictive value treated for immunologic test point inhibitor (ICIs) in order to further verify KMT2C, we pass through It downloads public database queuing message and carries out external certificate.We the website cBioPortal (http: // Www.cbioportal.org/ the queuing data of Rizvi et al. upload) has been downloaded, Rizvi queue incorporates 240 and receives to resist The Patients with Non-small-cell Lung of 1 single therapy of PD- (L) or the anti-CTLA-4 combined treatment of anti-PD- (L) 1+, specific patient's base Line data can refer to document.In 240 patients, KMT2C mutation patient has 25 (10.4%), and the patient of KMT2C mutation receives to exempt from Middle position PFS after epidemic disease treatment is long (middle position PFS:5.47 vs. 3.17 months, p=0.058) (Fig. 5) compared with KMT2C wild type patient. It is grouped further according to KMT2C and TP53 mutation status, 92 entitled TP53-WT/KMT2C-WT patients, 123 are TP53- MUT/KMT2C-WT patient, 7 are TP53-WT/KMT2C-MUT patient and 18 TP53-MUT/KMT2C-MUT patients.This Four groups of middle position PFS is respectively 2.47 months (95% CI 2.03-3.5), 2.57 months (95% CI, 1.63-NA), 4.2 months (95% CI 3.23-5.37), 7.33 months (95% CI, 2.50-NA) (Fig. 6), Cox multiplicity result further illustrated KMT2C/TP53 be mutated altogether be immunization therapy prognosis independent prediction risk factors (TP53-MUT/KMT2C-MUT vs TP53- WT/KMT2C-WT, HR:0.51,95%CI:0.27-0.96, p=0.036) (Fig. 7).

As it can be seen that KMT2C/TP53 gene mutation can be used as immunization therapy biomarker, PD-L1 ImmunohistochemistryMethods Methods are compared Artificial interpretation immunohistochemistry piece and TMB is needed to need for artificial this two o'clock of threshold value, the detection of gene mutation state It is more reliable.

Embodiment 5

It may be used to predict response of the NSCLC patient to the inhibitor for treating of PD-1, and and GMS in view of KRAS, TP53 gene Show positive correlation, KMT2C/TP53 in order to further illustrate the present invention be mutated altogether be immunization therapy prognosis independent prediction wind Dangerous factor, the present invention are mutated altogether for KRAS/TP53 and have carried out similar research.We the website cBioPortal (http: // Www.cbioportal.org/ the queuing data of Rizvi et al. upload) has been downloaded, Rizvi queue incorporates 240 and receives to resist The Patients with Non-small-cell Lung of 1 single therapy of PD- (L) or the anti-CTLA-4 combined treatment of anti-PD- (L) 1+.240 patients In, it is grouped further according to KRAS and TP53 mutation status, 45 entitled KRAS-WT/TP53-WT patients, 109 are KRAS-WT/TP53-MUT patient, 54 are that KRAS-MUT/TP53-WT patient and 32 KRAS-MUT/TP53-MUT suffer from Person.This four groups middle position PFS is respectively 2.6 months (95% CI 1.93-5.27), 3.6 months (95% CI 3.07- 5.37), 2.33 months (95% CI 1.93-4.37), 5.77 months (4.27-NA of 95%CI) (Fig. 8), multifactor point of Cox It is the independent prediction risk factors (KRAS-MUT/ of immunization therapy prognosis that analysis result, which further illustrates that KRAS/TP53 is mutated not altogether, TP53-MUT vs KRAS-WT/TP53-WT, HR:0.58,95%CI:0.33-1.04, p=0.067) because COX is returned P > 0.05(Fig. 9).

Embodiment 6

It may be used to predict response of the NSCLC patient to the inhibitor for treating of PD-1, and and GMS in view of PTPRD, TP53 gene Show positive correlation, KMT2C/TP53 in order to further illustrate the present invention be mutated altogether be immunization therapy prognosis independent prediction wind Dangerous factor, the present invention are mutated altogether for PTPRD/TP53 and have carried out similar research.We are in the website cBioPortal (http://www.cbioportal.org/) has downloaded the queuing data of Rizvi et al. upload, and Rizvi queue incorporates 240 Name receives the Patients with Non-small-cell Lung of anti-1 single therapy of PD- (L) or the anti-CTLA-4 combined treatment of anti-PD- (L) 1+.240 It in a patient, is grouped further according to PTPRD and TP53 mutation status, 91 entitled PTPRD-WT/TP53-WT suffer from Person, 119 are PTPRD-WT/TP53-MUT patients, and 8 are PTPRD-MUT/TP53-WT patient and 22 PTPRD- MUT/TP53-MUT patient.This four groups middle position PFS is respectively 2.47 months (95% CI 2.03-3.5), 3.8 months (95% CI 3.1-5.33), 2.79 months (95% CI, 2.1-NA), 6.33 months (4.17-NA of 95%CI) (Figure 10), Cox mostly because It is the independent prediction risk factors (PTPRD- of immunization therapy prognosis that element analysis result, which further illustrates that PTPRD/TP53 is mutated altogether, MUT/TP53-MUT vs PTPRD-WT/TP53-WT, HR:0.52,95%CI:0.30-0.92, p=0.026) (Figure 11).

Embodiment 7

It may be used to predict response of the NSCLC patient to the inhibitor for treating of PD-1 in view of HGF, TP53 gene, and be in GMS Reveal positive correlation, KMT2C/TP53 in order to further illustrate the present invention be mutated altogether be immunization therapy prognosis independent prediction risk Factor, the present invention are mutated altogether for HGF/TP53 and have carried out similar research.We the website cBioPortal (http: // Www.cbioportal.org/ the queuing data of Rizvi et al. upload) has been downloaded, Rizvi queue incorporates 240 and receives to resist The Patients with Non-small-cell Lung of 1 single therapy of PD- (L) or the anti-CTLA-4 combined treatment of anti-PD- (L) 1+.240 patients In, it is grouped further according to HGF and TP53 mutation status, 96 entitled HGF-WT/TP53-WT patients, 127 are HGF-WT/TP53-MUT patient, 3 are HGF-MUT/TP53-WT patient and 14 HGF-MUT/TP53-MUT patients. This four groups middle position PFS is respectively 2.57 months (95% CI 2.1-3.5), and 4.17 months (95% CI 3.3-5.43), 1.6 months (95% CI, 1.57-NA), 5.1 months (3.17-NA of 95%CI) (Figure 12), Cox multiplicity result was into one It is independent prediction risk factors (the HGF-MUT/TP53-MUT/ vs of immunization therapy prognosis that step, which illustrates that HGF/TP53 is mutated not altogether, HGF-WT/TP53-WT, HR:0.57,95%CI:0.29-1.12, p=0.104) (Figure 13), because the p of COX recurrence > 0.05。

Effect example

By being mutated altogether to KMT2C/TP53 respectively in embodiment 4-7, KRAS/TP53 is mutated, PTPRD/TP53 is mutated altogether, HGF/ The survival analysis research that TP53 is mutated altogether, the results show that position PFS is respectively 7.33 months in TP53-MUT/KMT2C-MUT patient (95% CI, 2.5-NA), position PFS is respectively 5.77 months (4.27-NA of 95%CI) in TP53-MUT/KRAS-MUT patient, TP53- Position PFS is respectively 6.33 months (4.17-NA of 95%CI) in MUT/PTPRD-MUT patient, position in TP53-MUT/HGF-MUT patient PFS is respectively 5.1 months (3.17-NA of 95%CI), it can be seen that the immunization therapy of TP53/KMT2C combination in terms of the median survival time Outcome prediction effect is best.

Meanwhile Cox proportional hazard model (cox proportional-hazards model), abbreviation Cox model be by A kind of semi-parametric regression model that Britain statistician D.R.Cox (1972) year proposes.The model with survive final result and existence when Between be dependent variable, influence of many factors to life cycle can be analyzed simultaneously, can not analyze the data for having truncation life span, and not It is required that the survival distribution type of estimated data, Cox model can handle influence of the Multiple factors to existence final result simultaneously, so being The most classic multiplicity method of Survival data is directed in clinical research.And Hazard ratio (hazard ratio, abbreviation HR) It is then most important concept in Cox model, in cancer research: ratio > 1 hazard is considered as bad prognostic Ratio < 1 factor, hazard is considered as good prognostic factor, and HR is smaller, then it is pre- to represent the group It is better afterwards that (the smaller group generation event risk that represents of HR is smaller, for example HR=0.51 is represented relative to control group, and experimental group occurs 49%) event risk has dropped, TP53-MUT/KMT2C-MUT group HR:0.51,95%CI:0.27-0.96, p=0.036, TP53-MUT/KRAS-MUT organizes HR:0.58,95%CI:0.33-1.04, p=0.067, TP53-MUT/PTPRD-MUT group 0.52,95%CI:0.30-0.92, p=0.026, TP53-MUT/HGF-MUT group 0.57,95%CI:0.29-1.12, p= 0.104.It is optimal from TP53-MUT/KMT2C-MUT combination from the point of view of HR angle.

Mode the above is only the implementation of the present invention is not intended to limit the scope of the invention, all to utilize this Equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it is relevant to be applied directly or indirectly in other Technical field is included within the scope of the present invention.

Sequence table

<110>to this medical science and technology (Shanghai) Co., Ltd.

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<130> 2019

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Claims (8)

1. the reagent of specific detection TP53 gene mutation and specific detection KMT2C gene mutation is non-small for predicting in preparation Cell lung cancer (NSCLC) patient is to the purposes in the kit or device of the sensibility of immunotherapy.

2. purposes according to claim 1, which is characterized in that the immunotherapy is using the immune of immunologic test point inhibitor Therapy.

3. purposes according to claim 1, which is characterized in that the prediction includes the following steps,

Step a) assesses the TP53 gene mutation in the tumor tissues of the patient；With

Step b) assesses the KMT2C gene mutation in the tumor tissues of the patient；With

Step c) is based on assessment result prediction cancer patient a), b) to the immunotherapy for using immunologic test point inhibitor Sensibility.

4. purposes according to claim 3, which is characterized in that the assessment refers to, the survey of comparison of tumor tissue and control tissue TP53 gene mutation and KMT2C gene mutation described in sequence data assessment.

5. one kind is for predicting non-small cell lung cancer (NSCLC) patient to the kit of the sensibility of immunotherapy, feature exists In including at least the reagent of specific detection TP53 gene mutation and specific detection KMT2C gene mutation.

6. one kind is for predicting non-small cell lung cancer (NSCLC) patient to the quick of the immunotherapy for using immunologic test point inhibitor The device of perception, which is characterized in that described device includes following three modules:

Evaluation module I), assess the TP53 gene mutation in the tumor tissues of the patient；

Evaluation module II), assess the KMT2C gene mutation in the tumor tissues of the patient；With

Prediction module III), be based on evaluation module I), II) assessment result prediction cancer patient to using immunologic test point to press down The sensibility of the immunotherapy of preparation.

7. one kind is for predicting non-small cell lung cancer (NSCLC) patient to the immunotherapy for using immunologic test point inhibitor The device of sensibility, which is characterized in that described device includes: memory, for storing program；Processor is executed for passing through The program of above-mentioned memory storage is to realize sensitivity of the prediction cancer patient to the immunotherapy for using immunologic test point inhibitor Property, the prediction include the following steps,

Step a) assesses the TP53 gene mutation in the tumor tissues of the patient；With

Step b) assesses the KMT2C gene mutation in the tumor tissues of the patient；With

Step c) is based on assessment result prediction cancer patient a), b) to the immunotherapy for using immunologic test point inhibitor Sensibility.

8. a kind of computer readable storage medium, which is characterized in that it includes program, the program can be executed by processor with Realize prediction non-small cell lung cancer (NSCLC) patient to the whole of the sensibility for the immunotherapy for using immunologic test point inhibitor Or part steps, all or part of the steps include,

Step a) assesses the TP53 gene mutation in the tumor tissues of the patient；With

Step b) assesses the KMT2C gene mutation in the tumor tissues of the patient；With

Step c) is based on assessment result prediction cancer patient a), b) to the immunotherapy for using immunologic test point inhibitor Sensibility.