CN106119398B - Biomarkers to predict responsiveness to pyrotinib therapy in breast cancer patients - Google Patents

Abstract

The invention discloses the use of specific sites on the PIK3CA gene and/or TP53 gene in preparing a kit for predicting the responsiveness of HER2-positive advanced breast cancer patients to pyrotinib treatment. The present invention analyzes the correlation between specific sites on the PIK3CA gene and/or TP53 gene and the responsiveness of pyrotinib to treatment by genotyping patients, and finds that by identifying specific sites on the PIK3CA gene and/or TP53 gene Whether it is wild-type or mutant can determine the therapeutic response of HER2-positive advanced breast cancer patients to pyrotinib. The research results of the present invention provide a reliable and sensitive method for determining the treatment plan for patients with HER2 positive advanced breast cancer.

Description

Biomarkers to predict responsiveness to pyrotinib therapy in breast cancer patients

technical field

The invention belongs to the field of biomedicine, and relates to a biomarker for predicting responsiveness of breast cancer patients to pyrotinib treatment and use thereof.

Background technique

HER2-positive breast cancer is a very dangerous subtype of breast cancer, and about 20%-30% of breast cancer patients are HER2-positive.

HER2, also known as human epidermal growth factor receptor-2, is a proto-oncogene expressed as HER2 protein on the cell membrane, responsible for transducing signals to promote cell growth and division. When the HER2 gene is overexpressed, too much HER2 protein appears on the surface of these cancer cells, which promotes the division and growth of cancer cells and forms HER2-positive tumors. HER2 positive often indicates that the tumor progresses rapidly, is prone to lymph node or blood metastasis, is not sensitive to endocrine therapy, and has a poor prognosis. According to statistics, even if patients with HER2-positive breast cancer receive conventional comprehensive treatment such as radiotherapy and chemotherapy, the survival time is only half of that of HER2-negative patients.

Pyrotinib is a novel oral irreversible HER1/HER2 receptor tyrosine kinase inhibitor. Studies have shown that pyrotinib can be used to treat HER2-positive breast cancer, but there are significant individual differences in clinical efficacy. Since the survival period of patients with advanced breast cancer is very limited, it is particularly important to select the most likely effective drug treatment as early as possible to prolong the survival of patients. In addition, targeted therapy drugs represented by pyrotinib are relatively expensive, and the price is dozens of times that of ordinary chemotherapy. From the perspective of health economics, it is also required to select patients who are most likely to benefit from targeted therapy. Therefore, finding predictors of pyrotinib efficacy and guiding individualized targeted therapy is a key clinical problem to be solved urgently.

Contents of the invention

The present invention provides a use of PIK3CA gene and/or TP53 gene in preparing a kit for predicting the responsiveness of HER2 positive advanced breast cancer patients to pyrotinib treatment.

Furthermore, the kit predicts the responsiveness of HER2-positive advanced breast cancer patients to pyrotinib treatment by detecting the genotype of specific loci of the PIK3CA gene and/or TP53 gene.

The research results of the present invention show that when the genotype of the specific site of the PIK3CA gene and/or the specific site of the TP53 gene is wild type, it is predicted that the HER2-positive advanced breast cancer patient has a good response to pyrotinib treatment; When the genotype of the specific locus of the PIK3CA gene and/or the genotype of the specific locus of the TP53 gene is a mutation, it is predicted that the HER2-positive advanced breast cancer patient will have poor response to pyrotinib treatment.

Further, the specific site of the above-mentioned PIK3CA gene is selected from one or more of the group consisting of the following sites: c.3140, c.1035; the specific site of the TP53 gene is selected from the group consisting of the following sites One or more of the groups of: c.706, c.659, c.375+2, c.318, c.497, c.392.

In the present invention, the wild-type genotype of the c.3140 site of the PIK3CA gene is AA, and the mutant genotype is AG; the wild-type genotype of the c.1035 site of the PIK3CA gene is TT, and the mutant genotype is TA; TP53 The wild-type genotype at c.706 of the gene is TT, and the mutant genotype is TA; the wild-type genotype at c.659 of the TP53 gene is AA, and the mutant genotype is AG; TP53 gene c.375+2 The wild-type genotype of the site is TT, and the mutant genotype is TG; the wild-type genotype of the c.318 site of the TP53 gene is CC, and the mutant genotype is CG; the wild-type gene of the c.497 site of the TP53 gene The genotype of the mutant type is CC, and the genotype of the mutant type is CG; the genotype of the wild type at c.392 of the TP53 gene is AA, and the genotype of the mutant type is AG.

Further, the kit includes reagents for detecting the genotype of specific sites of the PIK3CA gene and/or TP53 gene. The reagents can be the reagents required to detect the sites on the genes using any technique known in the art, as long as they can detect the genotypes of the specific sites of the PIK3CA gene and TP53 gene in the sample.

The methods commonly used in the prior art for detecting the genotype of a site on a gene include, but are not limited to, restriction fragment length polymorphism analysis, sequencing, single-strand conformation polymorphism analysis, chemical cleavage of mismatches, denaturation High performance liquid chromatography, polymerase chain reaction, arrays or combinations thereof. Such arrays include microarrays, including but not limited to planar microarrays or bead arrays; gene arrays; PCR arrays, including TaqMan OpenArray.

The invention provides a kit for predicting the responsiveness of HER2-positive advanced breast cancer patients to pyrotinib treatment, the kit includes a reagent for detecting the genotype of a specific site of PIK3CA gene and/or TP53 gene. The reagents can be the reagents required to detect the sites on the genes using any technique known in the art, as long as they can detect the genotypes of the specific sites of the PIK3CA gene and TP53 gene in the sample.

Further, the kit includes the use of restriction fragment length polymorphism analysis, sequencing, single-strand conformation polymorphism analysis, chemical cleavage of mismatches, denaturing high-performance liquid chromatography, polymerase chain reaction, and arrays to detect the PIK3CA gene and/or reagents used during genotyping of specific loci in the TP53 gene.

Further, in the present invention, the specific site of the PIK3CA gene is selected from one or more of the following sites: c.3140, c.1035; the specific site of the TP53 gene is selected from the following sites: One or more of the groups of: c.706, c.659, c.375+2, c.318, c.497, c.392.

Advantage of the present invention and beneficial effect are as follows:

(1) The present invention discovers for the first time that the specific sites of PIK3CA gene and/or TP53 gene are correlated with the responsiveness of HER2-positive advanced breast cancer patients to pyrotinib treatment, which provides a predictive index for individualized targeted therapy of clinical patients.

(2) The method of the present invention for predicting the therapeutic responsiveness to pyrotinib is simple, the source of the nucleic acid sample is convenient, and the peripheral blood can be extracted for detection. The method has low requirements for detection equipment and low cost, and is favorable for clinical promotion.

Description of drawings

Figure 1 shows the Kaplan-Meier curves for PIK3CA and TP53 wild-type patients versus PIK3CA or TP53 mutant patients.

specific implementation

The present invention will be further described below through specific examples. It should be noted that, unless otherwise specified, the examples of the present invention are only used to explain the present invention, and are not meant to limit the protection scope of the present invention.

Example 1 Correlation between Gene Locus and Pyrotinib Drug Effect

1. Research objects: 97 patients with HER2-positive advanced breast cancer. The selected patients were confirmed as HER2-positive advanced breast cancer patients by histology or cytology, and all patients provided informed consent.

2. Medication method: pyrotinib 400mg, once a day, orally. 28 days as a cycle, re-examination of imaging examination every 2 cycles to evaluate the curative effect.

3. Experimental method

3.1 DNA extraction from plasma

3.1.1 Separation of peripheral blood samples

(1) Collect 1-2 tubes (5mL/tube) of peripheral blood from the patient into EDTA anticoagulant tubes, gently invert up and down (to prevent cell rupture) 6-8 times and mix thoroughly, and perform the following treatment within 4-6 hours on the day of blood collection ;

(2) Centrifuge at 1600g for 10 minutes at 4°C. After centrifugation, divide the supernatant (plasma) into multiple 1.5mL/2mL centrifuge tubes, and the white blood cells in the middle layer cannot be absorbed during the absorption process;

(3) Centrifuge at 16,000g for 10 minutes at 4°C to remove residual cells, transfer the supernatant (plasma) to a new 1.5mL/2mL centrifuge tube, if the white blood cells cannot be sucked to the bottom of the tube, the required plasma after separation is obtained;

(4) After the plasma sample is processed, the separated plasma and remaining blood cells are stored in a -80°C refrigerator to avoid repeated freezing and thawing.

3.1.2 Plasma cell-free DNA extraction (using QIAamp Circulating Nucleic Acid Kit)

(1) Add 30μL proteinase K to a 1.5mL centrifuge tube;

(2) Add 300 μL plasma;

(3) Add 240 μL Buffer ACL and 1.68 μL Carrier RNA (0.2 μg/μL), vortex for 30 s, incubate at 60°C for 30 min, take out and oscillate properly during the incubation;

(4) Add 540 μL Buffer ACB, vortex for 15-30s, and place on ice or in a -20°C refrigerator for 5 minutes;

(5) Add 700 μL of plasma mixture to the filter column, and centrifuge at 7500 rpm for 30 s;

(6) The filter column is emptied at 8000rpm for 1min;

(7) Add 600μL Buffer ACW1, 8000rpm, 1min centrifugal washing;

(8) Add 700μL Buffer ACW2, 8000rpm, centrifuge for 1min to wash;

(9) Add 700 μL of absolute ethanol, 8000 rpm, and centrifuge for 1 min to wash;

(10) The filter column is emptied at 14.000rpm for 3min;

(11) Put the filter column into a new collection tube, open the lid, and put it in a metal bath at 56°C for 10 minutes;

(12) Put the column into a new centrifuge tube and add 60 μL Buffer AVE to redissolve for 3 minutes;

(13) Centrifuge at 14.000rpm for 1 min, and use Qubit (Invitrogen, the Quant-iTTMdsDNA HS AssayKit) to quantify the extracted plasma free DNA.

3.2 Library preparation and ultra-high depth sequencing

End repair of DNA fragments;

Add A to the end of the DNA fragment;

Connect Adapter library adapter: Library adapter (Adapter) refers to a designed base sequence, which is used to combine with primers during DNA library amplification to allow DNA amplification to proceed, and to combine with Standard Anchor during sequencing on the machine. It is beneficial for the combination of the probe and the site to be sequenced for assisted DNA sequencing.

The library was subjected to the first round of PCR amplification;

Library quality control after amplification and probe capture hybridization for the target region;

The hybrid library is subjected to the second round of PCR amplification;

Among them, the primer sequence is the universal adapter primer for library amplification:

Upstream primers:

5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3';

Downstream primers:

5'-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'.

The probe is aimed at all exon sequences of TP53 and PIK3CA, and the probe sequence is designed according to the two gene sequences, with a length of 120bp. The probe design is a technique well known to those skilled in the art.

Library quantification and quality control; Illumina HiSeq3000 on-machine sequencing

3.3 Bioinformatics analysis of off-board data

After obtaining the off-board data, the following biological information analysis is required to obtain the final mutation result

SOAPnuke filter: remove low-quality reads;

Align with the reference sequence to generate a bam file;

Mark repeats;

Sequences with poor alignment results were re-aligned and the quality value was corrected;

Remove mismatched sequences;

Analyze off-machine data QC: count chip capture efficiency, number of effective reads, average depth, repetition rate, coverage, and uncovered intervals, etc.;

Finding Variations: Call SNV/InDel/SV/CNV:

Call somatic SNV variation with mutect, varScan, somVar processes;

Use gatk, varScan, somVar processes to call somatic InDel mutation;

Call CNV with the contra.py process;

Call SV with the somVar process;

4. Experimental results

4.1 Genotyping

The statistics of the site mutations in the PIK3CA gene and TP53 gene in the 97 patients are shown in Table 1:

Table 1 Site mutation information

Gene wxya pHGVS_ad The proportion PIK3CA c.[3140A>G] p.[H1047R] 23/27 PIK3CA c.[1035T>A] p.[N345K] 4/27 TP53 c.[706T>A] p.[Y236N] 5/32 TP53 c.[659A>G] p.[Y220C] 6/32 TP53 c.[375+2T>G] 3/32 TP53 c.[318C>G] p.[S106R] 4/32 TP53 c.[497C>G] p.[S166*] 8/32 TP53 c.[392A>G] p.[N131S] 6/32

4.2 Correlation analysis of gene site mutations and response to pyrotinib treatment in patients with HER2-positive advanced breast cancer

The results showed that the remission rate (ORR=CR+PR) of pyrotinib in 27 patients with PIK3CA mutation was 0%, and the ORR in 70 patients with wild-type PIK3CA was 45.7% (32/70; p=0.114 chi-square test). Likewise, the ORR was 0% in 32 patients with TP53 mutation and 49.2% in 65 patients with wild-type TP53 (32/65; p=0.054 chi-square test). Combined analysis of PIK3CA and TP53 results, the ORR of patients with PIK3CA or TP53 mutations was 0%, while the ORR of patients with wild-type PIK3CA and TP53 was 60.0% (p=0.013, chi-square test), and there was a significant difference in mPFS between the two (14.9vs.46.1 weeks, p=0.015, log-rank test), the results are shown in Table 2 and Figure 1.

Table 2 analysis results

The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (3)

1. the reagent of the SNP site genotype of detection PIK3CA gene and/or TP53 gene is in preparation for predicting the HER2 positive Advanced breast cancer patient is to pyrroles for the purposes in the kit of Buddhist nun's therapeutic response, which is characterized in that the SNP of PIK3CA gene One or more in the group that following SNP site forms of site: c.3140, c.1035；The SNP site of TP53 gene Selected from following SNP site composition group in one or more: c.706, c.659, c.375+2, c.318, c.497, c.392；When the genotype of the SNP site of the SNP site and/or TP53 gene of PIK3CA gene is wild type, then prediction should HER2 positive advanced breast cancer patient is good for Buddhist nun's therapeutic response to pyrroles；The SNP site and/or TP53 gene of PIK3CA gene The genotype of SNP site when being saltant type, then predict the HER2 positive advanced breast cancer patient to pyrroles for Buddhist nun's therapeutic response Property is poor；PIK3CA gene c.3140 site wild type genotype be AA, mutant-type genotype AG；PIK3CA gene is c.1035 The wild type genotype in site is TT, mutant-type genotype TA；TP53 gene c.706 site wild type genotype be TT, Mutant-type genotype is TA；TP53 gene c.659 site wild type genotype be AA, mutant-type genotype AG；TP53 base Because the wild type genotype in c.375+2 site is TT, mutant-type genotype TG；The wild type base in TP53 gene c.318 site Because type is CC, mutant-type genotype CG；The wild type genotype in TP53 gene c.497 site is CC, and mutant-type genotype is CG；TP53 gene c.392 site wild type genotype be AA, mutant-type genotype AG.

2. purposes according to claim 1, which is characterized in that in the kit include detection PIK3CA gene and/or The reagent of the genotype of TP53 gene SNP site.

3. purposes according to claim 2, which is characterized in that more including the use of restriction fragment length in the kit State property analysis, sequencing, single-strand conformation polymorphism analysis, the chemical cracking of mispairing, denaturing high-performance chromatography, polymerase chain are anti- The reagent answered, used during genotype of the array to detect PIK3CA gene and/or TP53 gene SNP site.