JP2022102386A - Method of producing sample for gene analysis - Google Patents

Abstract

To provide a method of producing a sample for gene analysis for PCR, and a PCR method and a gene analysis method using the sample.SOLUTION: A method of producing a sample for gene analysis comprises a collected substance suspension step for suspending a collected substance, which is cells or a tissue collected from a subject, in nuclease-free water.SELECTED DRAWING: Figure 1

Description

Patent Law Article 30, Paragraph 2 Application Applicable https: // www. nature. com / https: // www. nature. com / articles / s41598-020-69221-6 Nature Research journal Scientific Reports Reiwa July 23, 2

The present invention relates to a method of preparing a sample for gene analysis without purifying nucleic acid by using cells or tissues collected at the time of treatment. By using this invention, for example, in the case of cancer treatment surgery, it is possible to rapidly perform gene analysis of collected cells and tissues.

Japanese Patent No. 6709319 describes a PCR method. Japanese Patent No. 672194 describes a cartridge for digital real-time PCR. As described above, the PCR method and the digital PCR method are already known.

For example, in the histological diagnosis of pancreatic tumor, rapid and accurate histopathological diagnosis may be difficult due to lack of specimens. Therefore, an excellent method for preparing a sample for gene analysis for PCR (particularly digital PCR) is desired so that KRAS mutations derived from tumors can be quickly detected.

Japanese Patent No. 6709319 Japanese Patent No. 672194

An object of the present invention is to provide a method for producing a sample for gene analysis for PCR, a PCR method using the sample, and a method for gene analysis. In particular, we will provide a method that can quickly prepare a sample for gene analysis even during treatment.

The present invention is basically PCR (particularly digital PCR) capable of detecting tumor-derived KRAS mutations by suspending a collection of cells or tissues collected from a subject in nuclease-free water. ) Based on the findings from the examples that the sample for gene analysis can be quickly prepared.

The first invention described herein relates to a method for producing a sample for gene analysis. This method involves a collection suspension step in which a collection of cells or tissues collected from a subject is suspended in nuclease-free water. The gene analysis sample thus prepared can be preferably used in the PCR method. Examples of PCR methods are the droplet digital PCR method or the emulsion PCR method.

A sample for gene analysis is a sample prepared for analyzing a gene. Preferred examples of samples for gene analysis are samples for PCR (particularly samples for digital PCR) and samples for DNA sequencers. Digital PCR (dPCR) is, for example, an analysis method in which a single DNA or cDNA sample is divided and PCR reactions are performed in parallel. For example, Japanese Patent No. 672194 describes digital real-time PCR. As described above, digital PCR (dPCR) is known. A DNA sequencer is described in JP-A-2002-153271 and JP-A-2018-535481. As described above, DNA sequencers are known. This gene analysis sample can be used for a DNA sequencer.

The harvested material may be a piece of cell or a piece of tissue remaining on the puncture needle. Further, the collected material may be obtained by suspending the cell pieces or tissue pieces remaining in the puncture needle and then centrifuging. A cell or tissue fragment is a portion of the cell or tissue of a patient of interest. The target site of the cell piece or tissue piece may be appropriately adjusted and may be various organs. As described above, even a minute sample or a residual sample for pathological diagnosis can be used as an excellent sample for gene analysis by using this method. The sampling method is endoscopic ultrasonographic puncture suction (EUS-FNA) or endoscopic biopsy. It is also possible by surgery to collect the harvest, or by fine needle aspiration during surgery. This sample may be 0.1 mg or more and 1 g or less, 1 mg or more and 100 mg or less, or 1 mg or more and 10 mg or less. The collected material does not need to be collected for the purpose of performing a genetic test, and may use a small amount of sample that adheres to the surgical instrument during the treatment.

Nuclease-free water is specially purified to avoid contamination of samples with nucleases, which are DNA and RNA degrading enzymes (DNase, which is a DNA degrading enzyme, and RNase, which is an RNA degrading enzyme). It is water. When a cell piece or tissue piece is suspended in nuclease-free water, osmotic pressure destroys the cell and releases genomic DNA into the nuclease-free water. In the present invention, the solution in which the genomic DNA is released may be used as it is as a sample for gene analysis by utilizing osmotic crushing. Therefore, the solution is preferably nuclease-free water. Of course, nuclease-free water from which genomic DNA has been released may be appropriately purified and used as a sample for gene analysis. The volume ratio of the sample to the nuclease-free water may be adjusted as appropriate. An example of the amount ratio of the sample to the nuclease-free water may be appropriately adjusted in consideration of the amount of the sample and the ease of adjustment, and when the sample is 1 part by weight, the nuclease-free water is 0. It may be 1 part by weight or more and 100,000 parts by weight or less, 0.1 part by weight or more and 10,000 parts by weight or less, 10 parts by weight or more and 10,000 parts by weight or less, 10 parts by weight or more and 1000 parts by weight or less. It may be 10 parts by weight or more and 100 parts by weight or less.

In the present invention, if the osmotic pressure is low, a nuclease-free water to which a nuclease-free reagent (for example, a buffer material or a pH adjuster) is appropriately added may be used as a solvent. In addition to water, physical crushing (ultrasonic waves, etc.) is also possible.

The sample may be mixed with nuclease-free water and then stirred or centrifuged. The conditions for centrifuging may be adjusted as appropriate. An example of the maximum centrifugal acceleration is 10 xg or more and 1 million xg or less, 100 xg or more and 100,000 xg or less, 100 xg or more and 10,000 xg or less, or 500 xg or more and 5000 xg or less. The maximum rotation speed for centrifugation may be 1 rpm or more and 100,000 rpm or less, 10 rpm or more and 10,000 rpm or less, 10 rpm or more and 1000 rpm or less, 50 rpm or more and 500 rpm or less, or 50 rpm or more and 300 rpm. It may be as follows. An example of the centrifugation time is 10 seconds or more and 10 hours or less, 1 minute or more and 2 hours or less, 1 minute or more and 1 hour or less, and 3 minutes or more and 30 minutes or less. The supernatant after centrifugation may be used as a sample for gene analysis, or the supernatant after centrifugation may be used to prepare a sample for gene analysis. In any case, it is preferable to prepare a sample for gene analysis without performing nucleic acid purification treatment. Since the nucleic acid purification process that is normally performed can be omitted in this way, gene analysis can be performed extremely quickly even during treatment.

The collection may also be tumor-derived cells or tissues collected from the subject. Examples of tumor-derived cells or tissues are pancreatic cancer cells or tissues. By detecting tumor-derived DNA (KRAS mutations found in pancreatic cancer, colon cancer, lung cancer, NRAS, BRAF, EGFR, PIC3CA, etc.), tumors can be obtained even if no morphological evidence of tumor cells can be obtained. The existence of the tumor can be diagnosed. Conventionally, nucleic acid purification is required in principle, but in the case of trace samples, it is often lost in the purification process and the amount required for genetic testing such as PCR cannot be obtained. However, according to the method of the present invention, even a small amount of sample can be effectively and rapidly analyzed for DNA. As described above, the gene analysis sample is preferably used for analyzing tumor-derived gene mutations.

As described above, gene analysis by PCR method, dPCR method, and gene analysis by DNA sequencer are known. Therefore, the above gene analysis sample can be used for gene analysis by a known method.

According to the present invention, it is possible to provide an excellent method for producing a sample for gene analysis for PCR, a PCR method using the sample, and a gene analysis method. According to the present invention, a sample for gene analysis can be quickly prepared, especially during a treatment.

FIG. 1 is a photo that replaces the drawing showing the encapsulated cells in the dPCR droplets. FIG. 2 is a photograph that replaces the drawing showing how genomic DNA is released into water. FIG. 3 is a graph that replaces the drawing showing the results of detecting wild-type or G12C mutations in KRAS using a droplet detector. FIG. 4 is a graph that replaces the drawing showing the results of measuring the number of KRAS copies of the wild type or G12C in FIG. 3 with the QuantaSoft software. FIG. 5 is a conceptual diagram for explaining the workflow of sampling and DNA preparation. FIG. 6 is an alternative to the drawing showing the dPCR plot of the KRAS G12 / G13 mutation assay in water-resuspended harvest tissue. FIG. 7 is a graph that replaces the drawing showing the correlation between the amplifyable DNA indexed by the number of copies of KRAS and the amount of template DNA.

Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the forms described below, and includes those modified appropriately by those skilled in the art from the following forms.

Cell lines Human pancreatic cancer cells (MIA PaCa-2; RCB2094) and non-cancerous skin fibroblasts (NB1RGB; RCB0222) were obtained from RIKEN Cell Bank (Japan) and 10% fetal bovine serum (GE Healthcare, Illinois, USA). It was grown using DMEM (MIA PaCa-2) and α-MEM (NB1RGB) medium (Fujifilm Wako Pure Drug, Japan) supplemented with 100 U / mL (Fuji Film Wako Pure Drug) and penicillin-streptomycin (Chicago). Cell lines were grown at 37 ° C. and 5% CO ₂ and passaged at a cell density of 70-80%. Cell counts were counted using a Countess (registered trademark) automated cell counter (Thermo Fisher Scientific, Waltham, Mass., USA).

Patients Twelve patients with resectable pancreatic disease were examined to investigate how to detect mutations in resected tissue. FNA (Fine Needle Aspiration) residual samples were obtained from 10 pancreatic tumor patients. This analysis was performed according to the Declaration of Helsinki. Written informed consent was obtained from all patients prior to enrollment.

Collection and preparation of fresh tissue from surgically resected specimens Within 30 minutes after surgical resection of pancreatic cancer in the operating room, small tissue specimens were obtained. Multiple tumor sites (typically 2-3 sites) were punctured and aspirated using a 22 gauge Cateran needle (Terumo, Japan, Tokyo) connected to a 10 mL syringe. The aspirated sample was suspended in 3 mL of phosphate buffered saline (PBS) containing 450 μL of undiluted solution from PAXgene Blood ccfDNA Tube (BD Life Sciences; Franklin Lake, NJ, USA) and stored at 4 ° C. for 1 week. The suspension was centrifuged at 1,000 xg for 10 minutes at room temperature and the pellet was resuspended in 12 μL of nuclease-free water using a 200 μL pipette tip with a truncated tip, which was immediately used as a PCR template.

Collection of FNA Residual Specimens After performing FNA biopsy sampling for cytopathology, FNA residual tissue was obtained using a 22-gauge Franseen biopsy needle (Acquire; Boston Scientific, Marlborough, Massachusetts, USA). The residual tissue remaining in the needle was collected in a 5.0 mL microtube by performing aspiration several times (typically 2-3 times), and then 3 mL of saline was released, and the PAXgene Blood was released. It was combined with 450 μL of undiluted solution from ccfDNA Tube by performing inversion and mixing several times and storing the contents at 4 ° C. for 1 week. In addition, a needle cleaning liquid fraction was collected to collect the cleaning tissue. At 4 ° C., each suspension was centrifuged at 1,000 xg for 10 minutes. Scrape off a portion of the remaining pellet fraction, resuspend in 12 μL of nuclease-free water using a 200 μL pipette tip with a cut tip, and resuspend the entire needle wash fraction in 12 μL of nuclease-free water. did. The fraction suspended in water again was directly used as a dPCR template.

The supernatant fraction obtained after centrifugation was directly charged into dPCR. Purified DNA was prepared and used as a control standard in conventional mutation analysis methods. The DNA in the supernatant fraction was purified using QiAmp MinElute cfDNA Mini Kit (manufactured by Qiagen®; Hilden, Germany), and the DNA in the pellet fraction was obtained from DNeasy Blood and Tissue Kit (registered trademark). Manufactured by).

Mutation detection assay using dPCR 20 microliters of resuspension, 10 μL of ddPCR Supermix for Solutions (without dUTP, Bio-Rad, Hercules, Calif., USA) and 1 μL of ddPCR KRAS Creating Mixture targeting KRAS exon2. It was mixed with # 1863506; Bio-Rad) and a template DNA solution, and then the mixture was vortexed 3 times at 2,500 rpm. The PCR mixture was mixed with 70 μL of Droplet Generation Oil (Bio-Rad) and localized using a QX200 droplet generator (Bio-Rad).

With this kit, seven KRAS mutations (G12A / C / D / R / S / V / G13D) could be screened with a frequency of> 0.2%, but no specific mutation could be determined. .. For tumors with KRAS Q61 mutations found by NGS, an additional assay was performed using the ddPCR KRAS Q61 Screening Kit (Q61K / L / R / H, Bio-Rad) to cut off the presence or absence of mutations> 0. It was evaluated at 5%. Again, it is not possible to determine a particular mutation.
Such mutation detection assays were performed using a protocol such as: 95 ° C. for 10 minutes, followed by 94 ° C. for 30 seconds for 40 cycles, and 55 ° C. for 60 seconds, followed by 98 ° C. for 10 minutes. Process (race plate, 2 ° C / sec at each step). The threshold of the absolute number of copies input during the reaction and the ratio of the mutant fractions were calculated using QuantaSoft (version 1.7; Bio-Rad) based on the Poisson distribution. Samples were considered mutant KRAS positive when at least 5 mutant droplets / reactions were detected using dPCR.

Tumor Specimens and Mutation Analysis Formalin-fixed paraffin-embedded (FFPE) tissue specimens and 10 μm or 4 μm thick unstained sections (resected tissue or FNA biopsy specimens, respectively) were prepared to validate the tumor mutation profile. .. Genomic DNA was isolated using GeneRead DNA FFPE Kit (Qiagen) and finally eluted in 30 μL elution buffer as described above. Purified DNA was quantified on a Qubit4 fluorometer (Thermo Fisher Scientific) using a Qubit dsDNA HS Assay Kit.

In addition, for somatic mutations in primary tumors of FFPE tissue specimens, Ion was designed using the Ion AmpliSeq Designer Website (https://www.ampriseq.com) using the targeted amplicon sequencing technology. Eight pancreatic cancer-related genes, namely KRAS, TP53, SMAD4, CDKN2A, GNAS, PIK3CA, BRAF, STK11, were profiled on the AmpliSeq Custom-Generation Sequence DNA panel (supplementary table). Details on sequencing analysis are given in the supplementary information.

Development of a method for detecting the minimum number of copies of tumor-derived mutant KRAS We verified a method for detecting mutants in small tissue samples using absolute quantification by dPCR. To generate input DNA from the sample, two methods were first tested to avoid loss during the nuclear purification step. In the first method, cells / tissues were encapsulated using a droplet generator and then the PCR reaction was performed directly. Serial dilutions were performed using two different cell lines, MIA PaCa-2 (homozygous KRAS G12C) and NB1RGB (wild type KRAS). The ratio of mutants to wild-type genes in the cell mixture was 20: 4,000, 100: 4,000, 500: 4,000, 1,000: 4,000. Suspended cells in 4 μL PBS were encapsulated directly into the emulsion using the QX200 system (FIG. 1). It was then used in the dPCR variant detection assay. As shown in FIGS. 3 and 4, the frequency of DNA detection after capture of encapsulated cells was not very high (12.9% on average in KRAS mutant cells, 2.9% on average in wild-type KRAS cells).

FIG. 1 is a photo that replaces the drawing showing the encapsulated cells in the dPCR droplets. The scale is 200 μm. As described above, cells were harvested, resuspended in dPCR reaction solution and mixed with droplet-generating oil using a QX200 droplet generator. The lower figure of FIG. 1 is an enlarged view of the upper figure of FIG.

Therefore, by alternative testing, cells were harvested and resuspended in nuclease-free water, resulting in an osmotic burst of cells. This can result in the release of genomic DNA into the liquid fraction (Fig. 2). The "crude" DNA was then used directly as a dPCR template without performing a DNA purification step (approximately 30 minutes), thus, through the assay, the mutational status of KRAS in fresh tumor samples within two and a half hours. It could be determined (dPCR was processed after a few minutes required to prepare tumor-derived DNA). The dPCR reaction proceeded without problems even with impure DNA, and the number of copies of KRAS detected was comparable to the number detected in the sample prepared by conventional DNA purification (Fig. 1C). This result suggests that the "underwater burst" method can be used to perform time-saving preparation steps and achieve highly efficient detection of a small number of DNA copies.

FIG. 2 is a photograph that replaces the drawing showing how genomic DNA is released into water. The scale is 500 μm. As shown in FIG. 2, when cells are ruptured with pure water, the ruptured cells are collected and resuspended in nuclease-free water, an osmotic burst of the cells occurs and the genomic DNA is released into the water. rice field. In this example, a "crude" solution containing genomic DNA was used as the dPCR template.

FIG. 3 is a graph that replaces the drawing showing the results of detecting wild-type or G12C mutations in KRAS using a droplet detector. The dPCR assay was performed using two novel DNA preparation methods, without a purification step. KRAS wild-type (fibroblasts; NB1RGB) and KRAS G12C (PDA; MIA-PaCa-2) cells were mixed with several dilution series (left panel). Wild-type or G12C mutations in KRAS were detected using a QX200 droplet detector as opposed to conventional DNA preparation methods using commercially available purification kits.

FIG. 4 is a graph that replaces the drawing showing the results of measuring the number of KRAS copies of the wild type or G12C in FIG. 3 with the QuantaSoft software.

Mutation detection analysis using fresh tissue punctured and aspirated from resected pancreatic tumor Next, the feasibility of detecting KRAS mutants using a small amount of resected tumor tissue was examined. Twelve patients with pancreatic tumors were enrolled and analyzed for fine needle aspirates from tumor and non-tumor sites of resected specimens. The aspirate was suspended in nuclease-free water following storage and spin-down. In the dPCR assay, KRAS G12 / G13 mutants were found in pancreatic cancer in 10 patients including pancreatic cancer derived from intraductal papillary mucinous neoplasm (IPMN), and the mutation was found in one pancreatic cancer patient. The body KRAS Q61H was expressed. In contrast, no KRAS mutant was detected in the tumors of patients with pancreatic neuroendocrine tumors (Table 1). Multiple KRAS variants were found in tumors obtained from one IPMN-derived carcinoma patient.

By using the "underwater burst" method, a reasonable number of KRAS G12 / G13 mutants were successfully detected in fresh puncture aspirates from 10 pancreatic cancers. In addition, one sample from patients expressing the KRAS Q61H mutation was found to have a KRAS Q61 mutation, and the number of KRAS G12 / G13 mutations was below the cutoff value (Table 1). There was 100% concordance for KRAS mutations in a small tumor cohort containing samples with wild-type KRAS. The lowest frequency of mutation to wild-type in these patients was 0.82% (the frequency of mutation alleles in primary tumor lesions was 12.8%, Table 1).

Detection of KRAS Mutations by dPCR Using Residual Tissue from Endoscopic Biopsy Samples We attempted to capture and verify dPCR-based driver mutations by the "underwater burst" method using tumor tissue pieces remaining on the FNA puncture needle. After submitting the pancreatic tumor biopsy sample to the pathology laboratory, scrape the residual tissue from the Petri dish and flush the needle with a stabilizing solution to collect the minimum amount of residual sample to test the "underwater burst" method. (Fig. 5). Body fluids were stored, transported, centrifuged, and then genetically analyzed. The pellets and supernatants suspended in water were then analyzed by a dPCR assay targeting KRAS mutations. In 9 of 10 patients, KRAS mutation was found in the residual tissue obtained after FNA (G12 / G13 mutant in 8 patients, Q61 mutant in 1 patient). G12 / G13 mutations were detected in 6 patients and Q61 mutations were detected in 1 patient in 7 samples of intraneedle rinse (Tables 2 and 6). In patient 7 diagnosed with pancreatic acinic cell carcinoma without the KRAS mutation, the level of the KRAS G12 / G13 mutation was found to be similar to the detection limit (0.2%) of the screening kit. In contrast, the mutation allele frequency of other samples with mutations in KRAS exceeded 10%. The residual tissue or needle flash portion of the FNA sample was also analyzed using the dPCR assay following DNA purification (Table 2). A strong correlation was observed between the number of amplified KRAS copies and the amount of template DNA (Table 3 and FIG. 7). An "underwater burst" assay using pellets from FNA residual tissue showed the same KRAS mutation allelic frequency as that of purified DNA, except for patient 2, but the supernatant required DNA purification.

FIG. 5 is a conceptual diagram for explaining the workflow of sampling and DNA preparation. After submitting the patient sample obtained by FNA for cytopathological diagnosis, the residual sample and needle washing solution were collected, centrifuged, and separated into a precipitate and a supernatant. DNA was prepared by the "underwater burst" method, in which the precipitate was centrifuged and suspended in water, followed by dPCR analysis. The supernatant was directly subjected to the dPCR reaction. These methods do not require DNA purification, and the time required from sample collection to obtaining genetic information is about two and a half hours. Purified DNA was also used as a control standard in the assay (see Table 2).

FIG. 6 is a dPCR plot of the KRAS G12 / G13 mutation assay in collected tissue resuspended in water (left panel). The plot graph shows the detection pattern of KRAS tissue obtained by centrifugation from FNA residual tissue or needle lavage fluid. The threshold (pink solid line) is manually extended to an amplitude (FAM mutation or HEX wild-type probe) that exceeds the maximum background intensity value by 2,000 or 1,000. The asterisk shows the results of the KRAS Q61 mutation assay for patient 9 (see Table 2, right panel).

FIG. 7 is a graph that replaces the drawing showing the correlation between the amplifyable DNA indexed by the number of copies of KRAS and the amount of template DNA.

To establish the fact that the KRAS mutations identified using this method are of tumor origin, the genotypes of pathological specimens from FFPE blocks were classified by targeted amplicon sequencing. The presence of KRAS mutations in pancreatic cancer tissue was pathologically demonstrated in all 9 samples in which mutant KRAS was detected by the "underwater burst" method. In one KRAS G12D tumor patient, no mutations were detected in the FNA lavage fluid, but mutations could be identified by using "underwater burst" of residual tissue in purified and supernatant DNA and FNA needles. It was completed (Table 2, Patient 8). Another patient with no KRAS mutations observed by "underwater burst" dPCR assay was pathologically diagnosed with pancreatic acinic cell carcinoma without KRAS mutations (patient 7). In summary, this mutation detection method and rapid and easy sample preparation enhance the feasibility of identifying KRAS mutations in small tissues.

Discussion Gene profiling of solid tumors enables a more effective understanding of the molecular pathways involved in tumor development and progression, and clinically relevant information on early diagnosis, pharmacological vulnerability and resistance to various types of cancer. Also provide. Safer capture of cancer cell or tissue sampling can make it difficult for pathologists to make a correct diagnosis. Recently, for lung and colorectal cancer patients, it has become more common to use biopsy-based genetic testing to select chemotherapeutic reagents, which are generally tumor cell-rich tissues. Needs. However, low tumor cell content and small tumor specimens can make molecular analysis difficult on their own. In addition, DNA extraction / purification is routinely performed for genetic testing, but the process is time consuming, costly, and sometimes significantly dilutes the target molecule. Therefore, we used specimens from patients with pancreatic cancer characterized by ultra-low tumor cell content and excessive fibrosis. This is a biological sample that is difficult not only for conventional immunohistochemical staining analysis but also for molecular analysis.
In this study, we evaluated a DNA preparation method without a purification step for genetic testing of FNA samples obtained from the pancreas using the dPCR platform. I tried the following two different methods. In the "cell encapsulation" method, target cells were encapsulated directly into the droplet, followed by dPCR. On the other hand, the "underwater burst" technique attempted to capture tumor-derived DNA by exposing it to pure water shortly before its localization during dPCR, following an osmotic burst of cancer cells. The latter method was found to be superior to the "intradroplet cell" method. In the "underwater burst" method, even a small number of cells with a homozygous KRAS mutation at codon 12 can be detected in a small amount of 20 cells (= 40 copies) in 4,000 normal cells having wild-type KRAS, which is a crude tumor. It has been shown that detection of dPCR-based direct driver mutations in tissues is feasible. In this method, after the core specimen was sent to the pathological laboratory, tumor lesion cells were obtained in 11 of 12 fine needle aspirations obtained from surgically resected pancreatic tissue and in 10 from FNA needles. Detection in 9 of the residual tumor cells demonstrated that the KRAS mutation was detected in the puncture aspirator, and thus we found that the method was clinically appropriate. These results indicate that the combination of the "underwater burst" technique and the dPCR technique has the potential to detect mutations with high sensitivity in specimens with low low tumor cell content such as pancreatic tumors.

FNA is the gold standard for pathological diagnosis in patients with various types of cancer, including pancreatic cancer, due to its high diagnostic accuracy. Multiple punctures may be required to avoid failure during pathological assessment due to tumor heterogeneity. Occasionally, the number of cancer cell assessments on which the report is based is limited, and false negatives can occur if the tumor tissue used for sampling is inadequate. On the other hand, there is a dilemma with FNA that there is a potential risk of bleeding and tumor cell dissemination at the puncture site. Detection of gene mutations may complement the limitations of pathological evaluation, and the usefulness of these policies has been demonstrated.

Gene panel testing based on next-generation sequencing (NGS) has become an extremely valuable tool in cancer diagnosis. This modality provides a large amount of information related to genetic variation from a single sample, and its operability is becoming easier. Nevertheless, high quality DNA is needed from abundant tumor tissue that requires multiple FNA punctures. In addition, sample preparation, library quantification and sequencing analysis take a lot of time. The detection limit for mutations is> 1% unless a library preparation process is used for new techniques such as molecular barcoding, which makes it more costly and time consuming than assays. Furthermore, in order to use the data in clinical practice, careful bioinformatics evaluation is required, such as for the purpose of error elimination and reporting. On the other hand, the sample required for the dPCR assay can be a very small amount (DNA 1-5 ng), and the detection limit of mutation is as low as 0.05%. The running cost of dPCR is low and is useful as an excellent screening method for identifying high-risk patients.

The most striking feature of this study was that the labor, cost, and time required for DNA purification could be saved simply by suspending the stored material in water and destroying the cells. Molecular tests, including dPCR, are not widespread in clinical practice. This new method will potentially play an active and important role in routine examinations. Due to the ease of sample preparation, analysis using the dPCR method has the potential to be an alternative to cytopathological examination. The only parameter that guarantees sample quality in the underwater burst method is currently the number of amplified KRAS copies. A strong correlation was observed between the number of copies and the amount of template DNA when the purification steps were included in the same sample set. Additional parameters such as DNA fragment size can facilitate accurate quality determination of crude samples.

We used a commercially available validated screening probe set to detect multiple KRAS codon 12/13 mutations using a small amount of tissue sample. There are some restrictions on the use of this probe set. First, false positives were observed in this study (0.27% in FNA residual tissue). The mutation frequency threshold determined by the assay manufacturer was 0.2%. The crude DNA used in this method contained impurities or fragmented DNA due to the possibility that a non-specific signal of mutation was generated by a degrading enzyme secreted from cells. In the future, it will be necessary to determine the cut-off value specific to this method using more tumor specimens in the clinical setting.

The second problem is that the screening probe set utilized was able to detect multiple KRAS mutations at codons 12, 13 or 61. It does not provide accurate information related to mutation-specific variability. Pancreatic tumors often undergo various clonal evolutions, so it is extremely important to determine each mutation pattern and accurately identify the presence of primary lesions and coexisting malignant or benign lesions. In addition, mutations in KRAS alone cannot provide genetic evidence for pancreatic cancer. Therefore, it is necessary to improve the protocol so that other driver genes such as TP53 and SMAD4 and tumor suppressor genes are used as analysis controls. To solve this problem, we are currently developing a new multiplex analysis method to identify mutations in major KRAS and other genes using 2D spatial information on fluorescence intensity in dPCR. The dPCR system used can distinguish between the two fluorescent colors, while the new dPCR platform has the ability to detect multicolored dyes simultaneously. These new tools may further enhance the usefulness of the assay during multiplex analysis, potentially enabling the detection of driver mutations across multiple genomic regions.

The number of patients involved in this study was minimal. Nonetheless, to further validate the feasibility of clinical use, conduct clinical studies with more patient samples and use FNA to examine various types of pancreatic tissue, ranging from benign to malignant tumor tissue. Seems necessary. In addition to pancreatic cancer, it is also necessary to detect unique driver mutations in other types of cancer, such as the BRAF V600E mutation observed during thyroid cancer, to open up the development potential for a wide range of clinical applications. .. This method clinically uses a limited amount of tissue for sampling and may be suitable for pathological diagnosis of tumors with minimal invasiveness. Direct amplification and detection of driver mutations compensates for conventional pathological diagnosis using tissue biopsy and cytopathological analysis, as observed in the evaluation of options using immune histological staining. Assessment of microsatellite instability with additional dPCR may provide more detailed information in the diagnosis and treatment of cancer.

In conclusion, we have developed a digital PCR-based ultrasensitive assay for detecting mutations that may also solve the problem of material shortages during cytopathological analysis. The results showed a high detection rate of KRAS mutations even in a small amount of FNA residual sample. Furthermore, using the "underwater burst" method, we have shown that digital PCR does not even require a DNA purification step to detect gene mutations. This simple and rapid protocol makes it possible to perform molecular analyzes with minimal invasiveness in cancer clinics.

The present invention can be used in the genetic analysis industry and the medical device industry.

Claims (8)

A method for producing a sample for gene analysis, which comprises a sample suspension step of suspending a sample, which is a cell or tissue collected from a subject, in nuclease-free water. The method according to claim 1.
The method, wherein the sample for gene analysis is a sample for digital PCR or a sample for a DNA sequencer. The method according to claim 1.
The method, wherein the harvest contains a piece of cell or tissue remaining on the puncture needle. The method according to claim 1.
The method, wherein the collected material is obtained by suspending a cell piece or a tissue piece remaining in a puncture needle and then centrifuging. The method according to claim 1.
The harvest is a tumor-derived cell or tissue collected from the subject.
The above-mentioned gene analysis sample is a sample for analyzing a tumor-derived gene mutation, a method. A PCR method comprising a step of producing a sample for gene analysis using the method for producing a sample for gene analysis according to any one of claims 1 to 5. The PCR method according to claim 6, which is a droplet digital PCR method or an emulsion PCR method. 6. The step of performing the PCR method according to claim 6 or 7.
Method for analyzing tumor-derived gene mutations.

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