Methods for cancer gene panel testing

Abstract

This provides methods to address the risks of sample mix-ups in cancer gene panel testing and contamination with nucleic acids from other individuals during sample preparation. [Solution] A method for creating an analysis report for a cancer gene panel test, comprising a computer generating an analysis report that includes one or more determination results selected from the determination results of whether multiple nucleic acid samples in the cancer gene panel test originate from the same patient, and the determination results of whether tumor cells are present in the normal sample of the cancer gene panel test.

Description

Technical Field

[0001] The present disclosure relates to the field of cancer gene panel testing.

Background Art

[0002] In gene panel testing that analyzes a set of multiple genes, since nucleic acid samples from multiple patients are processed simultaneously during the preparation process of the nucleic acid library for analysis, there is a risk of misidentification. Also, there is a risk of contamination with nucleic acids from others during sample preparation, such as when preparing thin sections of formalin-fixed paraffin-embedded specimens, and as a result, there is a possibility of returning incorrect results. Furthermore, in gene panel testing for hematopoietic tumors, when obtaining oral mucosa as a normal tissue sample, it is conceivable that tumor cells may be mixed into the normal tissue sample due to bleeding or the like. If gene abnormalities in tumor cells are found in the analysis results of the normal tissue sample, it may lead to incorrect result interpretation.

Summary of the Invention

[0003] In one aspect, the present disclosure relates to a method for creating an analysis report for cancer gene panel testing, including a computer generating an analysis report including one or more determination results selected from a determination result as to whether multiple nucleic acid samples in cancer gene panel testing are derived from the same patient and a determination result as to contamination of tumor cells in a normal tissue sample in cancer gene panel testing.

[0004] According to the present disclosure, it becomes possible to provide important information in result interpretation in cancer gene panel testing.

Brief Description of the Drawings

[0005] [Figure 1] FIG. 1 is an explanatory diagram of paired analysis in the cancer gene panel testing of the present disclosure. [Figure 2] FIG. 2 is an explanatory diagram of a method for determining whether multiple nucleic acid samples in the present disclosure are derived from the same patient. [Figure 3]Figure 3 shows a method for detecting gene mutations characteristic of tumor tissue in this disclosure, and the effect of contamination of a normal tissue sample with tumor cells on the detection method. [Figure 4] Figure 4 shows an example of the determination results presented in the analysis report in this disclosure regarding whether multiple nucleic acid samples originate from the same patient (cases where tumor DNA and normal DNA are determined to originate from the same patient, and cases where tumor DNA and tumor RNA are determined to originate from the same patient). [Figure 5] Figure 5 shows an example of the determination result for the presence of tumor cells in a normal tissue sample (when no contamination is determined) as presented in the analysis report in this disclosure. [Figure 6] Figure 6 shows an example of the results of determining the presence of tumor cells in a normal tissue sample, as presented in the analysis report in this disclosure (cases where no contamination is determined and cases where contamination is determined). [Figure 7] Figure 7 shows an example of the process for the cancer gene panel test described herein. [Modes for carrying out the invention]

[0006] Cancer gene panel testing is a test that simultaneously analyzes multiple cancer-related genes. The test results can be used for purposes such as cancer diagnosis, treatment selection, and prognosis prediction. Gene panel testing typically involves preparing a DNA library from the patient's nucleic acid sample, sequencing the prepared DNA library to obtain base sequence information, and analyzing the obtained base sequence information to detect genetic abnormalities.

[0007] In one embodiment, the cancer gene panel test is a hematopoietic malignancy gene panel test. The hematopoietic malignancy gene panel test targets hematopoietic malignancy-related genes and aims to detect hematopoietic malignancy gene abnormalities. The hematopoietic malignancy-related genes are based, for example, on the Hematopoietic Malignancy Genome Testing Guidelines of the Japanese Society of Hematology (http: / / www.jshem.or.jp / genomgl / ). The hematopoietic malignancy gene panel test may also include genes not included in the Hematopoietic Malignancy Genome Testing Guidelines as targets for testing.

[0008] The patient may have a hematopoietic malignancy. Examples of diseases for which hematopoietic malignancy gene panel testing is useful include: AML: acute myeloid leukemia MDS: myelodysplastic syndrome MPN: myeloproliferative neoplasms MDS / MPN: myelodysplastic syndrome / myeloproliferative neoplasms SM: systemic mastocytosis MLN-e:myeloid / lymphoid neoplasms with eosinophilia CML: chronic myeloid leukemia IBMFS:inherited bone marrow failure syndromes AA: aplastic anemia BPDCN:blastic plasmacytoid dendritic cell neoplasm ALL: acute lymphoblastic leukemia Aggressive B-NHL: Non-Hodgkin Lymphoma Indolent B-NHL T / NK-NHL HL: Hodgkin lymphoma CLL: chronic lymphocytic leukemia MM: multiple myeloma HDCN: histiocytic and dendritic cell neoplasms

[0009] Nucleic acid samples can be prepared from fresh samples (e.g., peripheral blood, bone marrow fluid, tissue, body cavity fluid) or formalin-fixed paraffin-embedded (FFPE) samples (e.g., sections or blocks). Normal tissue samples can be prepared from oral mucosa or nails. Nucleic acid samples include tumor DNA, normal tissue DNA, and tumor RNA. Tumor DNA and tumor RNA are used to detect acquired genetic abnormalities that occur specifically in cancer, while normal tissue DNA is used to subtract genetic abnormalities that an individual originally possesses from tumor analysis (paired analysis, Figure 1).

[0010] In this disclosure, gene mutations include single nucleotide variants (SNVs), short insertions or deletions (also known as indels), polynucleotide variants, insertions or deletions at the gene structure level, and structural abnormalities. Structural abnormalities include translocations, inversions, tandem duplications, deletions, and fusion genes. In this disclosure, gene mutation means a change from the human standard sequence (referred to as the reference sequence), and gene abnormality means a gene mutation characteristic of the tumor.

[0011] Methods for detecting gene mutations include, for example, chromosome analysis, FISH (fluorescent in situ hybridization), MLPA (Multiplex Ligation-dependent Probe Amplification), Sanger sequencing, ASO-PCR (allele-specific oligonucleotide PCR), RT-qPCR (reverse transcription quantitative PCR), whole genome sequencing (WGS), whole exome sequencing (WES), targeted sequencing, RNA sequencing, CGH (comparative genomic hybridization), array CGH, and SNP arrays. In one embodiment, gene mutations are detected by targeted sequencing. Gene mutations can also be detected using next-generation sequencers, though not limited to these methods.

[0012] Cancer gene panel testing may include examining whether gene mutations detected in normal DNA are detected in tumor DNA, and / or whether gene mutations detected in tumor DNA are detected in tumor RNA, in order to determine whether multiple nucleic acid samples originate from the same patient (Figure 2). If gene mutations detected in normal DNA are also detected in tumor DNA, the tumor DNA and normal DNA are determined to originate from the same patient. If gene mutations detected in tumor DNA are also detected in tumor RNA, the tumor DNA and tumor RNA are determined to originate from the same patient.

[0013] If samples are mixed up or contaminated, results based on the nucleic acids of another person will be returned. By determining the identity of the samples in gene panel testing and including that determination in the analysis report, high-quality information can be provided.

[0014] The cancer gene panel test may include examining whether gene mutations that suggest an association with cancer are detected in normal tissue DNA in order to determine the contamination of normal tissue samples with tumor cells. In the present disclosure, gene mutations that suggest an association with cancer may include gene mutations with a low frequency in the population frequency database and registered a predetermined number of times or more in the database of gene mutations related to cancer. Gene mutations that suggest an association with cancer may also include structural abnormalities selected from FLT3 tandem duplication and CALR deletion. Examples of the population frequency database include gnomAD, 1000 Genomes, ToMMo, and HGVD. An example of the database of gene mutations related to cancer is COSMIC (a database of somatic mutations related to cancer).

[0015] Gene mutations characteristic of the tumor region are gene mutations detected in tumor region DNA and not detected in normal region DNA (upper part of FIG. 3). If tumor cells are mixed into the normal region, it may be impossible to detect gene mutations characteristic of the tumor region (lower part of FIG. 3). By examining whether there are gene mutations that suggest an association with cancer among the gene mutations detected in tumor region DNA and also detected in normal region DNA (i.e., gene mutations not detected in the analysis using the normal region as a control but detected only in the analysis of the tumor region), it is possible to examine whether gene mutations that suggest an association with cancer are detected in normal region DNA. When gene abnormalities that suggest an association with cancer are not detected in the analysis using the normal region as a control but are detected only in the analysis of the tumor region, it is determined that contamination of the normal tissue sample with tumor cells is suspected. By determining the contamination of the normal tissue sample with tumor cells and including the determination result in the analysis result report, it is possible to avoid overlooking important gene abnormalities and provide high-quality test results.

[0016] The analysis report of the cancer gene panel test may include one or more determination results selected from the determination result of whether multiple nucleic acid specimens in the cancer gene panel test are derived from the same patient and the determination result of the contamination of tumor cells in the normal specimen of the cancer gene panel test. In one embodiment, the determination result of whether multiple nucleic acid specimens are derived from the same patient is the determination result of whether tumor DNA and normal DNA are derived from the same patient and / or the determination result of whether tumor DNA and tumor RNA are derived from the same patient. Examples of presenting the determination result of whether multiple nucleic acid specimens in the analysis report are derived from the same patient and the determination result of the contamination of tumor cells in the normal specimen are shown in FIGS. 4 to 6, but the method of presenting the determination result is not limited thereto.

[0017] In one embodiment, the cancer gene panel test (1) preparing a DNA library from the tumor DNA, normal DNA, and tumor RNA of the patient, respectively, (2) performing nucleotide sequencing of each prepared DNA library to obtain nucleotide sequence information, and (3) analyzing the obtained nucleotide sequence information for the detection of gene abnormalities, Here, the analysis of the nucleotide sequence information includes determining whether the tumor DNA and the normal DNA are derived from the same patient, determining whether the tumor DNA and the tumor RNA are derived from the same patient, and determining the contamination of tumor cells in the normal specimen, including one or more selected from and, (4) creating an analysis report including the gene abnormalities detected in (3) and one or more determination results obtained in (3), is included.

[0018] If the cancer gene panel test is a hematopoietic malignancy gene panel test, an interim report may be prepared for gene abnormalities for which rapid result return is desirable, and then a final report may be prepared for all gene abnormalities tested. "Genetic abnormalities for which rapid result return is desirable" are defined in the Hematopoietic Malignancy Genome Testing Guidelines of the Japanese Society of Hematology (http: / / www.jshem.or.jp / genomgl / ) and are called "Fast-track target gene abnormalities." Because it takes time to prepare a DNA library from tumor RNA for analysis of tumor RNA, in order to rapidly return Fast-track target gene abnormalities to medical institutions, analysis is performed as soon as the base sequence information of tumor DNA and normal DNA (for example, a FASTQ file, which is base sequence information output from a next-generation sequencer) is obtained, and the Fast-track target gene abnormalities are reported to the medical institution as an interim report. Subsequently, after obtaining the base sequence information of tumor RNA, analysis of tumor DNA, normal DNA, and tumor RNA is performed, and a final report is prepared. This enables comprehensive gene profiling based on hematopoietic malignancy genome testing guidelines, as well as rapid return of interim reports to medical institutions regarding gene abnormalities that fall under the Fast-track target gene abnormalities category.

[0019] In one embodiment, the cancer gene panel test is a hematopoietic malignancy gene panel test. (1) Prepare DNA libraries from the patient's tumor DNA, normal DNA, and tumor RNA, respectively. (2) Perform base sequence determination on each prepared DNA library to obtain base sequence information, and (3) Analyze the obtained base sequence information in order to detect gene abnormalities. Here, the analysis of the nucleotide sequence information is performed (3)-1. In order to detect genetic abnormalities for which rapid result return is desirable, analyze the base sequence information of DNA libraries derived from tumor DNA and DNA libraries derived from normal DNA, and (3)-2. After (3)-1 above, in order to detect genetic abnormalities in all subjects of testing, the base sequence information of the DNA library derived from tumor DNA, the DNA library derived from normal DNA, and the DNA library derived from tumor RNA shall be analyzed. including, and (4) Prepare an analysis report including the gene abnormalities detected in (3) above. Here, the preparation of the aforementioned analysis report is (4)-1. Prepare an interim analysis report including the gene abnormalities detected in (3)-1 above, and (4)-2. After (4)-1 above, prepare a final analysis report including the gene abnormalities detected in (3)-2 above. Includes. The analysis described in (3)-2 above is usually performed after the preparation of the interim analysis report described in (4)-1 above, but it may also be performed before preparation.

[0020] In one embodiment, the cancer gene panel test is a hematopoietic malignancy gene panel test. (1) Prepare DNA libraries from the patient's tumor DNA, normal DNA, and tumor RNA, respectively. (2) Perform base sequence determination on each prepared DNA library to obtain base sequence information, and (3) Analyze the obtained base sequence information in order to detect gene abnormalities. Here, the analysis of the nucleotide sequence information is performed (3)-1. In order to detect genetic abnormalities for which rapid result return is desirable, analyze the base sequence information of DNA libraries derived from tumor DNA and DNA libraries derived from normal DNA, and (3)-2. After (3)-1 above, in order to detect genetic abnormalities in all subjects of testing, the base sequence information of the DNA library derived from tumor DNA, the DNA library derived from normal DNA, and the DNA library derived from tumor RNA shall be analyzed. Includes, The analysis of the base sequence information in (3)-2 above is performed as follows: To determine whether tumor DNA and normal DNA originate from the same patient. To determine whether tumor DNA and tumor RNA originate from the same patient, and To determine the presence of tumor cells in a normal tissue sample. Includes one or more selected from, and (4) Prepare an analysis report including the gene abnormalities detected in (3) above. Here, the preparation of the aforementioned analysis report is (4)-1. Prepare an interim analysis report including the gene abnormalities detected in (3)-1 above, and (4)-2. After (4)-1 above, prepare a final analysis report that includes the gene abnormalities detected in (3)-2 above and one or more judgment results obtained in (3)-2 above. Includes. The analysis described in (3)-2 above is usually performed after the preparation of the interim analysis report described in (4)-1 above, but it may also be performed before preparation.

[0021] The sequencing of a cancer gene panel test, the analysis of the sequencing information, and the creation of the analysis report may be performed by computer. This disclosure includes a method for creating an analysis report for a cancer gene panel test, which includes a computer generating one or more determination results selected from the determination results of whether multiple nucleic acid samples in the cancer gene panel test originate from the same patient and the determination results of whether tumor cells are present in a normal sample, and generating an analysis report including the obtained one or more determination results.

[0022] This disclosure is a computer that (i) To perform nucleotide sequencing on each DNA library prepared from the patient's tumor DNA, normal DNA, and tumor RNA, and to obtain nucleotide sequence information, and (ii) Analyze the obtained base sequence information in order to detect gene abnormalities. Here, the analysis of the nucleotide sequence information is performed To determine whether tumor DNA and normal DNA originate from the same patient. To determine whether tumor DNA and tumor RNA originate from the same patient, and To determine the presence of tumor cells in a normal tissue sample. Includes one or more selected from, And, (iii) Generate an analysis report that includes the gene abnormalities detected in (ii) and one or more judgment results obtained in (ii). This includes methods for preparing analysis reports for cancer gene panel tests.

[0023] This disclosure or a computer (i) To perform nucleotide sequencing on each DNA library prepared from the patient's tumor DNA, normal DNA, and tumor RNA, and to obtain nucleotide sequence information, and (ii) Analyze the obtained base sequence information in order to detect gene abnormalities. Here, the analysis of the nucleotide sequence information is performed (ii)-1. In order to detect genetic abnormalities for which rapid result return is desirable, analyze the base sequence information of DNA libraries derived from tumor DNA and DNA libraries derived from normal DNA, and (ii)-2. After (ii)-1 above, in order to detect genetic abnormalities in all subjects of testing, the base sequence information of the DNA library derived from tumor DNA, the DNA library derived from normal DNA, and the DNA library derived from tumor RNA shall be analyzed. including, (iii) To generate an analysis report that includes the gene abnormalities detected in (ii) above, Here, the preparation of the aforementioned analysis report is (iii)-1. To generate an interim analysis report that includes the gene abnormalities detected in (ii)-1 above, and (iii)-2. After (iii)-1 above, generate a final analysis report including the gene abnormalities detected in (ii)-2 above. This includes methods for preparing analysis reports for cancer gene panel tests. The analysis described in (ii)-2 is usually performed after the generation of the interim analysis report described in (iii)-1, but it may also be performed before generation.

[0024] This disclosure or a computer (i) To perform nucleotide sequencing on each DNA library prepared from the patient's tumor DNA, normal DNA, and tumor RNA, and to obtain nucleotide sequence information, and (ii) Analyze the obtained base sequence information in order to detect gene abnormalities. Here, the analysis of the nucleotide sequence information is performed (ii)-1. In order to detect genetic abnormalities for which rapid result return is desirable, analyze the base sequence information of DNA libraries derived from tumor DNA and DNA libraries derived from normal DNA, and (ii)-2. After (ii)-1 above, analyze the base sequence information of the DNA library derived from tumor DNA, the DNA library derived from normal DNA, and the DNA library derived from tumor RNA in order to detect genetic abnormalities in all subjects of testing. Includes, The analysis of the base sequence information in (ii)-2 above is performed as follows: To determine whether tumor DNA and normal DNA originate from the same patient. To determine whether tumor DNA and tumor RNA originate from the same patient, and To determine the presence of tumor cells in a normal tissue sample. Includes one or more selected from, and (iii) To generate an analysis report that includes the gene abnormalities detected in (ii) above, Here, the preparation of the aforementioned analysis report is (iii)-1. To generate an interim analysis report that includes the gene abnormalities detected in (ii)-1 above, and (iii)-2. After (iii)-1, generate a final analysis report that includes the gene abnormalities detected in (ii)-2 and one or more judgment results obtained in (ii)-2. This includes methods for preparing analysis reports for cancer gene panel tests. The analysis described in (ii)-2 is usually performed after the generation of the interim analysis report described in (iii)-1, but it may also be performed before generation. [Examples]

[0025] 1. Identity of the specimen 1-1. Tumor DNA and Normal DNA We prepared two sets of DNA libraries: one derived from tumor DNA and the other from normal DNA, both from the same subject. We also prepared two sets of DNA libraries: one derived from tumor DNA and the other from normal DNA, both from different subjects. For each set, we sequenced the libraries using a next-generation sequencer (NextSeq 550Dx), and then used an analysis program to check whether gene mutations detected in normal DNA were also detected in tumor DNA. In the sets derived from the same subject, gene mutations detected in normal DNA were also detected in tumor DNA, confirming that this allows for the determination of sample identity.

[0026] 1-2. Tumor DNA and Tumor RNA We prepared sets of DNA libraries derived from tumor DNA and tumor RNA from the same subject, and sets of DNA libraries derived from tumor DNA and tumor RNA from different subjects. For each set, we sequenced the libraries using a next-generation sequencer (NextSeq 550Dx), and then used an analysis program to check whether gene mutations detected in tumor DNA were also detected in tumor RNA. In the sets derived from the same subject, it was found that gene mutations detected in tumor DNA were also detected in tumor RNA, confirming that this allows for the determination of sample identity.

[0027] 2. Contamination of normal tissue specimens with tumor cells To reproduce the contamination conditions, a FASTQ file was created containing a mixture of DNA from normal tissue and DNA from tumor tissue. This FASTQ file was used as the normal tissue sample where contamination occurred, and tumor tissue analysis was performed (paired pair analysis). Separately, analysis was performed on tumor tissue only, without specifying a normal tissue sample. By comparing the results of the paired pair analysis with the results of the tumor tissue-only analysis, it was confirmed that the presence of any of the following gene mutations in the tumor tissue-only analysis could be used to determine the contamination of the normal tissue sample with tumor cells. (1) SNPs that have a low frequency in the population frequency database (gnomAD / 1000 Genomes / ToMMo / HGVD) and have been registered more than the prescribed number of times in COSMIC (database of somatic mutations related to cancer) (all cancers or hematopoietic malignancies) (2) FLT3 tandem overlap and CALR deletion

Claims

1. A method for preparing an analysis report for a cancer gene panel test, comprising the computer generating an analysis report that includes one or more determination results selected from the determination results of whether multiple nucleic acid samples in the cancer gene panel test originate from the same patient, and the determination results of whether tumor cells are present in the normal sample of the cancer gene panel test.

2. The method according to claim 1, wherein the determination result of whether multiple nucleic acid samples originate from the same patient is the determination result of whether tumor DNA and normal DNA originate from the same patient and / or whether tumor DNA and tumor RNA originate from the same patient.

3. The method according to claim 1 or 2, wherein the cancer is a hematopoietic tumor.

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