Patent Application
20240043929
The present invention relates to a method for detecting the copy number of a specific nucleic acid per single cell.
Non Patent Literature (hereinafter, referred to as NPL) 1 discloses encapsulating genomic DNA extracted from cells in droplets and amplifying the genomic DNA by Polymerase Chain Reaction (PCR), although NPL 1 is not directed to a single-cell analysis. In NPL 1, a target sample and a reference sample are independently quantified in the same droplet. This analysis is achieved by distinguishing between the fluorescence signals of intercalators to distinguish between the lengths of amplicons.
NPL 2 discloses single-cell RT-PCR (reverse transcription PCR) targeting mRNA of a specific gene. In NPL 3, the presence of HIV-1 in CD4+ T cells is detected with high throughput by a reverse transcription reaction and a single-cell-in-droplet (scd) PCR assay.
NPL 4 discloses a single cell-based droplet digital PCR (sc-ddPCR) method. In this method, single cells are encapsulated in droplets and PCR is performed within the droplets using gene-specific primers and probes. One copy of the gene is artificially introduced into a cell.
NPL 5 discloses lysing a single cell within a droplet and combining the droplet with the single cell lysed therein with the droplet of the RT-PCR reaction solution. Patent Literature (hereinafter, referred to as PTL) 1 discloses analyzing, although not single cell analysis, the characteristics of DNA obtained from enriched fetal cells from maternal blood to determine its genetic status. PTL 1 discloses amplifying the DNA of the enriched fetal cells and analyzing the amplified DNA by using digital PCR. In addition, PTL 1 discloses that chromosome 21 is detected.
PTL 2 discloses that blood cells potentially containing fetal cells are each isolated at the single cell level, and chromosomal DNA is independently extracted from the isolated blood cells, and fractions containing the fetal-derived chromosomal DNA are identified in an after-the-fact manner.
NPL 6 discloses detecting an SRY gene on genomic DNA of fetal cells circulating in maternal blood by the above-described sc-ddPCR method.
NPL 7 discloses quantitative PCR using 1, 2, 4 . . . copies of a yeast genome weighed by using the piezoelectric effect as a template.
NPLs 8 and 9 will be described below.
PTL 1
Japanese Unexamined Patent Application Publication (Translation of PCT Application) No. 2014-533509
PTL 2
Japanese Patent Application Laid-Open No. 2018-102242
NPL 1
Laura Miotke, et al., “High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR”, Analytical chemistry, 86, 5, 2618-2624.
NPL 2
Dennis J. Eastburn, et al., “Identification and genetic analysis of cancer cells with PCR-activated cell sorting”, Nucleic Acids Research, 42, 16, e128.
NPL 3
Robert W. Yucha, et al., “High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay”, EBioMedicine, 20, 217-229.
NPL 4
Yuka Igarashi, et al., “Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy”, Molecular Therapy, Methods & Clinical Development, 6, 8-16.
NPL 5
Samuel C. Kim, et al., “Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis”, Analytical Chemistry, 90, 2, 1273-1279.
NPL 6
Taisuke Sato, et al., “Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis”, The Journal of Molecular Diagnostics, 22, 2, 2, 132-140.
NPL 7
Unoh Ki, et al., “A Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA”, Ricoh Technical Report, 2020, 44, 14-26. Retrieved from <https://jp.ricoh.com/technology/techreport/44/>
NPL 8
Ichiro Hanamura, et al., “Frequent gain of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation”, Blood, 108, 5, 1724-1732.
NPL 9
ISHIDA Tadao, “Tahatusei-kotujuishu: Senshokutai ijou to Rinshou byougata⋅Yogo (Multiple myeloma: chromosomal abnormalities, clinical forms, and prognosis),” [online], 2013-09-21, Clinical Hematology, 54, 10, 2, 1856-1866, Retrieved from <https://doi.org/10.11406/rinketsu.54.1856>
Digital PCR qualitatively indicates the presence or absence of a template within a minute compartment (for example, droplet). Therefore, digital PCR is not suitable for detecting a discrete copy number variation (CNV) for a specific nucleic acid within a minute compartment.
An object of the present invention is to provide a method capable of detecting the copy number of a specific nucleic acid per single cell in a population of cells (herein also referred to as “cell population”).
The present invention relates to a method for detecting the copy number of a specific nucleic acid per single cell as described below.
The present invention can detect the copy number of a specific nucleic acid per single cell in a cell population. For example, the present invention can detect a copy number variation (CNV) of a specific nucleic acid per single cell.
and
Hereinafter, in describing the CNV, the term “monoploid (monoploidy)” includes haploidization (i.e., monosomy) of all of a specific chromosome, as well as partially monoploid states due to deletions. The CNV includes all or part of a diploid chromosome becoming monoploid.
The term “triploid (triploidy)” includes triploidization (i.e., trisomy) of all of a specific chromosome, as well as partially triploid states due to duplication. The CNV includes all or part of a diploid chromosome becoming triploid. The CNV includes all or part of a diploid chromosome becoming to have the ploidy greater than that of triploid.
The CNV also includes ploidy changes due to duplication of all or part of a sex chromosome in a male, which is normally monoploid. In the following, monoploid, diploid, triploid and other polyploid are referred to as “polyploidy” in some cases. The CNV also includes changes resulting in the complete loss of all or part of a polyploid chromosome (biallelic deletion). Further, the CNV also includes changes that result in all or part of a chromosome that is not normally present.
In the present invention, detection of cells with aneuploid chromosomes includes the following: detecting that a cell population containing cells with euploid chromosomes also contains cells with aneuploid chromosomes. In the present invention, detection of cells with aneuploid chromosomes includes the following: detecting that a cell population containing cells with euploid chromosomes is a cell population that did not contain cells with aneuploid chromosomes, or contains aneuploid cells below the limit of detection. In the present invention, detection of cells with aneuploid chromosomes includes the following: detecting that a cell population contains cells with aneuploid chromosomes but does not contain cells with euploid chromosomes.
Overall flow
As illustrated in
As illustrated in
The DNA sample is, for example, a set of genomic DNA in a single cell, a reverse transcription product of RNA in a single cell, or DNA of a mitochondrial genome in a single cell (hereinafter referred to as “mitochondrial DNA”). In
In one aspect, the DNA sample is a set of genomic DNA in a single cell. A “set of genomic DNA” may be interpreted as genomic DNA as it is extracted. In one aspect, no whole genome amplification is performed on the set of genomic DNA. Allele dropout to be caused by the whole genome amplification can be thus avoided. The “set of genomic DNA” includes those unintentionally lost in part during cell lysis or other extraction procedures. In one aspect, the “set of genomic DNA” does not include mitochondrial DNA. However, for using a set of genomic DNA as the DNA sample, the reaction compartment may be contaminated with mitochondrial DNA.
In one aspect, the DNA sample is the reverse transcription product of RNA within a single cell.
In one aspect, the DNA sample is mitochondrial DNA in a single cell.
In one aspect, the DNA sample is a set of genomic DNA in a single cell and mitochondrial DNA in the same single cell. In this case, mitochondrial DNA may be used as a control for detecting the copy number of chromosomal genomic DNA.
In another aspect, the DNA sample is the nucleic acid of a microorganism or virus that has invaded a cell, or a nucleic acid that has been introduced into a cell.
As illustrated in
In
Flow of Detection
In step 25 after the start, a PCR premix is prepared. The premix contains a primer, a DNA polymerase, a deoxynucleoside triphosphate (dNTP), a buffer and necessary cofactors. In one aspect, the cofactors include magnesium ions. In one aspect, a cell lysis reagent is mixed with the premix in advance. Examples of the cell lysis reagent include sodium dodecyl sulfate, TRITON X-100 (TRITON is a registered trademark), and NP-40 (trademark). The premix includes necessary probes.
In step 26 illustrated in
In step 27 illustrated in
QX200 Droplet Generator (Bio-Rad Laboratories, Inc.), for example, can be used as a system for forming a single cell together with the premix into a droplet. The system for forming droplets is not limited thereto.
In one aspect illustrated in
In another aspect of step 27 illustrated in
In another aspect of step 27 illustrated in
In step 28 illustrated in
In step 29 illustrated in
In one aspect of step 25 illustrated in
In one aspect, the number of targets of the probes contained within the amplicon is one. In another aspect, the number of targets of the probes contained within the amplicon is two or more. As the plurality of probes, which bind to one amplicon, respectively include fluorescent materials that emit fluorescence of the same color, the fluorescence emitted from the amplicon can be enhanced.
Two-Color Probe Method
In this example, the target to which the probe of the first fluorescence wavelength is assigned, or a region containing the target, is used as a subject for copy number (CNV) detection. In this example, the target to which the probe of the second fluorescence wavelength is assigned, or a region containing the target, is used as a control. In one aspect, the control is a region with a relatively small CNV.
That is, as illustrated in
In one aspect illustrated in
Under ideal reaction conditions for the aspect illustrated in
Regarding cluster 40a illustrated in
Regarding cluster 40c illustrated in
In one aspect illustrated in
In another aspect illustrated in
The magnitude of the shifts on the scatter plot may be estimated by preliminary experiments. The ploidy of cells in each cluster on the scatter plot may be estimated by preliminary experiments.
The description returns to
In the scatter plot illustrated in
The description returns to
That is, in the example illustrated in
The type of fluorescent material (fluorophore dye) and quencher added to the probe is not limited. For example, the fluorescent material with the first fluorescence wavelength is FAM (trademark, Fluorescein amidite) and the fluorescent material with the second fluorescence wavelength is HEX (trademark, Hexachloro-fluorescein). Increased targets and enhanced signal
Regarding cluster 41a illustrated in
Regarding cluster 41b illustrated in
Regarding cluster 41c illustrated in
In one aspect illustrated in
In another aspect illustrated in
The magnitude of the shifts on the scatter plot may be estimated by preliminary experiments.
Clusters 44a and 44b illustrated in
The description returns to
With reference to
In the present embodiment, each region contains a plurality of targets, and a plurality of probes including fluorescent materials with identical fluorescence wavelengths are assigned to the plurality of targets, respectively. In addition, for quantifying amplicons for each region, the amplicons are quantified by measuring fluorescence from the plurality of probes collectively by color, regardless of the difference between the targets. This configuration enhances the fluorescence from the target assigned regions. Therefore, it is easier to separate the signal of a target from the background noise.
In one aspect, the number of targets in one region is 1 to 1,000. In one aspect, the number of targets in one region is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. The number of targets may be the same or different between regions. In each region, as many types of probes as there are targets are used. The same fluorescence wavelength is assigned to these probes.
In one aspect, a single combination of primer pair and probe specifies only one target on the genomic DNA. Using n types of probes in each region can detect n targets in this region.
Assignment of Three or More Colors to Three or More Regions
In the above embodiment, two fluorescence wavelengths are used. Further, the number of regions may be increased, and another fluorescence wavelengths may be assigned to an additional regions. In one aspect, a third fluorescence wavelength may be assigned to a chromosome other than chromosomes 18 and 21.
In one aspect, the number of sets of region and fluorescence wavelength is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In another aspect, the number of sets of region and fluorescence wavelength is one.
The type of fluorescent material (fluorophore dye) and quencher added to the probe is not limited. Examples of the fluorescent material include FAM, HEX, VIC, TAMRA, ROX, Cy5 and Cy5.5 (all trademarks).
The number of PCR cycles was 40. The PCR reaction reached a plateau through an exponential amplification phase. The reaction was saturated.
The upper part of
The middle part of
The lower part of
The upper part of
The middle part of
The lower part of
In each scatter of
The upper part of
The middle part of
The lower part of
In each scatter of
The upper part of
The lower part of
The upper part of
The lower part of
A PCR reaction solution was prepared by adding 2 μL of a cell suspension containing 10,000 cells (line GM22948 or line GM13721) and PBS to the PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, and the like in the PCR reaction solution are as follows.
The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.
In the present test example, 1, 5, 10, or 23 types of probes each including a fluorescent material (FAM) with the first fluorescence wavelength were assigned to chromosome 13. One of the above types of probes including the fluorescent material with the first fluorescence wavelength was assigned to each of 1, 5, 10, or 23 targets on chromosome 13. When the plurality of types of probes are used, the concentrations of the probes are the same, and the concentrations of the above probes (1, 5 or 10 types: 500 nM and 23 types: 575 nM) are the total concentrations of the plurality of types of probes. In addition, 1, 5, 10, or 25 types of probes each including a fluorescent material (HEX) with the second fluorescence wavelength were assigned to chromosome 18. One of the above types of probes including the fluorescent material with the second fluorescence wavelength was assigned to each of 1, 5, 10, or 25 targets on chromosome 18. When the plurality of types of probes are used, the concentrations of the probes are the same, and the concentrations of the above probes (1, 5 or 10 types: 1,000 nM and 25 types: 1,250 nM) are the total concentrations of the plurality of types of probes. The probes were mixed together in one droplet.
The upper parts of
The lower parts of
In each scatter of
As clearly seen from the difference in the positions of the solid and dashed arrows in these scatters, increasing the number of targets within one region (the targets correspond to probes of the same fluorescence wavelength) can determine copy number differences more easily.
Subsequently, for each scatter, the ratio (FAM/HEX) of fluorescence intensity at the first fluorescence wavelength with respect to fluorescence intensity at the second fluorescence wavelength was calculated for droplets each containing one cell, and summarized in a boxplot.
In all the graphs of
Internal Standard
In one aspect, ploidy measurements are made with an internal standard. In one aspect, cells that are euploid in the region to be detected are the internal standard for the CNV. For example, in Test Example 4 and the second half of Test Example 5, the copy number of the region to be detected in a heterogeneous cell population was measured. With respect to euploid clusters, the presence of clusters with CNVs was used to determine whether the clusters are triploidy or monoploidy.
In a heterogeneous cell population, the proportion of cells that are euploid in the target assigned region is greater than 0% and less than 100%. The ratio of euploid cells in the heterogeneous cell population is, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, 99.95, 99.96, 99.97, 99.98, or 99.99%.
In a heterogeneous cell population, the proportion of cells with CNVs of interest in the target assigned region is greater than 0% and less than 100%. The ratio of cells with CNVs in the heterogeneous cell population is, for example, 99.99, 99.98, 99.97, 99.96, 99.95, 99.9, 99.8, 99.7, 99.6, 99.5, 99, 98, 97, 96, 95, 90, 85, 80, 75, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.04, 0.03, 0.02, or 0.01%.
External Standard
In Test Examples 1 to 3 and the first half of Test Example 5, the copy number of the region to be detected in a homozygous cell population for polyploidy was measured. The CNVs of these cells were previously known. The following means can provide a copy number standard for the region to be detected in cells whose CNV is not identified in advance.
That is, in one aspect, ploidy measurements are made with an external standard. For example, in another emulsion system that is not mixed with the sample, PCR is performed simultaneously or before or after the sample measurement by using genomic DNA of cells whose ploidy is known in advance as a template, and the fluorescence intensity of the amplicon is measured. The cluster coordinates obtained from the fluorescence intensity are compared with the sample cluster coordinates.
Examples of such test examples with external standards are illustrated in the upper part (Diploidy) and the middle part (Triploidy) of
The following is an example of measuring the polyploidy of any one of the chromosomes of a cancer patient's tumor cells. As the treatment progresses smoothly, the originally present chromosomal mutations may disappear in all the cells to be measured. In this case, the region on the chromosome to be measured becomes euploid. Therefore, according to the above measurement method, an aneuploid cluster on the scatter disappears. In other words, only an euploid cluster remains among the various clusters.
On the other hand, when the disease of cancer patients significantly progresses, it is assumed that all the cells to be measured have chromosomal mutations. Alternatively, it is assumed that there will be significantly more aneuploid cells than euploid cells, and euploid cells may become undetectable. According to the above measurement method, the euploid cluster on the scatter disappear. In other words, only an aneuploid cluster will be detected among the various clusters.
Such an aneuploid cluster becomes a homogeneous population of cells with a specific ploidy in the region to be measured. Alternatively, the cluster becomes a heterogeneous population of cells with various ploidy.
In one aspect, genome polyploidy of tumor cells from cancer patients is measured with an external standard of ploidy. Using an external standard makes possible to measure cell ploidy without relying on euploid cells (healthy, non-cancerous cells) that should have been contained in a cell population as an internal standard. More accurate information on these ploidy is useful in determining the need for cancer treatment or in the diagnosis of complete remission.
The following is an example as a part of prenatal diagnosis in which fetal cells in amniotic fluid are to be measured. When fetal cells in the amniotic fluid are free of maternal cell contamination, the normal population of such cells would contain only fetal cells. Such a cell population is homogenous with respect to genome polyploidy.
In one aspect, genome ploidy of fetal cells is measured with an external ploidy standard. Using an external standard makes possible to measure cell ploidy without relying on euploid cells (in this case, cells of maternal origin) that should have been contained in a cell population as an internal standard.
When it can be confirmed in advance that fetal cells have been obtained from the amniotic fluid with 100% purity, using the above measurement method makes possible to examine whether the fetal cell population is heterogeneous or homogeneous with respect to genome ploidy. That is, the presence or absence of ploidy mosaicism in chromosomes of fetal cells can be checked.
In the present embodiment, there is a very clear linear relationship between the copy number of the genomic region to be measured and the center of fluorescence intensity of the cluster. Therefore, the obtainment of the fluorescence intensity from the external standard cells is not necessary each time the sample is measured. In other words, the ploidy of the region of the genome of the sample subjected to the measurement can be determined by comparing the fluorescence intensity information of the external standard obtained in advance with the fluorescence intensity information of the sample.
A PCR reaction solution was prepared by adding 10,000 cells (line GM22948) and 2 μL of a cell suspension containing PBS to the PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, and the like in the PCR reaction solution are as follows.
The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. Using the 23 separately prepared PCR reaction solutions, PCR was performed separately.
The lower part of
Therefore, in the present test example, the following medians of the negative cluster (the cluster located at the lower left in the scatter) were calculated for each of the 23 measurement results: the median of fluorescence intensity of the first fluorescence wavelength (FAM) (hereinafter also referred to as “FAM measurement value”) and the median of fluorescence intensity of the second fluorescence wavelength (HEX) (hereinafter also referred to as “HEX measurement value”). The average value of the 23 FAM measurement values was used as the FAM reference value for the negative cluster, and the average value of the 23 HEX measurement values was used as the HEX reference value for the negative cluster. Subsequently for each of the 23 measurement results, the calculation results “(FAM reference value+α)/(FAM measurement value+α)” and “(HEX reference value+α)/(HEX measurement value+α)” were used as correction values for correcting cluster shifts (variation in measurement results). Herein, α is a numeric value arbitrarily set in order to bring the ratio of “FAM reference value/FAM measurement value” and “HEX reference value/HEX measurement value” close to the true fluorescence intensity ratio in consideration of the fluorescence intensity detection limit of the measuring device. Herein, α is set to 2,000. All the measured values were multiplied by the obtained FAM correction value and HEX correction value to correct variations in the measurement results.
Table 1 shows the results of determining the measurement results using the obtained cutoff value. The sensitivity was 84.1% and the specificity was 99.3% when detecting the line GM13721 (cells with a partial region of one long arm of chromosome 13 being monoploid) from the cell population containing the line GM22948 (cells with diploid chromosome 13) and the line GM13721. As described above, the cutoff value set in this embodiment emphasizes specificity, and it is of course possible to set the cutoff value with emphasis on sensitivity.
The method of setting the cutoff value is not limited to the above examples. For example, ROC analysis may be used to calculate the cutoff value that maximizes sensitivity and specificity.
Detection of Mutation
In one aspect, detecting a CNV detects the presence of a single cell with a mutation in a population of cells. In one aspect, the mutation is a somatic or germline mutation. In one aspect, the mutation is a chromosomal or genetic mutation.
Detection of Chromosomal Mutation
In one aspect, when DNA sample contains a set of genomic DNA in a single cell, the presence of a single cell with a chromosomal mutation is detected in the cell population by detecting a CNV from the results of quantification of PCR amplicons. In one aspect, the chromosomal mutation is at least one of the following: aneuploidy over the entire length of a chromosome, partial aneuploidy of a chromosome, gene amplification, and gene deletion.
Gene amplification is the multiplication of a gene due to partial duplication of a chromosome. In one aspect, there is no limit to the number of times duplication occurs. Gene deletion is the loss of a gene due to partial deletion of a chromosome. In one aspect, the gene deletion is haplo- or null-type. In one aspect, gene amplification and deletion are polymorphism.
As described above, unless the cell population is dominated by cells with genomic aneuploidy, the cell population cell includes euploid cells. The cell population contains normal single cells with no chromosomal mutations at the location of a target genome. At least one of the single cells assigned to the respective reaction compartments (reaction droplets) described above have euploid chromosomes in the regions to be subjected to aneuploidy detection. On the scatter, a cluster of single cells with chromosomal mutations is shifted from the position of a cluster produced by normal single cells. Cluster 40a illustrated in
In addition, even when the cell population is dominated by cells with genomic aneuploidy, the ploidy of the cluster can be absolutely quantified by using the external standard described above. The external standard can also be applied to a heterogeneous cell population consisting of euploid and aneuploid cells.
Detected is a reaction droplet that has an amplicon amount different from that of a reaction droplet including a single cell not having a chromosomal mutation at the location of a target genome. Hereinafter, this is referred to as an aneuploid droplet. Clusters 40b and 40c illustrated in
Examples of chromosomal mutations that result in aneuploid droplets are the following. That is, a single cell with a detectable chromosomal mutation contains, for example, genomic DNA with any one of the following chromosomal mutations.
In one aspect, deletions exclude biallelic deletions, so-called nulls. In one aspect, a pair formed of a chromosome with a deletion part and a chromosome with a corresponding part being normal is to be detected.
In one aspect, data including information on whether or not an aneuploid droplet is detected is further generated. That is, data including information on whether or not a single cell with a chromosomal mutation is detected is generated. Selecting a cell population that generates single cells genetates useful data in specific diagnostic areas described below.
Application to Prenatal Diagnosis
In one aspect, single cells are obtained from either amniotic fluid or maternal blood. Specifically, a population of bulk cells are obtained from amniotic fluid or maternal blood. The cell population includes fetal cells. In one aspect, fetal cells in the cell population are enriched. A cell population includes maternal cells. In one aspect, the maternal cell is euploid in the region on the genomic DNA to be detected. In one aspect, the fetal cell is a fetal nucleated red blood cell (fetal nucleated erythroblasts, fNRBC) circulating in maternal blood, and the maternal cell is a white blood cells circulating in maternal blood. In one aspect, the fetal cell is a fetal cell floating in amniotic fluid and the maternal cell is a maternal cell floating in amniotic fluid.
In one aspect, data is provided for the diagnosis of trisomy 13, trisomy 18, trisomy 21, Turner syndrome, triple X syndrome, XYY syndrome, Klinefelter syndrome, Di George syndrome, Angelman syndrome, Prader-Willi syndrome, or cri-du-chat syndrome.
Application to Cancer Diagnosis
In one aspect, single cells are obtained from the blood of a cancer patient. Specifically, a population of bulk cells is isolated from the blood of a cancer patient. The cell population includes cancer cells consisting of peripheral circulating tumor cells (CTCs) and/or cancerous blood cells. In another aspect, the cell population includes cells obtained by biopsying a solid tumor and dispersing the biopsied tumor with a predetermined chemical and/or physical treatment. In one aspect, the cancerous blood cell is a cancerous plasma cell. In one aspect, these cancer cells in the cell population are enriched. The cell population further includes cells that are not cancer cells. In one aspect, a cell that is not cancer cell is euploid in the region on the genomic DNA to be detected. In one aspect, a cell that is not cancer cell is a normal white blood cell that circulates in the blood of a cancer patient. In one aspect, the normal white blood cell is a normal plasma cell.
In one aspect, data is provided for the diagnosis of myelodysplastic syndrome, multiple myeloma, idiopathic eosinophilia, chronic eosinophilic leukemia, acute nonlymphocytic leukemia, myeloproliferative neoplasm, chronic lymphocytic leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, or head and neck cancer.
Application Example to Multiple Myeloma
As described in NPLs 8 and 9, multiple myeloma (MM) is a type of hematopoietic malignancy, and is an disorder in which plasma cells undergo neoplasia. Chromosomal abnormalities are found in the majority of cases of MM. Trisomy on chromosomes 3, 5, 7, 9, 11, 15, and 19 are frequently reported. Monosomy on chromosomes 13, 14, 16, and 22 is frequently found. Of these, monosomy on chromosome 13 and deletion of the long arm of chromosome 13 (13q deletion) are examples of the most frequent chromosomal mutations. These chromosomal mutations are found in approximately 50% of new patients. Of these, 85% have a monoploid chromosome in its entirety or in a portion thereof to be detected, and 15% have intra-arm deletions, including the 13q14 deletion. Biallelic deletion is rare. In addition, deletion of 17p13 on the short arm of chromosome 17 is observed in about 10%. These are one of the poor prognostic factors of MM. Another poor prognostic factor is amplification of 1q21 on the long arm of chromosome 1. It is reported to be found in 43% of untreated MM patients.
In the present embodiment, the amount of residual tumors in a patient with at least one of these chromosomal abnormalities is monitored. Specifically, the extent to which plasma cells with such a chromosomal mutation (cancerous plasma cells) remain in the body is periodically measured during the post-treatment period.
In the present embodiment, a minimal residual disease (MRD) consisting of cells with the above-mentioned chromosomal mutation is measured. Among residual diseases, MRD is particularly difficult to measure with conventional techniques. The therapeutic effect can be deeply evaluated by measuring the MRD. Based on such evaluations, physicians decide to increase, decrease, discontinue, and resume treatment.
An example of an actual inspection method is described below. In one example, bone marrow fluid or blood taken from a patient is used as a sample. Red blood cells are removed from these liquids. Hemolysis, centrifugation, and/or a chip for hydraulic classification are used to remove red blood cells.
The sample is mixed with a predetermined probe-primer set and a PCR premix, and cells are lysed. A probe-primer set that has a target within the region of 13q is selected as a set to be detected. In one aspect, a probe-primer set that has a target at a location on chromosome 18 is selected as a control.
Cluster shifts as illustrated in
In one aspect, plasma cells, including circulating tumor cells, are enriched by using antibodies directed against plasma cell-specific antigens prior to measurement. As a result, cancerous plasma cells, namely tumor-forming plasma cells, present in the bone marrow fluid or blood can be detected with even higher sensitivity.
In another aspect, information on whether cells in the sample have at least one of deletion of 17p13 and amplification of 1q21 is obtained from the cluster shift. Such information helps to predict the prognosis of MM.
Application Examples to Myelodysplastic Syndrome and Other Hematopoietic Malignancies
Myelodysplastic syndrome (MDS) is a type of hematopoietic malignancy. At least 50% of MDS are caused by chromosomal mutations occurring in hematopoietic stem cells (specifically, aneuploidy over the entire length of a chromosome, partial aneuploidy of a chromosome, gene amplification, or gene deletion). Representative chromosomal mutations for MDS are deletion of the long arm of chromosome 5 (5q), monosomy of chromosome 7, and deletion of the long arm of chromosome 20 (20q). MRD is periodically measured by detecting cells with chromosomal mutations present in a sample.
Disorders associated with 5q deletion are independently defined as a type of MDS called 5q-syndrome. According to European and US studies, approximately 10% of MDS are 5q-syndrome. Lenalidomide (generic name) is effective for 5q-syndrome. Monitoring of the therapeutic effect of Lenalidomide is performed with the above cluster shift.
In another aspect, detection of other hematopoietic malignancies with chromosomal aneuploidies, particularly chromosomal deletions and amplifications, is performed with the above cluster shift.
Detection of Genetic Mutation
In one aspect, detecting CNV detects the presence of single cells with a genetic mutation. The genetic mutation is at least one of base substitutions, base insertions, and base deletions.
In one aspect, at least one of the single cells assigned to the respective reaction compartments (reaction droplets) is an internal control that has no detectable gene mutations at least at the location of a target genome. A cluster of reaction droplets that has an amplicon amount different from that of the reaction droplet serving as an internal control is shifted from the position of the cluster without a gene mutation. These droplets in such a cluster are detected as droplets with a mutation.
In another aspect, the fluorescence intensity of an external standard is measured in advance in another well or at another time. The fluorescence intensity information of the external standard is compared with the fluorescence intensity information at the center of each cluster. Based on such comparison, the copy number of the gene mutation in each cluster is measured. The copy number of a genetic mutation can be measured even in the following case: when a cell population is dominated by cells with the genetic mutation, and thus a cell with only the wild-type gene cannot be used as an internal control.
In one aspect, the genetic mutation is introduced into an unspecified region of a genome by a lentiviral vector or another vector. Examples of cells with vector-induced genetic mutations are chimeric antigen receptor T cells (CAR-T cells) and TCR-T cells. For the detection in the present embodiment, the target may be DNA introduced by a vector. For example, the copy number of DNA introduced by a vector may be detected.
Peripheral blood of a healthy subject was hemolyzed and added to the PCR premix to prepare a PCR reaction solution. The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.
In the present test example, 23 types of probes each including a fluorescent material (FAM) with the first fluorescence wavelength were assigned to chromosome 13. In addition, 25 types of probes each including a fluorescent material (HEX) with the second fluorescence wavelength were assigned to chromosome 18. The probes were mixed together in one droplet.
Next, peripheral blood of a healthy subject was hemolyzed, and CD45-positive cells were isolated by using EasySep Human CD45 Depletion Kit II (STEMCELL Technologies). The resulting CD45 cells were added to the PCR premix to prepare a PCR reaction solution. The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.
Also in the present test example, 23 types of probes each including a fluorescent material (FAM) with the first fluorescence wavelength were assigned to chromosome 13. In addition, 25 types of probes each including a fluorescent material (HEX) with the second fluorescence wavelength were assigned to chromosome 18. The probes were mixed together in one droplet.
Next, a cell population obtained by mixing the CD45-positive cells and cells of the human cultured cell line GM13721 at a predetermined ratio was added to the PCR premix to prepare a PCR reaction solution. The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. In the CD45-positive cell, chromosomes 13 and 18 are euploid, that is, diploid. In the human cultured cell line GM13721, a partial region of one long arm of chromosome 13 is monoploid, and chromosome 18 is euploid.
Also in the present test example, 23 types of probes each including a fluorescent material (FAM) with the first fluorescence wavelength were assigned to the defective region of chromosome 13. In addition, 25 types of probes each including a fluorescent material (HEX) with the second fluorescence wavelength were assigned to chromosome 18. The probes were mixed together in one droplet.
These results show that the CD45-positive cells and the human cultured cell line GM13721 are clearly distinguished from each other. This suggests that cells with abnormal copy numbers can also be detected from a cell population derived from human blood.
A cell population in which the CD45-positive cells and cells of the human cultured cell line GM13721 were mixed at a predetermined ratio was prepared in the same manner as in Test Example 8 above. The proportion of cells of the line GM13721 in the cell population was 0%, 0.1% or 3%. Each cell population was added to the PCR premix to prepare a PCR reaction solution. The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and cells were lysed in the respective droplets. Subsequently, PCR was performed under general conditions within each droplet. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. In the CD45-positive cell, chromosomes 13 and 18 are euploid, that is, diploid. In the human cultured cell line GM13721, a partial region of one long arm of chromosome 13 is monoploid, and chromosome 18 is euploid.
Also in the present test example, 23 types of probes each including a fluorescent material (FAM) with the first fluorescence wavelength were assigned to the defective region of chromosome 13. In addition, 25 types of probes each including a fluorescent material (HEX) with the second fluorescence wavelength were assigned to chromosome 18. The probes were mixed together in one droplet.
In the present test example, a dot having fluorescence intensity of 5,000 or more at the first fluorescence wavelength (FAM) or fluorescence intensity of 9,000 or more at the second fluorescence wavelength (HEX) was used as a positive droplet to be evaluated. Using the cutoff (y=0.84806x−1044) determined by the same manner as in Test Example 7, the ratio of abnormal droplets (droplets that is considered to include monoploid chromosome 13) with respect to the positive droplets was calculated.
Using a reverse transcription product of RNA in a single cell as DNA sample, the copy number of mRNA for the B2M gene and the copy number for the mRNA for the GAPDH gene were detected.
In the present test example, single cell-droplet RT-PCR was performed on the human cultured cell line GM22948. The premix contains primers for the B2M gene, primers for the GAPDH gene, probes for the B2M gene (FAM label), probes for the GAPDH gene (HEX label), and reverse transcriptase (SuperScript IV). The primers for both genes were designed to flank an intron in order to amplify the mRNA of the genes by PCR, rather than the genes in the genomic DNA.
Specifically, a PCR reaction solution was prepared by adding 10,000 cells (line GM22948) and 2 μL of a cell suspension containing PBS to the PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, and the like in the PCR reaction solution are as follows.
The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), the cells were lysed in each droplet, and the cells were allowed to stand at 50° C. for 10 minutes for reverse transcription. After denaturing the DNA at 95° C. for 10 minutes, PCR was performed under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.
No positive droplets were detected when the premix did not contain reverse transcriptase. That is, with the primers designed to flank the intron as described above, both genes in the genomic DNA were hardly amplified.
Using mitochondrial DNA as a DNA sample, the copy number of the ND1 gene was detected.
In the present test example, single cell-droplet PCR was performed on the human cultured cell line GM22948. Each droplet contains genomic DNA as well as mitochondrial DNA within a single cell. The premix contains primers for chromosome 13 (23 types), primers for ND1 gene of mitochondrial DNA, probes for chromosome 13 (23 types, FAM-labeled), and probes for the ND1 gene (HEX-labeled).
Specifically, a PCR reaction solution was prepared by adding 10,000 cells (line GM22948) and 2 μL of a cell suspension containing PBS to the PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, and the like in the PCR reaction solution are as follows.
The above PCR reaction solution was made into droplets by using QX200 Droplet Generator (BioRad Laboratories, Inc.), and the cells were lysed in each droplet. After denaturing the DNA at 95° C. for 10 minutes, PCR was performed under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.
Use of Mitochondrial DNA as Control
To detect CNVs in the target assigned regions, a control is assigned to a region considered to have a small CNV and be euploid. For cells with frequent chromosomal abnormalities, such as cancer cells, it is possible that a suitable chromosome cannot be found as a control. In such cases, mitochondrial DNA may be used as a control. That is, one of the fluorescence wavelengths to be used is selected, and a probe including a fluorescent material with the selected fluorescence wavelength is assigned to mitochondrial DNA.
For example, DNA sample contains a set of genomic DNA in a single cell and mitochondrial DNA in the single cell. The PCR reaction system includes a probe (including a fluorescent material with a first fluorescence wavelength) assigned to a region on genomic DNA and a probe (including a fluorescent material with a second fluorescence wavelength) assigned to mitochondrial DNA. The region includes one or more targets. In the reaction compartment, targets contained in the set of genomic DNA and in the mitochondrial DNA are amplified by PCR. Amplicons of the genomic DNA and mitochondrial DNA are quantified in each reaction compartment in a cycle during which amplification of the target contained in the mitochondrial DNA reaches a plateau and amplification of the target contained in the genomic DNA reaches an exponential amplification phase. At this time, fluorescence of the first and second fluorescence wavelengths is detected from one reaction compartment. This configuration can distinguish a reaction compartment, in which the target contained in the genomic DNA and the target contained in the mitochondrial DNA are amplified, from a reaction compartment, in which only a contaminating cell-free nucleic acid is amplified.
During droplet PCR, amplification using mitochondrial DNA as a template proceeds fast. Therefore, primers or probes are depleted early. This is because the copy number of mitochondrial DNA per cell is larger than the copy number of genomic DNA per cell. Therefore, the PCR is stopped at a cycle number where the reaction for mitochondrial DNA reaches a plateau. However, at such a cycle number, the amplification using the genomic DNA of the chromosome as a template is in the exponential amplification phase.
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This application is entitled to and claims the benefit of Japanese Patent Application No. 2020-216149 filed on Dec. 25, 2020, the disclosure of which including the specification and drawings is incorporated herein by reference in its entirety.
The detection method of the present invention is particularly advantageous for, for example, prenatal diagnosis and cancer diagnosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-216149 | Dec 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/047619 | 12/22/2021 | WO |
