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WO2019133752A1 - A method for predicting drug efficacy - Google Patents

A method for predicting drug efficacy Download PDF

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WO2019133752A1
WO2019133752A1 PCT/US2018/067746 US2018067746W WO2019133752A1 WO 2019133752 A1 WO2019133752 A1 WO 2019133752A1 US 2018067746 W US2018067746 W US 2018067746W WO 2019133752 A1 WO2019133752 A1 WO 2019133752A1
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kras
mutations
gene
csf
drug
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Inventor
Jia-Ming Chang
Yi-Ru Lee
Chiung-Wen Liou
Hsiang-Ping Huang
Yuan-Jen Huang
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Development Center for Biotechnology
DCB USA LLC
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Development Center for Biotechnology
DCB USA LLC
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to methods for predicting drug efficacies or effects.
  • Companion diagnosis may be used to determine whether the drug would he beneficial to the patient based on their biological characteristics (e.g., genetic profiles) that determine responders and non-responders to the therapy.
  • Companion diagnosis detects and assess biomarkers that can prospectively help predict likely outcome of the therapy (e.g., efficacies and toxicities).
  • companion diagnosis can also be used to monitor drug responses during treatment. This information may help doctors find new treatment strategies.
  • Companion diagnosis is relatively new and only a handful of drugs have established companion diagnosis. For example, companion diagnosis assessing the Her2 expression levels is helpful in deciding whether to use Herceptin for breast cancer treatment.
  • EGFR inhibitors can often shrink lung cancer. However, cancer cells eventually become resistant to the drug due to T970M mutation in the EGFR gene. Companion diagnosis of this mutation can help doctors switch the drugs to newer EGFR inhibitors that can work against ceils with the T790M mutation, such as osimertinib (Tagrisso®).
  • companion diagnosis has been shown to be helpful in selecting proper treatments for patients, there are only few established companion diagnoses at the moment.
  • Embodiments of the invention relate to diagnostic techniques for predicting therapeutic efficacy.
  • a method for predicting therapeutic efficacy of a drug in accordance with one embodiment of the invention includes analyzing a panel of genes to derive information for predicting whether a patient will respond to the drug.
  • the analyzing a panel of genes includes analysis of gene mutations, copy number variations, and/or expression levels.
  • the panel of genes comprises PIK3CA, KRAS, PTEN, BRAF, and CSF-1R.
  • the gene mutations may include E542K, E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R.
  • the drug may be a colony stimulating factor 1 receptor (CSF-1R) inhibitor.
  • CSF-1R inhibitor may be a small molecule drug, a biologic, or a nucleotide.
  • the nucleotide may be siRNA or miRNA.
  • the drug may be a drug targeting a protein translated from a gene in the panel of genes.
  • the drug may be a phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) inhibitor, a KRAS (also known as K-ras or Ki-ras) inhibitor, a phosphatase and tensin homolog (PTEN) inhibitor, or a BRAF (also known as B-Raf) inhibitor.
  • PIK3CA catalytic subunit alpha
  • KRAS also known as K-ras or Ki-ras
  • PTEN phosphatase and tensin homolog
  • BRAF also known as B-Raf
  • the analyzing makes use of multiplexing illumine, real-time polymerase chain reaction (PCR), next-generation sequencing (NGS), gene chips, microfluidics, flowcytometry, or a combination thereof.
  • PCR real-time polymerase chain reaction
  • NGS next-generation sequencing
  • the analyzing a panel of genes may be performed simultaneously in a multiplex format.
  • a method in accordance with one embodiment of the invention comprises using magnetic beads coupled with a probe to react with a sample to detect presence or absence of a target gene, wherein the probe can hybridize with a fragment of the target gene.
  • the target gene is KRAS and the probe is designed to detect G12D mutation in KRAS.
  • the probe is coupled with biotin for interaction with streptavidin-R-phycoerythrin to permit multiplexing illumine detection for fluorescence intensity and quantity.
  • the probe has the sequence of 5’-
  • FIG. 1 shows a schematic illustrating various factors involved in CSF-1R signaling. Based on analysis of gene databases from various cancer cells, it was found that PIK3CA and KRAS have high mutation rates in breast cancer and colorectal cancer. The incidence rates for PIK3CA mutations in various cancers are shown, as well as incidence rates for KRAS mutations in various cancers.
  • FIG. 2 shows a schematic illustrating various factors involved in CSF-1R signaling. Based on analysis of gene databases from various cancer cells, it was found that PTEN and BRAF have high mutation rates in glioma and thyroid cancer. The incidence rates for PTEN mutations in various cancers are shown, as well as incidence rates for BRAF mutations in various cancers.
  • FIG. 3 shows results from cross analyses of PIK3CA, KRAS, PTEN, and BRAF mutations. The results from these analyses revealed that among the colorectal cancers and thyroid cancers, the rates of having two or more mutations impacting these gene functions are as high as 40% among all different races.
  • FIG. 4 shows the results from hybridization between magnetic beads that are coupled with a probe and a target KRAS G12D sequence.
  • FIG. 5 shows results from probe specificity tests.
  • a Kras WT probe (0.2 nmol) is allowed to hybridize with either the wild-type Kras or a mutant Kras GAT (mGl2D). The results show that the probe of the invention is specific.
  • FIG. 6A shows results from testing optimal hybridization temperatures at 0.2 pmol of wild-type and mutant Kras. Magnetic beads with wild type Kras probe are hybridized with either the wild-type Kras or a mutant Kras GAT (mGl2D) sequence at different temperature and the median fluorescence intensity (MFI) values are measured to assess the hybridization. The results show that the hybridization proceed better and produce more stable results at higher temperatures, e.g., 60-80 °C.
  • FIG. 6B shows results from testing optimal hybridization temperatures at 0.05 pmol of wild-type and mutant Kras.
  • FIG. 6C shows results from testing optimal hybridization temperatures at 0.005 pmol of wild-type and mutant Kras.
  • Magnetic beads with a mutant Kras Mt probe are hybridized with a mutant Kras target sequence for different hybridization times and the median fluorescence intensity (MFI) values are measured to assess the hybridization.
  • MFI median fluorescence intensity
  • FIG. 7B shows results from testing for optimal hybridization times. Magnetic beads with a mutant Kras Mt probe are hybridized with a wild-type Kras target sequence for different hybridization times and the median fluorescence intensity (MFI) values are measured to assess the hybridization. The results show that a hybridization time of about 10 minutes proceed better and produce more stable results.
  • MFI median fluorescence intensity
  • Embodiments of the invention relate to diagnostic techniques for predicting therapeutic efficacy. Methods of the invention can be used as companion diagnosis to screen patients for subpopulations that would respond to a particular therapy, thereby increasing the probability of therapeutic success and avoiding waste of medical resources.
  • a method for predicting therapeutic efficacy of a drug may include the step of analyzing a panel of genes to derive information for predicting whether a patient will respond to the drug.
  • the analyzing a panel of genes may include analysis of gene mutations, copy number variations, and/or expression levels.
  • CSF-1R inhibitor therapy will be described.
  • the companion diagnosis may include analysis of genes that are involved in the CSF-1R signaling pathways.
  • genes for example, may include PIK3CA, KRAS, PTEN, and BRAF.
  • CSF-1R inhibition has been used to target tumor-associated macrophages in cancer therapy.
  • CSF-1R inhibition treatments vary among patients of different races, suggesting that different genetic backgrounds may play an important role.
  • PIK3CA, KRAS, PTEN, and BRAF mutations of genes immediately downstream from CSF-1R in the signaling pathways, such PIK3CA, KRAS, PTEN, and BRAF, may have impacts on the efficacy of CSF- 1R inhibitor therapy.
  • Mutations of these genes are frequently associated with various cancers.
  • several potential mutations including E542K, E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R, are found that might have impacts on CSF-1R inhibitor therapy.
  • CSF1 colony stimulating factor- 1
  • CSFIRV colony stimulating factor- 1 receptor
  • CSFIRV colony stimulating factor- 1 receptor
  • CSF-1R inhibition has been used to target tumor-associated macrophages in cancer therapy.
  • the effects of CSF-1R inhibition treatments seem to vary among patients of different races, suggesting that genetic backgrounds may play an important role. Therefore, analyzing genes and mutations related to CSF-1R signaling pathways may provide information to help predict therapy outcomes.
  • the cancer gene databases used for such analysis may include GENIE (AACR
  • cBioPortal platform (v.1.8.3) (http://www.cbioportal.org/index.do).
  • cBioPortal for Cancer Genomics is a tool developed at Memorial Sloan Kettering Cancer Center’s Computational Biology Center (cBio).
  • CSF-1R signal transduction related genes include CSF1R, PIK3CA, PTEN, KRAS, and BRAF.
  • the mutation rates of these genes were analyzed with respect to various cancers (glioma, oral cancer, thyroid cancer, lung cancer, breast cancer, stomach cancer, liver cancer, biliary tract cancer, colorectal cancer, ovary cancer, and uterus endometrial cancer) in patients of different races.
  • PIK3CA and KRAS have more than 5% mutation rates among all cancers.
  • the mutation rate of PIK3CA is about 31.3% in breast cancer, and the mutation rate for KRAS in non-small cell adenocarcinoma is about 27%.
  • the mutations of PIK3CA and KRAS are accompanied by increases in the CSF-1R activity. These mutations are also related to the lack of responses to treatments in the clinics.
  • PTEN and BRAF are the actors that interact directly with PIK3CA and KRAS, respectively. Therefore, mutations that affect PTEN (e.g., R130G, C71F/Y, and deletions) and BRAF (V600E) functions are expected to have a negative impact on the effects of CSF-1R inhibitors. Analyzing the potential impacts of these mutations on cancer treatments, it was found that these may affect up to 9% glioma patients among Caucasians and up to 50% thyroid cancers patients among Caucasians and Asians. (FIG. 2). Thus, these mutation analyses may be used to predict the percentages of patients that may not respond to the CSF-1R inhibitor treatments.
  • PTEN e.g., R130G, C71F/Y, and deletions
  • V600E V600E
  • CSF-1R signaling pathways play roles in CSF-1R inhibition treatments. These factors include CSF-1R, PIK3CA, PTEN, KRAS, and BRAF. Analyzing functional mutations that affect the functions of these factors may provide information to predict which patients would benefits from CSF-1R inhibition treatments and which patients would not.
  • Example 2 Use of cell lines to establish methods for analyzing gene mutations
  • genomic DNAs were obtained from lung cancer cells A549, H727, HCC-827, H1975, NCI-H146, H460, and H292.
  • gene fragments for CSF-1R, PIK3CA, KRAS, and PTEN were obtained using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the sequences of these gene fragments were determined using Sanger’s sequencing methods, and then the sequences were compared with the sequences of the same fragments from normal cells. The comparison would reveal any nucleotide differences. The mutation locations were further confirmed.
  • the procedures for sequence determination and analysis are as follows:
  • Genomic DNA extraction was performed using DNeasy Blood & Tissue Kit
  • the target lung cancer cells were collected in centrifuge tubes. The cells were washed with lx PBS to remove DMSO used to preserve cells in the frozen aliquots. The tubes were centrifuged, and the top clear solutions were discarded. To the cell pellets in the tubes were added 180m1 ATL lysis buffer (Qiagen) and 20m1 proteinase K. Mix and resuspend the cell pellets. Place the tubes in an oven at 56°C for 4 hours to digest proteins. After protein digestion, add 200m1 AL lysis buffer to the tubes and mix well. Then, add 200m1 pure ethanol and mix well.
  • TERTARA Polymerase
  • DNA sequencing was performed using Sanger’s method. Sequence analysis of the PCR products were performed using BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Each reaction uses 50 ng primers and l pl BigDye mixture, 10 ng PCR product, and I m ⁇ 5x reaction buffer. PCR was performed in GeneAmp PCR System 2700 thermocycler (Applied Biosystems, Foster City, CA). The reaction conditions are: 96°C initial denaturation for 1 minute, 96°C denaturation for 10 seconds, 50°C annealing for 10 seconds, repeat for 45 cycles, and then extend at 60°C for 4 minutes.
  • Biosystems which can sequence to 700-800 bps on average. Compare the sequences of CSF- 1R, PIK3CA, KRAS and PTEN from lung cancers cells with those from normal cells to determine the differences. Then, confirm the mutation locations.
  • H727, HCC-827, H1975, NCI-H146, H460, and H292 have been analyzed. Mutations in CSF-1R, PIK3CA, KRAS, and PTEN are found (FIG. 1 and FIG. 2). Results from these analyses shows that most genes are wild-type genes. Genes from lung cancer cells A549 and H727 have mutations at G12 (Table 2). These mutations can be accurately determined.
  • HCC-827, and H1975 mutations observed for CSF-1R, PIK3CA, KRAS and PTEN.
  • the results show that most genes are wild-type, and only KRAS from A549 and H727 have mutations at G12 location, PI3K from HCC-827 and H1975 have mutations at E545.
  • This example describes gene detection and analysis methods based on Luminex xMAP (Multi-Analyte Profiling; Luminex Corp., Austin, TX), gene-probe design, and hybridization techniques.
  • gene probes for the target DNA sequences e.g., CSF-1R, PIK3CA, KRAS, BRAF, and PTEN
  • the gene probes are used to detect specific target genes as follows. Select the gene probes that are specific for the target genes. Couple the selected gene probes on specific magnetic beads. Mix fluorescent magnetic beads for different assay samples. Add the amplified fragments of the assay samples and detect them with fluorescence labeled probes on the magnetic beads. Then, use flow cytometry to detect the types of magnetic beads and measure the fluorescence intensities.
  • This method can provide multiple genes detection and can achieve fast and accurate detection.
  • the methods can be used in the clinics.
  • KRAS mutations are often associated with colorectal cancer, pancreatic cancer, lung cancer, etc.
  • the most frequent KRAS mutations are found at positions G12 and G13.
  • the mutations at G12 are more common than those at G13, making the G12 mutations more relevant in the therapeutic responses.
  • An object of the invention is to provide methods for detecting G12 mutations in
  • Embodiments of the invention are based on the Liminex xMAP (Multi- Analyte Profiling) principle and make use of magnetic bead probes to detect gene mutations.
  • a method of the invention may include the steps of: expansion of the test sample, gene-probe design, and hybridization reactions. Briefly, a gene probe is coupled on magnetic beads and then added to the test sample that has been expanded (e.g., using PCR). The probe or beads may contain fluorescent tags, which would allow one to differentiate different magnetic beads using flow cytometry. The different magnetic beads permit differentiation of mutant or wild-type genes. In addition, the fluorescence intensities may be used to quantify the species. These methods allow one to perform gene diagnosis with speed and accuracy, as well as quantitation. [0053] An exemplary method will be described for illustration. However, one skilled in the art would appreciate that this example is for illustration only and that other modifications and variations are possible without departing from the scope of the invention.
  • the primers for Kras gene detection can be derived from the Kras sequence in the literature with some modifications.
  • a biotin can be attached to the 5’ end of the reverse primer, as shown in Table 4.
  • a wild-type (Wt) probe and a Kras G12D (GAT) mutant-type (Mt) probe may be designed, as shown in Table 5.
  • Wt wild-type
  • GAT Kras G12D
  • Mt mutant-type
  • DNA extracts are prepared from human cell lines or patient-derived xenograft
  • PDX samples Use specific modified primers and PCR to expand specific Kras gene fragments and tag them with fluorescence markers.
  • Beads with different codes are mixed well in 0.1M 2-
  • Kras G12D (GAT) mutant-type (Mt), are synthesized. The 5’ ends of these sequences are tagged with biotin. The two sequence fragments are 59 b.p. long, which serve as the standards for constructing the analysis platform.
  • the hybridization reactions are conducted in 96-well PCR plates. Each well is loaded with 2500 beads, which are mixed with the above-described synthetic sequences. The total volume per well is 50 ul.
  • the hybridization is performed in a PCR machine (Biometra Tadvanced). After hybridization, the samples in the PCR plate are transferred into a 96-well dark plate. With the aid of a magnetic plate, the upper layer clear solutions are discarded. Streptavidin-R-phycoerythrin (75 ul/well) was added to each well, and the binding reaction was allowed to proceed at room temperature for 30 minutes. The median fluorescence intensity (MFI) of the magnetic beads was measured using a Magpix equipment (Luminex).
  • MFI median fluorescence intensity
  • Kras G12D Mt probe are hybridized with different concentrations (0.005, 0.025, 0.05, 0.1, and 0.2 pmol) of the Kras G12D Mt sequence. The hybridization was performed at 95 °C for 5 minutes and then at 52°C for 30minutes. As shown in FIG. 4, Kras G12D Mt probe can detect Kras G12D Mt sequence in the range of 0.025-0.1 pmol, with a lowest detection limit of 0.025 pmol.
  • Wt probe are hybridized with different concentrations (0.005, 0.025, 0.05, 0.1, and 0.2 pmol) of the Kras Wt and Kras G12D Mt sequences. The hybridization was performed at 95 °C for 5 minutes and then at 52°C for 30minutes. As shown in FIG. 5, Kras Wt probe can hybridize with the Kras Wt sequence with a higher affinity than with the Kras G12D Mt sequence. Thus, the magnetic probes are specific.
  • Kras Wt probe are separately hybridized with low, medium, and high concentrations (0.005, 0.05, and 0.2 pmol) of the Kras Wt and Kras G12D Mt sequences, respectively.
  • the hybridization was performed at 95°C for 5 minutes and then at 23.3 - 75°C for 30minutes.
  • the MFI values show that Kras Wt probe can form more stable hybrids with both the Kras Wt and Kras G12D Mt sequences, as compared with at low hybridization temperatures. Therefore, a preferred hybridization temperature for a detection platform of the invention would use a relatively high hybridization temperature, such as 65-80°C, more preferably around 70°C (e.g., 70.6°C).
  • the Wt probe are separately hybridized with low, medium, and high concentrations (0.005, 0.05, and 0.2 pmol) of the Kras Wt and Kras G12D Mt sequences, respectively.
  • the hybridization was performed at 95°C for 5 minutes and then at 70.6°C to observe hybridizations at different reaction times (1, 5, 10, 15, 20, and 30minutes).
  • FIG. 7A and FIG. 7B with a hybridization time of 10 minutes, Kras G12D Mt probe at medium and high concentrations (0.05 and 0.2 pmol) can hybridize with Kras Wt and Kras G12D Mt sequences to produce the best MFI values, as compared with other hybridization time.
  • the hybridization time is preferably selected for 10 minutes for the detection platform of the invention.
  • the accuracy of the detection platform can be tested as follows. Human cell lines and patient-derived xenograph (PDX) samples are expanded with PCR to obtain the target gene fragments. Then, using the Quick Microbeads Kras Gene Detection Platform to detect the gene states. The detection samples have been analyzed and their gene sequences determined. Therefore, they can be used as the standards for assessing the detecting platform. Then, the samples are analyzed in 10 blind tests, each time with 10 PCR products and each sample is tested in quadruples. From these tests, it was found the detection platform or methods of the invention have an accuracy of 99% after testing 100 PCR products. Thus, a detection platform/method of the invention has the advantages of system stability and detection accuracy.

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Abstract

A method for predicting therapeutic efficacy of a drug includes analyzing a panel of genes to derive information for predicting whether a patient will respond to the drug. The analyzing a panel of genes includes analysis of gene mutations, copy number variations, and/or expression levels. The panel of genes comprises PIK3CA, KRAS, PTEN, BRAF, and CSF-1R. The gene mutations include E542K, E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R. The drug is a CSF-IR inhibitor.

Description

A METHOD FOR PREDICTING DRUG EFFICACY
BACKGROUND OF INVENTION
Field of the Invention
[0001] The present invention relates generally to methods for predicting drug efficacies or effects.
BACKGROUND OF INVENTION
[0002] Medical treatments are traditionally based on symptoms. Thus, patients having the same symptoms are presumed to have the same underlying cause, and therefore the same treatment should apply to all patients having the same symptoms. However, this approach ignores the fact that different patients may have different genetic backgrounds. Therefore, the same drugs may be effective for some patients, but they may not be effective for others. For patients who did not benefit from the treatments, they came out worse because they suffered from the adverse effects and they may also lose precious time/opportunities to have the proper treatments.
[0003] To improve the situation, there has been a push for personalized medicine or precision medicine. With precision medicine, one typically performs companion diagnosis to better understand the patients. Companion diagnosis may be used to determine whether the drug would he beneficial to the patient based on their biological characteristics (e.g., genetic profiles) that determine responders and non-responders to the therapy. Companion diagnosis detects and assess biomarkers that can prospectively help predict likely outcome of the therapy (e.g., efficacies and toxicities). In addition, companion diagnosis can also be used to monitor drug responses during treatment. This information may help doctors find new treatment strategies.
[0004] Companion diagnosis is relatively new and only a handful of drugs have established companion diagnosis. For example, companion diagnosis assessing the Her2 expression levels is helpful in deciding whether to use Herceptin for breast cancer treatment. In another example, EGFR inhibitors can often shrink lung cancer. However, cancer cells eventually become resistant to the drug due to T970M mutation in the EGFR gene. Companion diagnosis of this mutation can help doctors switch the drugs to newer EGFR inhibitors that can work against ceils with the T790M mutation, such as osimertinib (Tagrisso®). [0005] While companion diagnosis has been shown to be helpful in selecting proper treatments for patients, there are only few established companion diagnoses at the moment.
SUMMARY OF THE INVENTION
[0006] Embodiments of the invention relate to diagnostic techniques for predicting therapeutic efficacy.
[0007] One aspect of the invention relates to methods for predicting therapeutic efficacy of a drug. A method for predicting therapeutic efficacy of a drug in accordance with one embodiment of the invention includes analyzing a panel of genes to derive information for predicting whether a patient will respond to the drug. The analyzing a panel of genes includes analysis of gene mutations, copy number variations, and/or expression levels. The panel of genes comprises PIK3CA, KRAS, PTEN, BRAF, and CSF-1R. The gene mutations may include E542K, E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R.
[0008] In accordance with embodiments of the invention, the drug may be a colony stimulating factor 1 receptor (CSF-1R) inhibitor. The CSF-1R inhibitor may be a small molecule drug, a biologic, or a nucleotide. The nucleotide may be siRNA or miRNA.
[0009] In accordance with embodiments of the invention, the drug may be a drug targeting a protein translated from a gene in the panel of genes. The drug may be a phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) inhibitor, a KRAS (also known as K-ras or Ki-ras) inhibitor, a phosphatase and tensin homolog (PTEN) inhibitor, or a BRAF (also known as B-Raf) inhibitor.
[0010] In accordance with embodiments of the invention, the analyzing makes use of multiplexing illumine, real-time polymerase chain reaction (PCR), next-generation sequencing (NGS), gene chips, microfluidics, flowcytometry, or a combination thereof. The analyzing a panel of genes may be performed simultaneously in a multiplex format.
[0011] One aspect of the invention relates to a method/platform for gene diagnosis. A method in accordance with one embodiment of the invention comprises using magnetic beads coupled with a probe to react with a sample to detect presence or absence of a target gene, wherein the probe can hybridize with a fragment of the target gene. The target gene is KRAS and the probe is designed to detect G12D mutation in KRAS. The probe is coupled with biotin for interaction with streptavidin-R-phycoerythrin to permit multiplexing illumine detection for fluorescence intensity and quantity. The probe has the sequence of 5’-
TTGGAGCTGAT GGCGT AGGC A-3’ (SEQ ID NO: l) for detection of KRAS G12D mutation or 5’- TT GGAGCTGGT GGCGT AGGC A -3’ (SEQ ID NO:2) for detection of wild-type
KRAS, wherein the 5’ end of the probe is modified with an amino group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a schematic illustrating various factors involved in CSF-1R signaling. Based on analysis of gene databases from various cancer cells, it was found that PIK3CA and KRAS have high mutation rates in breast cancer and colorectal cancer. The incidence rates for PIK3CA mutations in various cancers are shown, as well as incidence rates for KRAS mutations in various cancers.
[0013] FIG. 2 shows a schematic illustrating various factors involved in CSF-1R signaling. Based on analysis of gene databases from various cancer cells, it was found that PTEN and BRAF have high mutation rates in glioma and thyroid cancer. The incidence rates for PTEN mutations in various cancers are shown, as well as incidence rates for BRAF mutations in various cancers.
[0014] FIG. 3 shows results from cross analyses of PIK3CA, KRAS, PTEN, and BRAF mutations. The results from these analyses revealed that among the colorectal cancers and thyroid cancers, the rates of having two or more mutations impacting these gene functions are as high as 40% among all different races.
[0015] FIG. 4 shows the results from hybridization between magnetic beads that are coupled with a probe and a target KRAS G12D sequence.
[0016] FIG. 5 shows results from probe specificity tests. A Kras WT probe (0.2 nmol) is allowed to hybridize with either the wild-type Kras or a mutant Kras GAT (mGl2D). The results show that the probe of the invention is specific.
[0017] FIG. 6A shows results from testing optimal hybridization temperatures at 0.2 pmol of wild-type and mutant Kras. Magnetic beads with wild type Kras probe are hybridized with either the wild-type Kras or a mutant Kras GAT (mGl2D) sequence at different temperature and the median fluorescence intensity (MFI) values are measured to assess the hybridization. The results show that the hybridization proceed better and produce more stable results at higher temperatures, e.g., 60-80 °C. FIG. 6B shows results from testing optimal hybridization temperatures at 0.05 pmol of wild-type and mutant Kras. FIG. 6C shows results from testing optimal hybridization temperatures at 0.005 pmol of wild-type and mutant Kras. [0018] FIG. 7 A shows results from testing for optimal hybridization times. Magnetic beads with a mutant Kras Mt probe are hybridized with a mutant Kras target sequence for different hybridization times and the median fluorescence intensity (MFI) values are measured to assess the hybridization. The results show that a hybridization time of about 10 minutes proceed better and produce more stable results.
[0019] FIG. 7B shows results from testing for optimal hybridization times. Magnetic beads with a mutant Kras Mt probe are hybridized with a wild-type Kras target sequence for different hybridization times and the median fluorescence intensity (MFI) values are measured to assess the hybridization. The results show that a hybridization time of about 10 minutes proceed better and produce more stable results.
DETAILED DESCRIPTION
[0020] Embodiments of the invention relate to diagnostic techniques for predicting therapeutic efficacy. Methods of the invention can be used as companion diagnosis to screen patients for subpopulations that would respond to a particular therapy, thereby increasing the probability of therapeutic success and avoiding waste of medical resources.
[0021] In accordance with embodiments of the invention, a method for predicting therapeutic efficacy of a drug may include the step of analyzing a panel of genes to derive information for predicting whether a patient will respond to the drug. The analyzing a panel of genes may include analysis of gene mutations, copy number variations, and/or expression levels.
[0022] Methods of the invention will be illustrated with limited number of examples.
However, one skilled in the art would appreciate that these examples are for illustration only and are not intended to limit the scope of the invention. In particular examples, CSF-1R inhibitor therapy will be described. For CSF-1R inhibitor therapy, the companion diagnosis may include analysis of genes that are involved in the CSF-1R signaling pathways. Such genes, for example, may include PIK3CA, KRAS, PTEN, and BRAF.
[0023] Cancer therapy resistance are found to be associated with the presence of colony stimulating factor- 1 (CSF1) and CSF1 receptor (CSF-lR)-driven tumor-infiltrating macrophages. Therefore, CSF-1R inhibition has been used to target tumor-associated macrophages in cancer therapy. However, the effects of CSF-1R inhibition treatments vary among patients of different races, suggesting that different genetic backgrounds may play an important role. [0024] By analyzing various gene databases for genes relating to CSF-1R actions, it was found that mutations of genes immediately downstream from CSF-1R in the signaling pathways, such PIK3CA, KRAS, PTEN, and BRAF, may have impacts on the efficacy of CSF- 1R inhibitor therapy. Mutations of these genes are frequently associated with various cancers. By analysis of mutations in these gene products that may impact their functions, several potential mutations, including E542K, E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R, are found that might have impacts on CSF-1R inhibitor therapy. By analyzing the occurrence rates of these mutations in various cancer cells, one can arrive at valuable information that can be used to predict therapeutic efficacy of CSF-1R inhibitor treatments.
[0025] Embodiments of the invention will be further illustrated with the following specific examples. One skilled in the art would appreciate that these examples are for illustration only and are not intended to limit the scope of the invention. One skilled in the art would appreciate that modifications and variations from these examples are possible without departing from the scope of the invention.
Example 1: Gene Mutations related to CSF-1R I nhibitor Actions
[0026] Cancer therapy resistance has been found to be associated with the presence of colony stimulating factor- 1 (CSF1)/CSF1 receptor (CSFIRVdriven tumor-infiltrating macrophages. Therefore, CSF-1R inhibition has been used to target tumor-associated macrophages in cancer therapy. However, the effects of CSF-1R inhibition treatments seem to vary among patients of different races, suggesting that genetic backgrounds may play an important role. Therefore, analyzing genes and mutations related to CSF-1R signaling pathways may provide information to help predict therapy outcomes.
[0027] Several human cancer gene databases were analyzed for genes, mutations in which can impact CSF-1R signaling and the effects of CSF-1R inhibition therapy. The analysis focuses on the impact of mutations on functions. Cross statistical analysis may be used to assess the rates of the gene mutations that are also present in cancer patients. Results from such analyses can be used to predict which drug would be suitable for the patients.
[0028] The cancer gene databases used for such analysis may include GENIE (AACR
Project Genomics Evidence Neoplasia Information Exchange), TCGA (The Cancer Genome Atlas), and ICGC (The International Cancer Genome Consortium). The analysis may use any suitable tool, such as the cBioPortal platform (v.1.8.3) (http://www.cbioportal.org/index.do). cBioPortal for Cancer Genomics is a tool developed at Memorial Sloan Kettering Cancer Center’s Computational Biology Center (cBio).
[0029] In this invention, analysis was performed on CSF-1R signal transduction related genes. These genes, for example, include CSF1R, PIK3CA, PTEN, KRAS, and BRAF. The mutation rates of these genes were analyzed with respect to various cancers (glioma, oral cancer, thyroid cancer, lung cancer, breast cancer, stomach cancer, liver cancer, biliary tract cancer, colorectal cancer, ovary cancer, and uterus endometrial cancer) in patients of different races.
Predicting the Effects of CSF-1R Inhibitors
[0030] By analyzing the signaling pathways associated with phosphorylation of the
CSF-1R protein, together with gene network analysis, on the cBioPortal platform, it was found that PIK3CA and KRAS, which are the closest to the CSF-1R in the signaling pathways (FIG. 1), have the highest mutation rates.
[0031] Analysis of the GEINE database for various cancer tissues, which are mostly from patients from the ET.S. and Europe, revealed that PIK3CA and KRAS have more than 5% mutation rates among all cancers. The mutation rate of PIK3CA is about 31.3% in breast cancer, and the mutation rate for KRAS in non-small cell adenocarcinoma is about 27%. The mutations of PIK3CA and KRAS are accompanied by increases in the CSF-1R activity. These mutations are also related to the lack of responses to treatments in the clinics.
Evaluating effects of point mutations in PIK3CA and KRAS on drug efficacies
[0032] That these mutations are related to drug efficacies may be confirmed by analysis of protein structures and literature information. The GEINE, and TCGA cancer databases may be used to analyze the mutation rates. It was found that in breast cancer tissues, PIK3CA mutations that impact protein functions (E542K, E545K, and H1047R) account for about 20% of the patients, regardless of the races. (FIG. 1).
[0033] The point mutations in KRAS (G12C, G12D, G12V, and G13D) that have impacts on drug efficacies are found to have higher mutation rates in colorectal cancer. The mutation rates are about 33% for all races. (FIG. 1). These rates of mutations can predict the percentages of patients who will not respond to CSF-1R inhibitor treatments. These mutations are also found in Asian biliary tract cancer and Caucasian lung cancers at the rates of 19% and 21%, respectively. (FIG. 1). These rates also can predict the percentages of patients who will not respond to CSF-1R inhibitor treatments.
[0034] In the CSF-1R signaling pathways, PTEN and BRAF are the actors that interact directly with PIK3CA and KRAS, respectively. Therefore, mutations that affect PTEN (e.g., R130G, C71F/Y, and deletions) and BRAF (V600E) functions are expected to have a negative impact on the effects of CSF-1R inhibitors. Analyzing the potential impacts of these mutations on cancer treatments, it was found that these may affect up to 9% glioma patients among Caucasians and up to 50% thyroid cancers patients among Caucasians and Asians. (FIG. 2). Thus, these mutation analyses may be used to predict the percentages of patients that may not respond to the CSF-1R inhibitor treatments.
Prediction of impacts on CSF-1R inhibitor treatment effects based on integrated mutation analysis
[0035] In order to better deal with unexpected situations in the clinics, cross analyses of PIK3CA, KRAS, PTEN, and BRAF mutations were performed. These analyses particularly look for two or more mutations that impact the functions of these genes. The results from these analyses revealed that among the colorectal cancers and thyroid cancers, the rates of having two of more mutations impacting these gene functions are as high as 40% among all different races. (FIG. 3). These results can be used to predict the percentages of patients that will not respond to CSF-1R inhibitor treatments.
CSF-1R Mutations
[0036] In addition to the above CSF-1R signaling pathway genes, mutations in CSF-1R itself is expected to impact the efficacy of CSF-1R inhibitor treatments. Based on analysis of genetic information at Taiwan Biobank, it was reported that among the population in Taiwan, 42% has an H362R mutation in CSF-1R. This mutation may lead to enhanced or attenuated response to CSF-1R inhibitor treatments.
[0037] The above analyses show that various factors in the CSF-1R signaling pathways play roles in CSF-1R inhibition treatments. These factors include CSF-1R, PIK3CA, PTEN, KRAS, and BRAF. Analyzing functional mutations that affect the functions of these factors may provide information to predict which patients would benefits from CSF-1R inhibition treatments and which patients would not.
Example 2: Use of cell lines to establish methods for analyzing gene mutations [0038] This example illustrates the use of cell lines to establish methods for the analysis of gene mutations. First, genomic DNAs were obtained from lung cancer cells A549, H727, HCC-827, H1975, NCI-H146, H460, and H292. Based on these genomic DNAs, gene fragments for CSF-1R, PIK3CA, KRAS, and PTEN were obtained using polymerase chain reaction (PCR). The sequences of these gene fragments were determined using Sanger’s sequencing methods, and then the sequences were compared with the sequences of the same fragments from normal cells. The comparison would reveal any nucleotide differences. The mutation locations were further confirmed. The procedures for sequence determination and analysis are as follows:
Preparation of genomic DNA
[0039] Genomic DNA extraction was performed using DNeasy Blood & Tissue Kit
(QIAGEN, Cat No. 69581). Briefly, the target lung cancer cells were collected in centrifuge tubes. The cells were washed with lx PBS to remove DMSO used to preserve cells in the frozen aliquots. The tubes were centrifuged, and the top clear solutions were discarded. To the cell pellets in the tubes were added 180m1 ATL lysis buffer (Qiagen) and 20m1 proteinase K. Mix and resuspend the cell pellets. Place the tubes in an oven at 56°C for 4 hours to digest proteins. After protein digestion, add 200m1 AL lysis buffer to the tubes and mix well. Then, add 200m1 pure ethanol and mix well.
[0040] Centrifuge the tubes in a microfuge for 15 seconds. Collect the top clear liquid and load it on a DNeasy Mini spin column (Qiagen). Centrifuge the spin column at 8000 rpm for 1 minute. Discard the lower liquid layer. Then, add 500m1 AW1 wash buffer, followed by centrifugation at 8000 rpm for 1 minute. Discard the lower liquid layer. Add 500m1 AW2 wash buffer, followed by centrifugation at 14000 rpm for 3 minutes. Discard the lower liquid layer. Centrifuge one more time at high speed (14000 rpm) for 3 minutes to remove the residual buffer.
[0041] Place the spin columns on top of 1.5 ml microcentrifuge tubes. Add 200m1 AE elute buffer on top of the columns. Allow the tubes to sit for 1 minute at room temperature before centrifuging the tubes at 8000 rpm for 1 minute. The liquid collected in the microfuge tubes are the samples containing the extracted genomic DNA.
Polymerase Chain Reaction (PCR)
[0042] The polymerase chain reactions were performed using PrimeSTAR GXL DNA
Polymerase (TAKARA). Specific primers for CSF-1R, PIK3CA, KRAS and PTEN are shown in TABLE 1. The amplification reaction conditions are described below. The desired DNA fragments were amplified and analyzed with electrophoresis to confirm the fragments and concentrations.
DNA Amplification
[0043] DNA sequencing was performed using Sanger’s method. Sequence analysis of the PCR products were performed using BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Each reaction uses 50 ng primers and l pl BigDye mixture, 10 ng PCR product, and I mΐ 5x reaction buffer. PCR was performed in GeneAmp PCR System 2700 thermocycler (Applied Biosystems, Foster City, CA). The reaction conditions are: 96°C initial denaturation for 1 minute, 96°C denaturation for 10 seconds, 50°C annealing for 10 seconds, repeat for 45 cycles, and then extend at 60°C for 4 minutes.
[0044] The PCR products were sequenced using ABI 3730XL DNA Analyzer (Applied
Biosystems), which can sequence to 700-800 bps on average. Compare the sequences of CSF- 1R, PIK3CA, KRAS and PTEN from lung cancers cells with those from normal cells to determine the differences. Then, confirm the mutation locations.
TABLE 1 : Primers
Figure imgf000011_0001
[0045] In sum, genes relating to CSF-1R signaling from lung cancer cell lines A549,
H727, HCC-827, H1975, NCI-H146, H460, and H292 have been analyzed. Mutations in CSF-1R, PIK3CA, KRAS, and PTEN are found (FIG. 1 and FIG. 2). Results from these analyses shows that most genes are wild-type genes. Genes from lung cancer cells A549 and H727 have mutations at G12 (Table 2). These mutations can be accurately determined.
[0046] The above example shows that one can analyze different cell lines for genetic information. Based on different genetic background information, one can predict or evaluate the effects of therapeutic treatments.
TABLE 2
Figure imgf000012_0003
Figure imgf000012_0001
[0047] Results from gene sequence analysis of lung cancer cell lines A549, H727,
HCC-827, and H1975, and mutations observed for CSF-1R, PIK3CA, KRAS and PTEN. The results show that most genes are wild-type, and only KRAS from A549 and H727 have mutations at G12 location, PI3K from HCC-827 and H1975 have mutations at E545.
Figure imgf000012_0002
[0048] Sequence analysis of lung cancer cell lines NCI-H146, H460 and H292, looking for mutations in CSF-1R, PIK3CA, KRAS and PTEN. Most genes analyzed are found to be wild-type, and only PI3K from H460 has mutation at E545
Example 3 : Development of detection methods for multiple genes
[0049] This example describes gene detection and analysis methods based on Luminex xMAP (Multi-Analyte Profiling; Luminex Corp., Austin, TX), gene-probe design, and hybridization techniques. First, gene probes for the target DNA sequences (e.g., CSF-1R, PIK3CA, KRAS, BRAF, and PTEN) are designed and synthesized. Then, the gene probes are used to detect specific target genes as follows. Select the gene probes that are specific for the target genes. Couple the selected gene probes on specific magnetic beads. Mix fluorescent magnetic beads for different assay samples. Add the amplified fragments of the assay samples and detect them with fluorescence labeled probes on the magnetic beads. Then, use flow cytometry to detect the types of magnetic beads and measure the fluorescence intensities.
[0050] This method can provide multiple genes detection and can achieve fast and accurate detection. The methods can be used in the clinics.
Example 4: Detection of KRAS gene mutations in cancers
[0051] In clinics, KRAS mutations are often associated with colorectal cancer, pancreatic cancer, lung cancer, etc. The most frequent KRAS mutations are found at positions G12 and G13. The mutations at G12 are more common than those at G13, making the G12 mutations more relevant in the therapeutic responses.
[0052] An object of the invention is to provide methods for detecting G12 mutations in
KRAS. Embodiments of the invention are based on the Liminex xMAP (Multi- Analyte Profiling) principle and make use of magnetic bead probes to detect gene mutations. A method of the invention may include the steps of: expansion of the test sample, gene-probe design, and hybridization reactions. Briefly, a gene probe is coupled on magnetic beads and then added to the test sample that has been expanded (e.g., using PCR). The probe or beads may contain fluorescent tags, which would allow one to differentiate different magnetic beads using flow cytometry. The different magnetic beads permit differentiation of mutant or wild-type genes. In addition, the fluorescence intensities may be used to quantify the species. These methods allow one to perform gene diagnosis with speed and accuracy, as well as quantitation. [0053] An exemplary method will be described for illustration. However, one skilled in the art would appreciate that this example is for illustration only and that other modifications and variations are possible without departing from the scope of the invention.
Primer and Probe Design
[0054] The primers for Kras gene detection can be derived from the Kras sequence in the literature with some modifications. For example, a biotin can be attached to the 5’ end of the reverse primer, as shown in Table 4. In addition, based on the target gene, a wild-type (Wt) probe and a KrasG12D (GAT) mutant-type (Mt) probe may be designed, as shown in Table 5. These primers are 21 nucleotide long, and the 5’ end of these primers are modified with an amino group.
TABLE 4: Kras primers
Figure imgf000014_0001
TABLE 5 : Probe designs for Kras detection
Figure imgf000014_0002
Sample varieties and Processing
[0055] DNA extracts are prepared from human cell lines or patient-derived xenograft
(PDX) samples. Use specific modified primers and PCR to expand specific Kras gene fragments and tag them with fluorescence markers.
Beads conjugated with Kras wild-type (Wt), KrasG12D (GAT) mutant-type (Mt) probe
[0056] Beads with different codes (e.g., Beads 012 and 078) are mixed well in 0.1M 2-
(N-morpholono) ethanesulfonic acid (MES) buffer. Each different coded beads are coupled with a specific probe (0.2 nmol) using 10 mg/ml l-ethyl-3-3 (3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The coupling reaction was performed at room temperature for 30 minutes. The EDC coupling reaction is repeated one more time. Then, 0.5 ml 0.02% Tween 20 is added and mixed well. The mixture is then centrifuged at 14000 rpm for 3 minutes. The upper layer clear solution is discarded. To the tube, 0.5 ml 0.1% SDS is added and mixed well. It was centrifuged again and the upper layer clear solution is discarded. A suitable volume of Tris-EDTA solution is added to re-dissolve the beads.
Hybridization
[0057] With the above-described procedures, two sequences, Kras wild-type (Wt) and
KrasG12D (GAT) mutant-type (Mt), are synthesized. The 5’ ends of these sequences are tagged with biotin. The two sequence fragments are 59 b.p. long, which serve as the standards for constructing the analysis platform.
[0058] The hybridization reactions are conducted in 96-well PCR plates. Each well is loaded with 2500 beads, which are mixed with the above-described synthetic sequences. The total volume per well is 50 ul. The hybridization is performed in a PCR machine (Biometra Tadvanced). After hybridization, the samples in the PCR plate are transferred into a 96-well dark plate. With the aid of a magnetic plate, the upper layer clear solutions are discarded. Streptavidin-R-phycoerythrin (75 ul/well) was added to each well, and the binding reaction was allowed to proceed at room temperature for 30 minutes. The median fluorescence intensity (MFI) of the magnetic beads was measured using a Magpix equipment (Luminex).
TABLE 6: Synthetic sequences of Kras wild-type (Wt) and KrasG12D (GAT) mutant-type (Mt)
Figure imgf000015_0001
Beads-Probe Coupling Efficiency
[0059] To assess the sensitivity of the probes, magnetic beads coupled with the
KrasG12D Mt probe are hybridized with different concentrations (0.005, 0.025, 0.05, 0.1, and 0.2 pmol) of the KrasG12D Mt sequence. The hybridization was performed at 95 °C for 5 minutes and then at 52°C for 30minutes. As shown in FIG. 4, KrasG12D Mt probe can detect KrasG12D Mt sequence in the range of 0.025-0.1 pmol, with a lowest detection limit of 0.025 pmol.
Probe Specificity
[0060] To assess the specificity of the probes, magnetic beads coupled with the Kras
Wt probe are hybridized with different concentrations (0.005, 0.025, 0.05, 0.1, and 0.2 pmol) of the Kras Wt and KrasG12D Mt sequences. The hybridization was performed at 95 °C for 5 minutes and then at 52°C for 30minutes. As shown in FIG. 5, Kras Wt probe can hybridize with the Kras Wt sequence with a higher affinity than with the KrasG12D Mt sequence. Thus, the magnetic probes are specific.
Optimal Hybridization Conditions
Hybridization temperature
[0061] To find the best hybridization temperature, magnetic beads coupled with the
Kras Wt probe are separately hybridized with low, medium, and high concentrations (0.005, 0.05, and 0.2 pmol) of the Kras Wt and KrasG12D Mt sequences, respectively. The hybridization was performed at 95°C for 5 minutes and then at 23.3 - 75°C for 30minutes. As shown in FIG. 6, at high hybridization temperatures, the MFI values show that Kras Wt probe can form more stable hybrids with both the Kras Wt and KrasG12D Mt sequences, as compared with at low hybridization temperatures. Therefore, a preferred hybridization temperature for a detection platform of the invention would use a relatively high hybridization temperature, such as 65-80°C, more preferably around 70°C (e.g., 70.6°C).
Hybridization Time/Duration
[0062] To find the best hybridization duration, magnetic beads coupled with the Kras
Wt probe are separately hybridized with low, medium, and high concentrations (0.005, 0.05, and 0.2 pmol) of the Kras Wt and KrasG12D Mt sequences, respectively. The hybridization was performed at 95°C for 5 minutes and then at 70.6°C to observe hybridizations at different reaction times (1, 5, 10, 15, 20, and 30minutes). As shown in FIG. 7A and FIG. 7B, with a hybridization time of 10 minutes, KrasG12D Mt probe at medium and high concentrations (0.05 and 0.2 pmol) can hybridize with Kras Wt and KrasG12D Mt sequences to produce the best MFI values, as compared with other hybridization time. Thus, with a shorter hybridization time (10 minutes instead of 30 minutes), one can obtain better detection signals. Accordingly, the hybridization time is preferably selected for 10 minutes for the detection platform of the invention.
Accuracy of the Detection Platform
[0063] The accuracy of the detection platform can be tested as follows. Human cell lines and patient-derived xenograph (PDX) samples are expanded with PCR to obtain the target gene fragments. Then, using the Quick Microbeads Kras Gene Detection Platform to detect the gene states. The detection samples have been analyzed and their gene sequences determined. Therefore, they can be used as the standards for assessing the detecting platform. Then, the samples are analyzed in 10 blind tests, each time with 10 PCR products and each sample is tested in quadruples. From these tests, it was found the detection platform or methods of the invention have an accuracy of 99% after testing 100 PCR products. Thus, a detection platform/method of the invention has the advantages of system stability and detection accuracy.
[0064] Embodiments of the invention have been illustrated with a limited number of examples. One skilled in the art would appreciate that these examples are for illustration only and are not meant to limit the scope of the invention because other modifications and variations are possible without departing from the scope of the invention. Therefore, the scope of protection should only be limited by the attached claims.

Claims

CLAIMS What is claimed is:
1. A method for predicting therapeutic efficacy of a drug, comprising: analyzing either an individual gene or a panel of genes to derive information for predicting whether a patient will respond to the drug.
2. The method according to claim 1, wherein the analyzing either an individual gene or a panel of genes includes analysis of gene mutations, copy number variations, and/or expression levels.
3. The method according to claim 2, wherein the panel of genes comprises PIK3CA, KRAS, PTEN, BRAF, and CSF-1R.
4. The method according to claim 3, wherein the gene mutations comprise E542K,
E545K, and H1047R mutations in PIK3CA, G12C, G12D, G12V, G13D mutations in KRAS, R130G and C71F/Y mutations or deletion in PTEN, V600E mutation in BRAF, and H362R mutation in CSF-1R.
5. The method according to claim 1, wherein the drug is a CSF-1R inhibitor or an
immunomodulatory agent.
6. The method according to claim 5, wherein the CSF-1R inhibitor or an
immunomodulatory agent is a small molecule drug, a biologic, or a nucleotide.
7. The method according to claim 6, wherein the nucleotide is siRNA or miRNA.
8. The method according to claim 1, wherein the drug comprises a drug targeting a protein translated from a gene in the panel of genes.
9. The method according to claim 8, wherein the drug is a PIK3CA inhibitor, a KRAS inhibitor, a PTEN inhibitor, or a BRAF inhibitor.
10. The method according to claim 1, the analyzing makes use of multiplexing illumine, real-time polymerase chain reaction (PCR), next-generation sequencing (NGS), gene chips, micro fluidics, flowcytometry, or a combination thereof.
11. The method according to claim 1, wherein the analyzing either an individual gene or a panel of genes is performed simultaneously in a multiplex format.
12. A method for gene diagnosis, comprising using magnetic beads coupled with a probe to react with a sample to detect presence or absence of a target gene, wherein the probe can hybridize with a fragment of the target gene.
13. The method according to claim 12, wherein the target gene is KRAS and the probe is designed to detect G12D mutation in KRAS.
14. The method according to claim 12, wherein the probe is coupled with biotin for
interaction with streptavidin-R-phycoerythrin to permit multiplexing illumine detection for fluorescence intensity and quantity.
15. The method according to claim 12, wherein the sample is expanded using polymerase chain reaction (PCR) prior to detection.
16. The method according to claim 15, wherein the polymerase chain reaction (PCR) uses 5’ -CTGAATATAAACTTGT GGTAGTTGGA-3’ (SEQ ID NO: 13) as a forward primer and 5’- TATCGTC AAGGC ACTCTTGC-3’ (SEQ ID NO: 14) as a reverse primer, wherein the reverse primer has a biotin coupled to the 5’ end.
17. The method according to claim 12, wherein the probe has the sequence of 5’- TTGGAGCTGATGGCGTAGGCA-3’ (SEQ ID NO: l) for detection of KRAS G12D mutation or 5’- TT GGAGCTGGTGGCGTAGGC A -3’ (SEQ ID NO:2) for detection of wild-type KRAS, wherein the 5’ end of the probe is modified with an amino group.
18. The method according to claim 15, wherein the hybridization of the magnetic beads with the sample is performed in a PCR reaction vessel, and wherein a hybridization temperature is at a temperature between 60-80°C and a hybridization duration between 1-30 minutes.
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