WO2021041762A1

WO2021041762A1 - Kit and methods to detect egfr variant iii

Info

Publication number: WO2021041762A1
Application number: PCT/US2020/048329
Authority: WO
Inventors: An Hsu; Pei-Yi Lin; Datsen George WEI; Shu-Jen Chen; Hua-Chien Chen
Original assignee: An Hsu; Lin Pei Yi; Wei Datsen George; Chen Shu Jen; Chen Hua Chien
Priority date: 2019-08-28
Filing date: 2020-08-28
Publication date: 2021-03-04

Abstract

Provided is a kit for detecting epidermal growth factor receptor variant III (EGFRvIII). The kit includes an EGFRvIII fusion-specific primer pair and an EGFRvIII fusion-specific probe with high specificity. Also provided is a method for detecting EGFRvIII, including generating an amplified target cDNA to hybridize with the EGFRvIII fusion-specific probe and detecting the probe-bound product to identify EGFRvIII in a biological sample.

Description

KIT AND METHODS TO DETECT EGFR VARIANT III

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority of Provisional Application No. 62/893, 152, filed on August 28, 2019, the content of which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[0002] The invention relates to a kit and a method for molecular diagnostics and genomics. Particularly, the invention relates to a kit and a method for molecular diagnostics and genomics of cancers.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created August 27, 2020, is named “ACTG-5PCT_SEQList_ST25.txt” and is 3,080 bytes in size.

BACKGROUND OF THE INVENTION

[0004] The EGFR gene codes for epidermal growth factor receptor, which is a tyrosine kinase receptor. When this receptor binds to its ligand, it dimerizes and activates downstream pathways/signaling molecules, such as the Ras/Raf/MAPK pathway, the PI3K pathway, phospholipase C, or STAT. The activation of these molecules and pathways promotes cell survival, growth, and proliferation.

[0005] Genomic alterations in EGFR may result in overexpression or constitutive activation of its downstream signaling pathways, ultimately leading to cancers. Among EGFR mutations, EGFR variant III (EGFRvIII) appears to be the most common deletion mutation found to date. EGFRvIII is the deletion of exons 2 to 7, resulting in the fusion of exons 1 and 8 in the EGFR gene, and it has been commonly found in 17% of all gliboastoma (GBM), as well as in lung cancer, breast cancer, and head and neck cancer. This deletion results in a mutated EGFR protein that is constitutively activated and incapable of binding to any ligand.

[0006] Several therapeutics targeting EGFR have been developed, including the first, second, and third-generation EGFR tyrosine kinase inhibitors (TKIs) and EGFR antibody therapies. Many cancer patients with EGFR mutations respond to these treatments. However, EGFRvIII harboring patients are resistant to them. Patients harboring EGFRvIII are typically resistant to EGFR targeted therapies, such as gefitinib, afatinib, erlotinib, dacomitinib and cetuximab. Therefore, it is important to develop a method for detecting EGFRvIII with high sensitivity and specificity in order to identify the patients who may be resistant to EGFR targeted therapies and require alternative treatments.

SUMMARY OF THE INVENTION

[0007] The present disclosure concerns a method for detecting EGFRvIII. The method includes the steps of:

(a) obtaining ribonucleic acids (RNA) from a biological sample;

(b) performing reverse transcription of the RNA to obtain complementary deoxyribonucleic acids (cDNA);

(c) amplifying the cDNA with an EGFRvIII fusion-specific forward primer and an EGFRvIII fusion-specific reverse primer to obtain an amplified product;

(d) mixing the amplified product with a probe to obtain a probe-bound product, wherein the probe has the nucleotide sequence of SEQ ID NO:l or a complementary sequence thereof; and

(e) detecting the probe-bound product to determine the presence of EGFRvIII.

[0008] To ensure sufficient production of the amplified product, an additional round of DNA amplification may be performed in the aforementioned step (c) using a pair of universal primers. Accordingly, in some preferred embodiments, in step (c) the cDNA is amplified first with the EGFRvIII fusion-specific forward primer and the EGFRvIII fusion-specific reverse primer and subsequently with a universal forward primer and a universal reverse primer to obtain the amplified product. In this setting, the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of the universal forward primer, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of the universal reverse primer.

[0009] In another aspect, a kit is also provided for detecting EGFRvIII according to the aforementioned method. The kit includes an EGFRvIII fusion-specific forward primer, an EGFRvIII fusion-specific reverse primer, and a probe having the nucleotide sequence of SEQ ID NO:l or a complementary sequence thereof.

[0010] In some preferred embodiments, the kit further includes a universal primer pair. In this setting, the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair.

[0011] In some embodiments, the kit further includes a reverse transcriptase for reverse transcription of the RNA isolated from the biological sample, and also includes a DNA polymerase for amplification of the cDNA generated by the reverse transcription.

[0012] The probe used in the method and the kit can target EGFRvIII with high specificity, ensuring accurate detection of EGFRvIII in biological samples. Moreover, the use of both an EGFRvIII fusion-specific primer pair and a universal primer pair for DNA amplification can increase the yield of the particular amplified product. Thus, application of the method and the kit in EGFRvIII detection can reduce the need for large sample volume.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiments, with reference to the attached drawings, in which:

[0014] FIG. 1 is a schematic illustration of the method for detecting EGFRvIII according to one embodiment of the invention; the detection is based on a single-amplified target-probe- barcoded magnetic bead (BMB) hybridization assay; and

[0015] FIG. 2 is a schematic illustration of the method for detecting EGFRvIII according to one embodiment of the invention; the detection is based on a double-amplified target-probe hybridization assay.

DETAILED DESCRIPTION

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

Definition [0017] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0018] The term “EGFRvIII” refers to a phenomenon where the EGFR gene has an in-frame deletion of 801 nucleotides, corresponding to exons 2-7, which results in the fusion of exons 1 and 8 in the EGFR gene. This EGFR mutation causes deletion of amino acids 6 through 273 in the extracellular domain of the EGFR protein, and adds a glycine at the fusion site. Thus, the terms “EGFRvIII” and “EGFRvIII fusion” are used interchangeably herein. The EGFRvIII gene has a “fusion junction,” which is the site where EGFR exon 1 (also called 5’ partner) fuses to EGFR exon 8 (also called 3’ partner). The fusion junction is located in a fusion region defined by a fusion sequence (also called a fusion junction sequence), which encompasses the sequence from the EGFR exon 1 and the sequence from EGFR exon 8.

[0019] EGFRvIII may be detected by identifying the fusion region in a DNA or in an RNA transcript of that DNA. In this disclosure, the fusion region to be identified is present in an RNA transcript.

[0020] The term “primer” refers to a synthetic single-stranded oligonucleotide that can be used to amplify a target nucleic acid having a specific length. As used herein, the terms “EGFRvIII fusion-specific primer” and “fusion specific primer” are used interchangeably, which refer to a DNA primer that is designed to amplify a target cDNA including a fusion junction originates from the EGFRvIII gene. The EGFRvIII fusion-specific primers are used in pairs, including an EGFRvIII fusion-specific forward primer capable of specifically binding to the 5 ’-end of a target cDNA, and an EGFRvIII fusion-specific reverse primer capable of specifically binding to the 3 ’-end of said target cDNA.

[0021] As used herein, the term “universal primer” refers to a DNA primer that is designed to amplify any DNA including the nucleotide sequence of the universal primer. The universal primers are used in pairs, including a universal forward primer and a universal reverse primer.

[0022] Unless defined otherwise, the term “probe” or “EGFRvIII fusion-specific probe” refers to a synthetic single-stranded DNA oligonucleotide that can hybridize to the fusion region originates from the EGFRvIII gene. [0023] As used herein, a “connector” or a “linker” refers to part of a molecule or part of a complex of molecules that connects one molecule to another. The connector or linker can act by covalent bonding, nucleic acid hybridization, or non-covalent interaction between a pair of molecules such as biotin-streptavidin interaction. In this disclosure, a “connector” is used to conjugate a primer with a detectable molecule such as a fluorescent molecule; and a “linker” is used to form linkage between a probe and a detectable molecule or a unique identifier such as a barcoded magnetic bead (BMB).

[0024] In the present disclosure, a method for detecting EGFRvIII is provided. The method includes the steps of:

(a) obtaining RNA from a biological sample;

(b) performing reverse transcription of the RNA to obtain cDNA;

(e) detecting the probe-bound product to determine the presence of EGFRvIII.

[0025] In step (a) of the disclosed method, RNA is prepared from a biological sample. The biological sample may be any sample obtained from an animal and a human subject. Examples of the biological samples include a formalin-fixed paraffin-embedded (FFPE) tissue section, blood, plasma, or cells. In some embodiments, the biological sample originates from a cancer patient. In some embodiments, the biological sample originates from a solid tumor, soft tissue sarcoma, or a hematological cancer. In some embodiments, the biological sample originates from a patient with glioblastoma, lung cancer, breast cancer, or head and neck cancer.

[0026] Preparation of total RNA from the biological sample can be carried out by various methods known in the art. One typical procedure is RNA extraction with organic solvents such as phenol/chloroform and precipitation by centrifugation. There are also commercially available kits for RNA isolation or purification. Once the RNA is obtained, a reverse transcriptase is used along with four kinds of deoxyribonucleoside triphosphates (dNTP, including dATP, dCTP, dTTP, and dGTP) to generate cDNA from the template RNA, a process called reverse transcription. The reverse transcription may be conducted using Superscript cDNA synthesis kit (Cat No: 11754050, Invitrogen).

[0027] In step (c) of the disclosed method, the cDNA is amplified with a DNA polymerase and a pair of EGFRvIII fusion-specific primers to obtain an amplified product for probe detection. The amplification may be conducted using a multiplex PCR kit (Cat No: 206143, Qiagen) which includes a DNA polymerase. The EGFRvIII fusion-specific primer pair may be provided as a regent before use.

[0028] In some preferred embodiments, in step (c) the cDNA is amplified first with the EGFRvIII fusion-specific forward primer and the EGFRvIII fusion-specific reverse primer and subsequently with a universal forward primer and a universal reverse primer to obtain the amplified product. In this setting, the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of the universal forward primer, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of the universal reverse primer.

[0029] In step (d) of the disclosed method, the amplified product is mixed with the probe so that a probe-bound product can form through nucleic acid hybridization. Because the probe is specifically designed based on the fusion sequence of the EGFRvIII fusion, the presence of EGFRvIII can be determined by detecting the probe-bound product.

[0030] Table 1 lists the specifically designed probe for detecting EGFRvIII. These probes have been demonstrated to be specific to the EGFRvIII fusion and does not cross-reacts with other sequences, allowing accurate detection of the EGFRvIII fusion. In some embodiments, both the probe having the sequence of SEQ ID NO: 1 and the probe having a complementary sequence are used to enhance detection efficiency.

TABLE 1

[0031] In some embodiments, EGFRvIII is the only target to be detected after one target cDNA is amplified and probed. In other embodiments, EGFRvIII in combination with other genomic mutations can be detected simultaneously after two or more target cDNAs with difference sequences are amplified in one reaction (called a multiplex amplification reaction) and/or probed in one reaction (called a multiplex hybridization reaction). When the disclosed method is performed in the multiplex setting, at least two probes are applied, i.e., one probe for detecting EGFRvIII and another probe for detecting other genomic mutations.

[0032] Typically, the probe and the amplified product are mixed at a specific temperature to facilitate probe hybridization. In some embodiments, the temperature for hybridization is between 35-50°C, 40-50°C, 40-45°C, or 45-50°C. In some embodiments, the hybridization is performed by using a thermomixer at a rotation speed between 700-1000 rpm, 750-1000 rpm, 800-1000 rpm, 900-1000 rpm, 700-750 rpm, 700-800 rpm, 750-800 rpm, or 800-900 rpm.

[0033] Detection of the probe-bound product may be accomplished by detecting the EGFRvIII fusion-specific primers, the universal primers, or the probe in said product. Thus, the primers or probes are usually modified to be detectable. They may be modified to have fluorescence or chemiluminescence activity or become chromogenic or colorimetric by being connected directly or indirectly to a detectable molecule. In some embodiments, one or both primers in the primer pair are connected to biotin or other compounds capable of binding to a streptavidin-conjugated detectable molecule. The detectable molecule may be a dye, a fluorescent molecule such as phycoerythrin (PE) or cyanines, or an enzyme for a chromogenic reaction such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). The enzyme used in a chromogenic reaction catalyzes the production of colored compounds in the presence of a chromogenic substrate.

[0034] In some embodiments, the probe for detecting EGFRvIII or other genomic mutations is connected to a unique identifier such that different genomic mutations can be detected simultaneously and distinguished from one another. The unique identifier may be an oligonucleotide with a unique sequence, or a microbead or a nanoparticle that includes a unique barcode on the surface. The barcode may be a geometric pattern that can be read by an optical scanner with a brightfield imaging system. In some embodiments, the microbead or nanoparticle is a magnetic particle. In some embodiments, the microbead or nanoparticle is made of synthetic polymers.

[0035] The unique identifier may be connected to the probe directly or through a linker. In some embodiments, the unique identifier is connected to the probe by direct chemical coupling and a covalent bond is formed therebetween. In some embodiments, the unique identifier is connected to the probe through a polymer linker. In some embodiments, the unique identifier is connected to the probe by hybridization between complementary nucleotide sequences.

[0036] The disclosed method can be performed on several technology platforms capable of running multiplex reactions, such as a microarray plate, a gene chip, microbeads, nanoparticles, a membrane, or a microfluidic device. In some embodiments, the probes are immobilized on a microarray plate, a gene chip, or a membrane at different positions, for example, in the form of an array of spots, each containing multiple copies of one type of probe. In other embodiments, the probes are coupled with microbeads (such as micro magnetic beads). In still other embodiments, the probes are coated on a substrate plate of a microfluidic device, in which different probes are placed in different regions of the substrate plate.

[0037] When the probes are immobilized on a DNA microarray plate, the microarray plate may further include a set of control spots, each containing multiple copies of a control probe. The control probe binds the cDNA of housekeeping genes such as beta-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and beta 2-microglobulin. Thus, the control spots can be used as an internal control to validate assay performance. In addition, the microarray plate may further include a set of anchor spots, each containing multiple copies of an anchor probe. The anchor probe is designed to be detected irrespective of the amplified products. Thus, the anchor spots can be used as a position indicator for nearby spots on the microarray plate.

[0038] In the present disclosure, a kit is also provided for detecting EGFRvIII according to the disclosed method. The kit includes an EGFRvIII fusion-specific forward primer, an EGFRvIII fusion-specific reverse primer, and a probe having the nucleotide sequence of SEQ ID NO:l or a complementary sequence thereof.

[0039] In some preferred embodiments, the kit further includes a universal primer pair. When the EGFRvIII fusion-specific primer pair is used in combination with the universal primer pair, the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of the universal forward primer, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of the universal reverse primer. [0040] In some embodiments, the kit further includes a reverse transcriptase for reverse transcription of the RNA isolated from the biological sample, and also includes a DNA polymerase for amplification of the cDNA generated by the reverse transcription.

[0041] In some embodiments, the kit further includes an internal control. The internal control may be a positive control sample where EGFRvIII is present or may be a negative control sample having no EGFRvIII fusion. In some embodiments, the internal control is a FFPE tissue section, blood, plasma, cells, nucleic acids, or oligonucleotides.

[0042] In some embodiments, when EGFRvIII is detected in a biological sample from a cancer patient, the patient is expected to be resistant to tyrosine kinase inhibitors and/or EGFR antibody therapies. The tyrosine kinase inhibitor may be an EGFR inhibitor, such as gefitinib, afatinib, erlotinib or dacomitinib. The EGFR antibody may be cetuximab. Examples

Example 1 Detection of EGFRvIII by a single amplified target-probe-BMB hybridization assay

[0043] Single-amplified target-probe-barcoded magnetic bead (BMB) hybridization assay can detect EGFRvIII. FIG. 1 shows the overall process of this assay, which includes the steps of obtaining RNA from a biological sample, reverse transcription of the RNA to obtain cDNA, PCR amplification of the EGFRvIII fusion region of the cDNA (i.e., the target cDNA) using an EGFRvIII fusion-specific primer pair to obtain an amplified product of the target cDNA, probe hybridization with the amplified target cDNA using a BMB- coupled probe and detection of the probe-bound product. Below is an example of this assay. Preparation of an EGFRvIII-fusion specific probe

[0044] Prior to the assay, a probe targeting EGFRvIII was designed based on the nucleotide sequence of the fusion region in the RNA transcript of the EGFRvIII gene (Table 2). The first 20 and the last 20 base pairs of the sequence in Table 2 are from exon 1 and exon 8 of the EGFR gene, respectively. For hybridization at the site underlined in Table 2, one probe was designed to have the sequence listed in Table 3. The probe was synthesized and modified with an amine group at the 5’ end by IDT (Integrated DNA Technologies, Inc, Coralville, IA). Subsequently, the fusion specific probe was coupled with BMBs having a specific identification number via amine-carboxyl bonding, forming a “probe-BMB” complex. TABLE 2

TABLE 3

PCR amplification using an EGFRvIII-fusion specific primer pair

[0045] To substitute clinical samples harboring EGFRvIII fusion, oligonucleotides having the sequence of SEQ ID NO: 3 (termed EGFRvIII fusion oligo) were synthesized by IDT to be used as a positive control template. The EGFRvIII fusion oligo was amplified by PCR with an EGFRvIII fusion-specific primer pair shown in Table 4. This primer pair, capable of binding to the 5 ’-end and the 3 ’-end of the EGFRvIII fusion oligo, was synthesized by IDT. The reverse primer in the primer pair was modified at the 5 ’-end with biotin for subsequent interaction with a streptavidin-phycoerythrin (SA-PE) conjugate (Thermo Fisher Scientific). The PCR was performed on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 30 thermal cycles using Platinum Taq DNA polymerase High Fidelity (Thermo Fisher Scientific) according to the manufacturer’s instructions.

TABLE 4

Probe hybridization and signal detection

[0046] The amplified product of the EGFRvIII fusion oligo was mixed with the probe-BMB in the same wells of a 96-well plate for hybridization. Hybridization was performed at 40°C for 10-30 minutes with agitation at about 700 rpm. After the hybridization, a fluorescent SA- PE conjugate was added to the wells to bind the biotin of the amplified product, and then the probe-BMBs were washed to remove unbound substances. An additional BMB (with an ID number of zero) bearing no probe was also added to the wells as a negative control. Finally, BioCode 2500 analyzer (Applied BioCode Inc., Taipei, Taiwan), equipped with a camera capable of both brightfield and fluorescence imaging, was used to read the barcodes of the BMBs and to detect the fluorescence signals of the BMBs.

[0047] Table 5 shows the fluorescence intensity of BMB92 and BMB0. According to Table 5, the fluorescence intensity of BMB92, indicating target-probe hybridization and the presence of EGFRvIII, was significantly higher than that of the BMB0, indicating no EGFRvIII fusion. The results show that the single-amplified target probe-BMB assay can be used to distinguish the presence and absence of EGFRvIII.

Example 2 Detection of EGFRvIII by a double amplified target-probe hybridization assay

[0048] Double-amplified target-probe hybridization assay is another method designed to detect EGFRvIII. FIG. 2 shows the overall process of this assay, which includes the steps of obtaining RNA from a biological sample, reverse transcription of the RNA to obtain cDNA, PCR amplification of the EGFRvIII fusion region of the cDNA (i.e., the target cDNA) using an EGFRvIII fusion-specific primer pair to obtain a first amplified product of the target cDNA, PCR amplification of the first amplified product using a universal primer pair to obtain a second amplified product of the target cDNA, probe hybridization with the amplified target cDNA and detection of the probe-bound product. Below is an example of this assay.

RNA extraction and reverse transcription

[0049] Both DNA and RNA are extracted from a FFPE tissue specimen from a cancerous patient by using RecoverAll total nucleic acid isolation kit (Cat No: AMI 975, Ambient Technologies) according to the manufacturer’s instructions. Reverse transcription of 100 ng of total RNA is carried out at 42°C for 30 to 60 minutes by using Superscript cDNA synthesis kit (Cat No: 11754050, Invitrogen) and random hexanucleotide primers, and 10 pL of cDNA product is obtained. PCR amplification using an EGFRvIII fusion-specific primer pair

[0050] Each primer in the EGFRvIII fusion-specific primer pair used in this assay is designed to have two segments. One segment, called a fusion specific segment, is used to bind the 5’- end or the 3 ’-end of the EGFRvIII fusion sequence. The fusion specific segment may have the sequence of SEQ ID NO:4 or 5 (Table 4). The other segment, called a universal segment, encompasses the nucleotide sequence of the universal primer to be used in the second round of PCR. The universal segment is always upstream, or at the 5’ position, relative to the fusion specific segment (FIG. 2). The universal primer may be any of the primers listed in Table 6, where each universal primer can be used as either the universal forward primer or the universal reverse primer.

TABLE 6

[0051] For fusion specific PCR, lOuL of the cDNA product is amplified on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 15-30 thermal cycles using multiplex PCR kit (Cat No: 206143, Qiagen) according to the manufacturer’s instructions, yielding a first amplified product in 10 pL.

PCR amplification using a universal primer pair

[0052] Since each fusion specific primer included the nucleotide sequence of a universal primer at the 5’ end, the first amplified product is able to be further amplified by PCR using a universal primer pair, including a universal forward primer with the sequence selected from SEQ ID NOs:6-15 and a universal reverse primer with the sequence selected from SEQ ID NOs:6-15. The universal reverse primer is biotinylated. For the second round of PCR, the first amplified product is diluted 100 folds in the final reaction mix and amplified on Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific) for 15-30 thermal cycles by using Platinum SuperFi II PCR Master Mix (Cat No: 12368010, Invitrogen) according to the manufacturer’s instructions, yielding a second amplified product in 10 pL.

Probe hybridization and signal detection

[0053] The second amplified product is placed to a 96-well PCR plate (Cat No: P46-4TI- 1000/C, 4titude). The second amplified product is denatured at 96°C for 5 minutes and transferred to pre-blocked wells, each of which is printed with an array of probe spots, including the spots of the EGFRvIII fusion-specific probe, the spots of control probes, and the spots of an anchor probe. Target-probe hybridization is performed at about 50°C for 15 minutes with vibration. After the hybridization, the wells are cooled and washed twice. A buffer containing a streptavidin-alkaline phosphatase conjugate is subsequently added to the wells to allow biotin-streptavidin interaction, and a substrate for the alkaline phosphatase is then added so that color products form at the position where a probe-target hybrid is present. By photographing the wells with a camera and identifying the position of color spots in the wells, the particular hybridization indicating the presence of EGFRvIII can be determined. The position of color spots can be analyzed by a computer.

Claims

WHAT IS CLAIMED IS:

1. A kit for detecting epidermal growth factor receptor variant III (EGFRvIII), comprising: an EGFRvIII fusion-specific forward primer and an EGFRvIII fusion-specific reverse primer; and a probe having the nucleotide sequence of SEQ ID NO:l or a complementary sequence thereof.

2. The kit of claim 1 , further comprising a universal primer pair, wherein the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of a universal forward primer in the universal primer pair, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of a universal reverse primer in the universal primer pair.

3. The kit of claim 1, wherein the EGFRvIII fusion-specific forward primer or the EGFRvIII fusion-specific reverse primer is connected to a detectable molecule.

4. The kit of claim 3, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

5. The kit of claim 2, wherein the universal forward primer or the universal reverse primer is connected to a detectable molecule.

6. The kit of claim 5, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

7. The kit of claim 1 , wherein the probe is immobilized on a microarray plate.

8. The kit of claim 7, wherein the microarray comprises a position indicator.

9. The kit of claim 1, further comprising a control probe for detecting a housekeeping gene.

10. The kit of claim 1, further comprising a reverse transcriptase and a DNA polymerase.

11. A method for detecting epidermal growth factor receptor variant III (EGFRvIII), comprising the steps of:

(a) obtaining ribonucleic acids (RNA) from a biological sample;

(c) amplifying the cDNA with an EGFRvIII fusion-specific forward primer and an EGFRvIII fusion-specific reverse primer to obtain an amplified product; (d) mixing the amplified product with a probe to obtain a probe-bound product, wherein the probe has the nucleotide sequence of SEQ ID NO: l or a complementary sequence thereof; and

(e) detecting the probe-bound product to determine the presence of EGFRvIII.

12. The method of claim 11, wherein in step (c) the cDNA is amplified first with the EGFRvIII fusion-specific forward primer and the EGFRvIII fusion-specific reverse primer and subsequently with a universal forward primer and a universal reverse primer to obtain the amplified product, wherein the EGFRvIII fusion-specific forward primer further encompasses the nucleotide sequence of the universal forward primer, and the EGFRvIII fusion-specific reverse primer further encompasses the nucleotide sequence of the universal reverse primer.

13. The method of claim 11, wherein the EGFRvIII fusion-specific forward primer or the EGFRvIII fusion-specific reverse primer is connected to a detectable molecule.

14. The method of claim 13, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

15. The method of claim 12, wherein the universal forward primer or the universal reverse primer is connected to a detectable molecule.

16. The method of claim 15, wherein the detectable molecule is a fluorescent molecule or an enzyme for a chromogenic reaction.

17. The method of claim 11, wherein in step (d) the probe and the amplified product are mixed at a temperature between 35-50°C.

18. The method of claim 11, wherein in step (d) the probe and the amplified product are mixed at a rotation speed between 700-1000 rpm.

19. The method of claim 11, wherein the probe is immobilized on a microarray plate.