Methods to detect genetic mutations in lung cancer

Abstract

The present disclosure relates in some aspects to methods and compositions for detecting genetic mutations in lung cancer samples. The invention related to an all-in-one test that utilizes multiplex targeting of all FDA-approved genetic mutations in lung adenocarcinoma within a single reaction well.

Description

106546-866311 (UTSD 4450)METHODS TO DETECT GENETIC MUTATIONS IN LUNG CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application 63 / 723,070, filed November 20, 2024, the contents of which are incorporated by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on November 19, 2025, is named “106546-866311 (UTSD 4550) Sequence Listing” and is 16,998 bytes in size.BACKGROUND

[0003] Lung cancer presents a significant global health challenge due to its heterogeneous nature and varied molecular profiles. Gene fusions involving ROS1, ALK, and RET have emerged as crucial biomarkers in non-small cell lung cancer (NSCLC), guiding targeted therapy with tyrosine kinase inhibitors (TKIs). However, fluorescence in situ hybridization (FISH) has limitations in sensitivity. Next generation sequencing (NGS) offers comprehensive analysis but requires significant amounts of tissue. Thus, improved methods to detect genetic mutations in lung cancer samples are urgently needed. Provided herein are methods and compositions that address such and other needs.BRIEF SUMMARY

[0004] In one aspect, a set of nine pairs of nucleotide sequences for detecting genetic mutations in a lung cancer sample is provided, wherein a first pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2, a second pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4, a third pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:6, a fourth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 8, a fifth pair of nucleotide sequences includes a nucleotide1107245884.2106546-866311 (UTSD 4450) sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10, a sixth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12, a seventh pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14, an eighth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16, and a ninth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18, where one nucleotide sequence of each pair further includes a tag.

[0005] Also provided herein, in some aspects, is a method for detecting genetic mutations in a lung cancer sample, the method includes i) obtaining or having obtained the lung cancer sample from a subject having or suspected of having a lung cancer, ii) extracting DNA from the sample, iii) mixing the DNA with a set of nucleic acid primers includes a first pair, a second pair, a third pair, a fourth pair, a fifth pair, and a sixth pair of nucleotide sequences and a DNA polymerase, iv) amplifying the DNA, v) determining the genetic mutations in the lung cancer sample based on the size of the amplified DNA, where the first pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2, where the second pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4, where the third pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 6, where the fourth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8, where the fifth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10, where the sixth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12,2107245884.2106546-866311 (UTSD 4450) where the seventh pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14, where the eighth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16, where the ninth pair of nucleotide sequences includes a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18, where one nucleotide sequence of each pair further includes a tag.

[0006] In some aspects, the lung cancer sample is selected from the group consisting of a formalin fixed paraffin embedded lung tissue sample, a fresh lung cancer biopsy specimen, a peripheral blood sample, and a plasma sample. In some aspects, the lung cancer sample is a formalin fixed paraffin embedded lung tissue sample.

[0007] In some aspects, the lung cancer is lung adenocarcinoma.

[0008] Also provided herein, in some aspects, is a method of treating a subject having or suspected of a lung cancer, the method comprising: obtaining a lung cancer sample from the subject; identifying the genetic mutations in a lung cancer sample according the methods described herein; and administering an agent effective to treat the lung cancer based on the genetic mutations in the lung cancer sample.

[0009] Also provided herein, in some aspects, is a kit for detecting genetic mutations in a lung cancer sample, the kit comprising a set of nucleic acid primers comprising a first pair, a second pair, a third pair, a fourth pair, a fifth pair, and a sixth pair, a seventh pair, an eight pair, and a ninth pair of nucleotide sequences and a DNA polymerase; wherein the first pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; wherein the second pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; wherein the third pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 6; wherein the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8; wherein the fifth pair of nucleotide sequences comprises a nucleotide sequence having at3107245884.2106546-866311 (UTSD 4450) least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; wherein the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; wherein the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; wherein the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; wherein the ninth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; and wherein one nucleotide sequence of each pair further comprises a tag.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. l is a diagram depicting the design of the Lung-FAST test. Gene targets are shown in capital letters. Color of fluorescent tags is shown (blue, green, black, orange, red). Numbers indicate the estimated size of amplicons for proper spacing.

[0011] FIG. 2 displays electropherograms of Lung-FAST amplified products as detected by fragment analysis. Distinct size and shape of allele-specific primers for BRAF and EGFR targets are shown (WT in blue, mutant in green).

[0012] FIG. 3 A displays RNA-Seq expression data from the UTSW Clinical NGS test for (from left to right) ALK, ROS1, and RET in cases from lung tissue.

[0013] FIG. 3B displays electropherograms of ROS 1 exon 20 (Ex20), exon 38 (Ex38), and exon 41 (Ex41).

[0014] FIG. 4 displays electropherograms of MET exon 14 skipping, detected as a shorter (left) Red / Green amplicon (bottom) vs the longer Red / Green amplicon in WT.

[0015] FIG. 5 displays electropherograms of Lung-FAST amplified products identifying examples of positive fusions for each gene. For each positive case, the 3’ peaks are higher than the 5’ peak. For each negative case, the 5’ peak is higher than the 3’ peaks.

[0016] FIG. 6A is a line graph displaying results from a limit of detection (LOD) analysis for ALK primers (SEQ ID NOs: 1-6) in cell lines bearing ALK fusions. The limit of detection is identified as the point where at least one of the 3' peaks is higher than the 5' peak.4107245884.2106546-866311 (UTSD 4450)

[0017] FIG. 6B is a line graph displaying results from a limit of detection (LOD) analysis for ROS1 primers (SEQ ID NOs: 7-12) in cell lines bearing ROS1 fusions. The limit of detection is identified as the point where at least one of the 3' peaks is higher than the 5' peak.

[0018] FIG. 6C is a line graph displaying results from a limit of detection (LOD) analysis for RET primers (SEQ ID NOs: 13-18) in cell lines bearing ROS1 fusions. The limit of detection is identified as the point where at least one of the 3' peaks is higher than the 5' peak.

[0019] FIGs. 7A-7D are representative electropherograms of Lung-FAST amplified products detecting ALK (FIG. 7A), ROS1 (FIG. 7B), and RET (FIG. 7C) fusions in lung cancer patients. Non-fusion controls are shown in FIG. 7D.DETAILED DESCRIPTION

[0020] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.I. Overview

[0021] The present application provides improved methods to detect and identify genetic mutations in lung cancer samples. Lung adenocarcinoma (LU AD) is difficult to treat due to its aggressive nature, frequent resistance, and drug side effects. Initial presentation for many patients is advanced disease in LUAD is very aggressive with 50% 1-year survival. Twenty- five targeted therapies for LUAD have been approved by the FDA and focus on 11 genes: EGFR, KRAS, BRAF, ERBB2, and fusions involving ALK, ROS1, MET, RET, and NTRK1 / 2 / 3. These drugs dramatically improve 1 year survival from 50% to 90%. Patients get onto these drugs through genetic testing through a variety of single gene tests. With nearly 18 biomarkers to test, it is not feasible to test one at a time. Next Generation Sequencing (NGS) detects multiple genetic variants simultaneously on one tumor specimen. However, NGS requires ten times more DNA, complex techniques, expensive equipment, and advanced bioinformatic analysis to conduct. In clinical practice, small, minimally invasive lung biopsies are rejected 30-40% of the time for being too small. This requires5107245884.2106546-866311 (UTSD 4450) rescheduling a biopsy and starting the process all over again which can delay results by 2 months.

[0022] The long time required to identify mutations in LUAD samples is a critical drawback. Cell free DNA testing is closing that gap to 5-7 days, but it is hard to know if a result is falsely negative. Furthermore, for tissue NGS testing, testing takes 2-3 weeks and 30-40% of minimally invasive biopsies are rejected for not enough cancer cell. PCR approaches are limited by 4-5 color channels, limited mutation detection, and are not easily adapted to new targets. Thus, there is a critical gap between PCR (faster, one to four genes) and NGS (slower, hundreds of genes).

[0023] If inappropriate therapy (like immunotherapy) is started before genetic results can be obtained, then outcomes are much worse. It is therefore vital to find the right drug for the right patient without extensive delays due to genetic testing.

[0024] To address these needs an all-in-one test was developed that utilizes multiplex targeting of all FDA-approved genetic mutations in LUAD within a single reaction well. The methods described herein require smaller and less expensive instrumentation that other standard genetic testing approaches.II. Terms and Definitions

[0025] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs.

[0026] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0027] “ About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 10% or less of the numerical value.

[0028] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature,6107245884.2106546-866311 (UTSD 4450) element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0029] As used herein, “and / or” refers to and encompasses all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0030] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0031] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0032] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0033] As used herein, “treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition.

[0034] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.7107245884.2106546-866311 (UTSD 4450)

[0035] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0036] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In one aspect, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another aspect, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In still another aspect, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another aspect, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In some aspects, the subject can be a human. In other aspects, the subject can be a human in need of repairing or regenerating a tissue or organ.

[0037] As used herein, “sample” refers to a sample form a subject that may be used for additional analysis. The sample may be a biosample, such as sputum, bronchoalveolar lavage (BAL), bronchial wash (BW), urine, other body fluid (BF), or tissue.

[0038] As used herein, “effective amount,” “effective dose,” or an “amount effective to”, refers to an amount that is effective in providing at least one desirable biological result.III. Nucleotide Sequences to Detect Genetic Mutations

[0039] In some aspects, provided herein is a set of nine pairs of nucleotide sequences for detecting genetic mutations in a lung cancer sample, comprising: a first pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; a second pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; a third pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:6; a fourth pair of nucleotide sequences comprising a nucleotide sequence having at least 85%8107245884.2106546-866311 (UTSD 4450) sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8; a fifth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; a sixth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; a seventh pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; an eighth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; and a ninth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; wherein one nucleotide sequence of each pair further comprises a tag.

[0040] In some aspects, the first pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 19 and 20 of the ROS proto-oncogene 1 (ROS / ) gene. In some aspects, the first pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:2. In some aspects the first pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO:2.

[0041] In some aspects, the second pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 36 and 38 of the ROS1 gene. In some aspects, the second pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:4. In some aspects the second pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO:3 and the nucleotide sequence set forth in SEQ ID NO:4.

[0042] In some aspects, the third pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 41 and 42 of the ROS1 gene. In some aspects, the third pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to9107245884.2106546-866311 (UTSD 4450)SEQ ID N0:5 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:6. In some aspects the third pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO:5 and the nucleotide sequence set forth in SEQ ID NO:6.

[0043] In some aspects, the fourth pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 4 and 5 of the ALK receptor tyrosine kinase (ALK) gene. In some aspects, the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO:8. In some aspects the fourth pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO:7 and the nucleotide sequence set forth in SEQ ID N0:8.

[0044] In some aspects, the fifth pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 21 and 22 of the ALK gene. In some aspects, the fifth pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 9 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 10. In some aspects the fifth pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO:9 and the nucleotide sequence set forth in SEQ ID NO: 10.

[0045] In some aspects, the sixth pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 25 and 26 of the ALK gene. In some aspects, the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 12. In some aspects the sixth pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO: 11 and the nucleotide sequence set forth in SEQ ID NO: 12.

[0046] In some aspects, the seventh pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 4 and 5 of the ret proto-oncogene RET) gene. In some aspects, the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least about 85%, at least10107245884.2106546-866311 (UTSD 4450) about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 14. In some aspects the seventh pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO: 13 and the nucleotide sequence set forth in SEQ ID NO: 14.

[0047] In some aspects, the eighth pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 16 and 17 of the RET gene. In some aspects, the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 16. In some aspects the eighth pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO: 15 and the nucleotide sequence set forth in SEQ ID NO: 16.

[0048] In some aspects, the ninth pair of nucleotide sequences is capable of hybridizing to a region of nucleic acids in exons 18 and 19 of the RET gene. In some aspects, the ninth pair of nucleotide sequences comprises a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homology to SEQ ID NO: 18. In some aspects the ninth pair of nucleotide sequences comprises the nucleotide sequence set forth in SEQ ID NO: 17 and the nucleotide sequence set forth in SEQ ID NO: 18.

[0049] It should however be understood that the present disclosure also contemplates a set of nucleotide sequences comprising any one pair among the nine pairs of nucleotide sequences detailed above, as well as any combination of two or more of the pairs detailed above.

[0050] In various aspects, it should also be understood that the present disclosure contemplates any individual nucleotide sequence making up a pair of sequences, as encompassing any sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence homology compared to the reference sequence, i.e. ., compared to any one of SEQ ID NOs: l-18.Methods for Detecting Genetic Mutations in Lung Cancer

[0051] In other aspects, provided herein is a method for detecting genetic mutations in a lung cancer sample, the method comprising: i) obtaining or having obtained the lung cancer sample from a subject having or suspected of having a lung cancer; ii) extracting DNA from the sample; iii) mixing the DNA with a set of nucleic acid primers comprising a first pair, a second pair, a third pair, a fourth pair, a fifth pair, and a sixth pair of nucleotide sequences 11107245884.2106546-866311 (UTSD 4450) and a DNA polymerase; iv) amplifying the DNA; v) determining the genetic mutations in the lung cancer sample based on the size of the amplified DNA, wherein the first pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; wherein the second pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; wherein the third pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 6; wherein the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8; wherein the fifth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; wherein the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; wherein the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; wherein the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; wherein the ninth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; wherein one nucleotide sequence of each pair further comprises a tag.

[0052] In some aspects, the lung cancer sample is selected from the group consisting of a formalin fixed paraffin embedded lung tissue sample, a fresh lung cancer biopsy specimen, a peripheral blood sample, and a plasma sample. In some aspects, the lung cancer sample is a formalin fixed paraffin embedded lung tissue sample.

[0053] In some aspects, the lung cancer is lung adenocarcinoma.Methods of Detection

[0054] In some aspects, the provided methods involve analyzing, e.g., detecting or quantifying, one or more sequences present in a PCR amplification product. In some12107245884.2106546-866311 (UTSD 4450) aspects, analyzing (e.g., detecting or quantifying) the one or more sequences in the PCR amplification product comprises detecting a complex comprising the one or more PCR amplification products and one or more tagged nucleotide sequences. In other aspects, detecting the one or more sequences in the PCR amplification product comprises detecting a complex comprising the one or more PCR amplification products and one or more fluorescently labeled nucleotide sequences. In some aspects, the fluorescently labeled nucleotide sequence comprises a fluorescent label selected from the group consisting of 6- Carboxyfluorescein (FAM), ATTO 488, Tetrachlorofluorescein (TET), ATTO 532, Hexachlorofluorescein (HEX), JOE, TYE 563, Cy 3, ATTO 550, Carboxytetramethyl- rhodamine (TAMRA), ATTO 565, ROX, ATTO Rho 101, TEX 615, and Texas Red-X. In some aspects, the fluorescently labeled nucleotide sequence comprises a fluorescent label consisting of 6-Carboxyfluorescein (FAM).

[0055] In some aspects, detecting the one or more sequences present in the PCR amplification product comprises separating the labeled PCR products by size using capillary electrophoresis. In this process, the PCR amplification products are injected into a capillary tube filled with a polymer matrix. An electric field is then applied, causing the fragments to migrate through the capillary at different rates based on their size. As the fragments pass through a detection window, a laser excites the fluorescent dyes, and a detector captures the emitted light. The resulting data is analyzed to determine the size of each fragment, which corresponds to specific PCR products.

[0056] In some aspects the PCR amplification product comprises an amplicon size of between about 100 base pairs (bp) and about 400 bp. In some aspects the PCR amplification product comprises an amplicon size of about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 150 bp, about 160 bp, about 170 bp, about 180 bp, about 190 bp, about 200 bp, about 210 bp, about 220 bp, about 230 bp, about 240 bp, about 250 bp, about 260 bp, about 270 bp, about 280 bp, about 290 bp, about 300 bp, about 310 bp, about 320 bp, about 330 bp, about 340 bp, about 350 bp, about 360 bp, about 370 bp, about 380 bp, about 390 bp, or about 400 bp. In some aspects, the PCR amplification product comprises an amplicon size provided in Table 1.IV. Methods of Treatment

[0057] Also provided herein are methods for treating a subject a subject having or suspected of a lung cancer, the method comprising: obtaining a lung cancer sample from the subject; identifying the genetic mutations in a lung cancer sample according to the methods13107245884.2106546-866311 (UTSD 4450) disclosed herein; and administering an agent effective to treat the lung cancer based on the genetic mutations in the lung cancer sample.

[0058] Depending upon the genetic mutations present in the lung cancer sample obtained from the subject, an effective dose of one or more of Afatinib, Dacomitinib, Erlotinib, Gefitinib, Osimertinib, Amivantamab, Mobocertinib, Trastuzumab Deruxtecan, Ado- Trastuzumab Emtansine, Debrafenib, Trametinib, Adagrasib, and Sotroasib may be administered to the subject. The agent effective to treat the lung cancer may be administered as appropriate for the agent.V. Kits

[0059] The present disclosure also provides kits for detecting genetic mutations in a lung cancer sample, the kit comprising a set of nucleic acid primers comprising a first pair, a second pair, a third pair, a fourth pair, a fifth pair, and a sixth pair, a seventh pair, an eight pair, and a ninth pair of nucleotide sequences and a DNA polymerase; wherein the first pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; wherein the second pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; wherein the third pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 6; wherein the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8; wherein the fifth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; wherein the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; wherein the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; wherein the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; wherein the ninth pair of nucleotide sequences comprises a nucleotide sequence having at14107245884.2106546-866311 (UTSD 4450) least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; and wherein one nucleotide sequence of each pair further comprises a tag.SEQUENCES15107245884.2106546-866311 (UTSD 4450)EXAMPLESExample 1:

[0060] Lung-FAST (Fragment Analysis for Translocations), a multiplex fragment analysis to detect RNA expression imbalance between 5’ and 3’ sections of translocated genes. Biologically, RNA from the kinase domain of ALK, ROS1, and RET genes are increased compared to 5’ exons when a translocation is present.

[0061] Primers targeting fusion breakpoints in ALK, ROS1, and RET were designed and used to amplify specific fragments from cell line RNA and patient samples. Capillary electrophoresis resolved PCR products based on fragment size and fluorescence intensity, identifying fusions.Table 1 : Primer design for Lung-FAST.16107245884.2106546-866311 (UTSD 4450)

[0062] We used a combination of PCR techniques to identify mutations associated with relevant FDA approved therapies. The selected mutations were targeted towards EGFR, KRAS, BRAF, HER2, MET, ALK, ROS1, RET, and NTRK1 / 2 / 3 (Table 2).Table 2: Drugs, genes and codons targeted by Lung-FAST with targeting method.17107245884.2106546-866311 (UTSD 4450)

[0063] We have developed allele-specific primers to several targets as detailed above (FIG. 1, Table 2). To increase target specificity, we used a combination of mismatch mutations upstream of the 3’ terminus. We found the following primer combinations produced strong on-target amplification with 28-210fold (8-10 CT PCR cycles) lower off- target amplification: EGFR L858 WT4’ A (WT-specific primer with the 4thbase pair from the end changed to a T) and EGFR_L858R_4’T (L858R-specific primer with the 4thbase pair from the end changed to a T) (FIG. 1).

[0064] This primer set was selected based on qPCR performance and later confirmed by capillary electrophoresis (FIG. 1). Here we could discriminate WT (blue) from mutant sequence (green) based on the color of the peak. Furthermore, we determined the limit of detection as 16 copies / reaction. This process has been repeated and verified by CE for 5 other allele-specific targets (Fig 4). Remaining AS-PCR targets have passed qPCR screening and are pending fluorescent tagging for CE assessment (Table 3, EGFR G719X and KRAS G12C). BRAF V600E, EGFR S768I, EGFR T790M, EGFR L861Q, and EGFR L858R primers were tested against 10 clinical lung specimens with 100% accuracy (FIG. 2). The clinical specimens were correctly identified by amplicon size and fluorescence signal (green “mutant” signal with no “mutant” signal in the wild type specimens).

[0065] EGFR exon 18: We will use the AS-primer strategy to screen for the three most common targetable variants: G719A / C / S (sensitivity = 99%) according to the COSMIC database. These G719X mutations account for 3% of all EGFR mutations and cause ligand independent EGFR constitutive phosphorylation and have an FDA-approved therapy: afatinib. Current allele-specific primer methods have been challenging, so published peptide nucleic acid (PNA) PCR clamp techniques will be used.

[0066] KRAS: G12C KRAS variants are common in lung cancer and have targeted drugs FDA approved (Adagrasib, Sotorasib). Therefore, we have developed allele-specific primers for these variants in the Lung-FAST genotyping test. As many hot spot variants can occur at codon 12 or 13, we have tested our AS-primers for interference from these other KRAS variants (G12A / S / R / V, G13D / C). A peptide nucleic acid (PNA) PCR clamp technique was used.

[0067] BRAF: The V600E variant turns on BRAF constantly and has FDA approval for targeted therapy in lung cancer (100% sensitivity). Allele-specific primers are best suited to detect these variants. Some previous allele-specific primers have been proposed (REF), however, these primers produced mutant signal in wild type controls. Therefore, we 18107245884.2106546-866311 (UTSD 4450) developed primers using methods of enhancing mutant allele-specificity with mismatch nucleotides to destabilize incorrect primer binding. For further sensitivity, we used PCR clamp technology to decrease amplification of WT sequence.

[0068] HER2: The FDA approved the breast cancer anti-HER2 (ERBB2) therapeutic antibody Trastuzumab for lung cancers with oncogenic “activating” exon 20 insertions. Unlike in breast cancer where HER2 is often amplified, amplifications by FISH or IHC testing have not predicted response to therapy. Uniquely, for lung cancer, 85-93% of HER2 variants are exon 20 insertions according to the DESTINY -LungOl Trial. Indels can be detected by fragment analysis, so we have developed HER2 exon 20 flanking primers in Lung-FAST.

[0069] There are 8 RNA-based molecularly targetable variant types in LU AD: ALK, ROS1, RET, NTRK1 / 2 / 3 fusions, and MET exon 14 skipping / amplification. MET amplification can be detected by DNA, but because simpler assays are more robust, the MET ex 14 flanking primers were used for amplification testing.

[0070] We have accomplished a fusion partner agnostic PCR approach for fusion detection. This has never been performed by capillary electrophoresis methods. We have exploited characteristic biology of oncogenic fusions such as increased mRNA expression of the 3’ end of the gene. The above fusions drive cancer, because kinase domain expression (3’ end of ALK / ROS1 / RET) is driven up 10-100 fold higher by the more highly expressed 5’ fusion partner (FIG. 3 A). Therefore, to detect ALK / ROS1 / RET fusions, we have developed primers on the 5’ and 3’ sides of fusion breakpoints and measured the ratio between 573’ sites. In contrast, MET inhibitors specifically target the exon 14 skipping event. Therefore, we have designed exon 13 and 15 primers that create a shorter PCR product when exon 14 skipping occurs. This shorter size PCR product was successfully resolved by capillary electrophoresis.Methods

[0071] PCR conditions and primer master mix preparation methods are provided in Table 3, Table 4, and Table 5.Table 3: TaqPath RT-PCR Master Mix.19107245884.2106546-866311 (UTSD 4450)Table 4: Thermocycler settings for PCR reaction.Table 5: 1 OX PCR Primer Mix.20107245884.2106546-866311 (UTSD 4450)Results

[0072] We have demonstrated that our MET exl4 skipping detection primers worked by placing flanking primers on exons 13 and 15, which would be a closer distance when exon 14 is skipped due to any variety of mutations. By labelling the exon 13 and 15 primers with different fluorophores, we intensify our confidence that the amplicon at the expected size resulted in exon 14 skipping regardless of the type of causative variant (intronic, exonic, or exon deletion).

[0073] We have demonstrated elevated RNA levels occur in LU AD fusions from our NGS clinical lab testing for ROS1, ALK, RET and NTRK2. In cell lines, EML4::ALK (HCC3122) and SLC34A4::ROS1 (HCC78) fusions demonstrated the 573’ allelic imbalance21107245884.2106546-866311 (UTSD 4450) as predicted. The peaks from the 3’ targets were >5x higher compared to the 5’ cDNA amplicon. Using a cut-off of 2x higher than the 5’ amplicon, we have estimated that the LoD was as low as 10% tumor cells (equivalent of 5% VAF seen in many NGS applications, FIG. 3 A). Furthermore, we have data (FIG. 3 A) demonstrating the 573’ ratio comparison differentiating wild type vs. ROS1 translocated RNA. In our results, the 5’ target (exon 20) is much lower than either of the 3’ targets (exons 38 and 41).

[0074] Single fusion primer-set efficiency is shown in Table 6.Table 6: Single fusion primer-set efficiency.

[0075] Efficiency of the Lung-FAST multiplex fragment analysis test is shown in Table 7.Table 7: Lung-FAST efficiency.* noted fusion positive with other fusions negative

[0076] Lung-FAST clinical accuracy on FFPE samples is shown in Table 8.Table 8: Lung-FAST Clinical Accuracy on FFPE samples22107245884.2106546-866311 (UTSD 4450)

[0077] Lung-FAST results from Representative clinical cases are shown in FIG. 5.

[0078] Limit of detection (LOD) analysis for ALK mutations are shown in FIG. 6A and Table 9.Table 9: LOD analysis for ALK mutations. LOD shown in bold.

[0079] Limit of detection (LOD) analysis for ROS1 mutations are shown in FIG. 6B and Table 10.Table 10: LOD analysis for ROS1 mutations. LOD shown in bold.23107245884.2106546-866311 (UTSD 4450)

[0080] Limit of detection (LOD) analysis for RET mutations are shown in FIG. 6C and Table 11.Table 11 : LOD analysis for RET mutations. LOD shown in bold.

[0081] Representative performance of the Lung-FAST test in detecting ALK, ROS1, and RET fusions in lung cancer patients is shown in FIGs. 7A-7D.

[0082] Conclusions: Lung-FAST offers a promising alternative to traditional methods, demonstrating robust performance in detecting ALK, ROS1, and RET fusions in lung cancer patients. While further optimization is needed to enhance sensitivity for less prevalent fusions, Lung-FAST presents a streamlined approach potentially reducing time and cost associated with molecular testing, thereby aiding targeted therapy decisions in clinical settings.24107245884.2

Claims

106546-866311 (UTSD 4450)CLAIMSWhat is claimed is:

1. A set of nine pairs of nucleotide sequences for detecting genetic mutations in a lung cancer sample, comprising: a first pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; a second pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; a third pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:6; a fourth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:8; a fifth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; a sixth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; a seventh pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; an eighth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; and a ninth pair of nucleotide sequences comprising a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; wherein one nucleotide sequence of each pair further comprises a tag.25107245884.2106546-866311 (UTSD 4450)2. A method for detecting genetic mutations in a lung cancer sample, the method comprising: i) obtaining or having obtained the lung cancer sample from a subject having or suspected of having a lung cancer; ii) extracting DNA from the sample; iii) mixing the DNA with a set of nucleic acid primers comprising a first pair, a second pair, a third pair, a fourth pair, a fifth pair, a sixth pair, a seventh pair, an eighth pair, and a ninth pair of nucleotide sequences and a DNA polymerase; iv) amplifying the DNA; v) determining the genetic mutations in the lung cancer sample based on the size of the amplified DNA, wherein the first pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; wherein the second pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; wherein the third pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:6; wherein the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 8; wherein the fifth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; wherein the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; wherein the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; wherein the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16;26107245884.2106546-866311 (UTSD 4450) wherein the ninth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; wherein one nucleotide sequence of each pair further comprises a tag.

3. The method of claim 1 or 2, wherein the lung cancer sample is selected from the group consisting of a formalin fixed paraffin embedded lung tissue sample, a fresh lung cancer biopsy specimen, a peripheral blood sample, and a plasma sample.

4. The method of any one of claims 1 to 3, wherein the lung cancer sample is a formalin fixed paraffin embedded lung tissue sample.

5. The method of claim 2, wherein the lung cancer is lung adenocarcinoma.

6. A method of treating a subject having or suspected of having a lung cancer, the method comprising: obtaining a lung cancer sample from the subject; identifying the genetic mutations in a lung cancer sample according to the method of claim 2; administering an agent effective to treat the lung cancer based on the genetic mutations in the lung cancer sample.

7. A kit for detecting genetic mutations in a lung cancer sample, the kit comprising a set of nucleic acid primers comprising a first pair, a second pair, a third pair, a fourth pair, a fifth pair, and a sixth pair, a seventh pair, an eight pair, and a ninth pair of nucleotide sequences and a DNA polymerase; wherein the first pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 1 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:2; wherein the second pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:3 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:4; wherein the third pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:5 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:6;27107245884.2106546-866311 (UTSD 4450) wherein the fourth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:7 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 8; wherein the fifth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO:9 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 10; wherein the sixth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 11 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 12; wherein the seventh pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 13 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 14; wherein the eighth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 15 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 16; wherein the ninth pair of nucleotide sequences comprises a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 17 and a nucleotide sequence having at least 85% sequence homology to SEQ ID NO: 18; and wherein one nucleotide sequence of each pair further comprises a tag.28107245884.2

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