IL304567B2 - Methods of detecting oncogenic pik3ca - Google Patents

Methods of detecting oncogenic pik3ca

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Publication number
IL304567B2
IL304567B2 IL304567A IL30456723A IL304567B2 IL 304567 B2 IL304567 B2 IL 304567B2 IL 304567 A IL304567 A IL 304567A IL 30456723 A IL30456723 A IL 30456723A IL 304567 B2 IL304567 B2 IL 304567B2
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cancer
nucleic acid
acid molecule
cytosine
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IL304567A
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IL304567A (en
IL304567B1 (en
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TULLER Tamir
LAZAROVITS Janin
ZARAI Yoram
FEUER Cynthia
Dassa Liat
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Oncodecipher Ltd
Univ Ramot
TULLER Tamir
LAZAROVITS Janin
ZARAI Yoram
FEUER Cynthia
Dassa Liat
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Application filed by Oncodecipher Ltd, Univ Ramot, TULLER Tamir, LAZAROVITS Janin, ZARAI Yoram, FEUER Cynthia, Dassa Liat filed Critical Oncodecipher Ltd
Priority to IL304567A priority Critical patent/IL304567B2/en
Publication of IL304567A publication Critical patent/IL304567A/en
Publication of IL304567B1 publication Critical patent/IL304567B1/en
Publication of IL304567B2 publication Critical patent/IL304567B2/en
Priority to PCT/IL2024/050707 priority patent/WO2025017560A1/en

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    • C12Q1/6883Nucleic 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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    • C12Q2600/156Polymorphic or mutational markers

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Description

METHODS OF DETECTING ONCOGENIC PIK3CA REFERENCE TO AN ELECTRONIC SEQUENCE LISTING id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"
[001] The contents of the electronic sequence listing (ONDP-RMT-P-001-IL.xml; Size: 369,781 bytes; and Date of Creation: July 18, 2023) is herein incorporated by reference in its entirety.
FIELD OF INVENTION id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"
[002] The present invention is in the field of cancer diagnostics.
BACKGROUND OF THE INVENTION id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"
[003] Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are involved in multiple cellular functions such as proliferation, cell growth, differentiation, adhesion, motility, intracellular trafficking, and survival. PI3Ks contain a conserved core composed of a C2 domain, a kinase domain, and a helical domain. Based on their substrate specificities and structural characteristics, PI3Ks are classified into four main classes: I, II, III and IV. Class I PI3Ks are heterodimeric molecules composed of a regulatory and a catalytic subunit and are divided to IA and IB subsets. Class IA PI3Ks are composed of a p110 catalytic subunit and a shorter regulatory subunit, mainly p85. The p110 catalytic subunit is known to have three different isoforms designated p110α, 110β, or 110δ. These catalytic subunits are encoded by separate genes; PIK3CA, PIK3CB, and PIK3CD for p110α, p110β, and p110δ, respectively. id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"
[004] The signaling mediated by PI3Ks, the PI3K/AKT signaling pathway, is known to play a key role in the progression of variety of cancers by inducing metabolism, cell growth, proliferation, angiogenesis, and metastasis. In fact, activation of the PI3K/AKT pathway occurs in approximately 30–50% of human cancers and results in resistance to various anti-cancer therapies. id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"
[005] Mutations in exon-coding regions of the PIK3CA gene, which lead to an amino acid substitution and result in increased activity of the p110α catalytic subunit, have been previously reported in variety of cancers. PIK3CAE545K is an example for a hotspot mutation in the coding region of exon 9, which results in suppressed inhibition of p110α by the p85 regulatory subunit. Another mutation, PIK3CAH1047, is present in the coding region of exon 20, and leads to enhanced affinity between the p110α subunit and the lipid membrane. PIK3CAE542K is another known mutation with significant oncogenic potential associated with elevated catalytic activity of the p110α subunit. id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"
[006] The correlation between activated PI3K/AKT signaling pathway and tumor progression has led to the recognition of PI3Ks as therapeutic targets in cancer. Idelalisib (PIK3CD inhibitor) was the first FDA-approved PI3K inhibitor for treatment of blood cancer, initially used in clinical practice in 2014. Today there are dozens of PI3K inhibitors, many of which are currently employed in clinical trials evaluation. Based on their mode of action, PI3K inhibitors are subdivided into dual PI3K/mTOR inhibitors, targeting both PI3K and the serine–threonine kinase mTOR due to their structural homology, pan-PI3K inhibitors, and isoform-specific inhibitors. Among the isoform-specific inhibitors, Alpelisib (BYL719), FDA-approved for treatment of HR-positive and HER2/neu-negative breast cancer in combination with endocrine therapy, and Serabelisib (MLN1117), which is undergoing clinical trials for several types of cancers, are PIK3CA inhibitors that selectively inhibit the p110α catalytic subunit. id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"
[007] Noncoding mutations in deep introns of tumor suppressor genes (TSGs), such as the telomere maintenance gene, POT1, were reported to contribute to tumorigenesis, specifically in blood cancers. Interestingly, these reported intronic mutations were overrepresented in TSGs but not in oncogenes ("Jung H et al., Comprehensive characterization of intronic mis-splicing mutations in human cancers. Oncogene. 2021; 40:1347-1361", herein incorporated by reference in its entirety). id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"
[008] There is a need for new biomarkers that may assist in strategic decision making in cancer. Specifically, understanding mechanisms involved in the PI3K/AKT signaling pathway in cancer, that are not related to known amino acid substitution mutations, and that can serve as either diagnostic or prognostic tools, or to affect the selection of a PIK3 inhibitor drug as a cancer therapy, are of great need.
SUMMARY OF THE INVENTION id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"
[009] The present invention provides methods of detecting oncogenic phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), diagnosing cancer and determining suitability of a subject suffering from cancer to be treated with a PI3K inhibitor comprising detecting the presence of a cytosine at position 1792035on chromosome 3 of hg38. Nucleic acid molecules and kits that differentiate between a cytosine and a guanine at position 179203524 on chromosome 3 of hg38 are also provided. id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"
[010] According to a first aspect, there is provided a method of detecting oncogenic phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in a sample, the method comprising, receiving a sample comprising a nucleic acid molecule and detecting the presence of a cytosine at position 179203524 on chromosome 3 in the nucleic acid molecule wherein the position is with respect to human genome build hg38, wherein the presence of the cytosine indicates the presence of oncogenic PIK3CA, thereby detecting oncogenic PIK3CA. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
[011] According to another aspect, there is provided a method of diagnosing cancer in a subject, the method comprising receiving a sample from the subject, detecting the presence of a cytosine at position 179203524 on chromosome 3 in a nucleic acid molecule from the sample wherein the position is with respect to human genome build hg38, wherein the presence of the cytosine indicates the subject suffers from cancer, thereby diagnosing cancer in a subject. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"
[012] According to another aspect, there is provided a method of selecting a subject suffering from cancer suitable for treatment with a phospatidylinositol-3 kinase (PI3K) inhibitor, the method comprising detecting the presence of a cytosine at position 1792035on chromosome 3 in a nucleic acid molecule derived from a cancer cell of the subject, wherein the position is with respect to human genome build hg38, thereby selecting a subject suffering from cancer suitable for treatment with a PIK3 inhibitor. id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"
[013] According to some embodiments, the nucleic acid molecule is a DNA molecule or a pre-mRNA molecule. id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"
[014] According to some embodiments, the DNA molecule comprises a PIK3CA genomic locus or the pre-mRNA comprises a PIK3CA transcript. id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"
[015] According to some embodiments, a PIK3CA gene and transcript comprises SEQ ID NO: 1 or 2 and wherein the cytosine is at position 55168 in the nucleotide sequence as set forth in SEQ ID NO: 1, or at position 54951 in the nucleotide sequence as set forth in SEQ ID NO: 2. id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16" id="p-16"
[016] According to some embodiments, the cancer is selected from: breast cancer, lung cancer, uterine cancer, brain cancer, head and neck cancer, kidney cancer, and thyroid cancer. id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"
[017] According to some embodiments, the cancer is breast cancer or lung cancer. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"
[018] According to some embodiments, the breast cancer is breast invasive carcinoma, and the lung cancer is lung squamous cell carcinoma. id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19" id="p-19"
[019] According to some embodiments, the detecting the presence of a cytosine comprises amplifying the nucleic acid molecule with at least one primer specific to the cytosine at position 179203524, wherein the specific primer does not amplify a nucleic acid molecule with a guanine at position 179203524. id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"
[020] According to some embodiments, the detecting the presence of a cytosine comprises sequencing the nucleic acid molecule or an amplified copy thereof. id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21" id="p-21"
[021] According to some embodiments, the method further comprises before the detecting receiving a sample comprising the nucleic acid molecule derived from a cancer cell of the subject. id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"
[022] According to some embodiments, the sample is a tumor sample comprising at least one cancerous cell or a liquid biopsy comprising at least one cell-free DNA molecule derived from a cancerous cell. id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23" id="p-23"
[023] According to some embodiments, the method further comprises after the detecting administering to a subject diagnosed with cancer at least one anticancer therapy. id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24" id="p-24"
[024] According to some embodiments, the method further comprises after the detecting administering to a suitable subject at least one PIK3 inhibitor. id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
[025] According to some embodiments, the PIK3 inhibitor is an inhibitor of the catalytic subunit of PIK3 (P110). id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26" id="p-26"
[026] According to some embodiments, the PIK3 inhibitor is a P110 specific inhibitor. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"
[027] According to some embodiments, the P110 is P110 isoform alpha (P110α). id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28" id="p-28"
[028] According to some embodiments, the PIK3 inhibitor is Alpelisib. id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"
[029] According to another aspect, there is provided a PIK3 inhibitor for use in treating cancer in a subject in need thereof, wherein the subject has been determined to comprise a cytosine at position 179203524 on chromosome 3, wherein the position is with respect to human genome build hg38. id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
[030] According to some embodiments, the determining comprises receiving a sample from the subject comprising a nucleic acid molecule derived from a cancer cell of the subject and detecting the presence of the cytosine at position 179203524 on chromosome 3 in the nucleic acid molecule. id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"
[031] According to another aspect, there is provided a nucleic acid molecule that differentiates between a cytosine and a guanine at position 179203524 on chromosome 3, wherein the position is with respect to human genome build hg38. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"
[032] According to some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence as set forth in any one of SEQ ID NO: 1-3 and does bind to a nucleotide sequence as set forth in any one of SEQ ID NO: 4-6. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"
[033] According to some embodiments, the nucleic acid molecule consists of not more than nucleotides. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"
[034] According to some embodiments, the nucleic acid molecule is for use in performing a method of the invention. id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35" id="p-35"
[035] According to another aspect, there is provided a kit comprising a nucleic acid molecule of the invention and at least one PIK3 inhibitor. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"
[036] According to some embodiments, the kit is for use in performing a method of the invention. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"
[037] According to some embodiments, the PIK3 inhibitor is Alpelisib. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"
[038] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"
[039] Figure 1: A sequencing chromatogram demonstrating PIK3CA:chr3:Intron 4:179203524:G:C (intron 4:G:C) mutated and non-mutated clone of A549 cells. The G to C mutation is highlighted in a circle. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"
[040] Figure 2: A bar graph showing normalized RNA levels of intron 4:G:C mutated clone compared to the non-mutated clone of A549 cells. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"
[041] Figure 3: A line graph exhibiting the proliferation rate of intron 4:G:C mutated clone compared to the non-mutated clone of A549 cells. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
[042] Figure 4: A line graph showing the proliferation rate of intron 4:G:C mutated clone compared to the non-mutated clone of A549 cells, in the presence, or the absence, of Alpelisib drug.
DETAILED DESCRIPTION OF THE INVENTION id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43" id="p-43"
[043] The present invention, in some embodiments, provides methods of detecting oncogenic phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), diagnosing cancer and determining suitability of a subject suffering from cancer to be treated with a PI3K inhibitor comprising detecting the presence of a cytosine at position 179203524 on chromosome 3 of hg38. Nucleic acid molecules and kits that differentiate between a cytosine and a guanine at position 179203524 on chromosome 3 of hg38 are also provided. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"
[044] By a first aspect, there is provided a method of detecting phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), the method comprising detecting the presence of a cytosine at position 179203524 on chromosome 3, thereby detecting PIK3CA. id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"
[045] By another aspect, there is provided a method of diagnosing cancer in a subject, the method comprising detecting the presence of a cytosine at position 179203524 on chromosome 3 in the subject, thereby diagnosing cancer in a subject. id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"
[046] By another aspect, there is provided a method of selecting a subject suitable for treatment with a phospatidylinositol-3 kinase (PI3K) inhibitor, the method comprising detecting the presence of a cytosine at position 179203524 on chromosome 3 in the subject, thereby selecting a subject suitable for treatment with a PI3K inhibitor. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"
[047] In some embodiments, the PIK3CA is oncogenic PIK3CA. In some embodiments, the PIK3CA is mutant PIK3CA. In some embodiments, the PIK3CA is a cancer driver. In some embodiments, the method is a method of detecting oncogenic PIK3CA. id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"
[048] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a prognostic method. In some embodiments, the method is a method of detecting cancer. In some embodiments, the method is a method of detecting an oncogene. In some embodiments, the method is a method of detecting an oncogenic mutation. In some embodiments, the method is a method of detecting a potential oncogene. In some embodiments, the method is a method of detecting a potential oncogenic mutation. In some embodiments, the method is a method of selecting a patient population. In some embodiments, the patient population is a population suitable for treatment. In some embodiments, treatment is treatment with a PI3K inhibitor. id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"
[049] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is in need of a method of the invention. In some embodiments, the subject is suspected of suffering from cancer. In some embodiments, the subject suffers from cancer. In some embodiments, the subject is naïve to treatment. In some embodiments, the subject has already received an anticancer treatment. In some embodiments, the anticancer treatment does not comprise a PI3K inhibitor. In some embodiments, the subject is in need of treatment. In some embodiments, the subject was not previously determined to have a mutation in PIK3CA. In some embodiments, a mutation in PIK3CA is a mutation in the coding region of PIK3CA. In some embodiments, the mutation in the coding region is a non-synonymous mutation. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"
[050] In some embodiments, the method comprises receiving a sample. In some embodiments, the method comprises obtaining a sample. In some embodiments, the sample is from the subject. In some embodiments, the sample comprises a cell. In some embodiments, the sample comprises a nucleic acid molecule. In some embodiments, the cell is from the subject. In some embodiments, the nucleic acid molecule is from the subject. In some embodiments, the cell is a cancerous cell. In some embodiments, the cell is at risk of being a cancerous cell. In some embodiments, the sample is a tumor sample. In some embodiments, the tumor sample is a biopsy. In some embodiments, the biopsy comprises at least one cancerous cell. In some embodiments, the sample comprises a plurality of nucleic acid molecules. In some embodiments, the sample comprises at least one nucleic acid molecule derived from a cancer cell. In some embodiments, the cancer cell is a cancer cell of the subject. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a bodily fluid sample. In some embodiments, the bodily fluid is selected from at least one of: blood, serum, plasma, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, lymph, breast milk, urine, interstitial fluid, vaginal fluid, cerebral spinal fluid and stool. In some embodiments, the bodily fluid is tumor fluid. In some embodiments, the biopsy is a liquid biopsy. In some embodiments, the liquid biopsy comprises at least one cell free DNA (cfDNA) molecule. In some embodiments, the at least one cfDNA molecule is derived from a cancerous cell. id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"
[051] In some embodiments, the detecting is in the sample. In some embodiments, the detecting is in the nucleic acid molecule. In some embodiments, the detecting is in the cell. In some embodiments, detecting comprises amplifying the nucleic acid molecule. In some embodiments, detecting comprises PCR. In some embodiments, detecting comprises sequencing. In some embodiments, sequencing is whole genome sequencing. In some embodiments, sequencing is next generation sequencing. In some embodiments, sequencing is massively parallel sequencing. In some embodiments, the detecting is with a molecule of the invention. In some embodiments, the detecting is with a nucleic acid molecule that is specific to a cytosine at position 179203524 on chromosome 3. In some embodiments, specific comprises not binding to a non-cytosine at position 179203524. In some embodiments, specific comprises not binding to quinine at position 179203524. In some embodiments, the molecule is a primer. In some embodiments, the primer binds to a molecule comprising cytosine at position 179203524 on chromosome 3 and does not bind to a molecule not comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, not comprising a cytosine is comprising a guanine. In some embodiments, the primer amplifies a molecule comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the primer does not amplify a molecule not comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the molecule is a probe. In some embodiments, the probe hybridizes to a molecule comprising cytosine at position 179203524 on chromosome 3 and does not hybridize to a molecule not comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the probe is integrated when amplifying a molecule comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the probe is not integrated when amplifying a molecule not comprising a cytosine at position 179203524 on chromosome 3. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"
[052] In some embodiments, the nucleic acid molecule comprises or consists of not more than 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid molecule comprises or consists of not more than 25 nucleotides. In some embodiments, the nucleic acid molecule comprises or consists of not more than 50 nucleotides. In some embodiments, the nucleic acid molecule comprises or consists of at least 5, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45 or 50 nucleotides. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid molecule comprises or consists of at least 8 nucleotides. In some embodiments, the nucleic acid molecule comprises or consists of at least 12 nucleotides. In some embodiments, the nucleic acid molecule comprises or consists of at least 20 nucleotides. id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"
[053] In some embodiments, the molecule is a nucleic acid molecule. The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"
[054] The terms "nucleic acid molecule" include but not limited to single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNA such as miRNA, siRNA and other short interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, small rRNA, hnRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, nucleic acids of infectious origin, amplification products, modified nucleic acids, plasmidical or organellar nucleic acids and artificial nucleic acids such as oligonucleotides. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"
[055] In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is cfDNA. In some embodiments, the DNA molecule comprises a PIK3CA genomic locus. In some embodiments, the DNA molecule comprises a PIK3CA gene. In some embodiments, the DNA molecule comprises a portion or fragment of a PIK3CA genomic locus. In some embodiments, the DNA molecule comprises a portion or fragment of the PIK3CA gene. In some embodiments, the portion or fragment comprises position 179203524 on chromosome 3. In some embodiments, the nucleic acid molecule is a pre-mRNA molecule. In some embodiments, the pre-mRNA is a pre-mRNA of PIK3CA. In some embodiments, the pre-mRNA is before splicing. In some embodiments, the nucleic acid molecule comprises introns. id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"
[056] In some embodiments, the PIK3CA gene comprises SEQ ID NO: 1. In some embodiments, the PIK3CA gene consists of SEQ ID NO: 1. In some embodiments, the PIK3CA pre-mRNA transcript comprises SEQ ID NO: 1. In some embodiments, the PIK3CA pre-mRNA transcript consists of SEQ ID NO: 1. In some embodiments, SEQ ID NO: 1 is transcript ENST00000263967.4. In some embodiments, mutation is mutation of the guanine at position 179203524 on chromosome 3 to cytosine. In some embodiments, position 179203524 on chromosome 3 is position 55168 in the nucleotide sequence as set forth in SEQ ID NO: 1. In some embodiments, a cytosine at position 179203524 on chromosome 3 is a cytosine at position 55168 of SEQ ID NO: 1. In some embodiments, a cytosine at position 55168 is mutation of the guanine at position 55168 to cytosine. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"
[057] In some embodiments, the PIK3CA gene comprises SEQ ID NO: 2. In some embodiments, the PIK3CA gene consists of SEQ ID NO: 2. In some embodiments, the PIK3CA pre-mRNA transcript comprises SEQ ID NO: 2. In some embodiments, the PIK3CA pre-mRNA transcript consists of SEQ ID NO: 2. In some embodiments, mutation is mutation of the guanine at position 179203524 on chromosome 3 to cytosine. In some embodiments, position 179203524 on chromosome 3 is position 54951 in the nucleotide sequence as set forth in SEQ ID NO: 1. In some embodiments, a cytosine at position 179203524 on chromosome 3 is a cytosine at position 54951 of SEQ ID NO: 2. In some embodiments, a cytosine at position 54951 is mutation of the guanine at position 54951 to cytosine. In some embodiments, the cytosine is within intron 4 of PIK3CA. In some embodiments, intron 4 of PIK3CA comprises SEQ ID NO: 3. In some embodiments, intron of PIK3CA consists of SEQ ID NO: 3. In some embodiments, position 179203524 on chromosome 3 is position 1984 in the nucleotide sequence as set forth in SEQ ID NO: 3. In some embodiments, a cytosine at position 1984 is mutation of the guanine at position 19to cytosine. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"
[058] In some embodiments, chromosome 3 is with respect to the human genome. In some embodiments, chromosome 3 is with respect to human genome build hg38. In some embodiments, position 179203524 on chromosome 3 is with respect to human genome build hg38. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"
[059] In some embodiments, the presence of the cytosine indicates the presence of oncogenic PIK3CA. In some embodiments, the presence of the cytosine indicates the PIK3CA is oncogenic. In some embodiments, the presence of the cytosine indicates the subject suffers from cancer. In some embodiments, the presence of the cytosine diagnoses the subject with cancer. In some embodiments, the presence of the cytosine indicates the subject is suitable to be treated with a PIK3 inhibitor. In some embodiments, the absence of the cytosine indicates the PIK3CA is non-oncogenic. In some embodiments, the absence of the cytosine indicates the subject does not suffer from cancer. In some embodiments, the absence of the cytosine indicates the subject is not suitable to be treated with a PIKinhibitor. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"
[060] As used herein "cancer" or "pre-malignancy" are diseases associated with cell proliferation. Non-limiting types of cancer include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cells tumors. In some embodiments, cancer is a solid cancer. In some embodiments, cancer is a hematopoietic cancer. In some embodiments, cancer is a tumor. In some embodiments, the cancer is selected from hepato-biliary cancer, cervical cancer, urogenital cancer (e.g., urothelial cancer), testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, ocular cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, hepatic cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer (e.g., triple negative breast cancer), renal cancer (e.g., renal carcinoma), skin cancer, head and neck cancer, leukemia and lymphoma. In some embodiments, the cancer is selected from: breast cancer, lung cancer, uterine cancer, brain cancer, head and neck cancer, kidney cancer, and thyroid cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the breast cancer is breast invasive carcinoma. In some embodiments, the lung cancer is lung squamous cell carcinoma. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"
[061] In some embodiments, the method further comprises administering at least one anticancer therapy to a subject diagnosed with cancer. In some embodiments, the method further comprises administering at least one PIK3 inhibitor to a subject determined to be suitable. In some embodiments, the anticancer therapy is a PIK3 inhibitor. In some embodiments, the administering is after the detecting. Anticancer therapies are well known in the art and any such therapy may be used. These include, but are not limited to radiation therapy, surgery, chemotherapy, immunotherapy, and targeted therapy. In some embodiments, the immunotherapy is immune checkpoint blockade/inhibition. In some embodiments, the immunotherapy is adoptive cell therapy or CAR therapy. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"
[062] As used herein, the terms "administering," "administration," and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, oral, intravenous, intramuscular, or intraperitoneal. id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"
[063] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"
[064] In some embodiments, the PIK3 inhibitor is a small molecule inhibitor. In some embodiments, the PIK3 inhibitor is nucleic acid molecule. In some embodiments, the nucleic acid molecule is an antisense molecule. In some embodiments, the nucleic acid molecule is reverse complementary to PIK3. In some embodiments, the nucleic acid molecule is selected from a small interfering RNA (siRNA), a small hairpin RNA (shRNA), an antisense oligonucleotide (ASO) and a gapmer. In some embodiments, the PIK3 inhibitor is an inhibitor of the catalytic subunit of PIK3. In some embodiments, the catalytic subunit is P110. In some embodiments, the PIK3 inhibitor is specific to PIK3. In some embodiments, the PIK3 inhibitor is a P110 specific inhibitor. In some embodiments, P110 is P110 isoform alpha (P110α). In some embodiments, the PIK3 inhibitor is Alpelisib. Examples of PIKinhibitors include, but are not limited to Alpelisib, Inavolisib, Risovalisib, Serabelisib, Copanlisib, RLY-2608, HS-10352, BPI-21668, Taselisib, TQ-B3525, MEN1611, Paxalisib, Gedatolisib, Ipatasertib, Capivasertib, and Miransertib. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
[065] In some embodiments, a composition is administered. In some embodiments, the composition comprises a PIK3 inhibitor. In some embodiments, the composition comprises a nucleic acid molecule of the invention. In some embodiments, the composition comprises a therapeutically effective amount of the PIK3 inhibitor. In some embodiments, the composition is a diagnostic composition. In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"
[066] As used herein, the term "carrier," "excipient," or "adjuvant" refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"
[067] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein. id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68" id="p-68"
[068] By another aspect, there is provided a PIK3 inhibitor for use in treating cancer in a subject in need thereof. id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"
[069] In some embodiments, the subject has been determined to comprise a cytosine at position 179203524 on chromosome 3. In some embodiments, the subject has been determined by a method of the invention. In some embodiments, the subject has been determined to comprise a nucleic acid molecule comprising a cytosine at position 1792035on chromosome 3. In some embodiments, a cell of the subject has been determined to comprise a nucleic acid molecule comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the cell is a cancerous cell. In some embodiments, a cancer of the subject has been determined to comprise a nucleic acid molecule comprising a cytosine at position 179203524 on chromosome 3. In some embodiments, the determining is within a sample from the cancer. In some embodiments, the determining is within a liquid biopsy from the subject. id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70" id="p-70"
[070] By another aspect, there is provided a nucleic acid molecule that differentiates between a cytosine and a guanine at position 179203524 on chromosome 3. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"
[071] In some embodiments, the nucleic acid molecule does not bind to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence present in SEQ ID NO: 1. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence as set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid molecule does not bind to SEQ ID NO: 2. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence present in SEQ ID NO: 2. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence as set forth in SEQ ID NO: 2. In some embodiments, the nucleic acid molecule does not bind to SEQ ID NO: 3. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence present in SEQ ID NO: 3. In some embodiments, the nucleic acid molecule does not bind to a nucleotide sequence as set forth in SEQ ID NO: 3. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"
[072] In some embodiments, the nucleic acid molecule does bind to SEQ ID NO: 4. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence present in SEQ ID NO: 4. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence as set forth in SEQ ID NO: 4. In some embodiments, the nucleic acid molecule does bind to SEQ ID NO: 5. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence present in SEQ ID NO: 5. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence as set forth in SEQ ID NO: 5. In some embodiments, the nucleic acid molecule does bind to SEQ ID NO: 6. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence present in SEQ ID NO: 6. In some embodiments, the nucleic acid molecule does bind to a nucleotide sequence as set forth in SEQ ID NO: 6. id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"
[073] In some embodiments, the nucleic acid molecule is a primer. In some embodiments, the nucleic acid molecule is a probe. In some embodiments, the nucleic acid molecule is reverse complementary to SEQ ID NO: 4. In some embodiments, the nucleic acid molecule is reverse complementary to SEQ ID NO: 5. In some embodiments, the nucleic acid molecule is reverse complementary to SEQ ID NO: 6. In some embodiments, the nucleic acid molecule comprises ggctcc. In some embodiments, the nucleic acid molecule comprises atggctcc. In some embodiments, the nucleic acid molecule comprises aaatggctcc (SEQ ID NO: 7). In some embodiments, the nucleic acid molecule comprises aggggg. In some embodiments, the nucleic acid molecule comprises attaaggggg (SEQ ID NO: 8). id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"
[074] In some embodiments, the nucleic acid molecule is for use in performing a method of the invention. In some embodiments, the nucleic acid molecule is for use in diagnosing cancer. In some embodiments, the nucleic acid molecule is for use in determining suitability of a subject. In some embodiments, the nucleic acid molecule is for use in identifying oncogenic PIK3CA. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"
[075] By another aspect, there is provide a kit comprising a nucleic acid molecule of the invention. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"
[076] By another aspect, there is provided a kit comprising a PIK3 inhibitor. id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"
[077] In some embodiments, the kit comprises instructions for performing a method of the invention. In some embodiments, the kit further comprises a PIK3 inhibitor. In some embodiments, the kit further comprises a nucleic acid molecule of the invention. In some embodiments, the kit is for use in a method of the invention. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"
[078] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"
[079] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"
[080] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B." id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81" id="p-81"
[081] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82" id="p-82"
[082] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents, unless the context clearly dictates otherwise. The terms "a" (or "an") as well as the terms "one or more" and "at least one" can be used interchangeably. id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83" id="p-83"
[083] Furthermore, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
[084] Wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of" and/or "consisting essentially of" are included. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"
[085] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"
[086] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"
[087] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
EXAMPLE 1: Discovery of a new mutation in the human PIK3CA gene id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
[088] A mutation in the fourth intron of PIK3CA gene was identified by the inventors. The mutation is located on chromosome 3 at position 179203524, with respect to the hgreference genome. The mutation changes the positive strand reference allele G to the positive strand alternative allele C (e.g., G to C mutation). According to hg38 coordinates, intron is located between position 179201541 and position 179203543. Thus, this mutation is located 19 bps from the 3' terminal side of the fourth intron (there are 19 nucleotides after it in the intron). The mutation is herein designated by the following ID: PIK3CA:chr3:Intron 4:179203524:G:C (or alternatively termed "intron 4:G:C" mutation). id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
[089] Table 1 summarizes the exon-intron map of the PIK3CA gene (transcript ENST00000263967.4, SEQ ID NO: 1). The mutation PIK3CA:chr3:Intron 4:179203524:G:C, is in intron 4, which consists of 2003 nucleotides, and is located between exon 4 and exon 5. The G that is mutated is nucleotide 1984 of intron 4 (SEQ ID NO: 3).
Table 1 – Intron-Exon map of isoform 1 of the PIK3CA gene.
Exon / Intron Start End LengthExon 1 179,148,357 179,148,603 2Intron 1 179,148,604 179,198,749 50,1Exon 2 179,198,750 179,199,177 4Intron 2 179,199,178 179,199,689 5Exon 3 179,199,690 179,199,899 2Intron 3 179,199,900 179,201,289 1,3Exon 4 179,201,290 179,201,540 2Intron 4 179,201,541 179,203,543 2,0Exon 5 179,203,544 179,203,789 2Intron 5 179,203,790 179,204,502 7Exon 6 179,204,503 179,204,588 Intron 6 179,204,589 179,209,594 5,0Exon 7 179,209,595 179,209,700 106 Intron 7 179,209,701 179,210,185 4Exon 8 179,210,186 179,210,338 1Intron 8 179,210,339 179,210,430 Exon 9 179,210,431 179,210,565 1Intron 9 179,210,566 179,218,209 7,6Exon 10 179,218,210 179,218,334 1Intron 10 179,218,335 179,219,195 8Exon 11 179,219,196 179,219,277 Intron 11 179,219,278 179,219,570 2Exon 12 179,219,571 179,219,735 1Intron 12 179,219,736 179,219,948 2Exon 13 179,219,949 179,220,052 1Intron 13 179,220,053 179,220,985 9Exon 14 179,220,986 179,221,157 1Intron 14 179,221,158 179,224,080 2,9Exon 15 179,224,081 179,224,187 1Intron 15 179,224,188 179,224,699 5Exon 16 179,224,700 179,224,821 1Intron 16 179,224,822 179,225,961 1,1Exon 17 179,225,962 179,226,040 Intron 17 179,226,041 179,229,271 3,2Exon 18 179,229,272 179,229,442 1Intron 18 179,229,443 179,230,003 5Exon 19 179,230,004 179,230,121 1Intron 19 179,230,122 179,230,224 1Exon 20 179,230,225 179,230,376 1Intron 20 179,230,377 179,234,093 3,7Exon 21 179,234,094 179,240,093 6,0 EXAMPLE 2: The in vitro effect of the intron 4:g:c mutation id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
[090] The intron 4:g:c mutation being in an intron does not directly alter the amino acid sequence of the PIK3CA protein. Further, the mutation is not predicted to alter a splice site and thus generate a mis-spliced protein. Rather, the mutation was computationally predicted to increase transcription of the PIK3CA pre-mRNA. In order to investigate the effect of the PIK3CA:chr3:Intron 4:179203524:G:C mutation, a human lung cancer cell line that has no known mutations in PIK3CA gene was identified using cancer cell line encyclopedia (CCLE) databases, literature and next generation sequencing (NGS) analysis. The A549 cell line (lung cell carcinoma) was edited by CRISPR technology to generate a stable homozygous clone that harbors this point mutation; PIK3CA:chr3:Intron 4:179203524:G:C. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"
[091] The sequence of the edited cells was confirmed by PCR and sequenced at the mutated region ( Fig. 1 ). Examination of the level of PIK3CA-transcribed mRNA in A549 edited cells harboring this mutation, by real-time PCR (RTqPCR), revealed ~52% increase compared to A549 cells that went through the identical CRISPR process and were not identified with this mutation, thus served as negative control cells ( Fig. 2 ). This increase correlates with the computational prediction model in which a ~60% increase in mRNA levels was predicted. The primers used for PCR were: Forward-CATGGAGGAGAACCCTTATG (SEQ ID NO: 9) and Reverse-CTTTCGGCCTTTAACAGAGC (SEQ ID NO: 10). id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
[092] The next objective was to examine whether this mutation affects cell proliferation. As discussed hereinabove, several oncogenic point mutations in PIK3CA are known and so it was hypothesized that increased expression of the wild-type protein would also be oncogenic. This was examined using a CellTiter-Glo® luminescent cell viability assay. As measured by the assay, there was a ~2.25-fold increase in the proliferation rate of the intron 4:G:C mutated cells, versus the non-mutated cells ( Fig. 3 ). Thus, this mutation is a driver mutation, that increases cell proliferation rate and is oncogenic. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"
[093] Alpelisib is an alpha-specific PI3K inhibitor indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, breast cancer patients, that harbor known mutations in exon-coding regions of the PIK3CA gene. Alpelisib is known to inhibit the proliferation of cells harboring these known mutations. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"
[094] As demonstrated in Figure 4 , addition of Alpelisib to cultured A549 cells homozygous for the intron 4:G:C mutation, diminished their proliferation rate. This reduction brought cell proliferation down to the level of the non-mutated cells. Thus, this mutation can be utilized as a criterion for selecting patients likely to be responsive to PI3K inhibitors, and specifically to alpha inhibitors, such as Alpelisib. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"
[095] PI3Ks are activated by phosphorylation of the 3-hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns) lipids in the plasma membrane. Phosphorylation assays are performed to quantify phosphorylation of PtdIns in the mutated cells as compared to the control cells. Increased phosphorylation of PtdIns lipids, indicating enhanced activity of PIK3CA in the intron 4:G:C mutated A549 cells is observed. Migration assays are also performed to test the migration capacity of the mutated cells versus the control cells. Migration and invasion capacity of the mutated cells is increased as compared to the non-mutated cells, indicating that this mutation increases the metastatic potential of the cells harboring it. Finally, a rodent model is constructed to test the mutation in vivo. Cells harboring the mutation and cells devoid of the mutation are injected into immunocompromised rodents and the growth of the resultant tumors is monitored. Tumor growth is also monitored when Alpelisib or other PI3K inhibitor are also administered. The mutated cells result in larger tumors which respond to the PI3K inhibitors.
EXAMPLE 3: The intron 4:g:c mutation in humans id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"
[096] It was surprisingly found that although this point mutation is in an intron coding region, it leads to a significant increase in the levels of PIK3CA-transcribed mRNA in vitro. To test if this phenomenon occurs in humans, data obtained from the cancer genome atlas (TCGA), a cancer genomics program that molecularly characterized over 20,000 primary cancer and matched normal samples spanning 33 cancer types, was analyzed for the presence of the intron 4:g:c mutation. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"
[097] Firstly, the mutation was indeed found in a wide variety of cancer samples from the database. The mutation was present in 4.9% of breast invasive carcinoma (BRCA) patients, 2.5% of lung squamous cell carcinoma (LUSC) patients, 2.4% of uterine corpus endometrial carcinoma (UCEC) patients, 2.4% of glioblastoma multiforme (GBM) patients, 2.1% of head and neck squamous cell carcinoma (HNSC) patients, 2.2% of kidney renal clear cell carcinoma (KIRC) patients, 0.8% of brain lower grade glioma (LGG) patients, 1.2% of lung adenocarcinoma (LUAD) patients, and 1.0% of thyroid carcinoma (THCA) patients. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
[098] Examination of data from the BRCA patients revealed a 1.2-fold increase in PIKCA mRNA levels in patients with the mutation as compared to patients without (P value < 1x10-). A 1.6-fold increase in PIKCA mRNA levels was observed in LUSC patients with the mutation (P value = 1.0x10-3). These results strongly indicate that the mutation does indeed increase transcription in human cancers. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
[099] It was observed that in some breast invasive carcinoma patients, the intron 4:G:C mutation was found to be simultaneously present with other known mutations in the exon-coding region of PIK3CA. For example, 3 patients were found to have the intron 4:G:C mutation and the E545K mutation; six patients were found to have the intron 4:G:C mutation and the H1047R mutation; three patients were found to have the intron 4:G:C and the E542K mutation; three patients were found to have the intron 4:G:C mutation and the Q546R mutation; and one patient was found to have the intron 4:G:C mutation and the H1047L mutation. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"
[0100] In conclusion, it was surprisingly found that a point mutation, in a non-translated region of the PIK3CA gene, has a significant oncogenic potential and induces an augmented cancerous phenotype in the cell harboring it, manifested by enhanced proliferation; a phenotype that is reversed by treatment with a PIK3CA inhibitor. These findings highlight this mutation as a diagnostic and prognostic tool, which can also be used as an onco-personalized selection tool of potential responders to PIK3 inhibitor therapy. id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101" id="p-101"
[0101] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims (28)

1. CLAIMS:
2. What is claimed is: 1. A method of detecting oncogenic phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in a sample, the method comprising, receiving a sample comprising a nucleic acid molecule and detecting the presence of a cytosine at position 179203524 on chromosome 3 in said nucleic acid molecule wherein said position is with respect to human genome build hg38, wherein the presence of said cytosine indicates the presence of oncogenic PIK3CA, thereby detecting oncogenic PIK3CA. 2. A method of diagnosing cancer in a subject, the method comprising receiving a sample from said subject, detecting the presence of a cytosine at position 179203524 on chromosome 3 in a nucleic acid molecule from said sample wherein said position is with respect to human genome build hg38, wherein the presence of said cytosine indicates the subject suffers from cancer, thereby diagnosing cancer in a subject.
3. A method of selecting a subject suffering from cancer suitable for treatment with a phospatidylinositol-3 kinase (PI3K) inhibitor, the method comprising detecting the presence of a cytosine at position 179203524 on chromosome 3 in a nucleic acid molecule derived from a cancer cell of said subject, wherein said position is with respect to human genome build hg38, thereby selecting a subject suffering from cancer suitable for treatment with a PIK3 inhibitor.
4. The method of any one of claims 1 to 3, wherein said nucleic acid molecule is a DNA molecule or a pre-mRNA molecule.
5. The method of claim 4, wherein said DNA molecule comprises a PIK3CA genomic locus or said pre-mRNA comprises a PIK3CA transcript.
6. The method of any one of claims 1 to 5, wherein a PIK3CA gene and transcript comprises SEQ ID NO: 1 or 2 and wherein said cytosine is at position 55168 in the nucleotide sequence as set forth in SEQ ID NO: 1, or at position 54951 in the nucleotide sequence as set forth in SEQ ID NO: 2.
7. The method of any one of claims 2 to 6, wherein said cancer is selected from: breast cancer, lung cancer, uterine cancer, brain cancer, head and neck cancer, kidney cancer, and thyroid cancer.
8. The method of claim 7, wherein said cancer is breast cancer or lung cancer.
9. The method of claim 8, wherein said breast cancer is breast invasive carcinoma and said lung cancer is lung squamous cell carcinoma.
10. The method of any one of claims 1 to 9, wherein said detecting the presence of a cytosine comprises amplifying said nucleic acid molecule with at least one primer specific to said cytosine at position 179203524, wherein said specific primer does not amplify a nucleic acid molecule with a guanine at position 179203524.
11. The method of any one of claims 1 to 10, wherein said detecting the presence of a cytosine comprises sequencing said nucleic acid molecule or an amplified copy thereof.
12. The method of any one of claims 3 to 11, further comprising before said detecting receiving a sample comprising said nucleic acid molecule derived from a cancer cell of said subject.
13. The method of claim 12, wherein said sample is a tumor sample comprising at least one cancerous cell or a liquid biopsy comprising at least one cell-free DNA molecule derived from a cancerous cell.
14. The method of any one of claims 2 to 13, further comprising after said detecting administering to a subject diagnosed with cancer at least one anticancer therapy.
15. The method of any one of claims 3 to 14, further comprising after said detecting administering to a suitable subject at least one PIK3 inhibitor.
16. The method of claim 15, wherein said PIK3 inhibitor is an inhibitor of the catalytic subunit of PIK3 (P110).
17. The method of claim 16, wherein said PIK3 inhibitor is a P110 specific inhibitor.
18. The method of claim 16 or 17, wherein said P110 is P110 isoform alpha (P110α).
19. The method of any one of claims 15 to 18, wherein said PIK3 inhibitor is Alpelisib.
20. A PIK3 inhibitor for use in treating cancer in a subject in need thereof, wherein said subject has been determined to comprise a cytosine at position 179203524 on chromosome 3, wherein said position is with respect to human genome build hg38.
21. The PIK3 inhibitor of claim 20, wherein said determining comprises receiving a sample from said subject comprising a nucleic acid molecule derived from a cancer cell of said subject and detecting the presence of said cytosine at position 1792035on chromosome 3 in said nucleic acid molecule.
22. A nucleic acid molecule that differentiates between a cytosine and a guanine at position 179203524 on chromosome 3, wherein said position is with respect to human genome build hg38.
23. The nucleic acid molecule of claim 22, wherein said nucleic acid molecule does not bind to a nucleotide sequence as set forth in any one of SEQ ID NO: 1-3 and does bind to a nucleotide sequence as set forth in any one of SEQ ID NO: 4-6.
24. The nucleic acid molecule of claim 22 or 23, consisting of not more than nucleotides.
25. The nucleic acid molecule of any one of claims 22 to 24, for use in performing a method of any one of claims 1 to 19.
26. A kit comprising a nucleic acid molecule of any one of claims 22 to 25 and at least one PIK3 inhibitor.
27. The kit of claim 26, for use in performing a method of any one of claims 15 to 19.
28. The kit of claim 26 or 27, wherein said PIK3 inhibitor is Alpelisib.
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