WO2021174262A1 - Méthodes d'évaluation de pronostic du cancer du sein et régimes de traitement basés sur l'état de méthylation de la région ins-igf2 - Google Patents

Méthodes d'évaluation de pronostic du cancer du sein et régimes de traitement basés sur l'état de méthylation de la région ins-igf2 Download PDF

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WO2021174262A1
WO2021174262A1 PCT/US2021/070206 US2021070206W WO2021174262A1 WO 2021174262 A1 WO2021174262 A1 WO 2021174262A1 US 2021070206 W US2021070206 W US 2021070206W WO 2021174262 A1 WO2021174262 A1 WO 2021174262A1
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igf2
methylation
ins
patient
breast cancer
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PCT/US2021/070206
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Daisy D. DE LEÓN
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Loma Linda University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to nucleic acid sequences whose DNA methylation levels are indicative of the status of certain cancers. More particularly, present disclosure relates to nucleic acid sequences whose DNA methylation levels are indicative of the status of breast cancers, such as Triple Negative Breast Cancer (TNBC), and can be used to assess response to treatment regimens.
  • TNBC Triple Negative Breast Cancer
  • BACKGROUND [0004] TNBC is the most aggressive type of breast cancer among different ethnic groups worldwide. DNA-based methylation is a process in which cytosines acquire a methyl group in 5' position only if they are followed by a guanine.
  • IGF2 Insulin-Like Growth factor 2
  • BC breast cancer
  • IGF2 is inhibited by tumor suppressor genes such as p53 and Pten, both important in normal cell and BC cell development. This growth factor inhibits apoptosis and promotes metastasis and chemoresistance, contributing to more aggressive tumors. Lack of expression or mutations in tumor suppressors contribute to higher IGF2 levels, not only for lack of suppression but also for gain of function such as in the case of mutated p53, which stimulates IGF2 instead of repressing the growth factor. IGF2 expression is increased by growth hormone, estrogen, progesterone, and prolactin, all important hormones in normal breast development and in BC progression. “Free” circulating IGF2 levels in humans are significantly correlated to breast tumor size and malignancy.
  • IGF2 expression is important in normal breast development. Increased IGF2 expression in the mammary gland contributes to BC malignancy.
  • the IGF2 gene is located on the short (p) arm of chromosome 11 at position 15.5. Methylation of the IGF2 gene regulatory regions occurs during the formation of an egg or sperm cell, and it is distinct and differentially modified depending on the parental origin of the allele.
  • the IGF2 gene has five promoters (P0, P1-P4) that generate distinct transcripts which vary by tissue type and developmental stages. In normal human tissues, the IGF2 gene is controlled by at least two differentially methylated regions (DMRs).
  • DMRs differentially methylated regions
  • IGF2 DMR IGF2 promoters
  • H19 DMR H19 DMR
  • the epigenetic information of the IGF2 gene is stored via heritable DNA methylation.
  • the organization of the IGF2 gene chromatin structure and its regulatory elements involves various long linear non-coding RNA and microRNAs at the early embryonic developmental stages.
  • the IGF2 DMR and the CpG island regions play a vital function in early embryonic development, and its methylation status is subject to modification by fetal environmental exposures such as famine, nutrition, and stress.
  • Abnormal regulation of IGF2 leads to various types of cancers and metabolic disorders like BC, pancreatic cancer, diabetes, and endocrine related disorders.
  • IGF2 protein levels are higher in BC tissues from African American women than in Caucasian women. Additionally, high IGF2 protein levels have been shown to be expressed in normal breast tissues of African American women, while little or no IGF2 may be detected in tissues from Caucasian women. Decreased DNA methylation of the IGF2 gene leads to different BC clinical features. In spite of these advances, there is currently no consensus regarding the methylation status of the IGF2 gene, and its relationship to the levels of IGF2 protein expressed in normal breast or in breast cancer tissues.
  • TNBC Triple Negative Breast Cancer
  • Applicant has recognized an unmet and urgent need for evaluating methylation status of the IGF2 gene to diagnose the status of breast cancers, such as Triple Negative Breast Cancer (TNBC).
  • TNBC Triple Negative Breast Cancer
  • compounds and methods addressing the shortcomings of the art, and may provide any number of additional or alternative advantages, including compositions and methods for evaluation of DNA methylation of a 257 base pair DNA region of the INS-IGF2 gene in breast cancer patients.
  • Hypomethylation levels of the INS-IGF2 region sequence (Chr11:2148098-2148354) increases IGF2 levels and promotes a more aggressive, less responsive breast cancer disease.
  • Assessment of the methylation of this region is a novel tool to improve the limited and ineffective treatment currently available for TNBC patients.
  • this region is designated the DaisyVin Differentially Methylated Region (INS-IGF2 DVDMR).
  • INS-IGF2 DVDMR DaisyVin Differentially Methylated Region
  • Lower methylation of this region increases IGF2 expression, proliferation of the cells, and a higher risk of metastasis, recurrence and chemoresistance.
  • Chemoresistance results from the increased IGF2 levels interfering with chemotherapeutic agents, such as paclitaxel, doxorubicin, Taxol and others.
  • chemotherapeutic agents such as paclitaxel, doxorubicin, Taxol and others.
  • the hypomethylation status of the INS-IGF2 DVDMR can be used to determine which treatment and follow up will be given to the patient. Higher levels of IGF2, driven by hypomethylation will make those patients less responsive to chemotherapy, and radiation.
  • assessing the hypomethylation of the INS-IGF2 DVDMR will provide critical information regarding the treatment to those patients because IGF2 will prevent the response to currently used chemotherapy agents.
  • the methylation of the INS-IGF2 DVDMR is determined by using methylation specific primers in a methyl PCR array.
  • the method can further include the steps of determining expression levels of insulin-like growth factor-II (IGF2) to assess response to treatment.
  • IGF2 insulin-like growth factor-II
  • assessing the INS-IGF2 DVDMR methylation status of the normal tissue surrounding the tumor can provide critical information to determine type of surgery, treatment, and follow-up.
  • the method can include the steps of determining expression levels of the IGF2 protein; and in response to increased expression of the IGF2 protein and hypomethylation status of the INS-IGF2 DVDMR, the patient can receive a modified treatment addressing the higher risk of recurrence and metastasis.
  • the expression level of IGF2 protein is determined by reverse phase protein array and Western blots.
  • the biological sample is obtained from a breast of the patient.
  • the biological sample is a blood sample from the patient.
  • Embodiments include diagnostic methods directed to determining DNA methylation of the INS-IGF2 DVDMR from a biological sample from a patient.
  • the hypomethylation status of the INS-IGF2 DVDMR is indicative of onset and/or progression of a breast neoplasm, such as an adenoma or adenocarcinoma.
  • One such method for detecting onset, or predisposition to the onset, of a breast neoplasm in a patient includes the steps of assessing a methylation level of CpG dinucleotides in SEQ ID NO: 1 or a complement thereof in a biological sample from the patient; comparing the assessed methylation level to a methylation level of CpG dinucleotides in SEQ ID NO: 1 or the complement thereof in a control sample from a human without a breast neoplasm; detecting a decreased level of CpG methylation in the assessed methylation level of CpG dinucleotides in SEQ ID NO: 1 or the complement thereof as compared to the methylation level in the control sample from the human without a breast neoplasm; and in response to the decreased level of CpG methylation in the assessed methylation level, identifying the patient as having an onset, or predisposition to the onset, of a breast neoplasm.
  • the step of assessing the methylation level of CpG dinucleotides in SEQ ID NO: 1 or the complement thereof in the biological sample from the patient further includes the steps of obtaining genomic deoxyribonucleic acid (DNA) from the biological sample from the patient; contacting the genomic DNA with a bisulfite salt to generate bisulfite converted genomic DNA; and amplifying bisulfite converted genomic DNA with primers directed to bisulfite converted SEQ ID NO: 1 or the complement thereof to assess methylation level of CpG dinucleotides in SEQ ID NO: 1 or the complement thereof.
  • DNA genomic deoxyribonucleic acid
  • the methylation level of CpG dinucleotides in SEQ ID NO: 1 or the complement thereof is assessed by one or more of methylation-specific PCR, methylation-sensitive single nucleotide primer extension, or methylated CpG island amplification.
  • the method further includes the steps of: determining a first expression level of insulin-like growth factor-II mRNA in the biological sample from the patient; comparing the first expression level of insulin-like growth factor-II mRNA in the biological sample from the patient to a second expression level of insulin-like growth factor-II mRNA in a control sample from a human without a breast neoplasm; and in response to the first expression level of insulin-like growth factor-II mRNA being greater than the second expression level of insulin-like growth factor-II mRNA and the decreased level of CpG methylation in the assessed methylation level, identifying the patient as having an aggressive breast cancer.
  • the aggressive breast cancer is triple negative breast cancer.
  • the method further includes the steps of determining a first expression level of insulin-like growth factor-II protein in the biological sample from the patient; comparing the first expression level of insulin-like growth factor-II protein in the biological sample from the patient to a second expression level of insulin-like growth factor-II protein in a control sample from a human without a breast neoplasm; and in response to the first expression level of insulin- like growth factor-II protein being greater than the second expression level of insulin-like growth factor-II protein and the decreased level of CpG methylation in the assessed methylation level, identifying the patient as having an aggressive breast cancer.
  • the aggressive breast cancer is triple negative breast cancer.
  • the measurement of DNA methylation levels of the INS-IGF2 DVDMR is combined with measurements of the levels of one or more proteins stimulated by IGF2 (IGF2 expression markers).
  • the measurement of DNA methylation levels of the INS-IGF2 DVDMR is used to assess chemotherapy response, as IGF2 levels impair the response to currently used chemotherapeutic agents.
  • the measurement of INS-IGF2 DVDMR methylation levels of the INS-IGF2 region will be use to design theranostic treatment that can be used to select and monitor the treatment regimens for patients with triple negative breast cancer.
  • Embodiments of the reagents are regulated by IGF2, highly expressed in breast cancer, and can be used in theranostic therapy. [0014] Described here are methods of diagnosing a patient with an aggressive breast cancer and treating the patient with one or more of a chemotherapeutic, immunotherapeutic, or a radiation agent.
  • One such method for treating a patient with breast cancer includes the steps of determining methylation of a differentially methylated region situated in-between exon 3 and exon 4 constituting a processed insulin-like growth factor-II gene sequence derived from a biological sample from a patient; in response to hypomethylation status of the differentially methylated region, identifying the patient as having an aggressive breast cancer; and administering to the patient an effective amount of one of more of a chemotherapeutic, immunotherapeutic, or a radiation agent.
  • the aggressive breast cancer is triple negative breast cancer.
  • the methylation of the differentially methylated region is determined by using methylation specific primers in a methyl PCR array.
  • the method further includes the steps of: determining expression level of insulin-like growth factor- II mRNA; and in response to increased expression of the insulin-like growth factor-II mRNA and hypomethylation status of the differentially methylated region, identifying the patient as having an aggressive breast cancer. In certain embodiments, the method further includes the steps of: determining expression level of insulin-like growth factor-II protein; and in response to increased expression of the insulin-like growth factor-II protein and hypomethylation status of the differentially methylated region, identifying the patient as having an aggressive breast cancer. [0015] Embodiments include kits for assaying a biological sample from a patient.
  • kits includes one or more polynucleotides that hybridize to a deoxyribonucleic acid (DNA) region defined by a portion of SEQ ID NO. 1 or a complement thereof; and at least one reagent for detection of DNA methylation.
  • the kit includes a compound that selectively mutates a non-methylated cytosine residue.
  • the kit includes (i) sodium bisulfite; (ii) primers that hybridize to the DNA region defined by the portion of SEQ ID NO. 1 or the complement thereof; and (iii) detectably-labelled probes that distinguish between methylated and unmethylated DNA that has been treated with bisulfite.
  • the kit includes one or more control DNA sequences representing methylated or unmethylated forms of the DNA region.
  • FIG. 1A-C are schematic diagrams of the human IGF2 gene structure, according to an embodiment.
  • FIG.1A is an IGF2 gene diagram showing the relative positions of the IGF2 introns and coding exons, according to an embodiment.
  • FIG. 1C presents the INS-IGF2 DVDMR BLAT search sequence (Chr11:2148098-2148354) (SEQ ID NO. 1), according to an embodiment.
  • FIGS. 2A-D are diagrams and graphical representations of the IGF2 gene structure and transcript variance in TNBC cell lines, according to an embodiment.
  • FIG.2A is a diagram showing specific transcripts generated from P1-P4 promoters, according to an embodiment.
  • FIG.2B shows the specific P1-P4 promoter transcript variance differentially expressed in breast cancer (BC) cell lines, according to an embodiment.
  • FIG.2C represents total endogenous IGF2 mRNA fold change expressed in breast cancer cell lines, using QPCR, according to an embodiment.
  • FIG.2D is a bar graph representing the levels of IGF2 protein being expressed in breast cancer cells assessed by Reverse Phase Protein Array (RPPA), according to an embodiment.
  • FIGS.2E-2F are graphical representations of the INS-IGF2 Transcript Variants (Long and Short), according to an embodiment.
  • FIG. 2E is a bar graph representing the INS-IGF2 Long (DQ104205) and Short (DQ104204) mRNA fold change expressed in African American (AA) and Caucasian (CA) TNBC Cells, according to an embodiment.
  • FIGS.3A-3C are an image of an EtBr Agarose gel of IGF2 CpG islands and DMROs and graphical representations of % methylation and IGF2 protein levels in paired normal/malignant breast tissues from AA women, according to an embodiment.
  • FIG.3A shows one of two ethidium bromide-stained agarose gels of Bisulfite g-DNA fragments produced by conventional PCR amplification of the CpG and DMRO regions of the IGF2 gene in paired breast tissues of AA women, according to an embodiment.
  • FIG.3B shows a comparative bar graph of the methylation of the INS-IGF2 DVDMR in paired tissues from AA women, in which average % MSRE Methylation is shown by Box and Whisker Plots, according to an embodiment.
  • FIG.3C is a bar graph showing the IGF2 levels from paired breast tissues from AA women assessed by Reverse Phase Protein Array (RPPA) assay, according to an embodiment.
  • RPPA Reverse Phase Protein Array
  • FIGS.4A-4C are an image of an EtBr Agarose gel of IGF2 CpG islands and DMROs and graphical representations of % methylation and IGF2 protein levels in paired normal/malignant breast tissues from CA women, according to an embodiment.
  • FIG.4A shows one of two ethidium bromide stained agarose gels of bisulfite g-DNA fragments produced by conventional PCR amplification of the CpG and DMRO regions of the IGF2 gene in paired breast cancer tissues from Caucasian American (CA) women.
  • CA Caucasian American
  • FIG.4B shows a comparative bar graph of the methylation of the INS-IGF2 DVDMR in paired tissues from CA women, in which average % MSRE Methylation shown by Box and Whisker Plots, according to an embodiment.
  • FIG.4C shows a bar graph of the IGF2 levels in the paired breast tissues from CA women assessed by the RPPA assay, according to an embodiment.
  • the Breast Cancer tissue samples details are shown in Table 1.
  • the “*” symbol denotes paired samples (Normal/Malignant) from same patient.
  • FIGS.5A-5C are an image of an EtBr Agarose gel of IGF2 CpG islands and DMROs and graphical representations of % methylation and IGF2 protein levels in paired normal/malignant breast tissues from VIET women, according to an embodiment.
  • FIG. 5A shows an ethidium bromide stained agarose gel of bisulfite g-DNA fragments produced by PCR amplification of the CpG and DMRO regions of the IGF2 gene in paired breast cancer tissues from Vietnamese women (VIET), according to an embodiment.
  • FIG.5B shows a comparative bar graph of the methylation of the INS-IGF2 DVDMR in paired tissues VIET by Methyl Sensitive Restriction Enzyme (MSRE) qPCR, in which average % MSRE Methylation shown by Box and Whisker Plots, according to an embodiment.
  • FIG. 5C is a bar graph of the IGF2 levels in paired breast tissues assessed by RPPA assay, according to an embodiment.
  • the TNBC tissue samples details are shown in Table 1.
  • the “*” symbol denotes paired samples (Normal/Malignant) from same patient.
  • FIG. 6A-6B are graphical representations of the methylation status of the INS-IGF2 DVDMR in paired breast cancer tissues exclusively obtained from TNBC tumors, according to an embodiment. Box and Whisker Plots next to each bar graph show the average (mean+/-SEM) of the % MSRE in AA and CA.
  • the TNBC tissue Samples details are listed in Table 2. The “*” symbol denotes paired samples (Normal/Malignant) from same patient.
  • FIG. 6A shows comparative bar graphs representing the methylation status of the INS-IGF2 DVDMR assessed by MSRE qPCR in paired TNBC tissues from AA women, according to an embodiment.
  • FIGS. 6C-6D are graphical representations of the methylation status of the INS-IGF2 DVDMR in paired TNBC tissues, according to an embodiment.
  • FIG. 6C is a bar graph representing the methylation status of the INS-IGF2 DVDMR in paired TNBC tissues from SKR women, according to an embodiment.
  • FIG. 6D is a bar graph representing average % MSRE Methylation shown by Box and Whisker Plots for SKR TNBC women, according to an embodiment.
  • FIG.7A is an image of the western blot assay representing the levels of IGF2 protein being expressed in Triple Negative Breast Cancer Cells (TNBC), according to an embodiment.
  • FIG.7B is a bar graph representing the levels of IGF2 protein being expressed in Triple Negative Breast Cancer Cells (TNBC) shown in Fig.7A.
  • TNBC Triple Negative Breast Cancer Cells
  • FIG.7A This Western blot (FIG.7A) demonstrates that the IGF2 present in breast cancer can be identified.
  • FIG. 8 is a bar graph representing the % methylation by methyl-sensitive restriction enzyme assay (MSRE) showing the % methylation levels of INS-IGF2 DMR levels in breast cancer cell lines by qPCR method, according to an embodiment. It shows that the level of methylation of the INS-IGF2 DVDMR in the TNBC cells correlates to the levels of IGF2 protein shown in FIGS. 7A and 7B. High methylation of the INS-IGF2 DVDMR corresponds to lower IGF2 and likewise, hypomethylation of the INS-IGF2 DVDMR corresponds to higher IGF2 protein expression. [0028] FIG.
  • MSRE methyl-sensitive restriction enzyme assay
  • FIG. 10 is a bar graph representing the IGF2 levels in paired breast tissues from Vietnamese patients as assessed by Reverse Phase Protein Array (RPPA) assay, according to an embodiment. Sample details are shown in Table 1.
  • the INS-IGF2 locus includes two alternatively spliced read-through transcript variants which align to the INS gene in the 5' region and to the IGF2 gene in the 3' region.
  • One transcript is predicted to encode a protein which shares the N-terminus with the INS protein but has a distinct and longer C-terminus, whereas the other transcript is a candidate for nonsense-mediated decay (NMD).
  • the INS-IGF2 gene is imprinted and is paternally expressed in the limb and eye.
  • IGF2 is one of the best characterized epigenetically regulated loci in the human genome. This gene is located on the short (p) arm of chromosome 11 at position 15.5.
  • IGF2 gene regulatory regions occurs during the formation of an egg or sperm cell, and it is distinct and differentially regulated depending on the parental origin of the allele.
  • IGF2 gene has four promoters (P1-P4) that generate distinct transcripts which vary by tissue type and developmental stages. In normal human tissues, the IGF2 gene is regulated by at least two differential methylated regions (DMRs). One DMR is located upstream of the IGF2 promoters (IGF2 DMR) and the second DMR is located upstream of the neighboring non-coding H19 gene (H19 DMR). Shifts in methylation established at these DMRs can lead to loss of imprinting and altered transcription of the IGF2 gene.
  • DMRs differential methylated regions
  • IGF2 DMR and the CpG island regions play a vital role in early embryo development and its methylation status is subject to modification by environmental exposures such as famine, nutrition, and stress.
  • Abnormal regulation of IGF2 leads to various metabolic disorders like breast cancer, pancreatic cancer, diabetes, and endocrine related disorders.
  • dysregulation of the IGF2 promoters occurs in breast and in other types of cancers.
  • the present disclosure is directed to a novel differentially methylated region (DMR), which consists of 257 base pair located in the INS-IGF2 gene between Exon 3 and Exon 4 of the chromosome region (Chr11:2148098–2148354).
  • DMR differentially methylated region
  • individual AA, CA, and VIET breast cancer tissue samples and related breast cancer cell lines showed differences in methylation patterns of INS-IGF2 DVDMR by Bisulfite and methyl sensitive QPCR array in normal versus malignant paired samples.
  • Embodiments include methods of screening for the onset or predisposition to the onset of a breast neoplasm in an individual by assessing the methylation status of the DNA region defined by INS-IGF2 DVDMR (Chr11:2148098–2148354) in a biological sample from the individual. A lower level of methylation of INS-IGF2 DVDMR relative to control levels is indicative of a neoplastic breast cell or a cell predisposed to the onset of a neoplastic state.
  • the method of screening for the onset or predisposition to the onset of a breast neoplasm in an individual includes assessing the methylation of one or more cytosine residues selected from those present as part of the INS-IGF2 DVDMR or a corresponding cytosine at position n+1 on the opposite DNA strand, in a biological sample from the individual.
  • a lower level of methylation of one or more of said residues relative to the methylation level of a corresponding residue in a control sample is indicative of a neoplastic breast cell or a cell predisposed to the onset of a neoplastic state.
  • the hypomethylation in the INS- IGF2 DVDMR is determined using a process containing the following steps: (i) treating the DNA derived from a biological sample with a compound that selectively mutates a non-methylated cytosine residue under conditions sufficient to induce mutagenesis; (ii) amplifying the DNA of step (i) using primers designed to amplify a DNA region defined by a portion of SEQ ID NO:1; (iii) sequencing the amplification product of step (ii) to identify the presence in the DNA from the test sample of one or more cytosine residues which have not undergone mutation relative to the corresponding mutated residues in DNA from a control sample.
  • the mutagenesis is induced with bisulfite or equivalent agent and unmethylated cytosine residues are converted to uracil.
  • the primers designed to amplify a DNA region defined by a portion of SEQ ID NO:1 include the following: SEQ ID NO.3: (F) 5’- AGGATGGGTTTTTGTTTGGTATT-3’ SEQ ID NO.4: (R) 5’- AAAAAAATTCATTTCCCCAAAAA-3’ [0037]
  • Onset means one or more cells of an individual exhibiting dysplasia.
  • the adenoma or adenocarcinoma may be well developed in that a mass of dysplastic cells has developed.
  • the adenoma or adenocarcinoma may be at a very early stage in that only relatively few abnormal cell divisions have occurred at the time of diagnosis.
  • Embodiments disclosed here can be used for the assessment of an individual's predisposition to the development of a neoplasm, such as an adenoma or adenocarcinoma.
  • Changed methylation levels of INS-IGF2 DVDMR can be indicative of an individual's predisposition to developing a neoplasia, such as the future development of an adenoma or adenocarcinoma or another adenoma or adenocarcinoma.
  • Changed methylation levels of INS-IGF2 DVDMR can be used to monitor the effectiveness of therapeutic or prophylactic treatment directed to modulating a neoplastic condition, such as adenoma or adenocarcinoma development.
  • Changed methylation levels of INS-IGF2 DVDMR can therefore be used in the diagnosis, prognosis, classification, prediction of disease risk, detection of recurrence of disease, and selection of treatment of a number of types of neoplasias.
  • kits for assaying a biological sample from a patient includes one or more polynucleotides that hybridize to a deoxyribonucleic acid (DNA) region defined by a portion of SEQ ID NO. 1 or a complement thereof; and at least one reagent for detection of DNA methylation.
  • the kit includes a compound that selectively mutates a non-methylated cytosine residue.
  • the kit includes (i) sodium bisulfite; (ii) primers that hybridize to the DNA region defined by the portion of SEQ ID NO.
  • the kit includes one or more control DNA sequences representing methylated or unmethylated forms of the DNA region.
  • Determining methylation status of INS-IGF2 DVDMR from a biological sample from a patient can be used to assess whether or not a biological sample taken from a mammal contains neoplastic cells or DNA derived from neoplastic cells. Determining methylation status of DNDMR from a biological sample from a patient can be used to estimate the risk or likelihood of a mammal developing a neoplasm. Determining methylation status of INS-IGF2 DVDMR from a biological sample from a patient can be used to select the appropriate anti-cancer treatment, such as one of more of a chemotherapeutic, immunotherapeutic, or a radiation agent.
  • Embodiments also include diagnostic kits for assaying biological samples containing one or more agents for detecting the methylation of one or more cytosine residues selected from the INS-IGF2 DVDMR and reagents useful for facilitating the detection by these agents. Further means may also be included, for example, to receive a biological sample.
  • the agent may be any suitable detecting compound known in the art.
  • this includes one or more nucleic acid molecules corresponding to a portion or the complete SEQ ID NO:1, or substantially similar nucleic acid molecule.
  • kits can also contain the standards (controls) against which the product amplified from the test sample is assessed.
  • these kits can also contain one or more amplification primer sets which primer sets are directed to amplify a portion of all of INS-IGF2 DVDMR or substantially similar sequences.
  • the primers sets are SEQ ID NOS 3 and 4.
  • a “neoplastic cell” refers to a cell exhibiting abnormal growth.
  • An example of abnormal cell growth is the uncontrolled proliferation of a cell. Another example is failed apoptosis in a cell, thus prolonging its usual life span.
  • the neoplastic cell may be a benign cell or a malignant cell.
  • the subject neoplasm is an adenoma or an adenocarcinoma.
  • an adenoma is generally a benign tumor of epithelial origin which is either derived from epithelial tissue or exhibits clearly defined epithelial structures. These structures may take on a glandular appearance.
  • DNA region refers to a specific section of genomic DNA. These DNA regions are specified by reference to a set of chromosomal coordinates, these being understood by the person of skill in the art. As detailed hereinbefore, the chromosomal coordinates for the DNA regions specified herein correspond to the December 2013 version of the UCSC Genome Browser, (GRCh38/hg38).
  • INS-IGF2 and the IGF2AS genes which code for non-translatable mRNAs.
  • the newly identified differentially methylated region (INS-IGF2 DVDMR) is localized in chr11:2148098-2148354).
  • the INS-IGF2 DVDMR located between exon 1 and exon 2 of the IGF2 gene showing chr11:214809821483545′-3′ _pad strand + sequence from Blat search in reference to human genome sequence; INS-IGF2 DVDMR BLAT search sequence (chr11:2148098-2148354) is provided as FIG. 1C.
  • a gene can be routinely identified by reference to its chromosomal location, via which its sequences can be routinely obtained. It should also be understood that reference to the DNA region Chr11:2148098–2148354 is interchangeably herein referred to by the name INS-IGF2 DVDMR. The 257-nucleotide reverse strand sequence of this locus is provided in SEQ ID NO:2. Any reference to each of the DNA regions detailed above should be understood as a reference to all forms of the molecules and to fragments or variants thereof. As would be appreciated by the person of skill in the art, some DNA regions are known to exhibit allelic variation between individuals or single nucleotide polymorphisms.
  • SNPs encompass insertions and deletions of varying size and simple sequence repeats, such as dinucleotide and trinucleotide repeats.
  • Variants include nucleic acid sequences from the same region sharing at least 90%, 95%, 98%, 99% sequence identity i.e. having one or more deletions, additions, substitutions, inverted sequences etc. relative to the DNA regions described herein. Accordingly, the present invention should be understood to extend to such variants which, in terms of the present diagnostic applications, achieve the same outcome despite the fact that minor genetic variations between the actual nucleic acid sequences may exist between individuals. Embodiments disclosed herein should therefore be understood to extend to all forms of DNA which arise from any other mutation, polymorphic or allelic variation.
  • methylation refers to the presence of a methyl group added by the action of a DNA methyl transferase enzyme to a cytosine base or bases in a region of nucleic acid, e.g. genomic DNA. As described herein, there are several methods known to those skilled in the art for determining the level or degree of methylation of nucleic acid.
  • methylation status refers to the presence, absence and/or quantity of methylation at a particular nucleotide, or nucleotides, within a DNA region.
  • the methylation status of a particular DNA sequence can indicate the methylation state of every base in the sequence or can indicate the methylation state of a subset of the base pairs (e.g., of cytosines) or the methylation state of one or more specific restriction enzyme recognition sequences within the sequence, or can indicate information regarding regional methylation density within the sequence without providing precise information of where in the sequence the methylation occurs.
  • the methylation status can optionally be represented or indicated by a methylation value or measurement.
  • a methylation value or measurement can be generated, for example, by quantifying the amount of intact DNA present following restriction digestion with a methylation dependent restriction enzyme. If a particular sequence in the DNA is quantified using quantitative PCR, an amount of template DNA approximately equal to a mock treated control indicates the sequence is not highly methylated whereas an amount of template substantially less than occurs in the mock treated sample indicates the presence of methylated DNA at the sequence. Accordingly, a value, i.e., a methylation value, for example from the above described example, represents the methylation status and can thus be used as a quantitative indicator of the methylation status. This is of particular use when it is desirable to compare the methylation status of a sequence in a sample to a threshold value.
  • An embodiment includes a method for identifying the aggressiveness of breast cancer in a subject patient in terms of percentage methylation in normal/tumor samples.
  • Methylation analysis may be performed using Sodium Bisulfite Analysis and Methylation Sensitive Restriction Enzyme (MSRE) digestion methods.
  • MSRE Methylation Sensitive Restriction Enzyme
  • INS-IGF2 DVDMR is hyper-methylated in normal breast tissues and hypo-methylated in BC tissues that significantly correlate with the lower/higher expression levels of IGF2 protein being expressed in those same samples performed by Western blots and Reverse phase protein array (RPPA). Based on this analysis, the aggressiveness of and risk in developing breast cancer can be determined in terms of the percentage methylation.
  • IGF2 is regulated by gene imprinting status in TNBC and undergoes various post translational modifications leading to the functional IGF2 mitotic protein which signals estrogen receptor- ⁇ and ⁇ (ESR ⁇ and ⁇ ), with Insulin receptor isoforms A and B (IRA and IRB), Insulin- like growth factor I and II (IGF1R and IGF-2R) tumor growth receptors leading to more aggressive breast cancer disease.
  • ESR ⁇ and ⁇ estrogen receptor- ⁇ and ⁇
  • IGF1R and IGF-2R Insulin- like growth factor I and II tumor growth receptors leading to more aggressive breast cancer disease.
  • hypomethylation of INS-IGF2 DVDMR correlates well with increased transcription and translation of IGF2 levels and can be effectively used as a prognostic biomarker for the predicting aggressive breast cancers.
  • the method may include analyzing genomic DNA for percentage methylation to distinguish between breast cancer and normal tissues by use of a sodium bisulphate and methyl PCR array using methylation specific primers for the newly identified differentially methylated region INS-IGF2 DMR.
  • the method may include analyzing IGF2 protein expression from the breast cancer and normal tissues by Reverse Phase Protein Array and Western Blots to correlate with a percentage methylation to the newly identified INS-IGF2 DMR.
  • this method of analyzing genomic DNA for percentage methylation using the sodium bisulphate and methyl PCR array may be compared with the methylation analysis using the MSRE digestion methods in order to identify normal cells and tumor breast cancer cells in the patient in terms of percentage methylation INS-IGF2 DMR and IGF2 protein expression in the normal cells and tumor breast cancer cells, and related cell lines.
  • the method may include analyzing RNA from the triple negative breast cancer cell lines.
  • Control level refers to the normal level, which is the level of methylation of the DNA region of a corresponding breast cell or cellular population which is not neoplastic or in another biological sample, for example blood plasma, from which DNA may be isolated for assay.
  • the normal or non-neoplastic methylation level may be determined using non-neoplastic tissues derived from the same individual, who is the subject of testing.
  • the normal or non-neoplastic methylation level may be determined using non- neoplastic tissues derived from one or more individuals.
  • the control level can also be a standard result which reflects individual or collective results obtained from individuals other than the patient at issue.
  • the standard results which provide the normal methylation level may be calculated by any suitable means which would be well known to the person of skill in the art. It should also be understood that the normal level may be determined from the subjects of a specific cohort and for use with respect to test samples derived from that cohort. Accordingly, there may be determined a number of standard values or ranges which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status. Said “normal level” may be a discrete level or a range of levels. A decrease in the methylation level of the subject genes relative to normal levels is indicative of the tissue being neoplastic.
  • Hypomethylation means that there are a fewer number of methylated CpG dinucleotides in the subject diagnosed than in a control sample, that is, either the proportion of DNA molecules methylated at a particular CpG site is lower or there are fewer number of separate CpG sites methylated in the subject as compared to control levels.
  • Embodiments described here are not limited by a precise number of methylated residues that are considered to be diagnostic of neoplasia in a subject, because some variation between patient samples will occur. Embodiments described here are not limited by positioning of the methylated residue. Nevertheless, a number of specific cytosine residues have been identified which undergo hypermethylation in the context of breast neoplasms.
  • Methylation status of the INS-IGF2 DVDMR can be performed on any suitable biological sample.
  • Biological sample means any sample of biological material derived from an animal such as, but not limited to, cellular material, biofluids (e.g.
  • the biological sample may be evaluated directly for methylation status or may require some form of treatment prior to testing.
  • a biopsy or surgical sample may require homogenization prior to testing or it may require sectioning for in situ testing of the qualitative expression levels of individual genes.
  • Breast tissues will be fast- frozen in liquid nitrogen and stored at -80 until processing by sonication.
  • the PureLinkTM Genomic DNA Mini kit from Invitrogen was used.
  • a cell sample may require permeabilization prior to testing.
  • the biological sample may not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to the sample.
  • a reagent such as a buffer
  • the biological sample may be directly tested or all or some of the nucleic acid present in the biological sample may be extracted prior to testing.
  • the sample may be partially purified or otherwise enriched prior to analysis.
  • the biological sample can be an individual cell or a group of cells present as a diffuse population of cells, a cell suspension, an encapsulated population of cells or a population of cells which take the form of tissue.
  • a chemotherapeutic, immunotherapeutic, or a radiation agent Described here are methods of identifying a patient with an aggressive breast cancer and treating the patient with one of more of a chemotherapeutic, immunotherapeutic, or a radiation agent.
  • the patient is identified by determining methylation of INS-IGF2 DVDMR derived from a biological sample from a patient.
  • the patient is identified as having an aggressive breast cancer in response to hypomethylation status of the INS-IGF2 DVDMR.
  • the aggressive breast cancer can be triple negative breast cancer.
  • the chemotherapeutic agent is one or more of anthracyclines, taxanes, capecitabine, gemcitabine, eribulin, cisplatin, carboplatin, or the targeted PARP inhibitors, olaparib and talazoparib.
  • the immunotherapeutic agent is a checkpoint inhibitor.
  • the checkpoint inhibitor is an anti–PD-1/PD-L1 agent.
  • the immunotherapeutic agent is one or more of, such as atezolizumab, pembrolizumab, and acituzumab govitecan-hziy.
  • the methylation of the differentially methylated region is determined by using methylation specific primers in a methyl PCR array.
  • the method can further include the steps of determining expression level of insulin- like growth factor-II mRNA; and in response to increased expression of the insulin-like growth factor-II mRNA and hypomethylation status of the INS-IGF2 DVDMR, identifying the patient as having an aggressive breast cancer.
  • the method can include the steps of determining expression level of insulin-like growth factor-II protein; and in response to increased expression of the insulin-like growth factor-II protein and hypomethylation status of the INS-IGF2 DVDMR, identifying the patient as having an aggressive breast cancer.
  • the expression level of insulin-like growth factor-II protein is determined by reverse phase protein array and Western blots.
  • the biological sample is obtained from a breast of the patient.
  • the biological sample is a blood sample from the patient.
  • Utilization of IGF2 as a biomarker improves diagnosis and treatment of BC patients. Described herein is an embodiment of a method of detecting methylation (by percentage) by methyl-sensitive restriction enzyme (MSRE) within the INS-IGF2 DVDMR by real-time PCR assay. Differentially methylated regions of the IGF2 gene corresponded to IGF2 protein expression in paired (Normal/Tumor) breast tissues and in BC cell lines.
  • MSRE methyl-sensitive restriction enzyme
  • Methylation analysis was performed using Sodium Bisulfite Analysis and Methylation Sensitive Restriction Enzyme digestion methods.
  • the methods described in the embodiments of the present disclosure focus on the DNA methylation patterns of the IGF2 gene in paired (normal/tumor) tissues obtained from African American (AA), Caucasian (CA), South Korean (SK), and Vietnamese (VIET) BC patients.
  • DNA methylation patterns of the IGF2 gene were also analyzed in several BC cell lines to determine the relationship between methylation of the IGF2 gene regulatory regions and the cellular expression levels of IGF2 protein.
  • Table 1 Paired Tissues from AA, CA, and VIET Breast Cancer Patients and Breast Cancer Cell Lines. S ample ID Receptor Status Age Ethnic G roup
  • AA African American
  • CA Caucasian
  • VIET Vietnamese breast cancer tissues were obtained from ILSbio (Integrated Laboratory Services-Biotech, 100 Radcliffe Drive, Chestertown, MD 21620 USA).
  • FIG.1A A modified diagram of the IGF2 gene and its transcript variant structure as described in the Human UCSC Genome Bioinformatics browser Dec. 2013 (GRCh38/hg38) Assembly is shown in FIG.1A.
  • the curated gene prediction from NCBI for the IGF2 gene includes processed exons and intron structure.
  • the IGF2 gene structure is shown to frame the newly identified DMR denoted in the red circle, named the INS-IGF2 DVDMR.
  • the INS-IGF2 DVDMR (DaisyVin Differentially Methylated Region) region is hypermethylated in normal breast tissues and hypomethylated in BC. This DMR has not been characterized or previously identified. It is located between exons 3 and 4 of the INS-IGF2 (Insulin-IGF2) gene, which is part of the IGF2 intergenic region of Chromosome 11. Also present in this region is the INS-IGF2 and the IGF2AS genes which code for non-translatable mRNAS.
  • the newly identified differentially methylated region (INS-IGF2 DVDMR) is represented in red (Chr11:2148098-2148354). This region is hypermethylated in normal breast tissues and hypomethylated in breast cancer.
  • the INS- IGF2 DVDMR is located between exons 3 and 4 of the INS-IGF2 gene. Furthermore, the INS- IGF2 DVDMR is also within exon 3 of the IGF2 anti sense (AS) (provided here as SEQ ID NOs:1 and 2). Both mRNAs (INS-IGF2 and IGF2 AS) are non-coding RNAs that do not encode proteins but are important in the translation of other mRNAs or epigenetic DNA modifications.
  • FIG. 1C shows the 5’-3’ strand sequence of the 257 base pairs INS-IGF2 DVDMR, localized in Chr11:2148098–2148354.
  • the diagram also shows the specific exons used to develop the primers to identify the IGF2 mRNA generated from each specific promoter (IGF2P1-IGF2P4).
  • it also shows the primer region developed from Exon 9 which recognizes all combined IGF2 mRNAs.
  • Arrows in P1-P4 promoters indicate the position of the forward and reverse primers designed using intron spanning assay. Capturing of Exon 9 measures the total IGF2 mRNA while the specific designed primers (P1-P4 forward and reverse) accurately measures IGF2 transcripts generated from P1-P4.
  • the INS-IGF2 DVDMR was identified utilizing DNA obtained from four different BC cell lines.
  • FIG. 2B shows a representative bar graph of the IGF2 P1-P4 transcript variant fold change analyzed by the intron spanning qPCR method.
  • the predominant IGF2 transcripts were expressed from P1 and P3 except in the Hs578T cells, which produced mRNA predominantly from P4 transcripts while transcripts from P1 and P3 where barely detectable.
  • the TNBC cell line CRL2335 (AA) expressed the highest IGF2 mRNA variant levels from P1 and P3 while the MDA-231 cells expressed high levels of transcripts from P4.
  • IGF2 protein levels were comparable in the CRL2335 (AA) and MDA-231cell lines, as illustrated in the embodiment shown in FIG. 2D.
  • MDA-MB-231 breast cancer cells (CA) expressed the second highest levels of total IGF2 protein even though it represented only 50% of the IGF2 mRNA when compared to the CRL2335 (AA) cell line (FIG. 2C).
  • cell lines MDA-MB-231 and Hs578t expressed similar amounts of protein but significantly lower levels of IGF2 mRNA.
  • the methylation levels of the INS-IGF2 DVDMR were compared to the expression of the INS-IGF2 mRNA short (DQ104204) and long (DQ104205) transcript variants.
  • DQ104204 mRNA short
  • DQ104205 long transcript variants.
  • high levels of the INS-IGF2 long and short transcript variants were detected in the CRL2335 and MDA-231 cells.
  • both cell lines expressed the highest levels of IGF2, as illustrated in FIG. 2D.
  • the MDA-468 cells only expressed low levels of the long INS-IGF2 transcript variant.
  • Hs578T cell line did not express either variant of the INS-IGF2 (FIG. 2E).
  • Ethidium bromide-stained agarose gels showing bisulfite g-DNA fragments produced by PCR amplification of the CpG and DMR regions of the IGF2 gene and the INS-IGF2 DVDMR obtained from paired breast tissues (Normal/Malignant) from AA (FIGS.3A – 3C), CA (FIGS.4A-4C) and VIET (FIGS.5A – 5C) patients are shown in panel A of each figure. No significant changes in the methylation pattern for any of the IGF2 CpG islands or DMR were observed in any of the three groups examined.
  • the INS- IGF2 DVDMR (257bp) is hypomethylated in malignant tissues as compared to the paired normal tissues in all three ethnic groups as shown in the agarose gels included in FIGS.3A, 4A, and 5A.
  • Universal Methylated Human DNA standard (182bp) and control primers were used to check the efficiency of bisulfite-mediated converted DNA by conventional PCR methods.
  • Table 2 Paired Tissues from AA, CA, and SKR TNBC Patients Samples S ample ID Receptor Status Age Ethnic G roup
  • the methylation patterns of the INS-IGF2 DVDMR for the same tissue samples analyzed in FIGS. 3A, 4A, and 5A were next examined.
  • 3C, 4C, and 5C represents the levels of IGF2 protein in paired Normal/Malignant breast tissues from AA, CA, and VIET, respectively. Higher IGF2 levels were detected in malignant tissues as compared to their paired normal tissue in all three groups. Thus, the IGF2 levels in the paired tissues are inversely correlated to the percent methylation levels detected in the INS-IGF2 DVDMR. Hypomethylation of the INS-IGF2 DVDMR leads to increased IGF2 protein levels in the malignant tissues while hypermethylation of the INS-IGF2 DVDMR lead to lower IGF2 protein levels in all normal breast tissues. These observations are significant because they suggest that similar mechanisms of IGF2 gene methylation occur in vitro (BC cells) and in vivo (paired breast tissues).
  • FIGS.6A – 6D show comparative bar graphs representing the methylation status of the INS-IGF2 DVDMR assessed by MSRE qPCR in paired TNBC tissues from AA women (FIG.6A), CA women (FIG.6B), and SKR women (FIG.6C).
  • FIG.6A Next to each MSRE Methylation graph of the pair samples (FIGS.6A- 6C) a Box and Whisker Plot shows the average (mean+/-SEM) of the paired samples MSRE in each ethnic group.
  • 6D shows all the Box and Whisker Plots comparing the methylation status of the INS-IGF2 DVDMR from paired TNBC samples of all four ethnic groups (AA, CA, VIET, and SKR).
  • This data demonstrates that the methylation of the INS-IGF2 DVDMR results obtained from TNBC samples are similar to those obtained from paired samples with combined different ER, PR and HER2 status.
  • the methylation pattern of the INS-IGF2 DVDMR is not dependent of the tissue receptor status. Regardless of which receptors are expressed, tumor samples are significantly hypomethylated when compared to paired normal tissues.
  • FIG.7A is an image of the western blot assay representing the levels of IGF2 protein being expressed in Triple Negative Breast Cancer Cells (TNBC), according to an embodiment.
  • FIG.7B is a bar graph representing the levels of IGF2 protein being expressed in Triple Negative Breast Cancer Cells (TNBC) shown in Fig.7A.
  • This Western blot (FIG.7A) demonstrates that the IGF2 present in breast cancer can be identified.
  • FIG. 8 is a bar graph representing the % methylation by methyl-sensitive restriction enzyme assay (MSRE) showing the % methylation levels of INS-IGF2 DMR levels in breast cancer cell lines by qPCR method, according to an embodiment.
  • MSRE % methylation by methyl-sensitive restriction enzyme assay
  • FIG. 9 is a bar graph representing the IGF2 levels in paired breast tissues from African Americans assessed by Reverse Phase Protein Array (RPPA) assay, according to an embodiment. Sample details are shown in Table 1.
  • FIG. 10 is a bar graph representing the IGF2 levels in paired breast tissues from Vietnamese patients as assessed by Reverse Phase Protein Array (RPPA) assay, according to an embodiment. Sample details are shown in Table 1. These TNBC samples of breast cancer tissues represent a different ethnic group to validate the observation that normal tissues have a higher DMDVR methylation while tumor tissues have a low DMDVR methylation status.
  • RPPA Reverse Phase Protein Array
  • This INS-IGF2 DVDMR consists of 257bp located in chromosome 11 and spans between bps 2148098 – 2148354 (GRCh38/hg38). Hypomethylation of the INS-IGF2 DVDMR correlated with the increase of both INS-IGF2 transcripts in all four cell lines. Unexpectedly, hypomethylation in this region also correlated closely with increased IGF2 mRNA and protein levels in all four cell lines. Interestingly, this novel observation regarding the correlation of hyper or hypo methylation of the INS-IGF2 DVDMR to decreased or increased IGF2 levels, respectively, was also observed in the paired breast tissue samples analyzed.
  • methylation profile of the INS-IGF2 DVDMR in paired breast tissues was distinct and could differentiate malignant breast tissue (hypomethylated) from normal adjacent breast tissue which was hypermethylated. This methylation pattern was observed in TNBC tissues and in all paired breast tissues examined regardless of ER, PR and HER2 receptor status. Similarly, the same methylation pattern in normal vs. malignant breast tissue was observed in paired tissues from four different ethnic groups examined. These results suggest that methylation of the INS-IGF2 DVDMR is a key regulator of IGF2 expression in BC. [0082] IGF2 promotes cell proliferation, inhibit apoptosis and stimulate transformation of BC cells.
  • IGF2 is also highly expressed in BC patients and “free” circulating IGF2 levels in humans are significantly correlated with breast tumor size.
  • Transgenic animal models with increased IGF2 expression show a significant increase in BC that develops at an early age and it is more aggressive.
  • IGF2 has previously been identified as an important biological factor contributing to higher BC mortality among AA women.
  • the present disclosure demonstrates that IGF2 levels inhibited or stimulated mitochondrial proteins in BC cell lines, preventing cell death and inducing chemoresistance. These studies also showed that mitochondrial proteins were significantly correlated with IGF2 levels in tissues from BC patients. Since mitochondria are important targets of chemotherapy, IGF2 expression by BC tumors may confer mitochondrial protection, thereby inducing chemoresistance and effectively reducing clinical treatment efficacy.
  • IGF2 may be a critical biological factor in BC that contributes to the development of chemoresistance and may increase BC mortality.
  • IGF2 represents a potential therapeutic target to decrease chemoresistance and improve survival among BC patients.
  • IGF2 expression in BC cells and in paired Normal-Malignant breast tissues are determined by the methylation of a novel region in the INS-IGF2 locus. The mechanisms underlying the methylation of the INS-IGF2 may be further explored to determine how they control IGF2 expression. Upregulation of IGF2 in terms of the methylation patterns of the INS-IGF2 DVDMR may have an important function in the tumorigenesis of the breast.
  • INS-IGF2 DVDMR may be a useful tool to identify women at risk of developing a more aggressive BC disease.
  • the examples are disclosed here to aid in the understanding of the embodiments of the invention, and are not intended and should not be construed to limit in any way the embodiments set forth in the claims which follow thereafter.
  • Materials and Methods [0085] Tissues and cell lines. The cell lines used in this study were obtained from the American Type Culture Collection (ATCC). The tissue samples for AA and Caucasian CA) patients were obtained from the Cooperative Human Tissue Network (CHTN) and the South Korean (SKR) TNBC gDNA samples were obtained as gift samples from Dr.
  • ATCC American Type Culture Collection
  • CHTN Cooperative Human Tissue Network
  • SBR South Korean
  • Dae-Kwang Kim Department of Medical Genetics, School of Medicine, Institute for Medical Genetics, Keimyung University, Hanvit Institute for Medical Genetics, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea.
  • the tissue samples from the Vietnamese (VIET) TNBC patients were obtained from ILSbio (Integrated Laboratory Services-Biotech, 100 Radcliffe Drive, Chestertown, MD 21620 USA).
  • ILSbio Integrated Laboratory Services-Biotech, 100 Radcliffe Drive, Chestertown, MD 21620 USA.
  • PureLinkTM Genomic DNA Mini kit from Invitrogen (Camarillo, CA, USA) from breast tissue samples of AA, CA, VIET, and SKR BC patients, and from BC cell lines.
  • the sodium bisulphate gDNA conversion was performed using EZ DNA Methylation-LightningTM Kit from Zymo Research (Irvine, CA) which converts unmethylated cytosine nucleotides into uracil nucleotides, while methylated cytosine remains unaltered.
  • PCR further converted uracils into thymines and methylated cytosines into cytosines using a probe specific methylation primer and HotStar DNA polymerase from (Qiagen) to amplify CpG, IGF-2 DMR and INS-IGF2 DVDMR PCR products using 580C as the annealing temperature.
  • RNA, cDNA, qPCR RNA was isolated using TRI REAGENT (Zymo Research, Inc.) and treated with DNase. Bio Rad’s iScript Synthesis Kit was used for cDNA synthesis. Specific primers for the IGF2 and INS-IGF2 short and long transcripts were designed with intron spanning assay primer designing tools.
  • Methylation primers were designed using the MethPrimer
  • Table 3. IGF2 primers for CPG islands, DMROs and IGF2 P1-4 promoters GF2 and INS-IGF2 mRNA transcript fold expression results were analyzed using the CFX Manager software Version 1.0.
  • Restriction Digestion and Methylation Real-Time PCR Assay To isolate DNA from BC tissues and cell lines, the PureLinkTM Genomic DNA Mini kit from Invitrogen (Camarillo, CA, USA) was used.
  • ⁇ Ct of 1.0 indicates that 50% of the template has been cleaved, 2.0 equals 75% cleavage.
  • RPPA Reverse Phase Protein Array for quantifying IGF II levels
  • Antibody signal for each spot was normalized to the corresponding signals from staining a different slide with the general protein stain SyproRuby, following the manufacturer’s instructions (Invitrogen, Camarillo, CA, USA).
  • Statistical analysis was determined by using one-way ANOVA and the Wilcoxon Signed Rank Test and paired T-Test, which were used for comparing the paired Normal/Malignant samples in GraphPad Prism 5. Values are expressed as the mean ⁇ SEM of at least 3 separate experiments done in triplicate. A P value ⁇ 0.05 was considered significant. Experiments depicted in FIGS. 3A, 4A and 5A (agarose gels) were not subjected to statistical analysis.

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Abstract

L'invention concerne des méthodes de diagnostic, de pronostic et de théranostic qui sont rendues possibles en déterminant l'état de méthylation d'une région d'ADN du gène INS-IGF2 à partir d'un échantillon biologique d'un patient. Les niveaux d'hypométhylation de cette séquence de région INS-IGF2 (Chrl 1:2148098-2148354) augmentent les taux d'IGF2 et favorisent une forme du cancer du sein plus agressive, moins sensible au traitement. L'évaluation de la méthylation de cette région détecte l'apparition, ou une prédisposition à l'apparition, d'un néoplasme du sein chez un patient et facilite également la sélection de meilleurs régimes de traitement pour des patients atteints de cancers du sein agressifs.
PCT/US2021/070206 2020-02-28 2021-02-28 Méthodes d'évaluation de pronostic du cancer du sein et régimes de traitement basés sur l'état de méthylation de la région ins-igf2 WO2021174262A1 (fr)

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Citations (3)

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US7981603B2 (en) * 2002-07-26 2011-07-19 The Johns Hopkins University School Of Medicine Methods for identifying cancer risk
US20150119350A1 (en) * 2012-03-26 2015-04-30 The United States Of America, As Represented By The Secretary, Dept. Of Health & Human Services Dna methylation analysis for the diagnosis, prognosis and treatment of adrenal neoplasms
WO2017008117A1 (fr) * 2015-07-14 2017-01-19 Garvan Institute Of Medical Research Procédé de diagnostic, de pronostic et de suivi d'un cancer du sein et réactifs correspondants

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US7981603B2 (en) * 2002-07-26 2011-07-19 The Johns Hopkins University School Of Medicine Methods for identifying cancer risk
US20150119350A1 (en) * 2012-03-26 2015-04-30 The United States Of America, As Represented By The Secretary, Dept. Of Health & Human Services Dna methylation analysis for the diagnosis, prognosis and treatment of adrenal neoplasms
WO2017008117A1 (fr) * 2015-07-14 2017-01-19 Garvan Institute Of Medical Research Procédé de diagnostic, de pronostic et de suivi d'un cancer du sein et réactifs correspondants

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KALLEDSOE, L ET AL.: "The insulin-like growth factor family and breast cancer prognosis: A prospective cohort study among postmenopausal women in Denmark", GROWTH HORMONE AND IGF RESEARCH, vol. 44, February 2019 (2019-02-01), pages 33 - 42, XP055850459, DOI: 10.1016/j.ghir. 2018.12.00 3 *
RADHAKRISHNAN, VK ET AL.: "Methylation of a newly identified region of the INS-IGF2 gene determines IGF2 expression in breast cancer tumors and in breast cancer cells", ONCOTARGET, vol. 11, no. 44, 3 November 2020 (2020-11-03), pages 3904 - 3920, XP055850431, DOI: 10.18632/oncotarget.27655 *
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