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EP3963092A1 - Contribution à un risque de cancer secondaire de mutations somatiques de facteurs du cancer dans les lésions d'endométriose - Google Patents

Contribution à un risque de cancer secondaire de mutations somatiques de facteurs du cancer dans les lésions d'endométriose

Info

Publication number
EP3963092A1
EP3963092A1 EP20799047.4A EP20799047A EP3963092A1 EP 3963092 A1 EP3963092 A1 EP 3963092A1 EP 20799047 A EP20799047 A EP 20799047A EP 3963092 A1 EP3963092 A1 EP 3963092A1
Authority
EP
European Patent Office
Prior art keywords
cancer
subject
endometriosis
mutation
combination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20799047.4A
Other languages
German (de)
English (en)
Inventor
Kenneth Ward
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Predictive Technology Group Inc
Original Assignee
Predictive Technology Group Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Predictive Technology Group Inc filed Critical Predictive Technology Group Inc
Publication of EP3963092A1 publication Critical patent/EP3963092A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • An aspect of the disclosure provides methods for obtaining a genetic material from a subject, the genetic material can be at least in part from an endometrial tissue, the subject can have or be suspected of having an endometriosis, a cancer, or a combination thereof; and detecting a presence or an absence of a mutation in a cancer driver gene in at least a portion of the genetic material, the detecting can comprise sequencing a nucleic acid sequence from the portion the genetic material, a cDNA thereof, an amplicon of any of these, or any combination thereof.
  • sequencing can comprise whole exome sequencing.
  • an endometrial tissue can comprise an endometrial lesion.
  • an endometrial lesion can be a benign endometrial lesion.
  • a mutation can comprise at least two mutations.
  • a presence or an absence of a first mutation can be detected in first cancer driver gene and a presence or an absence of a second mutation can be detected in a second cancer driver gene.
  • a cancer driver gene can comprise P53/PTEN.
  • a presence of a mutation in a cancer driver gene can identify an increased risk of developing an endometriosis, a cancer, or a combination thereof as compared to a subject lacking a mutation.
  • a presence of a mutation can at least partially identify a presence of an endometriosis, a cancer, or a combination thereof in a subject.
  • a subject may have previously received a diagnosis for an endometriosis.
  • a method can further comprise identifying a biological age of a subject upon receipt of a diagnosis for an endometriosis.
  • diagnosis for an endometriosis can be performed by histological analysis. In some embodiments, when a biological age can be identified as less than about 40 years of age, a subject may be identified as having an at least partially decreased risk of developing a cancer, and no further detecting may be performed.
  • a cancer can comprise a cancer of a reproductive system.
  • a cancer can comprise a breast cancer, an ovarian cancer, an endometrial cancer, a cervical cancer, a uterine cancer, a pelvic cancer, an abdominal cancer, a cutaneous melanoma, a non-Hodgkin’s lymphoma, or any combination thereof.
  • a mutation can comprise a single nucleotide polymorphism (SNP).
  • detecting can comprise hybridizing a probe to a portion of a genetic material.
  • a method can further comprise: measuring a total variant burden in at least a portion of a genetic material.
  • a presence or an absence of a mutation can be at least partially indicative of an endometriosis.
  • a presence or an absence of a mutation can be at least partially indicative of a cancer.
  • a mutation can be detected.
  • a mutation may not be detected.
  • a method can further comprise: obtaining a genetic material from a subject.
  • a subject can have a cancer.
  • a subject can have an endometriosis.
  • a subject can be at risk of developing an endometriosis.
  • a subject can suffer from pelvic pain.
  • a subject can suffer from infertility.
  • at least a portion of a genetic material can be from a blood sample.
  • at least a portion of a genetic material can comprise cell free DNA.
  • a genetic material can comprise endometrial tissue, uterine tissue, ovarian tissue, fallopian tissue, cervical tissue, vulvar tissue, or any combination thereof.
  • a method can further comprise treating a subject for endometriosis, a cancer, or a combination thereof.
  • treating can comprise prophylactic treating.
  • treating can comprise administering to a subject a pharmaceutical composition in unit dose form.
  • a method can further comprise administering a co-therapy to a subject.
  • a co-therapy can comprise chemotherapy, radiation, or a combination thereof.
  • a method can further comprise: comparing a result of a method to a reference.
  • a reference can comprise a derivative of a reference.
  • a reference can comprise a result of a method performed on a reference sample.
  • comparing can be performed by a computer processor.
  • comparing can be performed by a trained algorithm.
  • a reference can comprise a result obtained from genetic material of a subject diagnosed with an endometriosis, a cancer, or a combination thereof.
  • detecting can comprise sequencing at least a portion of WNT4, HDAC2, or a combination thereof.
  • sequencing can identify a presence or an absence of a risk allele T in homozygous or heterozygous form in WNT4, a presence or an absence of a mutation in HDAC2, or a combination thereof.
  • sequencing can identify a mutation in a plurality of genes.
  • a plurality of genes can share a biological pathway.
  • a biological pathway can comprise: an integral plasma membrane, a cytoskeletal part, a cell surface, a plasma membrane region, an extracellular matrix, a cell junction or any combination thereof.
  • a biological pathway can comprise a protein- protein interaction.
  • a subject can comprise an ethnicity that can be at least partially Caucasian, African American, Asian, Hispanic, or any combination thereof.
  • FIG. 1 shows three classes of endometriosis genetic markers.
  • FIG. 2 shows relative numbers of mutations in endometriosis lesions, endometriosis blood, and unaffected controls.
  • FIG. 3 shows results of a clinical follow-up showing cancer diagnosis may be positively associated with detection of a presence or one more driver mutations.
  • FIG. 4 shows a diagram showing a method and system as disclosed herein.
  • Methods as described herein may include preparing a sample for a diagnostic test. Methods may include methods of sample preparation. Methods may include methods of detection. Detection methods may include detecting a presence or absence of a mutation or a variant in one or more genes, detecting a total variant burden, detecting a presence of an endometrial polyp or lesion, or any combination thereof. Methods may include methods of sequencing genetic material. Methods may include diagnosing a disease or condition. Methods may include diagnosing more than one disease or condition, such as endometriosis and cancer. Methods may include identifying a subtype of endometriosis. Methods may include identifying a subtype of cancer. Methods may include identifying a risk of developing a disease or condition.
  • Methods may include obtaining a sample from a subject.
  • the sample may comprise genetic material.
  • the sample may be obtained from a tissue sample, a blood sample, or a combination thereof.
  • the sample may comprise cell-free DNA.
  • the sample may be obtained from an endometrial tissue.
  • the sample may be obtained from an endometriosis lesions of an endometrial tissue.
  • Genetic material can be obtained from a subject who may have cancer, endometriosis, or any combination thereof. Genetic material can be obtained from a tissue sample, a blood sample, a skin sample, a vaginal swab, a pap smear, a biopsy or any combination thereof.
  • the genetic material can be a tissue, for example, an endometrial tissue, a cervical tissue, a uterus tissue, a myometrium tissue, an ovarian tissue, a fallopian tube tissue, an adjacent tissue thereof, or any combination thereof.
  • Genetic material can be collected from a lesion or a polyp on the endometrial tissue.
  • genetic material can be collected from a cancer, such as endometrial cancer, or cervical cancer.
  • a cancer can be a precancerous sample, wherein the cells may have an increased risk to develop into a cancer.
  • Genetic material may be obtained from an endometrial tissue.
  • the endometrial tissue can include a cell from an endometrial tissue.
  • the endometrial tissue can comprise an endometriosis lesion.
  • the genetic material may include at least in part a cell-free DNA, an RNA, a genetic material extracted from a cell, genetic material from a tissue (such as an endometrial tissue).
  • Methods can comprise obtaining a genetic material from a subject.
  • the genetic material can be at least in part a cell-free DNA, an RNA, extracted from a cell, from an endometrial tissue, or any combination thereof.
  • the subject can have or be suspected of having an endometriosis, a cancer, or a combination thereof.
  • the method can comprise detecting a presence or an absence of a mutation in a cancer driver gene in at least a portion of the genetic material.
  • the detecting can comprise sequencing a nucleic acid sequence from at least the portion of the genetic material, a cDNA, an amplicon of any of these, or any combination thereof.
  • the genetic material can comprise at least a portion of: an exon, an intron, a regulatory element, a promotor, an operon, or any combination thereof.
  • the subject may be suspected of having endometriosis.
  • the subject may have endometriosis but not yet received a positive diagnosis.
  • the subject may have previously received a positive diagnosis for endometriosis.
  • the subject may not have previously received a positive diagnosed for endometriosis.
  • the subject may be suspected of having a cancer.
  • the subject may have a cancer but may not yet have received a positive diagnosis.
  • the subject may be asymptomatic for endometriosis.
  • the subject may be asymptomatic for cancer.
  • the subject may be at risk for endometriosis.
  • the subject may be at risk for cancer.
  • a sample obtained from a subject may be further processed.
  • a sample may be processed by dividing the sample into portions.
  • a sample may be processed for histological sectioning, staining, and analysis.
  • a sample may be processed for sequencing of genetic material.
  • a sample may be processed for isolating genetic material from the sample.
  • a sample may be processed for labeling a marker present in the sample.
  • a sample may be processed for a genetic analysis, such as sequencing, or for identification a presence or absence of one or more mutations in a sample.
  • a gene may be a cancer driver gene.
  • Sequencing may comprise whole exome sequencing. Sequencing may comprise high throughput sequencing. Sequencing may detect a presence or an absence of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations in a gene. Sequencing may detect a presence or an absence of at least about 2 mutations in a gene. Sequencing may measure a presence or an absence of one mutation in a gene. Sequencing may detect a presence or an absence of at least about 3 mutations in a gene.
  • Sequencing may detect a presence or an absence of a mutation in about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400 genes or more. Sequencing may detect a presence or an absence of a mutation in one gene. Sequencing may detect a presence or an absence of a mutation in at least two genes. Sequencing may detect a presence or an absence of a mutation in at least five genes. Sequencing may identify a presence or an absence of a mutation in at least about 5 genes.
  • Sequencing may comprise high throughput sequencing. Sequencing may comprise whole exome sequencing. Sequencing may comprise whole genome sequencing. Sequencing may comprise sequencing a portion of the genome. A sample may be enriched for a particular marker or genes and then sequencing may be performed on the enriched portion. A sample may be enriched for at least a portion of exomes from a genomic sample and then sequencing may be performed on the enriched portion. Genetic material may be extracted from a sample At least a portion of an exome may be extracted from a genome. Genetic material may be processed by physical fragmentation, an enzymatic assay, or a combination thereof to produce fragments of genetic material.
  • Genetic material such as one or more fragments may be labeled, such as labeled with an adapter, a primer, a fluorescent label, or any combination thereof.
  • a portion of fragments may be enriched or isolated from pool of fragments.
  • Sequencing may be performed on the enriched or isolated fragments.
  • Sequencing may comprise high throughput sequencing.
  • Sequencing may comprise nanopore sequencing.
  • Analysis of data obtained from sequencing may include identifying a presence or absence of a mutation in a gene (such as a cancer driver gene), a presence or absence of a variant in a gene (such as an SNP), a total variant burden, an expression level of a gene (such as a cancer driver gene), or any combination thereof.
  • Data obtained from sequencing may be compared (such as compared by a trained algorithm) to a reference (such as sequencing data obtained from a subject having endometriosis, having cancer, or both).
  • Sequencing can comprise preparing a sample.
  • DNA can be purified from a sample (e.g. tissue or blood). Quality of at least a portion of the DNA can be determined by different light absorbance values to provide a quality template for downstream processing and sequencing. At least a portion of the DNA can be preserved (e.g. by freezing) or used soon after purification. For target enrichment, at least a portion of the DNA can be fragmented (e.g. physically or enzymatically) and the exome can be captured. Several washing and elution steps can be included. Once the exome DNA has been enriched, the sample can be sequenced by a next generation sequencing technology (e.g. sequencing by synthesis) and the data can be analyzed by a bioinformatic computer program.
  • a next generation sequencing technology e.g. sequencing by synthesis
  • Identification of a mutation in at least one gene may identify a presence or a risk of developing a cancer, endometriosis, or both. Identification of a mutation in at least two genes may identify a presence or a risk of developing a cancer, endometriosis, or both. Identifying a presence or risk of a disease or condition may include identifying a total variant burden, one or more variants present in one or more genes, an age of receiving a positive diagnosis of endometriosis, or any combination thereof.
  • a presence of a mutation in a cancer driver gene may identify an increased risk of developing endometriosis, a cancer, or both.
  • a presence of a mutation in a cancer driver gene may identify a presence of endometriosis, a cancer, or both.
  • a presence of a mutation in a cancer driver gene may identify a subtype or staging of the endometriosis or the cancer.
  • Sequencing may identify one or more single nucleotide polymorphisms (SNP) in a portion of one or more genes. Sequencing may identify one or more rare variants, occurring in less than about 1%,
  • Sequencing may identify a presence or an absence of one or more mutations in a gene.
  • a mutation may be identified in a portion of a cancer driver gene.
  • a mutation may comprise a base substitution, a deletion, or an insertion.
  • a mutation may comprise a stop gain mutation, a stop loss mutation, a missense mutation, a splicing mutation, or any combination thereof.
  • Somatic mutations can accumulate during the clonal evolution of individual endometriosis lesions.
  • Endometriosis lesions can be a progressive, invasive, recurrent, and possibly metastatic.
  • Many endometriosis lesions can exhibit similar molecular features that may be observed in cancer (such as a resistance to apoptosis or stimulation of angiogenesis).
  • cancer can arise in an
  • endometriosis lesion a subset of cancer types can arise more commonly in women with endometriosis.
  • women having endometriosis may also develop or be at risk of developing: ovarian cancer (particularly clear-cell and endometrioid), breast cancer, endometrial cancer, cervical cancer, pelvic cancer, abdominal cancer, cutaneous melanoma, Non-Hodgkin's lymphoma, or any combination thereof.
  • the term“about” or“approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.
  • “about” can mean plus or minus 10%, per the practice in the art.
  • “about” can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value.
  • the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.
  • endometriosis can refer to any nonmalignant disorder in which functioning endometrial tissue is present in a location in the body other than the endometrium of the uterus, i.e. outside the uterine cavity or is present within the myometrium of the uterus.
  • endometrial tissue can refer to any nonmalignant disorder in which functioning endometrial tissue is present in a location in the body other than the endometrium of the uterus, i.e. outside the uterine cavity or is present within the myometrium of the uterus.
  • endometriosis can refer to any nonmalignant disorder in which functioning endometrial tissue is present in a location in the body other than the endometrium of the uterus, i.e. outside the uterine cavity or is present within the myometrium of the uterus.
  • it also includes conditions, such as adenomyosis/adenomyoma, that exhibit myometrial tissue in the lesions.
  • Endometriosis can include endometriosis externa, endometrioma, adenomyosis, adenomyomas, adenomyotic nodules of the uterosacral ligaments, endometriotic nodules other than of the uterosacral ligaments, autoimmune endometriosis, mild endometriosis, moderate endometriosis, severe
  • endometriosis endometriosis, superficial (peritoneal) endometriosis, deep (invasive) endometriosis, ovarian endometriosis, endometriosis-related cancers, and/or "endometriosis-associated conditions". Unless stated otherwise, the term endometriosis is used herein to describe any of these conditions.
  • endometriosis can comprise a nonmalignant disorder in which functioning endometrial tissue may be present outside the uterine cavity.
  • endometriosis can comprise endometriosis externa or adenomyosis externa.
  • symptoms of endometriosis can comprise pelvic pain, pelvic mass, alteration of menses, infertility, lesions on the bowel or bladder, pain during defecation, pain during urination, abdominal bloating, and rectal bleeding with menses, or any combination thereof.
  • Adenomyosis can cause symptoms in patients, usually late in the reproductive years.
  • symptoms can comprise: menorrhagia and intermenstrual bleeding, pain (e.g.
  • Treatments can include oral contraceptive steroids and GnRH agonists or antagonists.
  • surgery can be a treatment.
  • One or more symptoms of endometriosis can comprise pain, a presence of cramping, dysmenorrhea, dyspareunia, constipation, urinary tract issue, pelvic pain, pelvic inflammatory disease, pelvic adhesion, neoplasms, ovarian torsion, anovulation, a migraine, back pain, painful urination, painful defecation, an increased allergy, vaginal bleeding, blood in urine, blood in rectum, fatigue, nausea, diarrhea, bloating, pregnancy issues (e.g. difficulty getting pregnant, infertility), fever, chills or any combination thereof.
  • Diagnosis of endometriosis can comprise detecting a presence or an absence of one or more mutations in one or more genes of genetic material obtained from a subject. Detection may include sequencing, such an whole exome sequencing. Mutations may be in one or more cancer driver genes. Mutations may include rare variants - occurring in less than about: 1%, 0.1%, 0.01% of a population. Symptoms may include a physical inspection of the abdominal cavity of the patient. In some instances, diagnosis can comprise identification of a symptom or a plurality of symptoms of endometriosis. In some cases, diagnosis can comprise an imagine procedure (e.g. an ultrasound), a laparoscopy, or any combination thereof. In some cases, a diagnosis can comprise a histological examination of endometrial tissue.
  • Detection may include sequencing, such an whole exome sequencing. Mutations may be in one or more cancer driver genes. Mutations may include rare variants - occurring in less than about: 1%, 0.1%, 0.01% of a population.
  • Treatment can include a pain medication, for example NSAIDs, ibuprofen or naproxen sodium.
  • Other treatments can include, a hormone therapy (e.g. danazol), a contraceptive, a gonadotropin-releasing hormone (GnRH) agonist and antagonist (e.g.
  • Treatment can include a combination of a pain medication and a hormone therapy.
  • Treatment can include administration of a regenerative cell therapy, such as administration of stem cells.
  • a treatment can include gosarelin, nafarelin, a salt thereof or any combination thereof.
  • treatment can include surgery, a hysterectomy, or any combination thereof.
  • treating of endometriosis can include one or more of: reducing the frequency and/or severity of symptoms (such as pelvic pain), elimination of symptoms and/or their underlying cause, and improvement or remediation of damage.
  • treatment of endometriosis may include, for example, relieving the pain experienced by a woman suffering from endometriosis, and/or causing the regression or disappearance of endometriotic lesions. Treating may also include: improved fertility or ability to conceive, cessation of dyspareunia, absence of non-menstrual pelvic pain, or any combination thereof.
  • Biological samples obtained from individuals may be any sample from which a genetic material (e.g., nucleic acid sample) may be derived.
  • Genetic material may be obtained from endometrial tissue.
  • Genetic material may be obtained from any reproductive tissue (such as endometrial tissue, ovarian tissue, fallopian tissue, cervical tissue, vulvar tissue, uterine tissue, or any combination thereof.
  • Samples/Genetic materials may be from tissue, tissue biopsy, liquid biopsy, fine needle aspirate, buccal swab, saliva, blood, hair, nail, skin, cell, or any other type of tissue sample.
  • the genetic material comprises mRNA, cDNA, genomic DNA, or PCR amplified products produced therefrom, or any combination thereof.
  • the genetic material e.g., nucleic acid sample
  • the genetic material e.g., nucleic acid sample
  • Genetic material may comprise tumor DNA. Genetic material may be obtained from or proximal to a tumor site. Genetic material may be obtained from or proximal to an endometriosis lesion.
  • cell-free can refer to the condition of the nucleic acid sequence as it appeared in the body before the sample is obtained from the body.
  • circulating cell-free nucleic acid sequences in a sample may have originated as cell-free nucleic acid sequences circulating in the bloodstream of the human body.
  • nucleic acid sequences that are extracted from a solid tissue, such as a biopsy are generally not considered to be“cell-free.”
  • cell free nucleic acids can include cell free DNA or cell free RNA.
  • cell-free DNA may comprise fetal DNA, maternal DNA, or a combination thereof.
  • cell-free DNA may comprise DNA fragments released into a blood plasma.
  • the cell-free DNA may comprise circulating tumor DNA. In some cases, cell-free DNA may comprise circulating DNA indicative of a tissue origin, a disease or a condition.
  • a cell-free nucleic acid sequence may be isolated from a blood sample.
  • a cell-free nucleic acid sequence may be isolated from a plasma sample, urine, saliva, or synovial fluids.
  • a cell-free nucleic acid sequence may comprise a complementary DNA (cDNA). In some cases, one or more cDNAs may form a cDNA library.
  • the term“subject,” as used herein, may be any animal or living organism.
  • Animals can be mammals, such as humans, non-human primates, rodents such as mice and rats, dogs, cats, pigs, sheep, rabbits, and others.
  • a subject may be a dog.
  • a subject may be a human.
  • Animals can be fish, reptiles, or others.
  • Animals can be neonatal, infant, adolescent, or adult animals. Humans can be more than about: 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, or at least about 80 years of age.
  • the subject may have or be suspected of having a condition or a disease, such as endometriosis or related condition or cancer.
  • the subject may be a patient, such as a patient being treated for a condition or a disease, such as a patient suffering from endometriosis or suffering from cancer.
  • the subject may be predisposed to a risk of developing a condition or a disease such as endometriosis or cancer.
  • the subject may be in remission from a condition or a disease, such as a patient recovering from endometriosis or cancer.
  • the subject may be healthy.
  • the subject may be a subject in need thereof.
  • the subject may be a female subject or a male subject.
  • a sample comprising genetic material may be obtained from a subject, such as a subject in need thereof.
  • a sample 202 containing a genetic material may be obtained from a subject 201, such as a human subject.
  • a sample 202 may be subjected to one or more methods as described herein, such as performing an assay.
  • an assay may comprise sequencing, genotyping, hybridization, amplification, labeling, or any combination thereof.
  • One or more results from a method may be input into a processor 204.
  • One or more input parameters such as a sample identification, subject identification, sample type, a reference, or other information may be input into a processor 204.
  • One or more metrics from an assay may be input into a processor 204 such that the processor may produce a result, such as a diagnosis of endometriosis or cancer, a recommendation for treatment, or a combination thereof.
  • a processor may send a result, an input parameter, a metric, a reference, or any combination thereof to a display 205, such as a visual display or graphical user interface.
  • a processor 204 may (i) send a result, an input parameter, a metric, or any combination thereof to a server 207, (ii) receive a result, an input parameter, a metric, or any combination thereof from a server 207, (iii) or a combination thereof.
  • the term“sequencing” as used herein, may comprise high-throughput sequencing, next-gen sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing,
  • sequencing output data may be subject to quality controls, including filtering for quality (e.g., confidence) of base reads.
  • Exemplary sequencing systems include 454 pyrosequencing (454 Life Sciences), Illumina (Solexa) sequencing, SOLiD (Applied Biosystems), and Ion Torrent Systems' pH sequencing system.
  • a nucleic acid of a sample may be sequenced without an associated label or tag.
  • a nucleic acid of a sample may be sequenced, the nucleic acid of which may have a label or tag associated with it.
  • Sequencing can comprise next generation sequencing (NGS), a high throughput sequencing method.
  • Sequencing can comprise single molecule real time sequencing.
  • Sequencing can comprise nanopore sequencing.
  • Sequencing can comprise sequencing cell-free DNA.
  • Sequencing can comprise whole exome sequencing.
  • At least a portion of genetic material can be sequenced. Genetic material can comprise at least a portion of: an exon, an intron, a regulatory element, an operon, a promoter, or any combination thereof.
  • Sequencing can include sequencing genetic material, a cDNA thereof, an amplicon thereof, or any combination thereof.
  • Haplotype can mean a combination of genotypes on the same chromosome occurring in a linkage disequilibrium block. Haplotypes serve as markers for linkage disequilibrium blocks, and at the same time provide information about the arrangement of genotypes within the blocks. Typing of only certain variants which serve as tags can, therefore, reveal all genotypes for variants located within a block. Thus, the use of haplotypes greatly facilitates identification of candidate genes associated with diseases and drug sensitivity.
  • Linkage disequilibrium can mean that a particular combination of alleles (alternative nucleotides) or genetic variants for example at two or more different SNP (or RV) sites are non-randomly co-inherited (i.e., the combination of alleles at the different SNP (or RV) sites occurs more or less frequently in a population than the separate frequencies of occurrence of each allele or the frequency of a random formation of haplotypes from alleles in a given population).
  • the term “LD” can differ from "linkage,” which describes the association of two or more loci on a chromosome with limited recombination between them.
  • LD can also be used to refer to any non-random genetic association between allele(s) at two or more different SNP (or RV) sites.
  • Linkage disequilibrium block or “LD block” can mean a region of the genome that contains multiple variants located in proximity to each other and that are transmitted as a block.
  • Methods may include detecting a presence or an absence of a mutation in at least a portion of one or more genes.
  • a gene may comprise a cancer driver gene.
  • a gene may comprise a Tierl cancer driver gene.
  • Methods may including detecting a presence or an absence of a mutation in at least a portion of one or more of: ABL1, ACVR1, ACVR1B, ACVR2A, AJUBA, AKT1, ALB, ALK, AMER1, APC, APOB, AR, ARAF, ARHGAP35, ARID 1 A, ARID2, ARID5B, ASXL1, ASXL2, ATF7IP, ATM, ATR, ATRX, ATXN3, AXIN1, AXIN2, B2M, BAP1, BCF2, BCF2F11, BCOR, BRAF, BRCA1, BRCA2, BRD7, BTG2, CACNA1A, CARD11, CASP8, CBFB, CBWD3, CCND1, CD70, CD79B, CDH1, CDK12
  • MED 12 MEN1, MET, MGA, MGMT, MLH1, MSH2, MSH3, MSH6, MTOR, MUC6, MYC, MYCN, MYD88, MYH9, NCOR1, NF1, NF2, NFE2L2, NIPBL, NOTCH1, NOTCH2, NPM1, NRAS, NSD1, NUP133, NUP93, P53, PAX5, PBRM1, PCBP1, PDGFRA, PDS5B, PGR, PHF6, PIK3CA, PIK3CB, PIK3CG, PIK3R1, PIK3R2, PIM1, PLCB4, PLCG1, PLXNB2, PMS1, PMS2, POLE, POLQ, POLRMT, PPM ID, PPP2R1A, PPP6C, PRKAR1A, PSIP1, PTCH1, PTEN, PTMA, PTPDC1, PTPN11, PTPRC, PTPRD, RAC1, RAD21, RAF1, RARA,
  • the method may detect a presence or an absence of a subject of the preceding genes, such as at least 2 genes, at least 5 genes, at least 10 genes. In some cases, the method may detect a presence or an absence of a mutation in a plurality of genes, including TP53.
  • Methods may include detecting a mutation in a portion of one or more genes.
  • One or more genes may comprise ATM, FANCC, MREl lA, BRCA2, MLH1, PARP1, BRCA1, PMS2, CHEK1, CHEK2 , RAD51C, PARP2, MUTYH, ERCC4, PARP3, ATR, FANCG, RAD51B, FANCA, RAD51, RAD51D, BRIP1, FANCL , RAD52, MSH6, POLE, RAD54L, PALB2, POLD1, XRCC2, MSH2, NBN, CDK12, ARID 1 A, ATRX, or any combination thereof.
  • Methods may include detecting a mutation in a portion of one or more genes.
  • the method may include a method of diagnosing a presence or an absence of a condition (such as endometriosis or related condition) or a likelihood of developing the condition.
  • Methods may include diagnosing a presence or an absence of more than one condition (such as endometriosis and cancer) or a likelihood of developing more than one condition (such as endometriosis and cancer).
  • the method may include diagnosing the presence or the absence of the condition based at least in part on detecting a presence or and absence of one or more mutations.
  • the methods may include detecting a presence or an absence of one or more mutations in one or more of the following genes: ARID1A, ATM, ATR, BRCA1, BRCA2, BRIP1, CHEK2, FANCA, FANCC, or POLE.
  • the methods may include detecting a presence or an absence of one or more mutations in one or more of the following genes: ATM, BRCA2, BRIP1, CHEK2, or RAD5 ID.
  • the methods may include detecting a presence or an absence of one or more mutations in one or more of the following genes: ATM, RAD51C, MLH1, MUTYH, ARID1A, or ATRX.
  • the methods may include detecting a presence or an absence of one or more mutations in one or more of the following genes: BPTF, MTOR, ARFGEF2, FAT1, ATM, MAGI2, SVEP1, NOTCH1, or ABCB1.
  • a disease or condition may include a cancer.
  • a cancer may include a cancer of the reproductive system.
  • a cancer may include a breast cancer, an ovarian cancer, a cutaneous cancer, an endometrial cancer, a cervical cancer, a uterine cancer, a pelvic cancer, an abdominal cancer, a cutaneous melanoma, a non-Hodgkin’s lymphoma, or any combination thereof.
  • a cancer of the reproductive system can exclude 1, 2, 3, 4, 5 or more of the following: a breast cancer, an ovarian cancer, a cutaneous cancer, an endometrial cancer, a cervical cancer, a uterine cancer, a pelvic cancer, an abdominal cancer, a cutaneous melanoma, a non-Hodgkin’s lymphoma.
  • Detecting a presence or an absence of a mutation in a gene or portion thereof in a genetic material obtained from a subject may: (i) inform a presence or absence of one or more conditions the subject, (ii) convey a risk to the subject of developing one or more conditions, (iii) or any combination thereof.
  • detecting a presence or an absence of a mutation in a gene or portion thereof in a genetic material obtained from a subject may indicate a presence of or an increased risk of developing one or more conditions (such as cancer, a reproductive condition such as endometriosis, or a combination thereof) in the subject.
  • Detecting a presence or an absence of a mutation in a gene or portion thereof in a genetic material obtained from a subject may indicate an absence of or a decreased risk of developing one or more conditions in the subject. More than one mutation in a gene may be detected. A mutation in a plurality of genes may be detected.
  • XPG, XPV, MSH2, MSH6, MLH1, PMS2, MUTYH, P53, NTHL1 in genetic material obtained from a subject may indicate a presence or risk of developing a cancer in the subject.
  • Detecting a presence or an absence of one or more mutations in a gene in a genetic material obtained from a subject may indicate an increase in DNA damage repair or may indicate a decrease in DNA damage repair.
  • Detecting a presence or an absence of one or more mutations in a gene in a genetic material obtained from a subject may also inform a treatment or prevention course for one or more conditions. Detecting a presence or an absence of a mutation in a gene or portion thereof may indicate that a composition or set of compositions may be effective in treating or preventing one or more conditions (such as cancer, endometriosis, or a combination thereof) in the subject. Detecting a presence or an absence of a mutation in a gene or portion thereof of a genetic material obtained from a subject may indicate that a composition or set of compositions may be effective in treating the condition in the subject.
  • detecting a presence or an absence of a mutation in a gene or portion thereof of a genetic material from a subject may indicate that a compound or set of compounds may not be effective in treating or preventing a condition in the subject. In some cases, detecting a presence or an absence of a mutation in a gene or portion thereof of a genetic material from a subject may indicate that a compound or set of compounds may be effective in treating or preventing a condition in the subject.
  • Methods may include detecting a presence or an absence of one or more mutations in a genetic material.
  • a mutation may comprise one or more mutations.
  • a mutation may occur in one or more portions of a gene.
  • a mutation may occur in a gene body, a promoter region, or a combination thereof.
  • a mutation may comprise a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • a mutation may comprise a point mutation or a mutation to a portion of bases, such as a contiguous section of bases.
  • a mutation may comprise a base substitution, a deletion, an insertion, or any combination thereof.
  • a deletion may comprise deletion of one or more bases.
  • An insertion may comprise insertion of one or more bases.
  • Detecting may comprise detecting a gene expression level, such as a differential expression level as compared to a reference. Detecting may comprise detecting one or more variants, such as a single nucleotide polymorphism (SNP). Detecting may comprise detecting a copy number variation.
  • SNP single nucleotide polymorphism
  • Detecting may comprise sequencing, such as high throughput sequencing. Detecting may comprise an array-based assay. Detecting may comprise an imaging method.
  • a composition may be a pharmaceutical composition.
  • a composition may be employed to treat one or more conditions (such as a cancer or a reproductive condition such as endometriosis.
  • a condition may include cancer, endometriosis, or a combination thereof.
  • a treatment may comprise a pretreatment or prophylactic treatment.
  • Identifying a treatment for a subject may comprise detecting a presence or an absence of a genetic variant in genetic material from a subject.
  • a genetic variant may comprise a single nucleotide polymorphism (SNP).
  • a genetic variant may comprise a variation in copy number.
  • a genetic variant may comprise a genetic mutation.
  • a genetic variant can comprise a synonymous mutation, a non-synonymous mutation, a stop-gain mutation, a nonsense mutation, an insertion, a deletion, a splice-site variant, a frameshift mutation, or any combination thereof.
  • a genetic variant can comprise a protein damaging mutation.
  • a genetic variant may be a rare variant occurring in less than about: 1%, 0.5%, 0.1%, 0.05% or 0.01% of a population.
  • a genetic variant may have a minor allele frequency (MAF) of less than about 1% of a population.
  • a genetic variant may be selected from Table 1, Table 4 or any combination thereof. More than one genetic variant may be detected, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10 genetic variants or more.
  • a trained algorithm may include a nearest neighbor algorithm, a random forest algorithm, a support vector machine (SVM) algorithm, a decision tree algorithm, a linear regression algorithm, a logistic regression algorithm, a naive bayes algorithm, a kNN algorithm, any combination thereof or others.
  • a trained algorithm may utilize feature selection to identify one or more cancer driver mutations predictive of a presence or an absence of endometriosis, cancer or both, predictive of a risk of developing endometriosis, cancer or both, predictive of an efficacy of a treatment for endometriosis, cancer or both, predictive of a recurrence of endometriosis, cancer or both or any combination thereof.
  • a trained algorithm may rank or weight one or more cancer driver mutations.
  • a highly ranked or highly weighted cancer driver mutation may provide an identification of a presence or an absence of endometriosis, cancer or both in a sample at a greater accuracy than an cancer driver mutation that may be lower ranked or weighted.
  • a trained algorithm may identify a panel of one or more cancer driver mutations that may identify a presence or an absence of endometriosis, cancer or both at an accuracy of at least 80%, 85%, 90%, 95% or greater.
  • a panel may comprise a single cancer driver mutation.
  • a panel may comprise a plurality of cancer driver mutations.
  • a panel may comprise an cancer driver mutation in combination with another feature (such as an insertion, a deletion, a variation, a repetitive element, or a copy number change in a polymorphism), or another gene having a differential level of expression compared to a reference.
  • a panel may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250 cancer driver mutations or more.
  • a panel may comprise at least 5 cancer driver mutations.
  • a panel may comprise at least 10 cancer driver mutations.
  • a panel may comprise at least 20 cancer driver mutations.
  • a panel may comprise at least 50 cancer driver mutations.
  • a panel may comprise at least 100 cancer driver mutations.
  • a trained algorithm may receive a data set comprising information obtained from a medical questionaire or medical history obtained from a subject (such as an age of positive diagnosis of endometriosis), from a genetic -based assay, from a non-genetic based assay, from a cytological assay, from an immuno-based assay, from a sequencing assay, or any combination thereof.
  • a trained algorithm or other computer processing element may output a report, such as a printed report or an electronic report.
  • a report may comprise a result obtained from analyzing the sample.
  • a result may include a presence or absence of endometriosis, cancer, or both in the sample.
  • a report may comprise details of the sample, such as sample type, a collection media, a collection source, a panel of cancer driver mutations, genes or other markers assessed.
  • a report may comprise a recommendation, such as a recommendation of treatment or second diagnostic assay based on a result obtained.
  • Administration of a treatment can be affected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are known to those of skill in the art and can vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of
  • administration are known to those of skill in the art and can vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue.
  • routes of administration include oral administration, nasal administration, injection (such as intramuscular), and topical application.
  • Administration can refer to methods that can be used to enable delivery of a treatment, such as a treatment for endometriosis, cancer, or both. These methods can include topical administration (such as a lotion, a cream, an ointment) to an external surface of a surface, such as a skin. These methods can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration. In some instances, a subject can administer the treatment in the absence of supervision.
  • topical administration such as a lotion, a cream, an ointment
  • parenteral administration including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion
  • oral administration inhalation administration
  • intraduodenal administration rectal administration.
  • a subject can administer the treatment in the absence of supervision.
  • a subject can administer the treatment under the supervision of a medical professional (e.g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.).
  • a medical professional can administer the treatment.
  • a cosmetic professional can administer the treatment.
  • the treatment is given to a subject by injection, such an intramuscular injection.
  • Administration of a treatment may be given to a subject one time, such as a one-time treatment.
  • Administration of a treatment may be given to the subject two times or more, such as a two-time treatment, such as when a first-time treatment failed.
  • a composition can be administered or applied as a single dose or as divided doses.
  • compositions described herein can be administered at a first time point and a second time point.
  • a composition can be administered such that a first administration is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more.
  • a pharmaceutical composition described herein can compromise an excipient.
  • An excipient can comprise a pH agent (to minimize oxidation or degradation of a component of the composition), a stabilizing agent (to prevent modification or degradation of a component of the composition), a buffering agent (to enhance temperature stability), a solubilizing agent (to increase protein solubility), or any combination thereof.
  • An excipient can comprise a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a trigylceride, an alcohol, or any combination thereof.
  • An excipient can comprise sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HC1, disodium edetate, lecithin, glycerine, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof.
  • An excipient can be an excipient described in the
  • Non-limiting examples of suitable excipients can include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, a coloring agent.
  • an excipient can be a buffering agent.
  • suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • glycerophosphate calcium chloride, calcium hydroxide and other calcium salts or combinations thereof can be used in a pharmaceutical formulation.
  • an excipient can comprise a preservative.
  • suitable preservatives can include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • Antioxidants can further include but not limited to EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N- acetyl cysteine.
  • a preservatives can include validamycin A, TL-3, sodium ortho vanadate, sodium fluoride, N-a-tosyl-Phe- chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin,
  • phenylmethylsulfonyl fluoride diisopropylfluorophosphate, kinase inhibitor, phosphatase inhibitor, caspase inhibitor, granzyme inhibitor, cell adhesion inhibitor, cell division inhibitor, cell cycle inhibitor, lipid signaling inhibitor, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitor.
  • a pharmaceutical formulation can comprise a binder as an excipient.
  • suitable binders can include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the binders that can be used in a pharmaceutical formulation can be selected from starches such as potato starch, com starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatine; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone);
  • PEG polyethylene glycol
  • waxes waxes
  • calcium carbonate calcium phosphate
  • alcohols such as sorbitol, xylitol, mannitol and water or a combination thereof.
  • a pharmaceutical formulation can comprise a lubricant as an excipient.
  • suitable lubricants can include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the lubricants that can be used in a pharmaceutical formulation can be selected from metallic stearates (such as magnesium stearate, calcium stearate, aluminium stearate), fatty acid esters (such as sodium stearyl fiimarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffins, hydrogenated vegetable oils, leucine, polyethylene glycols (PEG), metallic lauryl sulphates (such as sodium lauryl sulphate, magnesium lauryl sulphate), sodium chloride, sodium benzoate, sodium acetate and talc or a combination thereof.
  • metallic stearates such as magnesium stearate, calcium stearate, aluminium stearate
  • fatty acid esters such as sodium stearyl fiimarate
  • fatty acids such as stearic acid
  • fatty alcohols glyceryl behenate
  • mineral oil such as paraffins, hydrogenated vegetable oils,
  • a pharmaceutical formulation can comprise a dispersion enhancer as an excipient.
  • suitable dispersants can include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • a pharmaceutical formulation can comprise a disintegrant as an excipient.
  • a disintegrant can be a non-efferve scent disintegrant.
  • suitable non- effervescent disintegrants can include starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • a disintegrant can be an effervescent disintegrant.
  • suitable effervescent disintegrants can include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • an excipient can comprise a flavoring agent.
  • Flavoring agents incorporated into an outer layer can be chosen from synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof.
  • a flavoring agent can be selected from the group consisting of cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.
  • an excipient can comprise a sweetener.
  • suitable sweeteners can include glucose (com syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like.
  • a composition may comprise a combination of the active agent, (e.g., leuprolide acetate), and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.
  • Carriers also include
  • protein excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates e.g., sugars, including monosaccharides, di-, tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers
  • exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffmose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose,
  • the methods detect one or more mutations in one or more cancer driver mutations. Detection may include sequencing, such as whole exome sequencing. Methods may detect one or more genetic variants. In some cases, the disclosure provides methods to detect one or more genetic variants (e.g., in Table 1, Table 4, or any combination thereof). In some cases, the methods include selecting a panel of the one or more genetic variants for detection. In some cases, a genetic variant in a panel may comprise two or more genetic variants defining a minor allele. In some instances, the detecting may comprise: DNA sequencing, hybridizing with a complementary probe, performing an oligonucleotide ligation assay, performing a PCR-based assay, or any combination thereof.
  • the panel may comprise at least: 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 150 200, 250, 300, 350, 400, 450, 500, or more genetic variants defining minor alleles disclosed herein.
  • the genetic variant to detect or detected has an odds ratio (OR) of at least: 0.1, 1, 1.5, 2, 5, 10, 20, 50, 100, 127, 130, 140, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,
  • a genetic variant may include single nucleotide polymorphisms (SNPs), insertion deletion polymorphisms (indels), damaging mutation variants, loss of function variants, synonymous mutation variants, nonsynonymous mutation variants, nonsense mutations, recessive markers, splicing/splice-site variants, frameshift mutation, insertions, deletions, genomic rearrangements, stop- gain , stop-loss, Rare Variants (RVs), translocations, inversions, and substitutions.
  • SNPs single nucleotide polymorphisms
  • indels insertion deletion polymorphisms
  • SNPs Genetic variants, for example SNPs, may be preceded and followed by highly conserved sequences that vary in less than 1/100 or 1/1000 members of the population. An individual may be homozygous or heterozygous for an allele at each SNP position.
  • a SNP may, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP is an amino acid "coding" sequence.
  • a SNP may arise from a substitution of one nucleotide for another at the polymorphic site. Substitutions can be transitions or transversions. A transition may be the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine. A transversion may be the replacement of a purine by a pyrimidine, or vice versa.
  • a synonymous codon change, or silent mutation is one that does not result in a change of amino acid due to the degeneracy of the genetic code.
  • a substitution that changes a codon coding for one amino acid to a codon coding for a different amino acid is referred to as a missense mutation.
  • a nonsense mutation may result in a type of non-synonymous codon change in which a stop codon is formed, thereby leading to premature termination of a polypeptide chain and a truncated protein.
  • a read-through mutation is another type of non-synonymous codon change that causes the destruction of a stop codon, thereby resulting in an extended polypeptide product.
  • An indel that occur in a coding DNA segment gives rise to a frameshift mutation.
  • one or more variant alleles of the disclosure can be associated with either an increased risk of having or developing a disease, a decreased risk of having or developing a disease, or any combination thereof.
  • Variant alleles that are associated with a decreased risk may be referred to as “protective” alleles
  • variant alleles that are associated with an increased risk may be referred to as "susceptibility" alleles, "risk factors", or "high-risk” alleles.
  • variants can be assayed to determine whether an individual possesses a variant allele that is indicative of an increased risk of having or developing a disease (i.e., a susceptibility allele)
  • other variants can be assayed to determine whether an individual possesses a variant allele that is indicative of a decreased risk of having or developing a disease (i.e., a protective allele).
  • the term "altered” may be used herein to encompass either of these two possibilities (e.g., an increased or a decreased risk/likelihood).
  • nucleic acid molecules may be double -stranded molecules and that reference to a site on one strand refers, as well, to the corresponding site on a complementary strand.
  • reference to an adenine, a thymine (uridine), a cytosine, or a guanine at a site on one strand of a nucleic acid molecule also defines the complementary thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule.
  • probes and primers may be designed to hybridize to either strand and variant genotyping methods disclosed herein may generally target either strand.
  • the method can comprise sequencing a nucleic acid sample obtained from a subject.
  • the subject may have a disease or be suspected of having a disease.
  • the sequencing may comprise a high throughput method.
  • the high throughput method can comprise nanopore sequencing.
  • the method can comprise detecting one or more genetic variants in a nucleic acid sample, wherein the one or more genetic variants are listed in Table 1, Table 4, or any combination thereof.
  • the nucleic acid sample can comprise RNA.
  • the RNA can comprise mRNA.
  • the nucleic acid sample can comprise DNA.
  • the DNA can comprise cDNA, genomic DNA, sheared DNA, cell free DNA, fragmented DNA, or PCR amplified products produced therefrom, or any combination thereof.
  • the one or more genetic variants can comprise a genetic variant defining a minor allele.
  • the one or more genetic variants can comprise at least about: 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, or more genetic variants.
  • the one or more genetic variants can comprise a synonymous mutation, a non-synonymous mutation, a stop-gain mutation, a nonsense mutation, an insertion, a deletion, a splice-site variant, a frameshift mutation, or any combination thereof.
  • the one or more genetic variants can be identified based on a predictive computer algorithm.
  • the one or more genetic variants can be identified based on reference to a database.
  • the method can further comprise identifying a subject as having a disease or being at risk of developing a disease, or any combination thereof.
  • variant genotyping the process of determining which specific nucleotide (i.e., allele) is present at each of one or more variant positions, such as a variant position in a nucleic acid molecule characterized by a variant.
  • the disclosure provides methods of variant genotyping, such as for use in screening for a disease or related pathologies or determining predisposition thereto or in genome mapping or variant association analysis, or any combination thereof.
  • Nucleic acid samples can be genotyped to determine which allele(s) is/are present at any given genetic region (e.g., variant position) of interest by methods well known in the art.
  • the neighboring sequence can be used to design variant detection reagents such as oligonucleotide probes, which may optionally be implemented in a kit format.
  • variant genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele-specific primer extension, allele-specific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, mass spectrometry with or with monoisotopic dNTPs
  • Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, electrospray mass spectrometry, and electrical detection.
  • detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, electrospray mass spectrometry, and electrical detection.
  • Various methods for detecting polymorphisms can include, but are not limited to, methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes, comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules and assaying the movement of polymorphic or wild-type fragments in polyacrylamide gels containing a gradient of denaturant using denaturing gradient gel electrophoresis (DGGE). Sequence variations at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or chemical cleavage methods.
  • DGGE denaturing gradient gel electrophoresis
  • a variant genotyping can be performed using the TaqMan assay, which is also known as the 5' nuclease assay.
  • the TaqMan assay may detect the accumulation of a specific amplified product during PCR.
  • the TaqMan assay may utilize an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye.
  • the reporter dye may be excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye may not emit a signal.
  • FRET fluorescence resonance energy transfer
  • the reporter dye and quencher dye may be at the 5' most and the 3' most ends, respectively, or vice versa.
  • the reporter dye may be at the 5' or 3' most end while the quencher dye is attached to an internal nucleotide, or vice versa.
  • both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter may be reduced.
  • the 5' nuclease activity of DNA polymerase may cleave the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product may be detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target variant-containing template which is amplified during PCR, and the probe may be designed to hybridize to the target variant site only if a variant allele is present.
  • TaqMan primer and probe sequences can readily be determined using the variant and associated nucleic acid sequence information provided herein. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the variants of the disclosure are useful in diagnostic assays for endometriosis and related pathologies and can be readily incorporated into a kit format. The disclosure also includes modifications of the Taqman assay well known in the art such as the use of Molecular Beacon probes and other variant formats.
  • a method for genotyping the variants can be the use of two oligonucleotide probes in an OLA.
  • one probe may hybridize to a segment of a target nucleic acid with its 3' most end aligned with the variant site.
  • a second probe may hybridize to an adjacent segment of the target nucleic acid molecule directly 3' to the first probe.
  • the two juxtaposed probes may hybridize to the target nucleic acid molecule and may be ligated in the presence of a linking agent such as a ligase if there is perfect complementarity between the 3' most nucleotide of the first probe with the variant site. If there is a mismatch, ligation may not occur.
  • the ligated probes may be separated from the target nucleic acid molecule and detected as indicators of the presence of a variant.
  • a method for variant genotyping may be based on mass spectrometry.
  • Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. variants can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternative variant alleles.
  • MALDI-TOF Microx Assisted Laser Desorption Ionization-Time of Flight mass spectrometry technology is exemplary for extremely precise determinations of molecular mass, such as variants.
  • Numerous approaches to variant analysis have been developed based on mass spectrometry.
  • Exemplary mass spectrometry-based methods of variant genotyping include primer extension assays, which can also be utilized in combination with other approaches, such as traditional gel-based formats and microarrays.
  • a method for genotyping the variants of the disclosure is the use of electrospray mass spectrometry for direct analysis of an amplified nucleic acid.
  • an amplified nucleic acid product may be isotopically enriched in an isotope of oxygen (O), carbon (C), nitrogen (N) or any combination of those elements.
  • the amplified nucleic acid may be isotopically enriched to a level of greater than 99.9% in the elements of O 16 , C 12 and N 14 .
  • the amplified isotopically enriched product can then be analyzed by electrospray mass spectrometry to determine the nucleic acid composition and the corresponding variant genotyping.
  • an amplified nucleic acid that is not isotopically enriched can also have composition and variant genotype determined by electrospray mass spectrometry.
  • variants can be scored by direct DNA sequencing.
  • the nucleic acid sequences of the disclosure enable one of ordinary skill in the art to readily design sequencing primers for such automated sequencing procedures.
  • Commercial instrumentation such as the Applied Biosystems 377, 3100, 3700, 3730, and 3730.times.1 DNA Analyzers (Foster City, Calif.), is commonly used in the art for automated sequencing.
  • Variant genotyping can include the steps of, for example, collecting a biological sample from a human subject (e.g., sample of tissues, cells, fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA, mRNA or both) from the cells of the sample, contacting the nucleic acids with one or more primers which specifically hybridize to a region of the isolated nucleic acid containing a target variant under conditions such that hybridization and amplification of the target nucleic acid region occurs, and determining the nucleotide present at the variant position of interest, or, in some assays, detecting the presence or absence of an amplification product (assays can be designed so that hybridization and/or amplification will only occur if a particular variant allele is present or absent).
  • the size of the amplification product is detected and compared to the length of a control sample; for example, deletions and insertions can be detected by a change in size of the amplified product
  • a variant genotyping can be used in applications that include, but are not limited to, variant-disease association analysis, disease predisposition screening, disease diagnosis, disease prognosis, disease progression monitoring, determining therapeutic strategies based on an individual's genotype, selecting a treatment, and stratifying a patient population for clinical trials for a treatment such as minimally invasive device for the treatment of disease.
  • FIG. 1 shows three classes of endometriosis genetic markers are shown above. By integrating genetic risk with clinical criteria, and area under the curve (AUC) of greater than about 90% may be reached.
  • AUC area under the curve
  • Methods as described herein may include detecting one or more SNPs described in the Table 1 above.
  • Somatic mutations may accumulate during the clonal evolution of individual endometriosis lesions.
  • Endometriosis lesions can be a progressive, invasive, recurrent, and possibly metastatic; many exhibit molecular features seen in cancer (for example, resistance to apoptosis and stimulation of angiogenesis).
  • Cancers may arise in endometriosis lesions.
  • ovarian cancer particularly clear-cell and endometrioid
  • breast cancer endometrial cancer
  • cervical cancer pelvic cancer
  • abdominal cancer cutaneous melanoma
  • Non-Hodgkin's lymphoma or any combination thereof.
  • Embodiment 1 A method comprising: (a) obtaining a genetic material from a subject, wherein the genetic material is at least in part from an endometrial tissue, and wherein the subject has or is suspected of having an endometriosis, a cancer, or a combination thereof; and (b) detecting a presence or an absence of a mutation in a cancer driver gene in at least a portion of the genetic material, wherein the detecting comprises sequencing a nucleic acid sequence from the portion the genetic material, a cDNA thereof, an amplicon of any of these, or any combination thereof,
  • Embodiment 2 The method of embodiment 1, wherein the sequencing comprises whole exome sequencing.
  • Embodiment 3 The method of any proceeding embodiment, wherein the endometrial tissue comprises an endometrial lesion.
  • Embodiment 4 The method of embodiment 3, wherein the endometrial lesion is a benign endometrial lesion.
  • Embodiment 5 The method of any proceeding embodiment, wherein the mutation comprises at least two mutations.
  • Embodiment 6. The method of any proceeding embodiment, wherein a presence or an absence of a first mutation is detected in a first cancer driver gene and a presence or an absence of a second mutation is detected in a second cancer driver gene.
  • Embodiment 7 The method of any proceeding embodiment, wherein the cancer driver gene comprises P53/PTEN.
  • Embodiment 8 The method of any proceeding embodiment, wherein a presence of the mutation in the cancer driver gene identifies an increased risk of developing the endometriosis, the cancer, or a combination thereof as compared to a subject lacking the mutation.
  • Embodiment 9 The method of any proceeding embodiment, wherein a presence of the mutation at least partially identifies a presence of the endometriosis, the cancer, or a combination thereof in the subject.
  • Embodiment 10 The method of any proceeding embodiment, wherein the subject has previously received a diagnosis for the endometriosis.
  • Embodiment 11 The method of embodiment 10, further comprising identifying a biological age of the subject upon receipt of the diagnosis for the endometriosis.
  • Embodiment 12 The method of embodiment 11, wherein the diagnosis for the endometriosis is performed by histological analysis.
  • Embodiment 13 The method of embodiment 11, wherein when the biological age is identified as less than about 40 years of age, the subject is identified as having an at least partially decreased risk of developing the cancer, and no further detecting is performed.
  • Embodiment 14 The method of embodiment 11, wherein when the biological age is identified as greater than about 45 years of age, the subject is identified as having an at least partially increased risk of developing the cancer, and the detecting is performed in a second endometrial tissue of the subject.
  • Embodiment 15 The method of any proceeding embodiment, wherein the cancer comprises a cancer of a reproductive system.
  • Embodiment 16 The method of any proceeding embodiment, wherein the cancer comprises a breast cancer, an ovarian cancer, endometrial cancer, a cervical cancer, a uterine cancer, a pelvic cancer, an abdominal cancer, a cutaneous melanoma, a non-Hodgkin’s lymphoma, or any combination thereof.
  • Embodiment 17 The method of any proceeding embodiment, wherein the mutation comprises a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • Embodiment 18 The method of any proceeding embodiment, wherein the detecting comprises hybridizing a probe to a portion of the genetic material.
  • Embodiment 19 The method of any proceeding embodiment, further comprising: measuring a total variant burden in at least a portion of the genetic material.
  • Embodiment 20 The method of any proceeding embodiment, wherein the presence or the absence of the mutation is at least partially indicative of the endometriosis.
  • Embodiment 21 The method of any proceeding embodiment, wherein the presence or the absence of the mutation is at least partially indicative of the cancer.
  • Embodiment 22 The method of any proceeding embodiment, wherein the mutation is detected.
  • Embodiment 23 The method of any proceeding embodiment, wherein the mutation is not detected.
  • Embodiment 24 The method of any proceeding embodiment, further comprising: obtaining the genetic material from the subject.
  • Embodiment 25 The method of any proceeding embodiment, wherein the subject has the cancer.
  • Embodiment 26 The method of any proceeding embodiment, wherein the subject has the endometriosis.
  • Embodiment 27 The method of any proceeding embodiment, wherein the subject is at risk of developing the endometriosis.
  • Embodiment 28 The method of any proceeding embodiment, wherein the subject suffers from pelvic pain.
  • Embodiment 29 The method of any proceeding embodiment, wherein the subject suffers from infertility.
  • Embodiment 30 The method of any proceeding embodiment, wherein at least a portion of the genetic material comprises is from a blood sample.
  • Embodiment 31 The method of any proceeding embodiment, wherein at least a portion of the genetic material comprises cell free DNA.
  • Embodiment 32 The method of any proceeding embodiment, wherein the genetic material comprises endometrial tissue, uterine tissue, ovarian tissue, fallopian tissue, cervical tissue, vulvar tissue, or any combination thereof.
  • Embodiment 33 The method of any proceeding embodiment, further comprising treating the subject for endometriosis, the cancer, or a combination thereof.
  • Embodiment 34 The method of embodiment 33, wherein the treating comprises prophylactic treating.
  • Embodiment 35 The method of embodiment 33, wherein the treating comprises administering to the subject a pharmaceutical composition in unit dose form.
  • Embodiment 36 The method of embodiment 33, further comprising administering a co-therapy to the subject.
  • Embodiment 37 The method of embodiment 36, wherein the co-therapy comprises
  • Embodiment 38 The method of any proceeding embodiment, further comprising: comparing a result of the method to a reference.
  • Embodiment 39 The method of embodiment 38, wherein the reference comprises a derivative of the reference.
  • Embodiment 40 The method of embodiment 38, wherein the reference comprises a result of the method performed on a reference sample.
  • Embodiment 41 The method of embodiment 38, wherein the comparing is performed by a computer processor.
  • Embodiment 42 The method of embodiment 38, wherein the comparing is performed by a trained algorithm.
  • Embodiment 43 The method of embodiment 38, wherein the reference comprises a result obtained from genetic material of a subject diagnosed with the endometriosis, the cancer, or a combination thereof.
  • Embodiment 44 The method of any proceeding embodiment, wherein the detecting comprises sequencing at least a portion of WNT4, HD AC 2, or a combination thereof.
  • Embodiment 45 The method of embodiment 44, wherein the sequencing identifies a presence or an absence of a risk allele T in homozygous or heterozygous form in WNT4, a presence or an absence of a mutation in HDAC2, or a combination thereof.
  • Embodiment 47 The method of embodiment 46, wherein the biological pathway comprises: an integral plasma membrane, a cytoskeletal part, a cell surface, a plasma membrane region, an extracellular matrix, a cell junction or any combination thereof.
  • Embodiment 48 The method of embodiment 46, wherein the biological pathway comprises a protein-protein interaction.
  • Embodiment 49 The method of any proceeding embodiment, wherein the subject comprises an ethnicity that is at least partially Caucasian, African American, Asian, Hispanic, or any combination thereof.
  • An endometrial tissue sample will be obtained from a subject suspected of having a cancer. A portion of the sample will be prepared for a whole exome sequencing analysis. The sequencing will detect a presence of a mutation in two cancer driver genes. The presence of the mutation in the two cancer driver genes will identify the subject having an increased risk of developing the cancer as compared to a subject lacking the mutation.
  • An endometrial tissue sample will be obtained from a subject suspected of having a cancer. A portion of the sample will be prepared for a whole exome sequencing analysis. The sequencing will detect a presence of a mutation in five cancer driver genes, including TICS. The presence of the mutation in the five cancer driver genes will identify the subject having a presence of the cancer.
  • An endometrial tissue sample will be obtained from a subject suspected of having an endometriosis. A portion of the sample will be prepared for a whole exome sequencing analysis. The sequencing will detect a presence of a mutation in five cancer driver genes. The presence of the mutation in the five cancer driver genes will identify the subject having a presence of the endometriosis and having an increased risk of developing the cancer as compared to a subject lacking the mutation.
  • An endometrial tissue sample will be obtained from a subject having received a positive diagnosis for endometriosis.
  • An age of positive diagnosis will be identified.
  • a portion of the sample will be prepared for sequencing, including whole exome sequencing analysis.
  • the sequencing will detect a presence of a mutation in two cancer driver genes.
  • the presence of the mutation in the two cancer drive genes in combination with the age of positive diagnosis will identify the subject having an increased risk of developing the cancer as compared to a subject lacking the mutation and having a different age of positive diagnosis.
  • An endometrial tissue sample and a blood sample will be obtained from a subject suspected of having an endometriosis. A portion of each sample will be prepared for sequencing. The sequencing will include whole exome sequencing and identifying a total variant burden. The presence of a mutation in two cancer driver genes and a level of total variant burden will identify the subject having a presence of the endometriosis and having an increased risk of developing the cancer as compared to a subject lacking the mutation and having a different level of total variant burden.
  • An endometrial tissue sample and a blood sample will be obtained from a subject suspected of having an endometriosis.
  • a portion of the sample will be prepared for sequencing.
  • the sequencing will include whole exome sequencing.
  • the sequencing will identify the presence of a mutation in a portion of two cancer driver genes and a presence of a rare variant SNP in a gene.
  • the presence of the mutation in the portion of the two cancer driver genes and the presence of the rate variant SNP in the gene will identifying the subject having a presence of endometriosis and having an increased risk of developing the cancer as compared to a subject lacking the mutation in the two cancer drive genes and the rare variant SNP.
  • OBJECTIVE To determine whether cancer driver mutations contribute to the development and progression of endometriosis and endometriosis associated cancers.
  • Endometriosis lesions might arise as an autotransplant, as a hamartoma, through metaplasia, or as a neoplasm. Some endometriosis lesions are progressive, invasive, and possibly metastatic, and cancers sometimes arise in endometriosis lesions. Recent studies have shown that somatic mutations accumulate during the clone evolution of individual endometriosis lesions. We conducted whole exome sequencing to investigate the presence of known cancer drive mutations in endometriosis lesions and to correlate these mutation with long term outcomes.
  • MATERIALS AND METHODS 276 woman (age 12 to 95) operated on at OHSU between 2003 and 2014 with a confirmed histologic diagnosis of endometriosis were considered. Exome sequencing was performed on DNA extracted from formalin-fixed paraffin-embedded tissue samples exhibiting endometriosis histology to varying degrees. Within a 5 to 16 year follow-up interval, 55/276 (20%) of these women had a subsequent diagnosis of cancer of OHSU.
  • RESULTS 113 Tier 1 cancer driver mutations (4 splicing, 15 stopgain and 94 missense) were seen in tissue from 66 women. 24% of the 276 surgical samples show at least one cancer driver mutation; 7.3% carried at least 2 cancer driver mutations, a single sample was observed to have 9 cancer driver mutations, and one sample had multiple deletions (runs of homozygosity) including a hemizygous drive mutation. The TP53 gene had the highest rate of cancer driver mutations with 5 mutations detected.
  • Somatic cancer drive mutations may be common in endometriosis lesions. When a cancer driver mutation is present in an endometriosis lesions, the risk of a secondary cancer may be elevated.
  • OBJECTIVE Examine whether somatic mutations found in endometriosis lesions are a risk factor for developing a clinically observed cancer.
  • SUBJECTS 276 consecutive women (ages 12 to 95) initially operated on at OHSU (between 2003 and 2014) with a confirmed histologic diagnosis of endometriosis.
  • METHODS Exome sequencing was performed on DNA extracted from formalin-fixed paraffin- embedded tissue samples. Microdissection was not performed, so the samples sequenced exhibited endometriosis histology to varying degrees. TIER1 mutations from the IntoGen catalog of cancer driver mutations (20,302 mutations in 644 genes) were considered. Pathology records at OSHU were searched to see if any of these women had a subsequent diagnosis of cancer confirmed at OHSU. 66 of the 276 surgical samples (24%) show at least one TIER 1 cancer driver mutation; 7.3% carried at least 2 mutations. A single sample was observed to have 9 TIER 1 cancer driver mutations.
  • FIG. 2 shows mutations are mostly somatic, with about 8X more mutations in endometriosis lesions as compared to endometriosis blood and with about 4X more mutations in endometriosis blood as compared to unaffected controls.
  • Table 3 described the primary cancer sites and number of driver mutations detected.
  • Methods as described herein may include detecting one or more SNPs of Table 4.
  • Endometriosis risk allele in WNT4 may interact with rare mutations in HDAC2 gene.
  • WNT4 can be a secreted protein that in humans can be encoded by the WNT4 gene, found on chromosome 1.
  • WNT4 may be associated with promoting female sex development and repressing male sex development.
  • HDAC2 may be a responsible for the removal of acetyl groups from lysine residues at the N-terminal region of the core histones (e.g. H2A, H2B, H3, H4 or any combination thereof).
  • the risk allele T in WNT4 was present in either homozygous of heterozygous form in 787 subjects (554 endometriosis cases and 233 controls). Eight endometriosis patients and none of the controls had histone deacetylase 2 ⁇ HD ACT) protein altering mutations identified. Table 5 shows the different HDA 2 variants identified in patients. The WNT4 alleles are indicated in Table 5 as being heterozygous or homozygous for the C allele or the T risk allele, for example, CT is heterozygous for the C and T alleles.
  • the woman with mutation in HDAC2 gene in the background of WNT4 risk allele T may be more likely to be susceptible to endometriosis.
  • the levels of HDAC1 and HDAC2 can be deregulated in endometriotic stromal cells.
  • HDAC1 and HDAC2 can be regulators of WNT and p53 pathways.
  • the deacetylase complex can interact with the WNT 4 chromatin in an HDAC-dependent manner and can lead to the suppression of the WNT4 gene and WNT4 dependent morphogenesis.
  • Women carrying the endometriosis risk allele T in WNT4 can be 15 times more likely to carry a HDAC2 mutation than a women with a wild type WNT4.
  • Table 5 shows the different mutations of the HDAC2 variants. Het indicates a heterozygous for the indicated gene.
  • EMT epithelial-to-mesenchymal transition
  • the data show low-frequency exome variants can cluster in the same cellular compartments and pathways.
  • Exome variants associated with endometriosis were enriched from cytoskeletal, cell membrane and extra cellular matrix genes. In some cases, proteins in the cytoskeleton, trans-membrane and extracellular matrix components can contribute to the pathogenesis of endometriosis. Genetic evidence may suggest that that the initiation of endometrioses can be linked to structural features of the cell.
  • Table 6 shows an example of enriched cellular components from the GO annotations in WebGestalt.
  • the cellular components were identified from whole exome sequencing of women diagnosed with endometriosis and comprises low frequency minor allele exon variants.
  • a study of Caucasian woman with surgically diagnosed endometriosis identified 1,113 low- frequency exome variants (MAF ⁇ 0.01) that showed association with endometriosis at the nominal significance threshold (p ⁇ 0.05).
  • An average burden of 3.1 variants was identified in the Caucasian patient population compared with 1.2 variants in a control population.
  • Exome sequencing was performed on 317 non-Caucasian women with surgically confirmed disease and 238 controls. Variant frequencies were compared against a published database of exome data (gnomAD, Broad Institute) consistency and variant burden in the non-Caucasian controls was determined using ethnically matched controls. The variant burden per individual was determined as the count of low-frequency variants.
  • a comparison of the 1,113 variants that showed nominal association with endometriosis in the Caucasian population identified 385 variants that showed MAF>1% in at least one non-European population (high ethnic or HE variants) while the remaining 728 variants showed MAF ⁇ 1% in all reported ethnicities (low ethnic or LE variants).
  • Haplotypic evidence demonstrate that at least a part of the 385 HE variants are present in the Caucasian population at least in part due to admixture.
  • Table 7 shows the average low-frequency -variant burden in cases and controls for both HE and LE variant groups. Additionally, there was an elevated genetic burden ( 1.34-3.16) for cases relative to controls in both groups of variants and across all non- Caucasian ethnic groups. This suggests that exome variants may have biologic effects. The average variant burden differs between ethnicities and generic risk may be estimated relative to the patient’s ethnic background. Low frequency exome variants associated with endometriosis have can have similar effects across ethnic groups.
  • Table 7 shows the average low-frequency-variant burden in cases and controls for both HE (high ethnic) and LE (low ethnic) variant groups in different ethnic populations.

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Abstract

L'invention concerne des méthodes de diagnostic d'une ou plusieurs pathologies, comprenant l'endométriose, le cancer, ou une combinaison de ceux-ci. Les méthodes peuvent également comprendre l'identification d'une probabilité ou d'un risque de développer une ou plusieurs pathologies. L'invention concerne des méthodes et des compositions pour le traitement d'une ou plusieurs pathologies, par exemple par administration d'une ou plusieurs compositions pharmaceutiques sous la forme de doses unitaires. Le traitement peut comprendre un traitement prophylactique. Les méthodes peuvent comprendre la détection d'une ou plusieurs mutations dans un matériau génétique prélevé chez un sujet, par exemple un sujet en ayant besoin.
EP20799047.4A 2019-05-02 2020-05-01 Contribution à un risque de cancer secondaire de mutations somatiques de facteurs du cancer dans les lésions d'endométriose Withdrawn EP3963092A1 (fr)

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