WO2020252721A1 - Marqueurs de méthylation de l'adn et leur utilisation dans la différenciation d'échantillons de biopsie de ganglions lymphatiques cancéreux suspectés - Google Patents

Marqueurs de méthylation de l'adn et leur utilisation dans la différenciation d'échantillons de biopsie de ganglions lymphatiques cancéreux suspectés Download PDF

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WO2020252721A1
WO2020252721A1 PCT/CN2019/091994 CN2019091994W WO2020252721A1 WO 2020252721 A1 WO2020252721 A1 WO 2020252721A1 CN 2019091994 W CN2019091994 W CN 2019091994W WO 2020252721 A1 WO2020252721 A1 WO 2020252721A1
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lymph node
sample
methylation
subject
genes
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Saraswati Sukumar
Mary Jo Fackler
Bradley Downs
Juanjuan Li
Shengrong SUN
Chuang CHEN
Jingping YUAN
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Renmin Hospital Of Wuhan University
Johns Hopkins University
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Renmin Hospital Of Wuhan University
Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death. In 2015, 2.4 million new cases were detected and 533,000 cases of deaths due to breast cancer worldwide [1] .
  • Breast cancer incidence is rising in the developing world because of longer life expectancies, decreased burden of infectious diseases, and changes in reproductive risk factors [2, 3] .
  • Patients with breast cancer in the underdeveloped world experience longer diagnostic delays than patients in developed countries, leading to later-stage presentations [4] .
  • optimal early detection strategies are not well-characterized in settings where population-based mammography screening is not yet available, primary care services are limited, and pathology and treatment services are available only at regional hospitals [5] .
  • Lymph node metastasis Breast cancer, a heterogeneous disease, presents with various pathological signs such as axillary lymph node metastasis.
  • the presence of axillary lymph node metastasis is associated with a high risk of recurrence and considered an important prognostic factor in the early stages of the disease [11] .
  • Staging of the axilla preoperatively in women presenting with enlarged lymph nodes is important since it will allow patients to be triaged to axillary lymph node dissection (ALND) with a positive result, or to sentinel node biopsy with a negative result.
  • Various imaging tests including ultrasound, mammography, and magnetic resonance imaging (MRI) can provide information on axillary lymph nodes preoperatively to assist staging and treatment.
  • MRI magnetic resonance imaging
  • PET Positron-emission tomography
  • PET/CT PET integrated with computed tomography
  • DNA methylation in gene promoters is a molecular modification of DNA that is tightly associated with loss of gene expression [15] .
  • the present inventors have previously shown that DNA methylation that occurs specifically in breast cancer can serve as a powerful marker for detection of breast cancer in body fluids such as nipple aspiration, ductal lavage fluid and blood, as well as in tissue such as is derived from core biopsy [16-20] .
  • the inventors performed extensive work to derive methylated gene marker panels [18, 19, 21-25] that are specific to pre-invasive and invasive ductal and lobular cancers [25] .
  • the present inventors provide a selection of a panel of one, two, or more DNA methylation markers that detects breast cancer in biological samples from enlarged or suspicious lymph nodes of subjects suspected of having breast cancer, with high sensitivity and specificity using DNA methylation techniques such as the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) .
  • QM-MSP Quantitative Multiplex Methylation-Specific PCR
  • the present invention provides a method for detecting the presence of one, two, or more methylated gene regions in a biological sample from a suspicious lymph node of a subject suspected of having breast cancer comprising: a) hybridizing nucleic acid obtained from the lymph node sample with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; and c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node samples of a) are hypermethylated compared to the level of methylation of a normal/benign lymph node sample or a sample from an enlarged lymph node caused by other pathological conditions.
  • the present invention provides a method for triaging a subject suspected of having breast cancer and having one or more suspicious lymph nodes into sentinel lymph node biopsy and pathological review comprising: a) hybridizing nucleic acid obtained in a biological sample from a suspicious lymph node of the subject with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node sample in a) are hypermethylated compared to the level of methylation of normal of a normal/benign lymph node sample or a sample from an enlarged lymph node caused by other pathological conditions; and d
  • Figure 1 Overview of the study workflow and sample selection for discovery and testing of breast cancer markers.
  • FFPE formalin fixed paraffin embedded
  • IDC invasive ductal carcinoma
  • DCIS ductal carcinoma in situ
  • QM-MSP quantitative multiplex methylation-specific PCR
  • AUC area under the ROC curve
  • FNA fine needle aspirate.
  • FIG. 1 Performance of the 10-gene marker panel to distinguish between benign and cancer tissue of the breast.
  • FFPE samples of IDC/DCIS and Benign/Normal Tissues were assayed by QM-MSP.
  • Histogram plots indicate the percent methylation (colored segment) and cumulative methylation (bar height, Y-axis) for each of the 10 markers in the panel.
  • Insets of Box plots show the median cumulative methylation of cancers (IDC/DCIS) versus benign/normal tissues.
  • ROC analyses indicate the discriminatory power of the 10-gene marker panel. Test cohort.
  • CMI cumulative methylation index
  • IDC invasive ductal carcinoma
  • ILC Invasive lobular carcinoma
  • DCIS ductal carcinoma in situ
  • FA fibroadenoma
  • Pap papilloma
  • UDH usual ductal hyperplasia
  • ROC receiver operator characteristic
  • AUC area under the ROC curve. Mann-Whitney P-values are indicated.
  • FIG. 3 Overview of the study workflow and sample selection for testing presence of tumor cells in the lymph node.
  • FNA fine needle aspirations
  • lymph nodes of patients who presented with suspicious lesions in their breast.
  • 29 had benign breast disease.
  • Pre-NAC neoadjuvant chemotherapy
  • lymph nodes were malignant and 12 were benign as determined by cytology alone.
  • FIG. 4 Is illustrative of the cancer detection method.
  • 40 contained cancer cells (cancer) and 28 did not (benign) by cytological analysis.
  • the different colors in the histogram represent the different methylated genes.
  • the percent methylation is stacked up to show cumulative methylation of the ten genes present in that sample.
  • the cancer-containing lymph nodes had high cumulative methylation (CM) levels, while benign samples had very little or undetectable methylation levels.
  • CM cumulative methylation
  • sensitivity was 92%and specificity was 93%. Detection of the methylated genes in 68 lymph node FNA samples using the 10-gene panel used for breast.
  • Box-Whiskers plots depict the percent methylation (Y-axis) for each of the 10-gene markers in FNA samples obtained from patients with Cancer versus Benign lymph nodes. Percent methylation in the benign/normal samples is shown. Mann-Whitney P-values are indicated. AUC was calculated using ROC to calculate the sensitivity and specificity at different threshold. Sample number (N) is shown below the X-axis.
  • FIG. 5 Performance of the 10 individual gene markers in the intraoperative sentinel lymph node FNA sample set.
  • a sensitivity of 86%and specificity of 100% was observed compared to histopathology (gold standard) .
  • Box-Whiskers plots depict the percent methylation (Y-axis) for each of the 10-gene markers in FNA samples obtained from patients with Cancer versus Benign lymph nodes. Percent methylation in the benign/normal samples is shown. Mann-Whitney P-values are indicated. Sample number (N) is shown below the X-axis. The assay performed with a sensitivity of 86%and specificity of 100%when compared to cytology.
  • the genomes of higher eukaryotes contain the modified nucleoside 5-methyl cytosine (5-meC) .
  • This modification is usually found as part of the dinucleotide CpG in which cytosine is converted to 5-methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme (31) .
  • This enzymatic conversion is the primary epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (32 -34) .
  • DNA methylation regulates normal cellular processes such as genomic imprinting, chromosomal instability, and X-chromosome inactivation.
  • DNA methylation occurs at the fifth carbon position of cytosine at dinucleotide 5′-CpG-3′ sites in or near gene promoters termed CpG islands or shores.
  • Methylation controls gene expression by down-regulating transcription either by directly inhibiting transcriptional machinery or indirectly through the recruitment of chromatin remodeling proteins.
  • Chromosomal methylation patterns change dynamically during embryonic development, and the correct methylation patterns have to be maintained throughout an individual's lifetime. Changes in methylation patterns are linked to aging, and errors in DNA methylation are among the earliest changes that occur during oncogenesis. Thus, the detection of methylation at gene promoters is important for diagnosing and/or monitoring patients with cancer.
  • Epigenetic alterations including DNA methylation, interrupt the DNA-RNA-protein axis which describes how genetic information is transcribed into messenger RNAs (mRNAs) .
  • mRNAs messenger RNAs
  • the correlation between genomic DNA variation, mRNA copy numbers and protein levels may be described by DNA methylation levels.
  • co-measurement of DNA methylation levels and corresponding down-stream mRNA levels can be important to understanding the mechanism of epigenetic cellular regulation.
  • the present invention is based on a methylated gene marker panel having between 2 and 20 gene regions that easily distinguishes between benign and cancer tissues obtained from the U.S., China and South Africa with high levels of sensitivity and specificity.
  • the gene panel is a 10-gene panel.
  • the gene panel is a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 gene panel, as well as permutations thereof.
  • the inventors Towards the goal of developing markers that are specific to malignant breast cancer, the inventors performed large-scale methylation arrays of breast cancer and normal tissue using Illumina arrays containing probes for 27K and 450K methylated CpGs in the genome. They also developed a sensitive and specific Quantitative Multiplex Methylation-Specific PCR (QM-MSP) technique to validate the newly discovered methylated gene markers [18, 19, 24, 25] . This method involves sodium bisulfite treatment of DNA, followed by a two-step reaction.
  • QM-MSP Quantitative Multiplex Methylation-Specific PCR
  • the present inventors have demonstrated the utility of assessing methylated markers in a biological or clinical sample from a suspected lesion, including samples from ductal lavage/ductoscopy fluids and cells, nipple fluids, and fine needle aspirates as well as tissues and biopsies, including core biopsies, using a panel of methylated gene markers for breast cancer screening in women with suspicious lesions, including those detected by palpation, mammography or ultrasound imaging.
  • the lymph node assay of the present invention is elegant, as it is simple in its execution, and thus could be mobilized in underserved regions of the world for the discrimination of suspicious breast mammograms or lesions, from actual breast cancer or benign lesions, by testing biological samples from a suspicious or enlarged lymph node of the subject.
  • the multi-gene panel lymph node assay of the present invention is broadly applicable to subjects, including females from diverse ethnicities and world regions, as no significant differences in cumulative methylation of the 10-gene marker and greater panel between samples from the United States, China or South Africa were found.
  • the inventive assay data and outcome attests to the strength of using a multi-gene panel of markers rather than a single gene marker.
  • the present invention provides a method for detecting the presence of one, two, or more methylated gene regions in a biological sample from a suspicious lymph node of a subject suspected of having breast cancer comprising: a) hybridizing nucleic acid obtained from the lymph node sample with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; and c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node sample of a) are hypermethylated compared to the level of methylation of a normal/benign lymph node sample or a sample from an enlarged lymph node caused by other pathological conditions.
  • the above method at step a) can comprise hybridizing nucleic acid obtained from the sample with 3, 4, 5, 6, 7, 8, 9, and 10 sets of QM-MSP primers and probes specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671 and permutations thereof.
  • the present invention provides a method for triaging a subject suspected of having breast cancer and having one or more suspicious lymph nodes into sentinel lymph node biopsy and pathological review comprising: a) hybridizing nucleic acid obtained in a biological sample from a suspicious lymph node of the subject with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node sample of a) are hypermethylated compared to the level of methylation of normal of a normal/benign lymph node sample or a sample from an enlarged lymph node caused by other pathological conditions; and d
  • the above method at step a) can comprise hybridizing nucleic acid obtained from the sample with 3, 4, 5, 6, 7, 8, 9, and 10 sets of QM-MSP primers and probes specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671 and permutations thereof.
  • the above methods for detection of breast cancer can detect increased methylation in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 different specific CpG regions of genes selected from the group consisting of AKR1B1, APC, ARHGEF7, CCND2, CDKL2, COL6A2, EVI1, GAS7C, GPX7, HIN1, HIST1H3C, HOXB4, MAL, RARB, RASGRF2, RASSF1, TM6SF1, TMEFF2, TWIST1, and ZNF671.
  • the present invention provides a method for monitoring disease or progression of disease in a subject having or suspected of having breast cancer comprising: a) hybridizing nucleic acid obtained in a biological sample from a suspicious lymph node of the subject with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node sample of a) are hypermethylated compared to the level of methylation of a previous lymph node sample from the subject and, d) triaging the subject into further or different treatment when any of the specific CpG regions of the one, two, or more genes of the lymph node sample
  • the present invention provides a method for detecting disease recurrence in a subject undergoing treatment or having been treated for breast cancer comprising: a) hybridizing nucleic acid obtained in a biological sample from a suspicious lymph node of the subject with one, two, or more QM-MSP primer and probe sets specific for the genes selected from the group consisting of AKR1B1, APC, CCND2, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1, TMEFF2, and ZNF671; b) performing QM-MSP on the lymph node sample from a) ; c) detecting if any of the specific CpG regions of the one, two, or more genes of the lymph node sample of a) are hypermethylated compared to the level of methylation of a previous lymph node sample from the subject; and, d) triaging the subject into a change in treatment when any of the specific CpG regions of one, two, or more genes
  • the samples of DNA of the inventive methods can be obtained from any lymph node in the body of the subject.
  • the lymph nodes sampled using the inventive methods can be from the pectoral lymph nodes, subclavian lymph nodes, axillary lymph nodes, supraclavicular lymph nodes, cervical lymph nodes, mediastinal lymph nodes, inguinal lymph nodes, retroperitoneal lymph nodes, pelvic lymph nodes, and mesentery lymph nodes, for example.
  • the samples can be taken from two or more different nodes from the same or different type of node.
  • the samples used in the present invention are axillary lymph nodes and other lymph nodes in the same draining system.
  • the samples of DNA of the inventive methods obtained from any lymph node can be used for staging of the lymph nodes preoperatively in women presenting with enlarged lymph nodes.
  • the staging will allow patients to be triaged to axillary lymph node dissection (ALND) , or other various lymph node dissection, with a positive result, or to sentinel node biopsy with a negative result.
  • ALND axillary lymph node dissection
  • the samples can be taken from the nodes and then formalin fixed for a period of time before applying the inventive methods.
  • the term "subject” refers to a human subject. Typically, the subject will be a human female, however the methods, are not limited to samples from females.
  • biological sample or “biological fluid” includes, but is not limited to, any quantity of a substance from a living or formerly living patient or mammal. Such substances include, but are not limited to, blood, serum, plasma, urine, cells, organs, tissues, bone, bone marrow, synovial tissue, chondrocytes, synovial macrophages, endothelial cells, and skin.
  • the sample can be a FFPE sample.
  • the sample is a fine needle aspirate of a sample of lymph nodes from a subject suspected to have breast cancer or tumor.
  • samples can be collected from such tissue sources like lymph nodes where the DNA amount is limiting, for example as little as about 50 to about 100 cells, as well as in larger samples, such as formalin-fixed paraffin-embedded sections of core biopsies.
  • QM-MSP primer and probe sets means the PCR primers and probes used to identify the methylated CpG regions methylated in the nucleic acid of the sample and disclosed in Table 1.
  • the detection of methylation of the CpG regions of the genes and the level of methylation detected in the samples from suspicious or enlarged lymph nodes of a subject suspected of having breast cancer is compared to the methylation levels of the CpG regions of the genes in normal or benign lymph nodes or enlarged lymph nodes due to other pathological conditions.
  • the level of methylation is elevated in the suspicious or enlarged lymph node sample compared to normal or benign lymph nodes or enlarged lymph nodes due to other pathological conditions, the breast lesion is diagnosed as having a high risk of cancer or malignancy.
  • the particular gene panel of the inventive methods were specifically chosen to identify those genes which were very highly methylated when malignant or cancerous, and had little or no methylation when in normal or benign lymph nodes. Moreover, when the level of methylation of the genes in the lymph node sample from a subject having mammographically or otherwise suspicious lesion are not significantly different from normal or benign lymph nodes or enlarged lymph nodes due to other pathological conditions, there is a low probability or risk of cancer in the lesion sampled.
  • the inventive methods herein employ two-step quantitative multiplex-methylation specific PCR (QM-MSP) .
  • QM-MSP quantitative multiplex-methylation specific PCR
  • the present inventors created this method and the methods are disclosed in U.S. Patent Nos. 8,062,849 and 9,416,404 which are hereby incorporated herein as if set forth in their entireties.
  • the QM-MSP technique combines the sensitivity of multiplex PCR with the quantitative features of quantitative methylation-specific PCR (Q-MSP) in such a way that a panel of genes whose hypermethylation is associated with a type of carcinoma can be co-amplified from limiting amounts of DNA derived from tissue or samples sources of the subject being tested.
  • the invention methods also provide quantitative definition of the extent of gene hypermethylation in normal appearing tissues on a gene-by- gene basis.
  • the inventive methods can be used to more powerfully discriminate between normal or benign tissues and malignant tissues and to monitor or assess the course of cancer development in a subject.
  • nucleic acid includes “polynucleotide, " “oligonucleotide, “ and “nucleic acid molecule, “ and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acids of the invention are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • nucleic acids used as primers in embodiments of the present invention can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al. (eds. ) , Molecular Cloning, A Laboratory Manual, 3 rd Edition, Cold Spring Harbor Laboratory Press, New York (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &Sons, NY (1994) .
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides) .
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl
  • isolated and purified means a protein that is essentially free of association with other proteins or polypeptides, e.g., as a naturally occurring protein that has been separated from cellular and other contaminants by the use of antibodies or other methods or as a purification product of a recombinant host cell culture.
  • biologically active means an enzyme or protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • the methods of the present invention can be used to diagnose, prognosticate, and monitor treatment of any disease or biological state in which methylation of genes is correlative of such a disease or biological state in a subject.
  • the disease state is breast cancer.
  • the type of breast cancer can be invasive ductal carcinoma or ductal carcinoma in situ.
  • the types of cancer diagnosis which may be made, using the methods provided herein, is not necessarily limited.
  • the cancer can be any cancer.
  • the term "cancer” is meant any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream.
  • the cancer is breast cancer.
  • the lymph node samples are taken from the subject to monitor disease.
  • the lymph node samples are taken from the subject to detect recurrence.
  • the lymph node samples are taken from the subject to monitor effectiveness of a treatment regimen.
  • the cancer can be a metastatic cancer or a non-metastatic (e.g., localized) cancer, an invasive cancer or an in situ cancer.
  • a metastatic cancer refers to a cancer in which cells of the cancer have metastasized, e.g., the cancer is characterized by metastasis of a cancer cells.
  • the metastasis can be regional metastasis or distant metastasis, as described herein.
  • inventive methods can provide any amount of any level of diagnosis, staging, screening, or other patient management, including treatment or prevention of cancer in a mammal.
  • treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the term “triaging the subject into lymph node biopsy of the suspicious lesion and pathological review” means that when the sample DNA has one, two or more markers methylated, the subject is then selected for FNA or biopsy of the suspect lymph node tissue which undergoes pathological review. In some embodiments, this includes sentinel lymph node biopsy, and surgical removal.
  • Stage 0 cancer cells are confined inside the breast ducts, to Stage 4 when cancer have disseminated to other organs of the body.
  • Stage 0 is also called ductal carcinoma in situ (DCIS) .
  • DCIS ductal carcinoma in situ
  • the cells divide but stay confined inside the duct where they could remain indolent or progress to a pathologically high grade which has a tendency to break through the ductal barriers and invade into the breast tissue a short time.
  • Stage I describes a very early stage of invasive cancer. At this point, tumor cells have spread to normal surrounding breast tissue, but are still contained in a small area. In Stage IA, a tumor measures up to 20 millimeters, and there is no evidence of cancer in the axillary lymph nodes.
  • Stage IB is a slightly more advanced step.
  • the tumor in the breast is less than 20 millimeters, but small clusters of cancer cells are observed in the lymph nodes; or no tumor is detectable in the breast but small clusters of cancer cells exist in the lymph nodes.
  • Stage II describes cancer that is larger and may or may not have spread to the lymph nodes.
  • stage III breast cancer the cancer has spread further into the breast or the tumor is a larger size than earlier stages and could be detectable in 5-9 lymph nodes.
  • stage IIIB the tumor has spread to the chest wall behind the breast. Cancer may also have spread to the skin, causing swelling or inflammation. It may have spread to as many as nine underarm (axillary) lymph nodes or to nodes near the breastbone.
  • stage IIIC there may be a tumor of any size in the breast, or no tumor present at all. Nevertheless, the cancer has spread to one of the following places-ten or more underarm (axillary) lymph nodes, lymph nodes near the collarbone, some underarm lymph nodes and lymph nodes near the breastbone, and the skin.
  • Stage IV is the most advanced stage of breast cancer. Breast cancer has spread to nearby lymph nodes and to distant parts of the body beyond the breast including the lungs, liver, brain or bones. Breast cancer may be stage IV when it is first diagnosed, or it can be a recurrence of a previous breast cancer that has spread despite initial successful treatment.
  • Breast cancer is defined histopathologically by its grade and histologic variations (invasive ductal carcinoma, lobular carcinoma, squamous etc. ) , and also by the gene markers it displays.
  • the status of the estrogen receptor (ER) , progesterone receptor (PR) , and human epidermal growth factor receptor 2 (HER2) are routinely performed in breast cancer specimens. The status of these receptors carry important implications. Treatment regimens are based largely on the status of these receptors.
  • breast cancer-specific survival is predictable on the ER/PR/HER2 subtypes and tumor grade. Using ER/PR+ and Her2-as a reference, ER/PR+, HER2-had the best survival and is susceptible to antagonists of ER-function.
  • ER/PR+ HER2+ breast cancer has seen considerable improvement with the development of antibody based therapy (Herceptin) and newer (TDM-1) therapies.
  • Herceptin antibody based therapy
  • TDM-1 newer
  • chemotherapy conferred significant overall and disease-free survival advantages.
  • ER/PR-, HER2- (triple negative) had the worst overall survival and the worst disease-free survival.
  • Subtype comparison revealed statistically significant differences in outcomes. The triple negative subtype has the worst overall and disease free survival.
  • Appropriate care in terms of breast cancer can constitute standard of care for treatment of breast cancer including, for example, surgery, surgery with post-operative radiation therapy, post-operative systemic therapy or chemotherapy depending on whether he tumor is hormone receptor negative or positive, the tumor is HER2/neu negative or positive, the tumor is hormone receptor negative and HER2/neu negative (triple negative) , and the size of the tumor.
  • the inventive methods can be used throughout the course of the disease progression and/or treatment.
  • neoadjuvant treatment if the subject is being treated with chemotherapy and/or radiation and surgery, a physician can examine the lymph nodes to see if they are or remain enlarged and then perform the test to see if there are still cancerous cells in the node or whether the enlargement is due to another pathological reason, such as inflammation due to infections.
  • a physician can also use the inventive methods for determining whether the cancer has become metastatic by testing enlarged lymph nodes at other sites in the subject.
  • nodes can be assayed using the inventive methods for determination of the effectiveness of the treatment and whether recurrence has begun.
  • no more treatment may be needed or postoperative therapy may include: tamoxifen therapy with or without chemotherapy; tamoxifen therapy and treatment to stop or lessen how much estrogen is made by the ovaries; drug therapy, surgery to remove the ovaries, or radiation therapy to the ovaries may be used; aromatase inhibitor therapy and treatment to stop or lessen how much estrogen is made by the ovaries; and drug therapy, surgery to remove the ovaries, or radiation therapy to the ovaries may be used.
  • postmenopausal women with hormone receptor positive tumors no more treatment may be needed or postoperative therapy may include: aromatase inhibitor therapy with or without chemotherapy; tamoxifen followed by aromatase inhibitor therapy, with or without chemotherapy.
  • postoperative therapy may include: chemotherapy.
  • postoperative therapy may include: chemotherapy and targeted therapy (trastuzumab) ; hormone therapy, such as tamoxifen or aromatase inhibitor therapy, for tumors that are also hormone receptor positive.
  • Drugs useful in the treatment of breast cancer include, but are not limited to: Abemaciclib; Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation) ; Ado-Trastuzumab Emtansine; Afinitor (Everolimus) ; Anastrozole; Aredia (Pamidronate Disodium) ; Arimidex (Anastrozole) ; Aromasin (Exemestane) ; Capecitabine; Cyclophosphamide; Docetaxel; Doxorubicin Hydrochloride; Ellence (Epirubicin Hydrochloride) ; Epirubicin Hydrochloride; Eribulin Mesylate; Everolimus; Exemestane; 5-FU (Fluorouracil Injection) ; Fareston (Toremifene) ; Faslodex (Fulvestrant) ; Femara (Letrozole) ; Fluorouracil Injection; Fulvestrant; Gemcitabine Hydro
  • “Complement” or “complementary” as used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • oligonucleotide useful as a probe or primer that selectively hybridizes to a selected nucleotide sequence is at least about 15 nucleotides in length, usually at least about 18 nucleotides, and particularly about 20 nucleotides in length or more in length.
  • Conditions that allow for selective hybridization can be determined empirically, or can be estimated based, for example, on the relative GC: AT content of the hybridizing oligonucleotide and the sequence to which it is to hybridize, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and sequence to which it is to hybridize (see, for example, Sambrook et al., "Molecular Cloning: A laboratory manual” (Cold Spring Harbor Laboratory Press 1989) ) .
  • “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • thymine (T) and uracil (U) may be considered equivalent.
  • Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
  • Probe as used herein may mean an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • a probe may be single stranded or partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
  • optically detectable DNA probe means an oligonucleotide probe that can act as a molecular beacon or an oligonucleotide probe comprising a fluorescent moiety or other detectable label, with or without a quencher moiety.
  • “Substantially complementary” used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99%identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
  • “Substantially identical” used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99%identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
  • an amplification “primer pair” as the term is used herein requires what are commonly referred to as a forward primer and a reverse primer, which are selected using methods that are well known and routine and as described herein such that an amplification product can be generated therefrom.
  • condition that allow generation of an amplification product or of “conditions that allow generation of a linear amplification product” means that a sample in which the amplification reaction is being performed contains the necessary components for the amplification reaction to occur.
  • conditions include, for example, appropriate buffer capacity and pH, salt concentration, metal ion concentration if necessary for the particular polymerase, appropriate temperatures that allow for selective hybridization of the primer or primer pair to the template nucleic acid molecule, as well as appropriate cycling of temperatures that permit polymerase activity and melting of a primer or primer extension or amplification product from the template or, where relevant, from forming a secondary structure such as a stem-loop structure.
  • selective hybridization refers to hybridization under moderately stringent or highly stringent conditions such that a nucleotide sequence associates with a selected nucleotide sequence but not with unrelated nucleotide sequences.
  • an oligonucleotide useful as a probe or primer that selectively hybridizes to a selected nucleotide sequence is at least about 15 nucleotides in length, usually at least about 18 nucleotides, and particularly about 21 nucleotides in length or more in length.
  • Conditions that allow for selective hybridization can be determined empirically, or can be estimated based, for example, on the relative GC: AT content of the hybridizing oligonucleotide and the sequence to which it is to hybridize, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and sequence to which it is to hybridize (see, for example, Sambrook et al., “Molecular Cloning: A laboratory manual” (Cold Spring Harbor Laboratory Press 1989) ) .
  • methylation-specific PCR can be used to evaluate methylation status of the target DNA.
  • MSP utilized primer and/or probe sets designed to be “methylated-specific” by including sequences complementing only unconverted 5-methylcytosines, or, on the converse, “unmethylated-specific” , complementing thymine’s converted from unmethylated cytosines. Methylation is then determined by the ability of the specific primer to achieve amplification. This method is particularly effective for interrogating CpG islands in regions of high methylation density, because increased numbers of unconverted methylcytosines within the target to be amplified increase the specificity of the PCR. In certain embodiments placing the CpG pair at the 3′-end of the primer also improves the specificity.
  • QM-MSP is a highly sensitive, specific and quantitative methylation assay. It combines the principles of conventional gel-based MSP, quantitative real time MSP, and multiplexed gel-based MSP into one format developed to enable quantification of methylated gene panels in clinical samples with limited DNA quantities available. It has an analytical sensitivity of 1 methylated copies in 100,000 unmethylated copies, which is nearly 10 fold higher than QMSP, and 100 fold higher than gel based MSP techniques.
  • QM-MSP is a two-step PCR approach for a multiplexed analysis of a panel of up to 14 genes in clinical samples with minimal quantities of DNA.
  • the External PCR reaction for up to 14 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This is a methylation-independent amplification step, to increase the number of DNA segments.
  • methylated (M) and unmethylated (U) primers are used which selectively amplify methylated and un-methylated DNA, and the amplicons are subsequently quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well. Methylation is reported on a continuous scale.
  • the assay is easily performed on fresh or fixed cytological samples including fine needle aspirates of lymph nodes as well as on cells or cell free nucleic acids from other tissues and biopsies.
  • quantitative real-time PCR methodology is adapted to perform quantitative methylation-specific PCR (QM-MSP) by utilizing a set of external primers pairs in round one (multiplex) PCR and internal primer pairs in round two (real time MSP) PCR.
  • QM-MSP quantitative methylation-specific PCR
  • each set of genes has one pair of external primers and two sets of three internal primers/probe (internal sets are specific for unmethylated or methylated DNA) .
  • the external primer pairs can co-amplify a cocktail of genes, each pair selectively hybridizing to a member of the panel of genes being investigated using the invention method.
  • Primer pairs are designed to exclude CG dinucleotides, or to employ a degenerate base which can recognize either C, T or U, and thereby render DNA amplification independent of the methylation status of the DNA sequence. Therefore, methylated and unmethylated DNA sequences internal to the binding sites of the external primers are co-amplified for any given gene by a single set of external primers specific for that gene.
  • the external primer pair for a gene being investigated is complementary to the sequences flanking the CpG regions that is to be queried in the second round of QM-MSP.
  • the sequences of external primers set forth in Table 1 above are used for multiplex PCR (first round PCR) of genes associated with primary breast cancer (Fackler M.J. et al, Int. J Cancer (2003) 107: 970-975; Fackler M.J. et al. Cancer Res (2004) 64: 4442-4452) .
  • Internal PCR primers used for quantitative real-time PCR of methylated and unmethylated DNA sequences are designed to selectively hybridize to the first amplicon produced by the first round of PCR for one or more members of the panel of DNA sequences being investigated using the invention method and to detect the methylation status, i.e., whether methylated (M) or unmethylated (U) , of the CpG regions in the first amplicons to which they bind.
  • methylation status i.e., whether methylated (M) or unmethylated (U)
  • a single gene or a cocktail of two or more genes can be co-amplified using distinguishable fluorescence labeled probes.
  • the probes used in the round two QM-MSP of the invention method are designed to selectively hybridize to a segment of the first amplicon lying between the binding sites of the respective methylation-status specific internal PCR primer pair.
  • Polynucleotide probes suitable for use in real-time PCR include, but are not limited to, a bi-labeled oligonucleotide probe, such as a molecular beacon or a TaqMan TM probe, which include a fluorescent moiety and a quencher moiety.
  • a molecular beacon the fluorescence is generated due to hybridization of the probe, which displaces the quencher moiety from proximity of the fluorescent moiety due to disruption of a stem-loop structure of the bi-labeled oligonucleotide.
  • Molecular beacons such as Amplifluor TM or TriStar TM reagents and methods are commercially available (Stratagene; Intergen) .
  • the fluorescence is progressively generated due to progressive degradation of the probe by Taq DNA polymerase during rounds of amplification, which displaces the quencher moiety from the fluorescent moiety.
  • the probe is degraded by the 5′-3′ exonuclease activity of the Taq DNA polymerase, and the fluorescence can be detected, for example by means of a laser integrated in the sequence detector.
  • the fluorescence intensity therefore, is proportional to the amount of amplification product produced.
  • fluorescence from the probe is detected and measured during a linear amplification reaction and, therefore, can be used to detect generation of the linear amplification product.
  • Amplicons in the 80-150 base pair range are generally favored because they promote high-efficiency assays that work the first time.
  • high efficiency assays enable quantitation to be performed using the absolute quantitation method. Quantitation of the copy number of unmethylated (U) and methylated (M) DNA amplicons for a specific gene eliminates the need to use actin as an estimate of DNA input, since U+M is taken to approximate the total number of copies of DNA amplicons for any given gene in the first amplicon product (derived from round one, multiplex PCR of QM-MSP) .
  • the quencher can quench the reporter fluorescence when the two dyes are close to each other, which happens in an intact probe.
  • Other reporter dyes include but are not limited to VIC TM and TET TM and these can be used in conjunction with 6-FAM to co-amplify genes by quantitative real time PCR.
  • unmethylated using a 6-FAM/TAMRA probe
  • unmethylated RARE using a VIC/TAMRA probe
  • the round two QM-MSP reactions are designed to be run as single gene reactions or in multiplex using automated equipment, as are other types of real time PCR.
  • Thermal cycling parameters useful for performing real time PCR are well known in the art and are illustrated in the Examples herein.
  • quantitative assays can be run using the same universal thermal cycling parameters for each assay. This eliminates any optimization of the thermal cycling parameters and means that multiple assays can be run on the same plate without sacrificing performance. This benefit allows combining two or more assays into a multiplex assay system, in which the option to run the assays under different thermal cycling parameters is not available.
  • a panel can be designed to query pathway-specific genes for their use as intermediate markers in specific trials of chemopreventive agents (Fackler M.J. et al. J Mammary Gland Biol Neoplasia (2003) 8: 75-89) .
  • the real-time PCR amplification in an invention two-step assay is, in fact, a group of real-time PCR reactions, which may be conducted together or using two separate aliquots of the first amplification product, for each of the first amplicons (i.e., for each DNA sequence in the panel that were selected for the assay) .
  • determination of the methylation status of a DNA sequence employs both a methylation-specific and an unmethylated DNA-specific internal primer pair for each amplicon contained in the first amplification product, one to determine if the gene is unmethylated and one to determine if the gene is methylated.
  • the real time PCR reactions in the second amplification step of the methods can conveniently be conducted sequentially or simultaneously in multiplex. Separate, usually dilute, aliquots of the first amplification reaction may be used for each of the two methylation status determining reactions.
  • the reactions can conveniently be performed in the wells of a 96 or 384 microtiter plate.
  • the methylated and unmethylated status determining second reactions for a target gene may be conducted in adjacent wells of a microtiter plate for high throughput screening.
  • several genes, for example 2 to 5 genes may be simultaneously amplified in a single real time PCR reaction if the probes used for each first amplicon are distinguishably labeled.
  • one, two to ten, or more distinguishable fluorescence signals from the second amplification product (s) may be accumulated to determine the cumulative incidence or level of methylation of the DNA sequences, especially of CpG regions therein, in the several genes included in the assay.
  • These cumulative results are compared with the cumulative results similarly obtained by conducting the two step QM-MSP assay on comparable DNA sequences (e.g., promoter DNA sequences) obtained from comparable normal tissue of the same type or types as used in the assay.
  • any of the known methods for conducting cumulative or quantitative “real time PCR” may be used in the second amplification step so long as the first amplicons in the first amplification product are contacted with one or more members of a set of polynucleotide probes that are labeled with distinguishable optically detectable labels, one or more members of the set being designed to selectively hybridize to one or more of the DNA sequences being tested, while the set cumulatively binds to the various DNA segments being tested contained in the first amplicons of the first amplification product.
  • first amplicons may also be contacted with such a set of probes and one or more members of a set of DNA sequence-specific methylation status-dependent inner primer pairs, wherein the set of inner primer pairs collectively bind to the various first amplicons in the first amplification product.
  • additional genes can be co-amplified provided that each gene primer set incorporates a different color fluorescent probe.
  • sample values are extrapolated from the standard curve for target and reference DNAs. This is called absolute quantitation.
  • a comparable normal DNA sample means that the plurality of genomic DNA sequences that is being tested for methylation status, such as in a mammal, is matched with a panel of genomic DNA sequences of the same genes from a “normal” organism of the same species, family, and the like, for comparison purposes.
  • the normal sample would be, for example lymph node tissue from normal lymph nodes or nodes which are enlarged, but for other pathological reasons, such as infection with bacteria or parasites.
  • a substantial cumulative increase or decrease in the methylation level in the test sample as compared with the normal/benign sample is a reliable indicator of the presence of the condition being assayed.
  • methylation is evaluated using a MethyLight method.
  • the MethyLight method is based on MSP, but provides a quantitative analysis using quantitative PCR (see, e.g., Eades et al. (2000) Nucleic Acids Res., 28 (8) : E32. doi: 10.1093/nar/28.8. e32) .
  • Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region.
  • the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences.
  • Quantitation can be made in reference to a methylated reference DNA.
  • One modification to this protocol to increase the specificity of the PCR for successfully bisulphite-converted DNA (ConLight-MSP) uses an additional probe to bisulphite-unconverted DNA to quantify this non-specific amplification (see, e.g., Rand et al. (2002) Methods 27 (2) : 114-120) .
  • the MethyLight methods utilize technology, which is based on the cleavage of a dual-labeled fluorogenic hybridization probe by the 5′ nuclease activity of Taq-polymerase during PCR amplification (Eads et al. (1999) Cancer Res., 59: 2302-2306; Livak et al. (1995) PCR Meth. Appl., 4: 357-362; Lee et al. (1993) Nucleic Acids Res., 21: 3761-3766; Fink et al. (1998) Nat. Med., 4: 1329-1333) .
  • the use of three different oligonucleotides in the technology offers the opportunity for several sequence detection strategies.
  • the methods described herein can involve nested PCR reactions and the reagents (e.g., primers and probes) for such nested PCR reactions.
  • methylation is detected for one, two, three, four, five, or six genes (gene promoters) . Since bisulfite conversion of a DNA changes cytosine resides to uracil, but leave 5-methyl cytosine residues unaffected, the forward and reverse strands of converted (bisulfite-converted) DNA are no longer complementary. Accordingly, it is possible to interrogate the forward and reverse strands independently (e.g., in a multiplex PCR reaction) to provide additional specificity and sensitivity to methylation detection.
  • assaying of a single target can involve a two-plex multiplex assay, while assaying of two, three, four, five, or six target genes can involve four-plex, six-plex, 8-plex, 10-plex, or 12-plex multiplex assays.
  • the assays can be divided into two multiplex reactions, e.g., to independently assay forward and reverse strands.
  • the grouping of assays need not be by forward or reverse, but can simply include primer/probe sets that are most compatible for particular PCR reaction conditions.
  • breast and other cancers can be identified, and/or staged and/or a prognosis therefor determined by the detection/characterization of the methylation state on the forward and/or reverse strand of gene promoters whose methylation (or lack thereof) is associated with a cancer.
  • methylation (forward strand and/or reverse strand) of one or more of the genes shown herein for each cancer can be determined to identify, and/or stage, and/or provide a prognosis for the indicated cancer.
  • methylation status of all of the genes shown for a particular cancer can be determined in a single multiplex PCR reaction.
  • the inventive methods can be used to assess the methylation status of multiple genes, using very small quantities of DNA.
  • a cumulative score of hypermethylation among multiple genes better distinguishes normal or benign from malignant tumors in lymph node samples as compared to the value of individual gene methylation markers.
  • an amplification “primer pair” as the term is used herein requires what are commonly referred to as a forward primer and a reverse primer, which are selected using methods that are well known and routine and as described herein such that an amplification product can be generated therefrom.
  • Breast cancer samples included invasive ductal carcinoma (IDC) , invasive lobular carcinoma (ILC) , and ductal carcinoma in situ (DCIS) .
  • IDC invasive ductal carcinoma
  • ILC invasive lobular carcinoma
  • DCIS ductal carcinoma in situ
  • Two non-cancer groups were studied: normal breast and benign breast disease.
  • the cancer FFPE blocks ranged in age from 2-28 years (median 4 years)
  • the benign/normal blocks ranged in age from 2-19 years (median 2 years) .
  • a large majority of our FFPE samples were 2-10 years old [cancer (73%) , benign (89%) ] .
  • QM-MSP was used in the training and test sets for marker selection and evaluation. Twenty-five individual markers were assayed using DNA from FFPE tissues in the training cohort. Marker selection criteria required first, that markers show considerably higher median methylation in cancer than in benign/normal tissue (P ⁇ 0.002, based on the Mann-Whitney test) . Secondly, to further minimize the risk of false positives, benign and normal samples were required to be methylated at lower levels and less frequently compared to cancer samples. Specifically, we calculated the 75th percentile of methylation separately in tumor and benign samples, discarding any markers in which the 75th percentile of normal methylation was high, or where the difference between normal and tumor was small (Table 2) . The selected markers were then evaluated as a panel in the training set.
  • CMI Cumulative Methylation Index
  • Percent methylation (minimum, 25th and 75th percentile, and maximum) is shown for each of 25 genes analyzed by QM-MSP in the training cohort of FFPE breast cancer and benign/normal samples. Sixteen markers showed considerably higher median methylation in cancer than in benign/normal (P ⁇ 0.01, based on the Mann-Whitney test) . To further minimize the risk of false positives, each remaining marker was examined to determine that levels of methylation in benign and normal samples were low. Specifically, the 75th percentile of methylation was calculated separately in tumor and benign samples, discarding any markers in which the 75th percentile of normal methylation was high, or where the difference between normal and tumor was small. The ten genes that were finally selected are denoted in red. Ca. cancer; B, benign; ns, not significant.
  • LN positive lymph node detection
  • Women may be excluded for any condition that in the opinion of the investigator may make it safe to take part (e.g. a previous or concomitant malignancy, comorbidity where stopping a concomitant medication is not in the best interest of the patient) .
  • the FNA is performed on one ultrasound visible breast lesion and one or more palpable lymph nodes per patient, using the method described by Fabian et. al. [29, 30] which is standard all over the world and used by the breast imagers.
  • the cells are smeared onto 4-10 uncharged slides.
  • the syringe is then washed in 10 ml of Cytolyt TM fixative solution.
  • the standard post biopsy instructions, both verbal and written are provided to all patients.
  • the cells are smeared onto 4-10 uncharged slides.
  • a single slide from each patient (in batches of 10) , is stained with May-Grunwald-Giemsa and epithelial cells are counted.
  • the slides are provided to the cytopathologist to determine their diagnosis (benign or malignant) , the cytopathologist then creates a report, de-identifies the slide with a lab number and keeps the lab personnel blinded to the diagnosis.
  • All the subjects recruited have imaging detected suspicious breast lesion and at least one enlarged palpable lymph node.
  • ROC analysis is performed, reporting the area under the ROC curve along with bootstrap confidence bands.
  • Clinical considerations lead us to target a sensitivity of at least 0.90. Accordingly, in addition to the ROC analysis, we fixed sensitivity at 0.90, selecting the threshold on cumulative methylation to achieve that level, and reporting the conditional specificity along with the BTII confidence interval proposed by Zhou and Qin (35) .
  • Sample size requirements were calculated to control the width of the confidence interval for specificity, when sensitivity is fixed at 90%.
  • We resampled pilot data FNA biopsies from primary tumors or lymph nodes) to achieve a range of sample sizes, calculating the width of the BTII 90%confidence interval for each. Results are shown in the table below.
  • Candidate gene markers were among those previously identified by the inventors as having frequent measurable hypermethylation in histopathological subtypes of ER/PR/HER2-positive and triple negative breast cancer (24, 25) .
  • Nine of the 25 markers were discarded based on their inability to significantly distinguish between cancers (Invasive cancer/DCIS) and benign/normal breast tissues.
  • the parameters defined in the training set were locked for analysis in the independent test cohort of 222 cancerous, benign, and normal breast tissues using QM-MSP.
  • the marker panel was significantly more methylated in IDC/ILC/DCIS samples compared to benign/normal tissues (P ⁇ 0.0001; Figure 2) .
  • FIG. 4 Is illustrative of cancer detection.
  • 40 contained cancer cells (cancer) and 28 did not (benign) by cytological analysis.
  • the different colors in the histogram represent the different methylated genes.
  • the percent methylation is stacked up to show cumulative methylation of the ten genes present in that sample.
  • the cancer-containing lymph nodes had high cumulative methylation (CM) levels, while benign samples had very little or undetectable methylation levels.
  • CM cumulative methylation
  • sensitivity was 92%and specificity was 93%. Detection of the methylated genes in 68 lymph node FNA samples using the 10-gene panel used for breast.
  • Box-Whiskers plots depict the percent methylation (Y-axis) for each of the 10-gene markers in FNA samples obtained from patients with Cancer versus Benign lymph nodes. Percent methylation in the benign/normal samples is shown. Mann-Whitney P-values are indicated. AUC was calculated using ROC to calculate the sensitivity and specificity at different threshold. Sample number (N) is shown below the X-axis.
  • a sensitivity of 86%and specificity of 100% was observed compared to histopathology (gold standard) .
  • Box-Whiskers plots depict the percent methylation (Y-axis) for each of the 10-gene markers in FNA samples obtained from patients with Cancer versus Benign lymph nodes. Percent methylation in the benign/normal samples is shown. Mann-Whitney P-values are indicated. Sample number (N) is shown below the X-axis. The assay performed with a sensitivity of 86%and specificity of 100%when compared to cytology.
  • Unger-Saldana K Challenges to the early diagnosis and treatment of breast cancer in developing countries. World J Clin Oncol 2014, 5 (3) : 465-477.
  • Baylin SB DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2005, 2 Suppl 1: S4-11.

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Abstract

Le procédé comprend un système basé sur un marqueur de méthylation rapide et précis basé sur un marqueur de méthylation pour distinguer rapidement entre un tissu bénin/normal et un tissu du sein cancéreux dans un échantillon provenant d'un ganglion lymphatique suspect chez un sujet. Les procédés sont fournis pour aider au triage des sujets suspectés d'avoir un cancer pour les diriger vers une biopsie accélérée et un examen pathologique dans un contexte de ressources limitées.
PCT/CN2019/091994 2019-06-20 2019-06-20 Marqueurs de méthylation de l'adn et leur utilisation dans la différenciation d'échantillons de biopsie de ganglions lymphatiques cancéreux suspectés Ceased WO2020252721A1 (fr)

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WO2023059654A1 (fr) * 2021-10-05 2023-04-13 Personalis, Inc. Essais personnalisés pour la surveillance personnalisée d'un cancer
CN115961048A (zh) * 2023-01-04 2023-04-14 广州禾信康源医疗科技有限公司 一种基因甲基化检测引物组合、试剂及其应用
WO2024129928A3 (fr) * 2022-12-13 2024-08-08 The Johns Hopkins University Marqueurs de méthylation pour la détection et la surveillance du cancer du col de l'utérus
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US12371746B2 (en) 2013-01-17 2025-07-29 Personalis, Inc. Methods and systems for genetic analysis
US12516385B2 (en) 2014-10-30 2026-01-06 Personalis, Inc. Methods for using mosaicism in nucleic acids sampled distal to their origin
US12571039B2 (en) 2016-05-27 2026-03-10 Personalis, Inc. Methods and systems for genetic analysis
US12217830B2 (en) 2019-11-05 2025-02-04 Personalis, Inc. Estimating tumor purity from single samples
US12512183B2 (en) 2019-11-05 2025-12-30 Personalis, Inc. Estimating tumor purity from single samples
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CN115851923A (zh) * 2022-05-25 2023-03-28 广州市基准医疗有限责任公司 用于检测结直肠癌淋巴结转移的甲基化生物标记物及其应用
WO2024129928A3 (fr) * 2022-12-13 2024-08-08 The Johns Hopkins University Marqueurs de méthylation pour la détection et la surveillance du cancer du col de l'utérus
CN115961048A (zh) * 2023-01-04 2023-04-14 广州禾信康源医疗科技有限公司 一种基因甲基化检测引物组合、试剂及其应用
CN115961048B (zh) * 2023-01-04 2025-06-03 广州禾信康源医疗科技有限公司 一种基因甲基化检测引物组合、试剂及其应用

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