Patent Application
20140371096
Fine needle aspiration (FNA) is currently the best diagnostic tool for the pre-operative evaluation of a thyroid nodule, but it is often inconclusive as a guide for subsequent surgical management because 15-20% of fine needle aspirations yield indeterminate results. Recent studies have demonstrated that detecting mutations in BRAF, RAS, RET/PTC, and PAX8/PPARy in clinical fine needle aspiration samples contributes to the diagnostic accuracy of fine needle aspiration cytology. Unfortunately, current assays are still insufficiently sensitive and specific.
Genetic gains and losses in thyroid cancers have been studied. Although DNA copy number changes are frequent in benign follicular adenomas, DNA copy number changes and large chromosomal aberrations are much less common in papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTCs). FVPTCs and PTCs are particularly difficult to diagnose because morphological classification is subject to significant inter-observer and even intra-observer variation. Characteristic objective measures for diagnosing such tumors is urgently required.
As described below, the present invention features compositions and methods for characterizing thyroid lesions (e.g., benign follicular adenomas (FAs), papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTCs)).
In one aspect, the present invention provides a method for molecularly characterizing a thyroid lesion, the method including detecting in a biological sample of the lesion characteristic DNA copy number variation at one or more of chromosomes 7, 12, and 22, thereby characterizing the lesion as having benign or malignant potential.
In another aspect, the present invention provides a method for characterizing a thyroid lesion, the method including detecting in a biological sample of the lesion characteristic DNA copy number variation at one or more of chromosomes 7, 12, and 22 by one or more of techniques such as, for example, SNP array analysis, PCR analysis, hybridization, fluorescence in situ hybridization, quantitative Real-time genomic PCR analysis, gene expression array analysis, or transcriptome array analysis, thereby characterizing the lesion as having benign or malignant potential.
In another aspect, the present invention provides a method for molecularly characterizing a thyroid lesion, the method including detecting in a biological sample of the lesion characteristic DNA copy number variation at one or more of chromosomes 7, 12, and 22, thereby characterizing the lesion as a benign follicular adenoma, a classic papillary thyroid carcinoma or a follicular variant papillary thyroid carcinoma.
In another aspect, the present invention provides a method for distinguishing a follicular adenoma from other thyroid lesions, the method including detecting in a thyroid lesion a segmental amplification in chromosomes 7 and 12, such that the presence of said amplification at chromosomes 7 and/or 12 is indicative that the lesion is a follicular adenoma.
In yet another aspect, the present invention provides a method for distinguishing adenomatoid nodules or follicular variant papillary thyroid carcinoma from other thyroid lesions, the method comprising detecting in a thyroid lesion a chromosome 12 amplification, such that the presence of the chromosome 12 amplification is indicative of adenomatoid nodules or follicular variant papillary thyroid carcinoma.
In various embodiments of any of the above-delineated aspects, the method may identify a characteristic DNA copy number variation that could not be identified by karyotyping.
In various embodiments of any of the above-delineated aspects, the method may further include detecting a mutation in a Ras gene. In various additional embodiments, the mutation may be H-ras or N-ras.
In various embodiments of any of the above-delineated aspects, the method may further include detecting an increase in telomerase expression or activity. In various additional embodiments, telomerase activity may be detected in an HTERT assay.
In various embodiments of any of the above-delineated aspects, the molecular characterization is not by karyotyping.
In various embodiments of any of the above-delineated aspects, detection of the copy number variation may be by one or more techniques such as, for example, SNP array analysis, PCR analysis, hybridization, fluorescence in situ hybridization, quantitative Real-time genomic PCR analysis, gene expression array analysis, or transcriptome array analysis.
In various embodiments of any of the above-delineated aspects, the characteristic DNA copy number variation is a segmental amplification at chromosome 12 that is indicative of a follicular adenoma.
In various embodiments of any of the above-delineated aspects, the method distinguishes a follicular adenoma from a classic papillary thyroid carcinoma or a follicular variant papillary thyroid carcinoma.
In various embodiments of any of the above-delineated aspects, the characteristic DNA copy number variation is chromosome 12 amplification that identifies the lesion as being benign or as having no or little malignant potential.
In various embodiments of any of the above-delineated aspects, amplification at chromosome 12 is detected by measuring the expression or activity of any one or more markers selected from the group consisting of NDUFA12, NR2C1, FGD6, VEZT, MIR331, RPL29P26, LOC729457, METAP2, USP44, CD163L1, LOC727815, BICD1, FGD4, DNM1L, YARS2, UTP20, ARL1, SPIC, WNK1, DRAM, RAD52, HSPD1P12, CERS5, LIMA1, MYBPC1, CHPT1, SYCP3, PKP2, CCDC53, HAUS6, PLIN2, LOC729925, YPEL2, DHX40, CLTC, PTRH2, TMEM49, MIR21, TUBD1, PLIN2, RPS6 KB1, HEATR6, LOC645638, LOC653653, LOC650609, CA4, USP32, SCARNA20, C17orf64, and APPBP2.
In various embodiments of any of the above-delineated aspects, amplification at chromosome 12 is detected by measuring the expression or activity of any one or more markers selected from the group consisting of NDUFA12, NR2C1, FGD6, VEZT, MIR331, RPL29P26, LOC729457, METAP2, USP44, and CD163L1.
In various embodiments of any of the above-delineated aspects, amplification at chromosome 12 is detected by measuring the expression or activity of any one or more markers selected from the group consisting of NDUFA12, NR2C1, FGD6, VEZT and GDF3.
In various embodiments of any of the above-delineated aspects, the characteristic DNA copy number variation is a chromosome 22 deletion, and presence of the deletion is indicative of a premalignant state leading to invasive disease.
In various embodiments of any of the above-delineated aspects, the biological sample is a tissue sample, biopsy sample, or fine needle aspirant.
In various embodiments of any of the above-delineated aspects, RNA or genomic DNA may be isolated from the sample prior to analysis.
In various embodiments of any of the above-delineated aspects, detection of the amplification on chromosome 12 indicates that said follicular adenoma is unlikely to progress to thyroid cancer.
The invention provides characterizing thyroid lesions using DNA copy number variations to determine their benign or malignant potential. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 (NDUFA12) nucleic acid molecule” is meant a polynucleotide encoding a NDUFA12 polypeptide. See, NCBI Gene ID 55967. Exemplary NDUFA12 nucleic acid molecules are provided at NCBI Accession Nos. NM—001258338.1 and NM—018838.4, as well as below:
By “nuclear receptor subfamily 2, group C, member 1 (NR2C1) nucleic acid molecule” is meant a polynucleotide encoding a NR2C1 polypeptide. See, NCBI Gene ID 7181. Exemplary NR2C1 nucleic acid molecules are provided at NCBI Accession Nos. NM—003297.3, NM—001032287.2, and NM—001127362.1, as well as below:
By “FYVE, RhoGEF and PH domain containing 6 (FGD6) nucleic acid molecule” is meant a polynucleotide encoding a FGD6 polypeptide, as summarized in NCBI Gene ID 55785. An exemplary FGD6 nucleic acid molecule is provided at NCBI Accession No. NM—018351.3, as well as below:
By “vezatin, adherens junctions transmembrane protein (VEZT) nucleic acid molecule” is meant a polynucleotide encoding a VEZT polypeptide, as summarized in NCBI Gene ID 55591. An exemplary VEZT nucleic acid molecule is provided at NCBI Accession No. NM—017599.3, as well as below:
By “growth differentiation factor 3 (GDF3) nucleic acid molecule” is meant a polynucleotide encoding a GDF3 polypeptide, and as summarized in NCBI Gene ID 9573. An exemplary GDF3 nucleic acid molecule is provided at NCBI Accession No. NM—020634.1, as well as below:
By “microRNA 331 (MIR331) nucleic acid molecule” is meant a polynucleotide encoding a microRNA. An exemplary MIR331 nucleic acid molecule is provided at NCBI Accession No. NR—029895.1, as well as below:
By “ribosomal protein L29 pseudogene 26 (RPL29P26) nucleic acid molecule” is meant a polynucleotide encoding a RPL29P26 pseudogene. An exemplary RPL29P26 nucleic acid molecule is provided at NCBI Accession No. gi1224589803:c95861652-95861038, as well as below:
By “hypothetical protein LOC729457 (LOC729457) nucleic acid molecule” is meant a polynucleotide encoding a hypothetical LOC729457 polypeptide. An exemplary LOC729457 nucleic acid molecule is provided at NCBI Accession No. gi189161190:c32151164-32150334, as well as below:
By “methionyl aminopeptidase 2 (METAP2) nucleic acid molecule” is meant a polynucleotide encoding a METAP2polypeptide. An exemplary METAP2nucleic acid molecule is provided at NCBI Accession No. NM—006838.3, as well as below:
By “ubiquitin specific peptidase 44 (USP44) nucleic acid molecule” is meant a polynucleotide encoding a USP44polypeptide. An exemplary USP44 nucleic acid molecule is provided at NCBI Accession No. NM—001042403.1, as well as below:
By “CD163 molecule-like 1 (CD163L1) nucleic acid molecule” is meant a polynucleotide encoding a CD163L1polypeptide. An exemplary CD163Llnucleic acid molecule is provided at NCBI Accession No. NM—174941.4, as well as below:
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By “biologic sample” is meant any tissue, cell, fluid, or other material derived from an organism.
By “characteristic DNA copy number variation” is meant that the number of DNA copies on a chromosome varies (i.e., is increased or decreased) relative to the number of DNA copies present in a healthy control cell or organism.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include thyroid lesions (e.g., benign follicular adenomas (FAs), papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTCs)).
The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “invasive disease” is meant a neoplasia or carcinoma that has metastasized or that has a propensity to metastasize.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any analyte (e.g., polypeptide, polynucleotide) or other clinical parameter that is differentially present in a subject having a condition or disease as compared to a control subject (e.g., a person with a negative diagnosis or normal or healthy subject). For example, characteristic DNA copy number variation on any one or more of chromosomes 7, 12, or 22, or an alteration in the expression level of a NDUFA12, NR2C1, FGD6, VEZT and/or GDF3 polypeptide or polynucleotide. In another embodiment, an amplification or deletion of a portion of a chromosome is a marker of the invention.
By “molecularly characterize” is meant detect using assays or tools of molecule biology. Such methods do not include chromosomal karyotyping or cytological methods.
By “mutation” is meant an alteration in the sequence of a polynucleotide or polypeptide relative to a reference sequence. A reference sequence is typically the wild-type sequence.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
By “periodic” is meant at regular intervals. Periodic patient monitoring includes, for example, a schedule of tests that are administered daily, bi-weekly, bi-monthly, monthly, bi-annually, or annually.
By “premalignant state” is meant the state of a cell prior to malignancy.
By “malignant potential” is meant a propensity to become malignant.
By “benign potential” is meant a propensity to remain benign.
By “severity of neoplasia” is meant the degree of pathology. The severity of a neoplasia increases, for example, as the stage or grade of the neoplasia increases.
By “Marker profile” is meant a characterization of the expression or expression level of two or more polypeptides or polynucleotides.
“Primer set” means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard of comparison. For example, the characteristic DNA copy number or level of NDUFA12, NR2C1, FGD6, VEZT and GDF3 polypeptide or polynucleotide level present in a patient sample may be compared to the level of said polypeptide or polynucleotide present in a corresponding healthy cell or tissue or in a neoplastic cell or tissue that lacks a propensity to metastasize.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
By “thyroid lesion” is meant any abnormality present in the thyroid of a subject. Such abnormalities include indeterminate thyroid lesions, as well as benign follicular adenomas (FAs), papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTCs).
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
In general, the invention provides compositions and methods for characterizing thyroid lesions (e.g., benign follicular adenomas (FAs), papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTCs)).
The invention is based, at least in part, on the discovery that thyroid tumor subtypes show characteristic DNA copy number variation (CNV) patterns when analysed using high-resolution single nucleotide polymorphism (SNP) arrays for the genomic characterizations of thyroid tumors. In order to maximize the statistical power of the initial analysis, the three tumor subtypes most commonly leading to an ambiguous pre-operative diagnosis: papillary thyroid carcinomas (PTC), follicular variant papillary thyroid carcinomas (FVPTCs), and follicular adenomas (Fas) were selected for characterization. Follicular carcinomas (FCs) are much less common, and were therefore not included in our initial genome-wide screen.
Fine needle aspiration is the best diagnostic tool for pre-operative evaluation of thyroid nodules, but is often inconclusive as guide for surgical management. As detailed below, thyroid tumor subtypes show characteristic DNA copy number variation (CNV) patterns. The present invention provides for the characterization of such profiles, thereby improving preoperative classification. The study cohorts included benign follicular adenomas (FA), classic papillary thyroid carcinomas (PTC) and follicular variant papillary thyroid carcinomas (FVPTC), the three subtypes most commonly associated with inconclusive preoperative cytopathology.
Tissue and FNA samples were obtained from subjects that underwent partial or complete thyroidectomy for malignant or indeterminate thyroid lesions. Pairs of tumor tissue and matching normal thyroid tissue derived DNA were compared using 550K SNP arrays and significant differences in characteristic DNA copy number variation patterns were identified between tumor subtypes.
Segmental amplifications in chromosomes 7 and 12 were more common in follicular adenomas than in papillary thyroid carcinomas or follicular variant papillary thyroid carcinomas. Additionally, a subset of follicular adenomas and follicular variant papillary thyroid carcinomas showed deletions in Ch22. The present study also identified five CNV-associated genes capable of discriminating between follicular adenomas and papillary thyroid carcinomas/follicular variant papillary thyroid carcinomas. These genes correctly classified 90% of cases. These five chromosome 12 genes were validated by quantitative genomic PCR and gene expression array analyses on the same patient cohort. The five-gene signature was then successfully validated against an independent test cohort of benign and malignant tumor samples. Finally, a feasibility study was performed on matched FA-derived intraoperative FNA samples. This study correctly distinguished follicular adenomas harboring the chromosome 12 amplification signature from follicular adenomas without the chromosome 12 amplification. Thus, thyroid tumor subtypes possess characteristic genomic profiles. These profiles provide for the identification of structural genetic changes in thyroid tumor subtypes.
The present invention provides a number of diagnostic assays that are useful for the identification or characterization of a thyroid lesion. In one embodiment, a thyroid tumor subtype possesses a characteristic genomic profile that identifies it as a benign follicular adenoma (FA), classic papillary thyroid carcinoma (PTC) or follicular variant papillary thyroid carcinoma. To separate the thyroid lesions into subtypes characteristic DNA copy number variation patterns are identified. Such patterns include characteristic DNA copy number variation at one or more of chromosomes 7, 12 and 22. Characterizing the thyroid tumor by subtype is useful for preoperative classification.
In certain embodiments, alterations in chromosomes 7, 12, and 22 are assayed in combination with telomerase activity or expression levels. Human telomerase is a specialized ribonucleoprotein composed of two components, a reverse transcriptase protein subunit (hTERT) (J. Feng, Science 269, 1236-1241 (1995); T. M. Nakamura, Science 277, 911-912 (1997)), as well as several associated proteins. Telomerase directs the synthesis of telomeric repeats at chromosome ends, using a short sequence within the RNA component as a template. Telomerase is considered to be an almost universal marker for human cancer, its effect on telomere length playing a crucial role in evading replicative senescence. Telomerase refers to the ribonucleoprotein complex that reverse transcribes a portion of its RNA subunit during the synthesis of G-rich DNA at the 3′ end of each chromosome in most eukaryotes, thus compensating for the inability of the normal DNA replication machinery to fully replicate chromosome termini. The human telomerase holoenzyme minimally comprises two essential components, a reverse transcriptase protein subunit (hTERT), and the “RNA component of human telomerase.” The RNA component of telomerase from diverse species differ greatly in their size and share little sequence homology, but do appear to share common secondary structures, and important common features include a template, a 5′ template boundary element, a large loop including the template and putative pseudoknot, referred to herein as the “pseudoknot/template region,” and a loop-closing helix. Human telomerase activity is described for example by V. M. Tesmer Mol Cell Biol. 19(9):6207-160 (1999) and US Patent Application No. 20110257251, which is incorporated herein by reference in its entirety for all purposes.
In other embodiments, characteristic DNA copy number variation is used in combination with HRas (Omim No. 190020; Cytogenetic location: 11p15.5, Genomic coordinates (GRCh37): 11:532,241-535,549) or Nras (Omim No. 164790; Cytogenetic location: 1p13.2 Genomic coordinates (GRCh37): 1:115,247,084-115,259,514).
While the examples provided below describe methods of detecting characteristic DNA copy number variation using SNP array analysis, quantitative Real-time genomic PCR analysis, gene expression array analysis, or transcriptome array analysis, the skilled artisan appreciates that the invention is not limited to such methods. Characteristic DNA copy number variation levels are quantifiable by any standard method, such methods include, but are not limited to real-time PCR, bisulfite genomic DNA sequencing, restriction enzyme-PCR, DNA microarray analysis based on fluorescence or isotope labeling, and mass spectroscopy.
In one embodiment, a desired genomic target (e.g., portions of chromosomes 7, 12 and/or 22) is analysed.
Characteristic DNA copy number variation or gene set copy number or expression can be measured using the polymerase chain reaction (PCR). The amplified product is then detected using standard methods known in the art. In one embodiment, a PCR product (i.e., amplicon) or real-time PCR product is detected by probe binding. In one embodiment, probe binding generates a fluorescent signal, for example, by coupling a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates (e.g., TaqMan® (Applied Biosystems, Foster City, Calif., USA), Molecular Beacons (see, for example, Tyagi et al., Nature Biotechnology 14(3):303-8, 1996), Scorpions® (Molecular Probes Inc., Eugene, Oreg., USA)). In another example, a PCR product is detected by the binding of a fluorogenic dye that emits a fluorescent signal upon binding (e.g., SYBR® Green (Molecular Probes)).
The characteristic DNA copy number variation defines the profile of a thyroid carcinoma. The DNA copy number present in a biological sample is compared to a reference. In one embodiment, the reference is the DNA copy number present in a control sample obtained from a patient that does not have a carcinoma. In yet another embodiment, the reference is a reference level or a standardized curve.
Methods for measuring DNA copy number as described herein is used, alone or in combination with other methods, to characterize the thyroid carcinoma. In one embodiment the carcinoma is characterized to determine its stage or grade. Grading is used to describe how abnormal or aggressive the neoplastic cells appear, while staging is used to describe the extent of the neoplasia.
The present invention features diagnostic assays for the characterization of thyroid lesions (e.g., benign follicular adenomas, papillary thyroid carcinomas, and follicular variant papillary thyroid carcinomas). In addition to detecting DNA copy number changes, polypeptide and polynucleotide markers may also be used as diagnostics. In one embodiment, levels of any one or more of the following markers: NDUFA12, NR2C1, FGD6, VEZT and GDF3 are measured in a subject sample and used to characterize a thyroid lesion. In other embodiments, levels of any one or more of NDUFA12, NR2C1, FGD6, VEZT and GDF3 are characterized in a subject sample. Standard methods may be used to measure levels of a marker in any biological sample. Biological samples include tissue samples (e.g., cell samples, fine needle aspiration, biopsy samples). Methods for measuring levels of polypeptide include immunoassay, ELISA, western blotting and radioimmunoassay. Elevated levels of any of NDUFA12, NR2C1, FGD6, VEZT and GDF3 alone or in combination with one or more additional markers are used to characterize a thyroid lesion. The increase in NDUFA12, NR2C1, FGD6, VEZT and GDF3 levels may be by at least about 10%, 25%, 50%, 75% or more. In one embodiment, any increase in a marker of the invention can be used to characterize a thyroid lesion.
Any suitable method can be used to detect one or more of the markers described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, preferably, quantify the markers. These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g., polynucleotides or polypeptides) are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.
Detection methods may include use of a biochip array. Biochip arrays useful in the invention include protein and polynucleotide arrays. One or more markers are captured on the biochip array and subjected to analysis to detect the level of the markers in a sample.
Markers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multiwell microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker. Capture can be on a chromatographic surface or a biospecific surface. For example, a sample containing the markers may be used to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluant, such as phosphate buffered saline. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash.
Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. In one embodiment, mass spectrometry, and in particular, SELDI, is used. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.
Mass spectrometry (MS) is a well-known tool for analyzing chemical compounds. Thus, in one embodiment, the methods of the present invention comprise performing quantitative MS to measure the serum peptide marker. The method may be performed in an automated (Villanueva, et al., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with MS operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS). Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979 and references disclosed therein.
In an additional embodiment of the methods of the present invention, multiple markers are measured. The use of multiple markers (e.g., two or more of NDUFA12, NR2C1, FGD6, VEZT and GDF3) increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification and patient monitoring. The process called “Pattern recognition” detects the patterns formed by multiple markers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of a certain disease, a particular stage of cancer-progression, or a positive or adverse response to drug treatments. While particular embodiments have been disclosed with respect to the detection of specific amplification of chromosome 12 and/or 7 by the use of specific markers (e.g., NDUFA12, NR2C1, FGD6, VEZT and GDF3), it is contemplated within the scope of the disclosure that any marker or markers residing within the copy number variation region may be used.
Expression levels of particular nucleic acids or polypeptides are correlated with thyroid carcinoma, and thus are useful in diagnosis. Antibodies that bind a polypeptide described herein, oligonucleotides or longer fragments derived from a nucleic acid sequence described herein (e.g., an NDUFA12, NR2C1, FGD6, VEZT and GDF3 nucleic acid sequence), or any other method known in the art may be used to monitor expression of a polynucleotide or polypeptide of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of thyroid carcinoma. In particular embodiments, an increase in expression of a NDUFA12, NR2C1, FGD6, VEZT and GDF3 polypeptide is indicative of thyroid carcinoma or the propensity to develop thyroid carcinoma. In other embodiments, a 2, 3, 4, 5, or 6-fold change in the level of a marker of the invention is indicative of thyroid carcinoma. In yet another embodiment, an expression profile that characterizes alterations in the expression two or more markers is correlated with a particular disease state (e.g., thyroid carcinoma). Such correlations are indicative of thyroid carcinoma or the propensity to develop thyroid carcinoma. In one embodiment, a thyroid carcinoma can be monitored using the methods and compositions of the invention.
In one embodiment, the level of one or more markers is measured on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of thyroid carcinoma or the propensity to develop thyroid carcinoma. The level of marker in a subject having thyroid carcinoma or the propensity to develop such a condition may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or more relative to the level of such marker in a normal control.
The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of thyroid carcinoma.
As indicated above, the invention provides methods for aiding a human cancer diagnosis using one or more markers, as specified herein. These markers can be used alone, in combination with other markers in any set, or with entirely different markers in aiding human cancer diagnosis. The markers are differentially present in samples of a human cancer patient and a normal subject in whom human cancer is undetectable. Therefore, detection of one or more of these markers in a person would provide useful information regarding the probability that the person may have thyroid carcinoma or regarding the aggressiveness of the thyroid carcinoma.
The detection of a marker, a molecular profile, or a characteristic DNA copy number variation is correlated with a probable diagnosis of cancer. The correlation may take into account the amount of the marker or markers in the sample compared to a control amount of the marker or markers (e.g., in normal subjects or in non-cancer subjects such as where cancer is undetectable). A control can be, e.g., the average or median amount of marker present in comparable samples of normal subjects in normal subjects or in non-cancer subjects such as where cancer is undetectable. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. As a result, the control can be employed as a reference standard, where the normal (non-cancer) phenotype is known, and each result can be compared to that standard, rather than re-running a control.
Accordingly, a marker profile may be obtained from a subject sample and compared to a reference marker profile obtained from a reference population, so that it is possible to classify the subject as belonging to or not belonging to the reference population. The correlation may take into account the presence or absence of the markers in a test sample and the frequency of detection of the same markers in a control. The correlation may take into account both of such factors to facilitate determination of cancer status.
In certain embodiments of the methods of qualifying cancer status, the methods further comprise managing subject treatment based on the status. The invention also provides for such methods where the markers (or specific combination of markers) are measured again after subject management. In these cases, the methods are used to monitor the status of the cancer, e.g., response to cancer treatment, remission of the disease or progression of the disease.
The markers of the present invention have a number of other uses. For example, they can be used to monitor responses to certain treatments of human cancer. In yet another example, the markers can be used in heredity studies. For instance, certain markers may be genetically linked. This can be determined by, e.g., analyzing samples from a population of human cancer subjects whose families have a history of cancer. The results can then be compared with data obtained from, e.g., cancer subjects whose families do not have a history of cancer. The markers that are genetically linked may be used as a tool to determine if a subject whose family has a history of cancer is pre-disposed to having cancer.
Any marker, individually, is useful in aiding in the determination of cancer status. First, the selected marker is detected in a subject sample using the methods described herein. Then, the result is compared with a control that distinguishes cancer status from non-cancer status. As is well understood in the art, the techniques can be adjusted to increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician.
While individual markers are useful diagnostic markers, in some instances, a combination of markers provides greater predictive value than single markers alone. The detection of a plurality of markers (or absence thereof, as the case may be) in a sample can increase the percentage of true positive and true negative diagnoses and decrease the percentage of false positive or false negative diagnoses. Thus, preferred methods of the present invention comprise the measurement of more than one marker.
As reported herein, a number of markers (e.g., a characteristic DNA copy number variation, NDUFA12, NR2C1, FGD6, VEZT and GDF3) have been identified that are associated with various thyroid lesions (e.g., benign follicular adenomas, papillary thyroid carcinomas, and follicular variant papillary thyroid carcinomas). Methods for assaying the characteristic DNA copy number variation or the expression of NDUFA12, NR2C1, FGD6, VEZT and GDF3 gene or polypeptide expression are useful for characterizing thyroid carcinoma. In particular, the invention provides diagnostic methods and compositions useful for identifying a molecular profile that characterizes a thyroid lesion.
The polypeptides and nucleic acid molecules of the invention are useful as hybridizable array elements in a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.
Proteins (e.g., NDUFA12, NR2C1, FGD6, VEZT and GDF3) may be analyzed using protein microarrays. Such arrays are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the invention, or a fragment thereof. In particular, such microarrays are useful to identify a protein whose expression is altered in thyroid carcinoma. In one embodiment, a protein microarray of the invention binds a marker present in a subject sample and detects an alteration in the level of the marker. Typically, a protein microarray features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the invention) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).
The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a homogenized tissue sample (e.g. a tissue sample obtained by biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.
To produce a nucleic acid microarray, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.), incorporated herein by reference. Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient as a tissue sample (e.g. a tissue sample obtained by biopsy). For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the microarray.
Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 3° C., more preferably of at least about 37 C., and most preferably of at least about 42 C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 C., more preferably of at least about 42.degree. C., and most preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences simultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997). Preferably, a scanner is used to determine the levels and patterns of fluorescence.
After a subject is diagnosed as having a thyroid lesion, the lesion is characterized to determine its subtype and or its benign or malignant potential. If the thyroid lesion is benign and is unlikely to have malignant potential, no treatment may be necessary. However, the lesion may be monitored periodically (annually, biannually) to confirm that no malignancy is presence. If the thyroid lesion has malignant potential a method of treatment (e.g., surgery) is selected. Such treatment may be combined with any one or a number of standard treatment regimens.
The diagnostic methods of the invention are also useful for monitoring the course of a thyroid cancer in a patient or for assessing the efficacy of a therapeutic regimen. In one embodiment, the diagnostic methods of the invention are used periodically to monitor the characteristic DNA copy number variation or the copy number or expression of a gene set (e.g., NDUFA12, NR2C1, FGD6, VEZT and GDF3). In one example, the thyroid carcinoma is characterized using a diagnostic assay of the invention prior to administering therapy. This assay provides a baseline that describes the DNA copy number prior to treatment. Additional diagnostic assays are administered during the course of therapy to monitor the efficacy of a selected therapeutic regimen.
The invention also provides kits for the diagnosis or monitoring of a thyroid carcinoma in a biological sample obtained from a subject. In various embodiments, the kit includes materials for SNP array analysis, quantitative Real-time genomic PCR analysis, gene expression array analysis, or transcriptome array analysis. In yet other embodiments, the kit comprises a sterile container which contains the primer or probe; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids. The instructions will generally include information about the use of the primers or probes described herein and their use in diagnosing a thyroid carcinoma. Preferably, the kit further comprises any one or more of the reagents described in the diagnostic assays described herein. In other embodiments, the instructions include at least one of the following: description of the primer or probe; methods for using the enclosed materials for the diagnosis of a neoplasia; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Using Illumina 550K SNP arrays, genome-wide DNA copy number changes were investigated in 39 thyroid tumors (14 FAs, 13 FVPTCs, and 12 PTCs) with paired normal thyroid tissue samples from the same patients as controls (See Table 1 and Table 2 for clinical patient information).
An unsupervised hierarchical cluster analysis of segmented and smoothed copy number estimates for each sample was performed, summarized at 25,000 by intervals, and the 10% of segments with the greatest sample-to-sample variation in copy number were selected. These regions were not evenly distributed throughout the genome, but were concentrated over several chromosomes, most notably 7, 12 and 22, although all chromosomes were represented to some extent, as shown in
These tumors exhibited a genomic amplification pattern/profile predominantly involving chromosomes 7 and 12, which is consistent with previous studies although the rate observed here is higher than previous estimates (see, e.g., references 8, 12, and 15). Most of the PTCs and FVPTCs clustered together in the center of the heatmap, identified as cluster 2, where few CNVs were observed, which is consistent with the observation that PTCs tend to be relatively stable genomically (see, e.g., references 10 and 16). Finally, in cluster 3, a distinct subset of FVPTCs and FAs were characterized by large deletions in Ch22q, which are indistinguishable from monosomy 22 because of the lack of probes on the acrocentric chromosome 22p arm. Two of the samples with the chromosome 7 and 12 amplifications also harbored this deletion. Upon analysis of clinical and pathological parameters, the Ch22 deletion pattern was found to be associated with younger patients (32 years vs. 46 years, P<0.01, by 2-sided t-test). No other significant associations with clinical indices or specific histopathological features, such as, for example, tumor stage or degree of encapsulation, were observed. All cases showing a BRAF mutation, including 2 cases of FVPTC, were in cluster 2.
Statistical analysis was performed to identify significant CNVs as genomic amplifications and deletions (see, e.g.,
Chromosomal amplifications were more frequent in FAs than in FVPTCs or in PTCs (P<0.01, Chi-square test, see, e.g.,
To identify genes in which copy number differed by tumor type, the original segmented data was mapped to genes and analyzed by an ANOVA, and the Type I error was controlled by the Benjamini-Hochberg false discovery rate and maintained at a level less than 10%. A total of 1209 genes for which DNA copy number showed significant differences (adjusted P<0.05) between FAs and FVPTCs/PTCs were found. The majority of these genes were located on chromosomes 7, 12, and 17. The dominant CNV pattern was determined to be low level but widespread copy number gain of Ch12 in FAs, as illustrated in
To obtain a gene set whose CNVs could distinguish benign FAs from malignant PTCs and FVPTCs, the top 10 ranked genes on Ch12 were selected, ordered according to their statistical significances, and their mean copy number changes within each sample were calculated. This resulted in a significant difference in mean copy number change (P<0.001). Discrimination between classes (e.g., FAs, PTCs, and FVPTCs) was optimal at a cutoff of 0.07 for mean log fold copy number change. A 10-gene set, including, for example, the genes NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 (NDUFA12), nuclear receptor subfamily 2, group C, member 1 (NR2C1), FYVE, RhoGEF and PH domain containing 6 (FGD6), vezatin, adherens junctions transmembrane protein (VEZT), microRNA 331 (MIR331), ribosomal protein L29 pseudogene 26, hypothetical protein LOC729457, methionyl aminopeptidase 2 (METAP2), ubiquitin specific peptidase 44 (USP44), and CD163 molecule-like 1 (CD163L1), was identified that could accurately classify 11 out of 14 FAs and 24 out of 25 PTCs and FVPTCs (see, e.g.,
The chromosome 12 copy number changes were validated in order to: 1) provide a technical validation of the Ch12 signature using an independent, PCR-based assay; and 2) investigate if the CNV-signature found in FAs was in fact FA-specific, or also present in FCs/HCs and FVPTCs on the one hand, or in ANs on the other, given the morphological similarities between these follicular neoplasms. The genes NDUFA12, NR2C1, FGD6, VEZT (the top 4 ranked genes according to their statistical significance by ANOVA) and GDF3 (located at 12p13.31, a region showing amplifications in FAs and deletions in FVPTCs) were selected for validation, and the average copy number levels across the five genes was used to obtain a single estimated value for each sample. The Genbank annotation for these five genes can be found in Table 4.
Based on the distributions of the five gene score in benign and malignant tumors on the SNP array (see, e.g.,
In order to determine the clinical applicability of detecting CNVs in thyroid FNA samples, given the expected contamination with blood and white blood cells (WBCs), a small FNA feasibility study was performed. Matching FNAs were available from 18 of the FA cases considered under the present study. All FNA samples were obtained intraoperatively after surgical isolation of the target lesion and stored in 95% ethanol. FNA samples were enriched for epithelial cells using magnetic beads, resulting in a total of 10 matching FNA samples with detectable amounts of DNA, as determined by achieving identifiable real-time PCR threshold cycle numbers. The results of the successful QPCR assays of this subset are shown in
The somatic genomic alterations in one benign (FAs) and two malignant (PTC and FVPTC) thyroid tumor subtypes were characterized. These three tumor subtypes were the focus of the analysis because they are the most commonly associated with a suspicious but inconclusive preoperative cytopathology. The much more limited FC samples were reserved for a validation of the screening results. In total, 39 thyroid tumor/normal pairs, including 14 FAs, 13 FVPTCs, and 12 PTCs, were analyzed using the Illumina 550K SNP Array platform. This is believed to be the first study to report genome-wide DNA copy number profiles comparing FA, PTC and FVPTC thyroid tumors based on a high-resolution SNP array analysis.
The most frequent genomic aberrations occurred in FAs, and included amplifications of chromosomes 7 and 12, which is consistent with prior CGH and array-CGH studies (see, e.g., references 8, 12, 15). Importantly, the frequency of such events in FAs as determined in the present study is much higher than previously estimated using lower resolution techniques. Conversely, with the notable exception of Ch22 deletions observed in several FVPTCs, both PTCs and FVPTCs showed relatively few copy number changes. This is consistent with the notion that these are relatively stable, from a genomic standpoint, neoplasms at least in their initial, well differentiated stages (see, e.g., references 10, 14, 16,).
The unsupervised hierarchical cluster analysis of detected CNVs clearly shows distinct patterns, which are identified in
Although the number of cases showing Ch22 deletions is small, the consistency of the Ch22deletion patterns seen in several FAs and FVPTCs suggests that this genetic lesion may also represent a distinct subset of these tumors. In this context, it is worth noting that large Ch22 deletions and monosomy 22 have been associated with subsets of malignant follicular neoplasms (see. e.g., references 27, 28), and may therefore be indicative of precursor lesions. However, with the exception of a statistically significant association of the Ch22 deletion cluster with younger age, there was no apparent correlation of any clinical or pathological parameter with a particular CNV cluster. Of note, the 2 FVPTCs harboring BRAF mutations were in the PTC-associated cluster 2, supporting the notion that FVPTCs may broadly belong to either follicular or papillary tumors, each with its distinct molecular and clinical signatures.
The most striking result of the present study arose from a gene-by-gene comparison of copy number in the 14 benign and 25 malignant lesions of the discovery cohort. As seen in the cluster analysis in
To confirm this result by independent methodologies, five genes, NDUFA12, NR2C1, FGD6, VEZT and GDF3, were selected for validation using quantitative Real-time genomic PCR (QPCR). The gene expression array data for the same samples was also analyzed to determine if the amplification on Ch12 could be detected by such an approach as well. Both copy number changes, as assessed by QPCR, and gene expression, as assessed by transcriptome array, supported the presence of gene amplifications on Ch12 in FAs. In addition, a number of genes identified in an integrated analysis of gene expression and DNA copy number showed concordant results between DNA copy number change and gene expression levels (e.g., the above described 50 gene superset). Not surprisingly, Ch12 was over-represented in this set, but similar results were observed in other regions as well.
Ch12 copy number changes were also confirmed in an independent test cohort that included both benign and malignant tumors, which again showed amplification in FAs, while other tumor subtypes, regardless of dignity (e.g., tumor dignity means malignant versus benign) or presence or absence of oncocytic cells, generally did not. This suggests that FAs with amplifications on Ch12 are less likely to progress to thyroid cancer, since that genetic change would not be expected to disappear as FAs progressed. Accordingly, the present disclosure may provide the ability to positively identify FAs with a low chance of malignant progression, which would be an important adjunct to our current set of diagnostic tests that are focused on identifying oncogenic mutations and translocations in malignant thyroid tumors.
In light of these results, tumor pathology was assessed to determine if any distinct morphological patterns matching the Ch12 CNVs could be identified. Both initial blinded and subsequent open reviews failed to identify a morphological subset in our FA cohort. It is also noteworthy that among our samples in the morphological continuum ranging from AN to FA to FVPTC, small numbers of both ANs and FVPTCs harbored the Ch12 amplification characteristic of FAs, which may support a reevaluation of these lesions based on molecular traits in addition to morphological characteristics. It remains to be seen if the 5 genes that we used to represent chromosome 12 have any functional roles in thyroid tissues or thyroid neoplasia, since they were selected based on the structural chromosomal changes detected by the above described CNV analysis.
Finally, an initial feasibility study was performed to determine the Ch12 amplification signature could be detected in cytological specimens. The principal challenge in applying the above described quantitative genomic PCR assay to FNA samples is the unavoidable presence of varying amounts of blood contamination. To address this challenge, the archival FNA samples were fractionated using a commercially available magnetic bead separation approach, and the epithelial cell enrichment lead to the correct classification of all 10 amplifiable DNA preparations, as shown in
In summary, the present disclosure provides a high-resolution analysis of somatic copy number aberrations in FA, PTC and FVPTC thyroid tumors. According to the techniques herein, distinct genomic patterns of copy number changes associated with benign and malignant thyroid tumors, of which the gene copy number gains in Ch12 were the most distinctive, were limited to benign tumors. These amplifications were verified using Realtime-PCR of genomic DNA and transcriptome arrays of the same 39 tumor-normal paired thyroid samples, and the specificity of this result was validated on an additional independent test set of benign and malignant thyroid tumors. The results demonstrated the diagnostic feasibility of assessing CNV signatures in thyroid FNA samples.
Since FAs are a common source of inconclusive pre-operative cytopathology results, the techniques herein, which provide a molecular signature (e.g., Ch12 amplifications) that positively identifies a subset of follicular neoplasms with no malignant potential, represents an important diagnostic adjunct to the currently available tests for oncogenic genetic changes in thyroid cancers. Similarly, the ability to identify the presence of Ch22 deletions in FAs is a useful diagnostic indicative of a premalignant state that may ultimately lead to invasive disease. The present disclosure illustrates the value of the molecular characterization of benign thyroid tumors and well-differentiated thyroid cancer, which continue to confound the pre-operative diagnosis of thyroid nodules, and may help justify the clinical development of molecular assays based on an epithelial cell-enriched fraction of the standard FNA sample.
The results described herein above were obtained using the following methods and materials.
Tissue Samples and DNA Isolation:
Cases were identified that underwent partial or complete thyroidectomy for malignant or indeterminate thyroid lesions at the Johns Hopkins Medical Institutions between 2000 and 2008 and from whom tissue had been immediately snap frozen in liquid nitrogen within one hour of surgery and stored at −80° C. until use. Initial case selection was based on review of the official surgical pathology reports identifying thyroid tumor subtypes falling into the scope of this study. Cases were then selected for availability of adequate matching tumor and normal tissue and passing quality controls for both DNA and RNA. The study pathologist (WW) reviewed both the official archival permanent H&E sections to confirm the original diagnoses as well as the research cryosections to confirm tumor content of the analyzed sample. The diagnoses of thyroid tumors in this study was based on the criteria described in the 2004 World Health Organization (WHO) monograph on endocrine tumors (see, e.g., reference 29). None of these cases had oncocytic features. Each tumor tissue block used for nucleic acid isolation was confirmed to contain more than 70% tumor cells on H&E-stained cryosections (see, e.g., reference 30).
SNP Array Analyses:
DNA from 39 thyroid tumor-normal paired samples was genotyped using the Illumina 550K SNP Array (Illumina, San Diego, Calif.). DNA samples were assessed for quality both by NanoDrop Spectrophotometry and agarose gel electrophoresis. Samples judged to be of sufficient quality were assayed at the Center for High-throughput Microarray Analysis at the Johns Hopkins University School of Medicine.
CNV Detection:
BeadStudio (I lumina Inc., San Diego, Calif.) software routines were applied to normalize the SNP array data and export signal intensity (R value) and SNP location information for each SNP probe. DNA abundance was calculated as the geometric mean of the signal intensities from each allelic pair, R=(IA2+IB2)1/2, so that the logged R-ratio, Rlr=log2(Rtumor)-log2(Rnormal) represented log fold copy number. Circular Binary Segmentation (CBS), as implemented in the Bioconductor R package, DNAcopy, was applied to estimate the boundaries of segments of constant copy number, and to calculate the mean log fold copy change estimate for each such segment (see, e.g., reference 31). The hybrid approach was adopted to control the amount of smoothing, using sensitive settings in the CBS algorithm in order to detect small, focal events. A second smoothing algorithm was used to combine adjacent segments if the difference in mean log fold copy change was less than 0.25, and the intervening segment of normal copy number covered less than 10% of the total genomic region spanned by the segments under consideration, to prevent excessive segmentation of much larger changes.
Statistical Significance Analysis of Genomic Amplifications and Deletions:
Statistically significant changes were identified by comparing the observed, segmented copy number changes to a null distribution obtained by permuting genomic locations and repeating the segmenting and smoothing steps. Segments of a given log fold copy number change were deemed significant if they extended over a sufficient number of SNPs, selected to control type I error rates at no more than 10%. Specific segment length criteria were derived for log fold changes above 0.25 and below −0.25, as illustrated in
Real-Time Quantitative PCR (qPCR):
Reactions were preformed in triplicate using 1 ng of genomic DNA in a 150 reaction that contained 1 μM of each amplification primer in Real-time SYBR PCR Master Mix (Bio-Rad). Samples were amplified on an Applied Biosystems 7900HT Sequence Detection System and the data was collected and analyzed with SDS 2.3 software. Standard curves were constructed using serial two-fold dilutions of genomic DNA from a normal individual and used to estimate the PCR amplification efficiency, which was confirmed at >97% for each gene to insure the comparability with reference genes. The DNA content of each sample for target genes was normalized to that of Alu, a repetitive genomic element for which the copy number per haploid genome is similar among all human cells (see, e.g., reference 32). Each sample was run in triplicate to ensure quantitative accuracy, and the medians of the threshold cycle numbers (Ct) were taken. The relative copy number changes in the thyroid tumor/normal pairs were reported as T:N ratios and calculated using the 2-AACt method (see, e.g., reference 33). A 130 by Ch21 segment (Ch21: by 27423633-27423762) was chosen for Real-time PCR analysis to compare 3 DNA samples obtained from Down Syndrome patients (Ch21 trisomy) to a DNA sample with normal copies as a genomic amplification control; and a 87 by chromosome X segment (ChX: by 12057855-12057941) to compare normal thyroid tissue samples from 9 males and from 3 females as a genomic hemizygous deletion control.
Real-Time Quantitative PCR of FNA Samples:
All FNA samples were obtained intraoperatively after surgical isolation of the target lesion. All samples were collected with Institutional Review Board approval as part of an ongoing research protocol. The samples were placed immediately into 95% ethanol and stored at −20° C. A total of 18 FNA samples that matched FA tissue samples in this study were available for the subsequent assays. The FNA samples were enriched for epithelial cells using magnetic beads coated with anti-human epithelial antigen antibodies provided in the Dynal Epithelial Enrich kit (Life Technologies, Grand Island, N.Y.) in accordance with the manufacturer's instructions. Genomic DNA was isolated using Lyse and Go PCR reagent according to the manufacturer's instructions (Thermo Scientific, Rockford, Ill.). For the real-time PCR, the same primer sets (see Table 5 below) and amplification protocol as used for thyroid tissue samples were used to assay genomic DNA from the FNA samples. The normalized Ct value (i.e., -delta Ct(Target-Alu)) was calculated to represent the copy number relative to internal Alu sequence signal in thyroid FNA samples. For reference, 3 white blood cell samples from patients with benign thyroid disease (multinodular hyperplasia) were used as normal control of Ch12 copy numbers.
RNA Isolation and Expression Array Analysis:
RNA samples were prepared from the same 39 thyroid tumor-normal tissue samples used for SNP arrays, using the Qiagen RNeasy Kit (Qiagen, Valencia, Calif.). The quantity and integrity of extracted RNA was evaluated by ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, Del.) and Bio-Rad Experion RNA Assay (Bio-Rad, Hercules, Calif.), respectively. Microarray hybridizations were performed in the Microarray Core Facility at Johns Hopkins University School of Medicine. For each sample, 500 ng total RNA was used for transcriptome analysis using the HumanHT-12 v3 Expression BeadChip kit (Illumina, San Diego, Calif.), which targets ˜25,000 annotated genes with more than 48,000 probes. Arrays were processed as per the manufacturer's instructions. Hybridization signals were analyzed using BeadStudio Gene Expression Module v.3 (Illumina) (see, e.g., reference 34). Quantile normalization and statistical analysis of the gene array data were carried out using the Limma (see, e.g., reference 35) package and customized scripts in R/Bioconductor (see, e.g., reference 36).
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of the following U.S. Provisional Application No. 61/568,923, filed Dec. 9, 2011, the entire contents of which are incorporated herein by reference.
This work was supported by the following grant from the National Institutes of Health, Grant No: R01 CA107247-04. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/68811 | 12/10/2012 | WO | 00 | 6/9/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61568923 | Dec 2011 | US |
