WO2009111881A1 - Biomarqueurs pour diagnostic du cancer différencié de la thyroïde - Google Patents

Biomarqueurs pour diagnostic du cancer différencié de la thyroïde Download PDF

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WO2009111881A1
WO2009111881A1 PCT/CA2009/000306 CA2009000306W WO2009111881A1 WO 2009111881 A1 WO2009111881 A1 WO 2009111881A1 CA 2009000306 W CA2009000306 W CA 2009000306W WO 2009111881 A1 WO2009111881 A1 WO 2009111881A1
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biomarkers
thyroid
thyroid cancer
galectin
sample
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PCT/CA2009/000306
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English (en)
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Sam Michael Wiseman
Steven Jones
Obi Lee Griffith
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British Columbia Cancer Agency Branch
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers

Definitions

  • the present invention relates generally to panels of biomarkers for diagnosis of thyroid cancer. More specifically, methods employing biomarkers are described herein, together with tests, assays or kits for diagnosis of thyroid cancer, classification of subtypes of thyroid cancer, and prognostic assessment of thyroid cancer.
  • Thyroid nodules are extremely common and are palpable in 5% of the general population. Ultrasound examination of the thyroid is more accurate than palpation and can diagnose nodules in up to half of individuals evaluated. It is the increased utilization of ultrasound that is believed to be partially responsible for a rise in the incidence of thyroid cancer cases diagnosed in the United States. This rising thyroid cancer incidence has been reported to be a consequence of an increase in the rate of diagnosis of small papillary carcinomas (or papillary thyroid carcinoma).
  • Fine needle aspiration biopsy is currently considered the critical initial diagnostic test for evaluation of thyroid nodules.
  • Gharib [3] reviewed greater than 18,000 thyroid fine needle aspiration biopsies carried out at the Mayo Clinic and reported an average sensitivity of 83%, specificity of 92%, and an overall accuracy of 95%.
  • An indeterminate thyroid fine needle aspiration biopsy includes those biopsies classified as 'follicular neoplasm' and ⁇ urthle cell neoplasm' and arises as a direct result of overlapping cytomorphologic characteristics exhibited by benign and malignant follicular thyroid lesions.
  • Only approximately 20% of cases with a fine needle aspiration biopsy diagnosis of follicular neoplasm are eventually proven by histopathologic evaluation to be cancer. Additionally, a diagnosis of Hurthle cell neoplasm will eventually be diagnosed as a cancer in approximately 25% of cases.
  • the presence of focal nuclear atypia can also lead to an indeterminate fine needle aspiration biopsy diagnosis that may often be described as 'suspicious for papillary carcinoma'.
  • PCR polymerase chain reaction
  • RNA based diagnostic assays for determining thyroid cancer
  • Galectins are involved in many of the biologic functions of the cell including: growth, differentiation, adhesion, mRNA processing, and apoptosis [23,24].
  • Galectin-3 a chimera type galectin that contains a non-lectin portion connected to a lectin domain, as being consistently expressed in the cytoplasm of malignant thyrocytes [25-27].
  • the gene encoding the Galectin-3 protein (LGALS3) was also found to be up-regulated in thyroid cancer compared to benign thyroid lesions, by a significant number of studies [6].
  • Galectin-3 immunocytochemical expression has been investigated as an adjunctive test to fine needle aspiration biopsy to improve the accuracy of thyroid cancer diagnosis.
  • Galectin-3 expression in 1009 thyroid specimens was a highly sensitive, specific, and accurate marker for thyroid cancer diagnosis [27]. Not all investigators have been able to reproduce these promising results. In a cohort of benign and malignant thyroid tumors Galectin-3 expression was unable to distinguish follicular adenomas and follicular carcinoma [28].
  • CK19 low molecular weight cytokeratin 19
  • Another marker is low molecular weight cytokeratin 19 (CK19), the utility of which is controversial as a diagnostic marker for thyroid cancer [1].
  • Most reports have described a high rate of CK19 expression by small papillary carcinomas [1].
  • CK19 A thyroid tumor meta-review and meta-analysis study conducted by Griffith et al. identified the gene encoding CK19 (KRT19) as being significantly upregulated in thyroid cancer [15].
  • KRT19 nuclear factor
  • VEGF vascular endothelial growth factor
  • cytokines vascular endothelial growth factor
  • VEGF plays an important role in tumor neovascularization, or angiogenesis, which is essential for cancer growth and spread [33].
  • VEGF has generally not been utilized as a diagnostic marker for thyroid cancer.
  • the current literature evaluating VEGF expression in benign and malignant thyroid tumors is conflicting and results are highly dependent upon the specific thyroid tumor cohort evaluated. Lewy-Trenda et al. reported VEGF expression, utilizing an immunohistochemical technique, in 40% of papillary thyroid carcinoma, 44% of follicular thyroid carcinoma, 50% of follicular adenoma, and 12% of goiters [34].
  • VEGF expression was higher in follicular adenoma (13 cases) than follicular thyroid carcinoma (12 cases) [35].
  • a multi-cancer type study measuring VEGF mRNA expression utilizing DNA microarrays found that the median level of expression, relative to normal thyroid tissue, was higher in benign tumors and nodular hyperplasia than in either thyroid cancer or thyroid cancer metastases [36].
  • VEGF levels evaluated by immunohistochemistry and PCR, to be either unchanged or higher in thyroid cancer compared to benign lesions [37,38]. Caution must be exercised when comparing and contrasting the results of studies that measure VEGF levels in human tissues.
  • VEGF-A vascular endothelial growth factor-A
  • VEGF-B vascular endothelial growth factor-B
  • VEGF-C vascular endothelial growth factor-B
  • VEGF-D vascular endothelial growth factor-A
  • VEGF-A vascular endothelial growth factor-A
  • VEGF-A165 vascular endothelial growth factor-A165
  • VEGF-A189 vascular endothelial growth factor
  • VEGF-A206 vascular endothelial growth factor-A
  • Aurora-A is a member of the Aurora kinase family and is a biomarker primarily associated with the centrosome in mitotic cells where it interacts with and phosphorylates several substrates (including Eg5, TPX2, and TACC3) and is involved in spindle formation and stability [40].
  • substrates including Eg5, TPX2, and TACC3
  • Aurora-A protein expression has been significantly higher in cases of papillary thyroid carcinoma compared to normal matched tissues obtained from the same patients.
  • a further biomarker is P16, which is a cyclin-dependent kinase inhibitor that causes the cell cycle to arrest in G1 phase by competing with cyclin D for binding to CDK4 and preventing the cyclin D-CDK4 complex from phosphorylating Rb [42].
  • P16 is a cyclin-dependent kinase inhibitor that causes the cell cycle to arrest in G1 phase by competing with cyclin D for binding to CDK4 and preventing the cyclin D-CDK4 complex from phosphorylating Rb [42].
  • P16 is a cyclin-dependent kinase inhibitor that causes the cell cycle to arrest in G1 phase by competing with cyclin D for binding to CDK4 and preventing the cyclin D-CDK4 complex from phosphorylating Rb [42].
  • the Androgen Receptor is a nuclear receptor transcription factor that regulates the cellular actions of androgens, the male sex steroids [44].
  • AR Androgen Receptor
  • AR RNA in 100% of goiters, 56% of follicular adenomas, 100% of follicular thyroid cancers, and 75% of papillary thyroid cancers.
  • Immunohistochemistry on fresh tissue detected AR in 50% of normal thyroid specimens, 1 goiter, and 50% of neoplastic thyroids [47].
  • AR status has not been evaluated utilizing immunocytochemistry on aspiration biopsy specimens from thyroid tumors, though it has been evaluated in aspiration biopsy specimens from other human tumor types [48].
  • HBME-1 The anti-human mesothelial cell mouse monoclonal antibody (HBME-1 ) was originally raised against an unidentified membrane antigen that exists in the micovilli of mesothelioma cells and, like Galectin-3 and CK19, has been extensively studied for its utility as a diagnostic marker for thyroid cancer [13,14].
  • Mctinen et al. evaluated HBME-1 expression in a cohort of 463 thyroid tumors and found strong expression in all papillary thyroid cancers (145 of 145 cases) and all follicular thyroid cancers (27 of 27 cases) and observed either no reactivity or focal staining (for a third of cases) in goiters and papillary hyperplasia [49]. lto et al.
  • HBME-1 the most sensitive and specific marker for thyroid cancer diagnosis and a panel of all 3 markers could correctly distinguish the follicular variant of PTC from all other follicular lesions in 83.6% of cases [19].
  • Prasad et al. evaluated expression of Galectin-3, CK19, HBME-1 , CITED-1 , and fibronectin-1. They found an immunohistochemical panel consisting of tumors expressing Galectin-3, HBME-1 and fibronectin-1 was 100% sensitive for diagnosing carcinomas [20].
  • Galectin-3, CK19, HBME-1 , P16, ERK, and RET in a 90 thyroid tumor cohort (37 cancers and 53 benign lesions). They found that simultaneous expression of Galectin-3, CK19, and HBME-1 had a sensitivity of 54% and a specificity of 100% for diagnosing cancer. They also found simultaneous expression of P16, ERK and RET had a sensitivity of 51% and specificity of 89% for diagnosing cancer [21].
  • Park et al. examined expression of Galectin-3, CK19, HBME-1 , High Molecular Weight Cytokeratin, Cyclin D1 , and p27 [22].
  • Benign and malignant thyroid tumors encompass a large and heterogeneous group of histologic diagnoses. Inclusion of medullary and anaplastic carcinomas, which are usually diagnosed by cytomorophologic evaluation, in thyroid diagnostic marker study cohorts may not be necessary. Apart from its histologic appearance, medullary thyroid cancer is readily diagnosed by calcitonin staining. Further, an immunohistochemical panel of 8 markers to distinguishes differentiated thyroid cancer and anaplastic thyroid cancer has been reported [11 ,54]. However, studies evaluating differentiated thyroid cancer diagnostic markers often fail to address the histologic heterogeneity and due to an insufficiently sized cohort. Most thyroid cancer diagnostic marker panel studies have a relatively small cohort size [19-21 ,52,53].
  • the present invention provides a method of diagnosing differentiated thyroid cancer in a subject. The method comprises the steps: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising Galectin-3, P16 and androgen receptor; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the present invention provides a method of diagnosing differentiated thyroid cancer in a subject wherein the method comprises the steps: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising Galectin-3, P16 and HBME-1 ; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the present invention provides a method of diagnosing differentiated thyroid cancer in a subject, the method comprising the steps: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising CK19 and Vascular Endothelial Growth Factor; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the present invention provides a kit for the diagnosis of differentiated thyroid cancer in a subject.
  • the kit comprises reagents, and instructions for their use, for determining in a tissue sample levels of a plurality of biomarkers, the plurality of biomarkers comprising: (a) Galectin-3, P16 and androgen receptor; (b) Galectin-3, P16 and HBME-1 ; or (c) CK19 and Vascular Endothelial Growth Factor.
  • the present invention provides a method of confirming a differentiated thyroid cancer diagnosis made by histopathology or cytomorphology assessment of a sample.
  • the method comprises the steps: determining the levels of a plurality of biomarkers in the sample, the plurality of biomarkers comprising: (a) Galectin-3, P16 and androgen receptor; (b) Galectin-3, P16 and HBME-1 ; or (c) CK19 and Vascular Endothelial
  • immunohistochemistry and immunocytochemistry methods allow for rapid evaluation of discriminatory molecular markers at the protein level and are routinely carried out in most clinical laboratories. Evaluation of molecular markers by immunocytochemistry can be done relatively easily, inexpensively, reproducibly, and could potentially serve as a useful adjunct to cytomorphology for improving the accuracy of thyroid cancer diagnosis.
  • biomarker panels described herein promotes more accurate preoperative classification and diagnosis of thyroid lesions and thus can improve the clinical management of patients who present with nodular thyroid disease.
  • Figure 1 is illustrates sample tissue microarray core immunohistochemistry for papillary carcinoma cores exhibiting increased expression of: AR (A); Aurora-A (B); CK19 (C);
  • Figure 2 is a hierarchical clustering of 35 biomarkers.
  • Figure 3 is a hierarchical clustering of 7 biomarkers.
  • Figure 4 is a multidimensional scaling (MDS) plot from Random Forests classification of benign versus malignant thyroid tumors.
  • a method of using a plurality of biomarkers for diagnosing differentiated thyroid cancer is described herein. Further, methods of classifying subtypes of thyroid cancer, assessing the prognosis of thyroid cancer, or developing treatments for thyroid cancer are also described. The plurality of biomarkers could be useful in tests, assays or kits.
  • panel as applied herein to biomarkers, it is meant a plurality of biomarkers intended for use together through concurrent, simultaneous, parallel or serial determination of either qualitative or qualitative measurement or assessment.
  • differentiated thyroid cancer it is meant to encompass all histologic forms, types and subtypes of papillary, follicular, and Hurthle cell carcinoma originating from thyrocytes. This group represents the vast majority of thyroid cancers. Certain forms of thyroid cancer not generally included as differentiated thyroid cancer, for example medullary carcinoma (which originates from c cells) and anaplastic carcinoma, which originates from differentiated thyroid cancer, but is distinguishable therefrom.
  • level or “levels” with reference to a biomarker or a plurality of biomarkers is meant to encompass a score, quantitative measurement, a qualitative assessment, or other acceptable observation obtained when a biomarker or observation correlated to a biomarker is assessed.
  • a method of diagnosing differentiated thyroid cancer in a subject comprises the steps of: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising Galectin-3, P16 and androgen receptor; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the plurality of biomarkers of the method can further comprise: Cytokeratin 19,
  • a method of diagnosing differentiated thyroid cancer in a subject comprises the steps of: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising Galectin-3, P16 and HBME-1 ; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the plurality of biomarkers can further comprise: Cytokeratin 19, Vascular Endothelial Growth Factor, androgen receptor, and/or Aurora-A.
  • a method of diagnosing differentiated thyroid cancer in a subject comprises the steps of: obtaining a thyroid tissue sample from the subject; determining, in the sample, a level of each of a plurality of biomarkers in the sample, the plurality of biomarkers comprising CK19 and Vascular Endothelial Growth Factor; and comparing the levels determined against a reference to determine if the levels indicate differentiated thyroid cancer.
  • the plurality of biomarkers can further comprise Galectin-3.
  • the plurality of biomarkers can still further comprise: androgen receptor, p16, Aurora-A, and/or HBME-1.
  • the method of diagnosing differentiated thyroid cancer can comprise obtaining a thyroid tissue sample by fine needle aspiration biopsy.
  • a method of confirming a differentiated thyroid cancer diagnosis made by histopathology or cytomorphology assessment of a sample comprises the steps of: determining the levels of a plurality of biomarkers in the sample, the plurality of biomarkers comprising: (a) Galectin-3, P16 and androgen receptor; (b) Galectin-3, P16 and HBME-1 ; or (c) CK19 and Vascular Endothelial Growth Factor; and comparing the levels determined against a reference to determine if the levels confirm a differentiated thyroid cancer diagnosis.
  • the methods can comprise using immunohistochemistry or immunocytochemistry to determine if the levels indicate differentiated thyroid cancer.
  • the reference used in the methods can be determined from a level of the biomarker in known non-cancerous thyroid tissue or known differentiated thyroid cancer tissue. The reference can also be determined from a level of the biomarker in the subject's own noncancerous tissue.
  • a kit is provided for the diagnosis of differentiated thyroid cancer in a subject.
  • the kit comprises reagents, and instructions for their use, for determining in a tissue sample levels of a plurality of biomarkers, the plurality of biomarkers comprising: (a) Galectin-3, P16 and androgen receptor; (b) Galectin-3, P16 and HBME-1 ; or (c) CK19 and Vascular Endothelial Growth Factor.
  • the plurality of biomarkers can also comprise (a) and further comprise
  • the plurality of biomarkers can also comprise (b) and further comprise Cytokeratin 19, Vascular Endothelial Growth Factor, androgen receptor, and/or Aurora-A.
  • the plurality of biomarkers can comprise (c) and further comprise Galectin-3.
  • the plurality of biomarkers can comprise (c), Galectin-3 and further comprise Aurora-A, HBME-1 , P16 and/or androgen receptor. [0047]
  • the level of each member of a plurality of biomarkers is determined. The levels determined are compared against a reference to determine if the levels indicate differentiated thyroid cancer.
  • Determining a level of individual biomarkers, or a plurality of biomarkers can be achieved using any number of different techniques known in the art. Such techniques could include, but are not limited to, determination of biomarker changes at the level of DNA (e.g. amplification, deletion, duplication etc.), RNA expression (by detection of various biomarker transcripts, including alternative transcripts, using, for example, PCR-based/amplification approaches, microarrays, exon arrays or other transcriptome-based techniques) and protein (including, for example, proteomics-based approaches).
  • determination of biomarker changes at the level of DNA e.g. amplification, deletion, duplication etc.
  • RNA expression by detection of various biomarker transcripts, including alternative transcripts, using, for example, PCR-based/amplification approaches, microarrays, exon arrays or other transcriptome-based techniques
  • protein including, for example, proteomics-based approaches.
  • Some of the most important characteristics required for a diagnostic test to have clinical utility are: ease of usage, reproducibility, and reliability.
  • a variety of methodologies can be utilized to evaluate diagnostic immunocytochemical marker expression in fine needle aspiration biopsy specimens from thyroid tumors.
  • the levels of the plurality of biomarkers are determined using immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • determining the level of a biomarker can be achieved by direct or indirect immunohistochemical detection of a biomarker using an appropriate antibody and detection reagents as known in the art.
  • tissue is treated to rupture the membranes, usually by using a kind of detergent, such as Triton X-100. Some antigens also need an additional step for unmasking, resulting in better detection results.
  • a labeled antibody e.g. FITC conjugated antiserum
  • tissue antigen i.e. the biomarker
  • a secondary antibody is reacted with the primary antibody.
  • the secondary antibody can be a labeled antibody, which is labeled, for example, with a fluorescent dye or an enzyme.
  • a biotinylated secondary antibody can also be used.
  • the biotinylated secondary antibody is detected with an enzymatic avidin or steptavidin conjugate, for example streptavidin-horseradish peroxidase.
  • This exemplary conjugate can be reacted with 3,3'- Diaminobenzidine (DAB) to produce a brown staining wherever the primary and secondary antibodies have detected the biomarker.
  • DAB 3,3'- Diaminobenzidine
  • Immunocytochemistry-based methods incorporating appropriate antibodies to the markers and detection reagents are also known in the art. Similar to IHC, immunocytochemical methods determine the level of a biomarker using antibodies which can specifically bind to the biomarker of interest. Primary antibodies or antisera can be used for detection. A direct method can be used which incorporates a detectable tag (for example: a fluorescent molecule, gold particles, etc.,) directly to the antibody that is then allowed to bind to the biomarker in a cell. [0055] Alternatively, an indirect method can be used. In one such method, the biomarker is bound by a primary antibody which is then amplified by use of a secondary antibody which binds to the primary antibody.
  • a detectable tag for example: a fluorescent molecule, gold particles, etc.
  • the second antibody can incorporate a detectable tag, as described above.
  • a tertiary reagent could be used.
  • a tertiary reagent could be bound to the secondary antibody could contain an enzymatic moiety.
  • the enzymatic moiety converts the substrate into a detectable reaction product (e.g. a dye) in the same location as the biomarker.
  • a detectable reaction product e.g. a dye
  • DAB (3,3'-Diaminobenzidine).
  • these reagents produces a detectable reaction product after exposure to the appropriate enzyme (e.g., horseradish peroxidase conjugated to an antibody reagent).
  • the secondary antibody may be covalently linked to a fluorophore which is detected in a fluorescence or confocal microscope.
  • a number of proteomic techniques which can detect biomarkers are known in the art. Such techniques include, but are not limited to: western blot analysis, enzyme linked immunosorbent assay (ELISA) or mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) is a mass spectrometry method for detecting biomarkers. In proteomics, MALDI can be used for the identification of proteins isolated through gel electrophoresis: SDS-PAGE, size exclusion chromatography, and two-dimensional gel electrophoresis.
  • ELISA enzyme linked immunosorbent assay
  • MALDI Matrix-assisted laser desorption/ionization
  • Determination of a biomarker can also be undertaken at the RNA level.
  • RNA blot is a method routinely used in molecular biology to check for the presence of a RNA sequence in an RNA sample.
  • Northern blotting combines agarose gel electrophoresis for size separation of RNA with methods to transfer the size-separated RNA to a filter membrane for probe hybridization.
  • the probe can be labeled so that it can be detected, usually by incorporating radioactivity or tagging the probe with a fluorescent or chromogenic dye.
  • Hybridized probe and by correlation the biomarker, can be visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
  • a microarray consists of an arrayed series of thousands of microscopic spots of oligonucleotides containing a specific sequence that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions.
  • Probe-target hybridization can be detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • the probes are attached to a solid surface by a covalent bond to a chemical matrix.
  • the solid surface can be glass or a silicon chip, or microscopic beads.
  • Microarrays can be used to detect DNA (as in comparative genomic hybridization), or detect RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins.
  • the process of measuring gene expression via cDNA is called expression analysis or expression profiling.
  • Exon junction array or "exon array”.
  • Exon arrays use probes specific to the expected or potential splice sites of predicted exons for a gene.
  • Exon arrays employ probes designed to detect each individual exon for known or predicted genes, and can be used for detecting different splicing isoforms.
  • RNA Ribonucleic acid
  • Determination of a biomarker can also be undertaken at the DNA level.
  • Detection of DNA can be achieved using methods known in the art, such as Southern blot analysis, microarrays or other techniques known in the art.
  • a Southern blot combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization.
  • the probe can be labeled so that it can be detected, usually by incorporating radioactivity or tagging the probe with a fluorescent or chromogenic dye.
  • the hybridized probe, and by correlation the biomarker can be visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
  • DNA microarrays consists of an arrayed series of thousands of microscopic spots of oligonucleotides containing a specific sequence that are used as probes to hybridize a cDNA sample (called target) under high-stringency conditions.
  • Probe-target hybridization can be detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • the probes are attached to a solid surface by a covalent bond to a chemical matrix.
  • the solid surface can be glass or a silicon chip, or microscopic beads.
  • DNA can also be detected using polymerase chain reaction (PCR), or other similar techniques known in the art.
  • PCR can be used to quantitatively measure starting amounts of DNA, cDNA or RNA.
  • fluorescent dyes such as Sybr Green
  • fluorophore-containing DNA probes such as TaqMan
  • biomarkers of the invention could be measured in specimens such as FNAB (a preferred embodiment), formalin-fixed paraffin embedded (FFPE) tissue, tissue microarrays (TMA), fresh-frozen or freshly-obtained thyroid biopsy material.
  • specimens such as FNAB (a preferred embodiment), formalin-fixed paraffin embedded (FFPE) tissue, tissue microarrays (TMA), fresh-frozen or freshly-obtained thyroid biopsy material.
  • a number of references or standards would be considered appropriate.
  • a biomarker level could be compared with the level of the biomarker in tissue known to be non-cancerous thyroid tissue.
  • An individual's own tissue could be used as a reference for comparison, or a population- derived value may be obtained.
  • Local reference standards can be established if it is found that epidemiological variation exists among populations studied. Standards established within a group having a common demographic may also be established based on, for example, age, sex, smoking status, and other potentially influencing factors.
  • Statistical methods can be used to define the range of values for a reference standard.
  • a range could be values within one standard deviation of the mean, and preferably values within two standard deviations of the mean.
  • a level of biomarker within such a range may indicate that a tissue sample is non-cancerous.
  • a level of biomarker outside of the defined range would indicate that the tissue is differentiated thyroid cancer tissue.
  • An alternate reference for comparison of the biomarker level could be determined by establishing the level of biomarker in tissue known to be differentiated thyroid cancer tissue.
  • Statistical methods can be used to define the range of values for the alternate reference.
  • a range could be values within one standard deviation of the mean, and preferably values within two standard deviations of the mean.
  • Biomarker levels may be compared with a threshold level beyond which a positive indication is determined.
  • a threshold level of change for example, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 125%, 150%, 200%, 300%, 400% or 500% increase, or a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% or 100% decrease, relative to a reference standard may be set or pre-determined to allow a comparison from which a positive or negative indication can be derived.
  • biomarker When a biomarker is up-regulated as an indication of a positive result, this can be easily distinguished from background (low-level values). Background expression levels may be used to form a "cut-off' above which increased staining will be scored as significant positive expression. Positive expression may be represented by a high level of antigen in tissue, or by a high proportion of cells from within a tissue that give a positive signal.
  • a biomarker can be determined to be statistically different from a reference standard or background if the mean or median expression level of the biomarker in a group forming a reference standard versus a group representing a positive diagnosis of thyroid cancer is to be statistically significant, as was the case in the present invention.
  • Common tests for statistical significance include, among others, t-test, ANOVA 1 Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ration. Individual samples (of unknown status) can be compared with data from the reference group (negative control), and/or compared with data obtained from a positive control group known to have cancer.
  • the phrases "indicative of cancer” or “diagnosis indicative of differentiated thyroid cancer” when referring to levels of biomarkers or an expression pattern of biomarkers indicates an expression pattern which is diagnostic or confirmatory of disease such that the biomarker levels or expression pattern is found significantly more often in subjects with a disease than in subjects without the disease (as determined using routine statistical methods setting confidence levels at a typical minimum, such as at 95%).
  • an expression pattern which is indicative of disease is found in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more in subjects who have the disease, and is found in less than 10%, less than 8%, less than 5%, less than 2.5%, or less than 1% of patients who do not have the disease.
  • the phrase may also indicate an expression pattern which is diagnostic of disease such that the expression pattern more properly categorizes expression patterns of individuals with disease as compared with control expression patterns of individuals without disease using statistical algorithms for class prediction. Such comparisons would readily be understood by a person skilled in the art, and could be implemented using computerized means, such as for example commercially available programs available from Silicon Genetics (e.g. GeneSpringTM).
  • a positive diagnosis could be based upon comparison of levels found in the population with respect to the expression that is seen in cancer compared to benign.
  • Each marker may have a different scoring system and these variables can be evaluated both continuously and/or with different cut-off points to see what results in the best performance.
  • a reference standard may easily be based on the expression by cancer and benign lesions seen in acceptable sized cohorts. Cut-off points to be utilized can be specified for each marker evaluated, based on population cohorts.
  • differentiated thyroid cancer can be diagnosed if the levels of the plurality of biomarkers being determined, in comparison to the reference, indicate, or on balance indicate, that the tissue sample is differentiated thyroid cancer.
  • Biomarkers described herein can be used to support results and data obtained in conventional ways, such as by histopathology or cytomorphology examination of stained tissue sections. Should a conventionally derived diagnosis require confirmation, the panels of biomarkers described herein can provide such confirmation
  • the prognosis of thyroid cancer can be observed and trends over time can be followed based on the described panels of biomarkers. Such observations can be used to determine prognosis or outcome of a lesion in a patient harbouring a malignant thyroid lesion. For example, the responsiveness to surgery or alternative treatment interventions may be observed and correlated to the marker panels described herein so that at the time a test assay is conducted, decisions can then be made as to the possible outcome for each method of treatment. Personalized treatment plans can be undertaken, depending on the outcome of the assessment of a biomarker panel. In this way, patients and health care providers have more information on which to make treatment decisions, as well as personal decisions.
  • biomarkers/biomarker panels of the invention may also useful for a) disease sub-classification of malignant thyroid lesions, b) determining the prognosis or outcome of a lesion/patient harbouring a malignant thyroid lesion, c) predicting the response to surgery or other treatment intervention and/or d) as potential therapeutic targets alone or in combination to enable the development of targeted agents/drugs for treatment of DTC or thyroid cancer in general.
  • kits comprising reagents for detecting or measuring the expression levels of a single biomarker or a panel of biomarkers in a sample tissue, could be useful for the diagnosis/classification of lesions as either malignant (DTC) or benign.
  • panels of biomarkers to be detected or measured include, for example: Galectin-3, Cytokeratin 19, Vascular Endothelial Growth Factor (VEGF), Androgen Receptor (AR), p16, Aurora-A, and HBME-1 ; Galectin-3, P16 and AR; Galectin-3, P16 and HBME-1 ; Galectin-3, CK19 and VEGF; or CK19 and VEGF.
  • the levels of various pluralities of biomarkers were determined to be indicative of differentiated thyroid cancer.
  • a large panel of immunohistochemical markers were studied and 35 markers (8 down-regulated and 27 up-regulated) were found to be able to discriminate between benign and malignant thyroid tumors.
  • Some examples of the plurality of biomarkers useful in a method according to the present invention include, for example: Galectin-3, P16 and AR; Galectin-3, P16 and HBME-1 ; Galectin-3, CK19 and VEGF; or Galectin-3, CK19, VEGF, P16, AR, HBME-1 and Aurora-A. Further biomarkers for use with the invention, and combinations thereof, are described below.
  • TMA tissue microarray block
  • a Leica microtome was used to cut serial 4-mm sections from the TMA blocks that were then transferred onto adhesive-coated glass slides for immunohistochemistry. The dilutions, antigen retrieval methods, and other details for 54 antibodies have been previously described by Wiseman et al. [11], and are incorporated herein by reference. [0088] All antibodies were optimized for thyroid tissue according to the manufacturer's instructions and for each appropriate positive and negative controls were utilized. Two pathologists that were blinded to all clinical data examined the stained TMA sections at high power magnification in order to evaluate expression of the molecular markers. The scoring systems utilized were based on previously published reports of immunohistochemical studies evaluating these markers and have been previously described [11] and/or are summarized in Table 1 and Table 2. Examples of antibodies useful for detection of particular biomarkers of interest include:
  • IGFBP2 Insulin-like growth factor binding protein 2
  • IGFBP5 Insulin-like growth factor binding protein 5
  • MRAS Muscle RAS oncogene homolog
  • 3 >40% cells positive (strong staining intensity)
  • 2 5-40% cells positive (moderate staining intensity)
  • IGFBP2, IGFBP5 and MRAS were cytoplasmically stained, and antigen retrieval was with CC1 (EDTA Buffer) VENTANA.
  • Figure 1 shows microarray cores of papillary carcinoma exhibiting increased expression of AR (A), Aurora-A (B), CK19 (C), Galectin-3 (D), HBME-1 (E), P16 (F), and decreased expression of VEGF (G) (Original magnification X400). All scores were recorded in a standardized TMA case map that corresponded to each TMA section (Microsoft Excel; Microsoft, Redmond, WA) and all data was processed by custom TMA-deconvoluter software (developed by O. L. G. using the Perl programming language). The deconvoluted data was then transferred into a master database, which also included all collected clinical and pathologic data, for statistical analysis.
  • This test does not require the marker scores to be grouped but instead uses the actual semi-quantitative scores.
  • the specimens and markers were also clustered using a hierarchical clustering algorithm and heat maps generated to visualize the data using the 'gplots' library (version 2.3.0) for the R programming language (version 2.3.1 ).
  • the entire set of markers were also evaluated for their utility in classification (benign versus malignant) using the Random Forests (RF) classifier algorithm.
  • RF Random Forests
  • An out-of-bag cross-validation was used to assess classification performance and Gini variable importance was determined for each marker.
  • a 'combination analysis' was carried out in which all possible two-gene and three-gene combinations of a subset of the markers (e.g.
  • the final study cohort was composed of 100 individuals diagnosed with benign thyroid lesions and 99 individuals diagnosed with PTC, follicular carcinoma (FTC), or Hurthle cell carcinoma (HCC). Six patients diagnosed with medullary carcinoma, and one of their associated lymph node metastases, were excluded from the study cohort. Thus, there were 23 lymph node metastasis specimens, each of which corresponded to a study cancer patient, that were also evaluated.
  • FTC follicular carcinoma
  • HCC Hurthle cell carcinoma
  • marker scores were grouped as either negative/low (score ⁇ i ) or positive/high (score ⁇ 2).
  • the table shows the number of patient specimens staining negative or positive and the percent positive for benign versus malignant tumors.
  • the Random Forests algorithm was able to achieve a good classification of patients into their correct diagnostic group using the 35 biomarkers shown in Table 6.
  • the molecular marker score has an estimated sensitivity of 87.9%, specificity of 94.0% and overall accuracy of 91.0%. Specifically, this translates to an estimated misclassification of 6 out of 100 benign and 12 of 96 malignant specimens. This performance is graphically illustrated in Figures 2 and 3 by the good separation of benign (indicated by light portion of the side bar) versus the malignant (dark portion of the side bar) specimens in the hierarchical clustering heat maps.
  • the multidimensional scaling (MDS) plot also shows a good separation of benign (Bs) versus malignant (Ms) based on the Random Forests proximity measurements ( Figure 4).
  • Bs benign
  • Ms malignant
  • Figure 4 Random Forests proximity measurements
  • Figure 2 illustrates that all 35 significant markers (by MU test) were submitted to a simple hierarchical clustering method and a heat map of marker expression was generated. Both patient samples and markers were clustered according to marker expression level. The key indicates marker score values of 0 - 4 according to the scoring systems. Along the bottom axis, all markers are listed. Along the left axis, benign samples are indicated by a light bar and malignant by a dark bar. The light bar on the horizontal top axis indicates up- regulated markers and the dark bar on the horizontal top axis indicates down-regulated markers. Separation between benign and malignant was observed but separation improved when only the most discriminatory markers (determined by RF variable importance) were included (Figure 3).
  • Figure 3 illustrates that the seven most discriminatory markers (variable importance greater than ⁇ 5 in RF classifier) were submitted to a simple hierarchical clustering method and a heat map of marker expression was generated. Both patient specimens and markers were clustered according to marker expression level. The key indicates marker score values of 0 - 3 according to the scoring systems. Along the bottom axis, the significant markers are listed. Without the noise of the less informative markers, hierarchical clustering was better able to separate the benign specimens (light bar on the left axis) from the malignant specimens (dark bar on the left axis). The light bar on the horizontal top axis indicates up- regulated markers and the dark bar on the horizontal top axis indicates down-regulated markers.
  • FIG. 4 illustrates that the 199 thyroid specimens were visualized using a multidimensional scaling plot based on the random forest dissimilarity measurement. Tumors are labelled by their pathology class ( 1 B' for benign and 'M 1 for malignant). A multidimensional scaling (MDS) plot from Random Forests classification of benign versus malignant thyroid tumors is shown.
  • MDS multidimensional scaling
  • VEGF unlike the other 6 top diagnostic markers identified in the current study, exhibited significantly decreased expression in cancer compared to benign lesions. Whether a marker that is down-regulated in cancer has as much clinical relevance and applicability as a diagnostic marker that is up-regulated in cancer will require further study.
  • top markers Galectin-3, CK19 and VEGF
  • RF classifier When only the top three markers (Galectin-3, CK19 and VEGF) were submitted to the RF classifier we obtained an estimated sensitivity 84.8%, specificity of 92.0% and overall accuracy of 88.4% with estimated misclassification of 8 benign and 15 malignant samples. Therefore, the top markers, especially the top 3, are predominantly responsible for the overall performance of the classifier.
  • the combination analysis also showed that there are several different two- or three- marker combinations from the top 7 that can achieve performance nearly as good as the complete marker set.
  • Table 7 summarizes the performances of the top 3 markers, top 7 markers and
  • biomarkers include, for example: Galectin-3, Cytokeratin 19, Vascular Endothelial Growth Factor (VEGF), Androgen Receptor (AR), p16, Aurora-A, and HBME-1 ; Galectin-3, P16 and AR; Galectin-3, P16 and HBME-1 ; Galectin-3, CK19 and VEGF; or CK19 and VEGF.
  • VEGF Vascular Endothelial Growth Factor
  • AR Androgen Receptor
  • the measurement of the expression level of a specific biomarker, either alone or in combination with other biomarkers, in thyroid lesions can be useful for the diagnosis/classification of these lesions as either malignant (DTC) or benign.
  • Some examples of the specific biomarker are shown in Table 6 and include, for example: the androgen receptor (AR); Aurora-A; VEGF; or P16.
  • AR protein expression is increased (+) in malignant (DTC) versus benign thyroid lesions.
  • Aurora-A protein expression is increased (+) in malignant (DTC) versus benign thyroid lesions.
  • VEGF protein expression is decreased (-) in malignant (DTC) versus benign thyroid lesions.
  • P16 protein expression is increased (+) in malignant (DTC) versus benign thyroid lesions.
  • a method for diagnosing differentiated thyroid cancer is described below.
  • a sample of thyroid tissue is obtained from a subject using fine needle aspiration biopsy.
  • the tissue is fixed in 2% paraformaldehyde, Bouins solution, or other fixative from 30 minutes to overnight and subsequently embedded in paraffin.
  • the tissue is sectioned in 5-10 micrometers slices.
  • the slices are incubated 2-3 times in xylene for 10 minutes each, and incubated twice in 100% ethanol for 2 minutes each. Hydration is undertaken by placing in 95%, 70%, 50%, 30% ethanol for 2 minutes each.
  • Slides are placed in a 0.25 M Tris-HCI at pH 7.5 buffer for 5 minutes.
  • a sample of thyroid tissue is obtained from a subject by biopsy of the thyroid tissue.
  • the sample is prepared for flow cytometry using standard techniques and incubated with a primary antibody directed against the biomarker of interest.
  • the sample is washed and blocked.
  • the sample is incubated with a secondary antibody directed against the primary antibody and labeled with a fluorescent tag.
  • the sample is washed and detection the fluorescent sample is conducted using flow cytometry. Compare the fluorescent biomarkers detection to biomarker references previously determined from cancerous or non-cancerous tissue obtained from the subject.
  • a plurality of flow cytometric markers are used for detection and comparison of all desired biomarkers.
  • a sample thyroid tissue is obtained from a subject by biopsy of the thyroid tissue.
  • the sample is prepared for DNA microarray analysis using standard techniques to generate cDNA probes.
  • cDNA probes are hybridized to a DNA array.
  • Detection the hybridization signal is undertaken using fluorescent markers and laser confocal devices.
  • the signal intensities are analysed to determine if the biomarkers of interest are up-regulated, down-regulated, or unchanged. Comparison is made of the intensities to biomarker references previously determined from tissue samples of known differentiated thyroid cancer.
  • Galectin-3 is a presurgical marker of human thyroid carcinoma. Cancer Res. 15;58:3015-3020, 1998.
  • VEGF vascular endothelial growth factor

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Abstract

La présente invention concerne un procédé de diagnostic du cancer différencié de la thyroïde chez un sujet. Le procédé comprend : l'obtention d'un échantillon de tissu de la thyroïde du sujet; la détermination, dans l'échantillon, du niveau de chaque élément d'une pluralité de biomarqueurs dans l'échantillon, et la comparaison des niveaux déterminés par rapport à une référence afin de déterminer si les niveaux indiquent un cancer différencié de la thyroïde. La pluralité de biomarqueurs peut être un groupe comprenant : (a) la galectine-3, la P16 et le récepteur androgène; (b) la galectine-3, la P16 et le HBME-1; ou (c) la cytokératine 19 et le facteur de croissance endothéliale vasculaire; chacun d’entre eux pouvant être utilisé conjointement avec d'autres biomarqueurs.
PCT/CA2009/000306 2008-03-13 2009-03-12 Biomarqueurs pour diagnostic du cancer différencié de la thyroïde WO2009111881A1 (fr)

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US10672504B2 (en) 2008-11-17 2020-06-02 Veracyte, Inc. Algorithms for disease diagnostics
US10934587B2 (en) 2009-05-07 2021-03-02 Veracyte, Inc. Methods and compositions for diagnosis of thyroid conditions
WO2011018288A1 (fr) * 2009-08-13 2011-02-17 Basf Se Moyens et procédés pour diagnostiquer des désordres de la thyroïde
CN102472744A (zh) * 2009-08-13 2012-05-23 巴斯夫欧洲公司 诊断甲状腺疾病的手段和方法
CN102472744B (zh) * 2009-08-13 2015-11-25 巴斯夫欧洲公司 诊断甲状腺疾病的手段和方法
US10731223B2 (en) 2009-12-09 2020-08-04 Veracyte, Inc. Algorithms for disease diagnostics
WO2011079846A2 (fr) 2009-12-30 2011-07-07 Rigshospitalet Classification d'arnm de néoplasie folliculaire thyroïdienne
EP2366800A1 (fr) 2010-03-01 2011-09-21 Centrum Onkologii-Instytut im M. Sklodowskiej-Curie Oddzial w Gliwicach Kit, méthode et l'utilisation pour le diagnostic de cancer papillaire de la thyroïde en utilisant un profil d'expression génique
US10260103B2 (en) 2012-11-27 2019-04-16 Pontificia Universidad Catolica De Chile Compositions and methods for diagnosing thyroid tumors
US11976329B2 (en) 2013-03-15 2024-05-07 Veracyte, Inc. Methods and systems for detecting usual interstitial pneumonia
US11639527B2 (en) 2014-11-05 2023-05-02 Veracyte, Inc. Methods for nucleic acid sequencing
US10534005B2 (en) 2015-05-04 2020-01-14 Universita'delgi Studi Di Milano-Bicocca Method for the in vitro diagnosis of thyroid diseases
WO2017064664A3 (fr) * 2015-05-04 2017-05-26 Universita' Degli Studi Di Milano - Bicocca Procédé de diagnostic in vitro de maladies thyroïdiennes
US11217329B1 (en) 2017-06-23 2022-01-04 Veracyte, Inc. Methods and systems for determining biological sample integrity
CN111808950A (zh) * 2020-06-02 2020-10-23 中南大学湘雅医院 一种甲状腺乳头状癌miRNA标志物及其应用
CN111808950B (zh) * 2020-06-02 2023-11-14 中南大学湘雅医院 一种甲状腺乳头状癌miRNA标志物及其应用
CN114264828B (zh) * 2022-01-28 2023-09-08 中国科学院基础医学与肿瘤研究所(筹) 鉴别良性甲状腺结节与甲状腺癌的生物标志物及其应用
CN114264828A (zh) * 2022-01-28 2022-04-01 中国科学院基础医学与肿瘤研究所(筹) 鉴别良性甲状腺结节与甲状腺癌的生物标志物及其应用
WO2023179263A1 (fr) * 2022-03-22 2023-09-28 西湖欧米(杭州)生物科技有限公司 Système, modèle et trousse d'évaluation de degré ou de probabilité de malignité de nodules thyroïdiens
CN115144599B (zh) * 2022-09-05 2023-01-06 西湖大学 蛋白组合在制备对儿童甲状腺癌进行预后分层的试剂盒中的用途及其试剂盒、系统
CN115144599A (zh) * 2022-09-05 2022-10-04 西湖大学 蛋白组合在制备对儿童甲状腺癌进行预后分层的试剂盒中的用途及其试剂盒、系统

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