CA2534871C - Three-gene test to differentiate malignant from benign thyroid nodules - Google Patents

Abstract

Methods of identifying malignant thyroid tissue comprising testing a thyroid tissue sample for the expression of at least two genes chosen from CCND2, PCSK2, and PLAB. Kits for use in the disclosed methods are also provided.

Description

THREE-GENE TEST TO DIFFERENTIATE MALIGNANT FROM BENIGN
THYROID NODULES
[0011 Work leading to this invention was supported at least in part by National Cancer Institute grants CA16058 and CA16059. The United States government has certain rights in this invention.
Field of the Invention

[002] This invention generally relates to tests for determining whether tissue is malignant. In particular, the tests relate to thyroid tissue, and more particularly, to thyroid nodules. The tests generally involve testing for the expression of two or more of the three genes identified and known in the art as CCND2, PCSK2, and PLAB. In some embodiments, the testing involves assaying for the expression of at least two of the three, and in other embodiments, the testing involves assaying forme expression of all three. The test involves measuring and comparing the relative expression levels of the genes in sample tissues and in normal or non-malignant thyroid tissues ("controls"), wherein differences between the expression levels of the genes indicative of the presence or absence of malignancy.
Background of the Invention

[003] Thyroid cancer derived from the follicular epithelial cell is the most common endocrine cancer. Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) account for the great majority of all thyroid malignancies (1). An estimated 7% of the adult population (275,000 in 1999 in the United States alone) develops clinically significant thyroid nodules during their lifetime (2). The advent of thyroid ultrasound now allows for an increasing number of nodules to be diagnosed, and it is novv reccgnized that nodules are present in an estimated 50% of the 22135321.2 general population and are detected at a subclinical level Because only 10% of these nodules will be a true malignancy, preoperative testing to differentiate benign from malignant nodules has been developed (3,4), Currently, fine needle aspiration (FNA) biopsy is the best diagnostic tool available for preoperative diagnosis.
The FNA-based cytological diagnosis can be straightforward. Hovitever, approximately 20% (ranging from 9.2-42%) of all FNA will result in an inconclusive or suspicious outcome, especially if a follicular proliferation is found; the differentiation between a benign follicular neoplasia, especially follicular adenomas (FAs), and FTC.
based on the morphological features on FNA cytology is virtually impossible (5-8).

[004] Therefore, because of the obvious difficulty in such preoperative diagnoses, surgical removal of the involved thyroid gland is routinely performed for diagnostic purposes in the seftirig of thyroid nodules and follicular cytology.
However, in only 10-20% of these cases would a follicular thyroid malignancy be found on final histology, resulting in unnecessary surgery for the vast majority of patients (4-6,8,9). More importantly, false-negative cytologies can lead to delayed treatment with potentially serious consequences for the patient (10).

[005] Regarding the obvious limitation of FNA cytology in the preoperative diagnosis, there is a clinical need for new, reliable preoperative markers to distinguish benign from malignant thyroid nodules. Nonetheless, whereas numerous assays have been developed in an attempt to reduce these inconclusive preoperative diagnoses, none has yet proven more successful than FNA cytology in the clinical setting (4,11-13). A possible underlying cause for this clinical problem is the continued limited understanding of the biological relationship of the different 22135321,2 2 benign thyroid neoplasias to each other and to thyroid carcinoma, despite much research in this field (11, 14-17).
[0061 Therefore, to directly address the clinically relevant issue, we sought to elucidate further the molecular differences between benign follicular neoplasia and FTC. We took a global expression array approach to dissect out the minimal number of genes that can play a fundamental role in the early steps of FTC
carcinogeriesis, thus, not only giving new biological insight, but also allowing us to differentiate FTC, even at the minimally invasive stage, from benign follicular neoplasia by evaluating expression of a limited set of genes. The use of objective molecular markers will serve as an adjunct in the preoperative diagnosis of follicular thyroid cancer.
SUMMARY OF THE INVENTION
[007] In various embodiments, the invention provides methods for identifying malignant thyroid tissue and methods for differentiating between malignant and non-malignant neoplasms of thyroid tissue. Accoding to the various embodiments, a thyroid tissue sample is tested for the expression of at least two genes chosen from CCND2, PCSK2, and PLAB, wherein the level of expression is determined by measuring the amount of mRNA corresponding to the gene of interest. Figures 5, 7, and 9, respectively, each show one embodiment of each the mRNA sequences of interest. In some embodiments, the thyroid tissue sample is tested for the expression of CCND2 and PCSK2. In other embodiments, the thyroid tissue sample is tested for the expression of CCND2 and PLAB. And in yet other embodiments, the thyroid tissue sample is tested for the expression of PCSk2 and PLAB. In some embodiments according to the invention, the thyroid tissue sample is tested for the expression of CCAID2, PLAB, and PCSK2. In yet other embodiments, the expression of other genes such as those noted herein, may be used to assist in the identification of malignant tissue_ A variety of methods and tools are known in the art for measuring levels of expression, including direct measurement of mRNA
levels. The examples provided herein are not intended to be limiting, and other methods as described in the references noted herein may also be used in carrying out the invention.
[008] In some embodiments, a determination of the presence of malignant thyroid tissue is obtained wherein the level of expression of two or more of the genes CCN'D2, PCSK2, and PLAB show changes as follows when compared with normal thyroid tissue or tissue having otherwise benign nodules: decreased expression of COND2, decreased expression of PCSK2 and increased expression of PLAB. In other embodiments, variations in the levels of expression of at least two of the three genes are indicative of the presence of malignancy, according to the examples provided herein.
poq The invention also provides kits for identifying malignant thyroid tissue comprising means for assaying a thyroid tissue sample for the expression of at least two genes chosen from CCAID2, PCSK2, and AAR In some embodiments, the kits comprise at least two of the following: (a) a container containing at least one CCNO2 primer; (b) a container containing at least one PCSK2 primer; and (c) a container containing at least one PLAB primer.

CA. 2,534,871 Agent Ref. 70619/00002 [010] Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
[011] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
[012] The accompanying drawings are incorporated in and constitute a part of this specification, and together with the description, serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[013] Figure 1: Supervised hierarchical cluster analysis based on a set of 80 genes differentiates FTC from FA. Expression values of each gene across all samples were linearly scaled (standardized) to have a mean of 0 and SD of 1.
These standardized values were used to calculate the correlation between genes, based on the distance metric (1-correlation). The average linkage model was used for merging nodes. Over and under expression are indicated by shading.
[014] Figure 2: Classification of 24 FTCs and 31 benign thyroid nodules (training set and validation set) by linear discriminant analysis. The two groups of samples (12 FTCs and 12 FAs) in the training set can be distinguished perfectly based on the expression of CCND2 and PCSK2 (A). PCSK2 and PLAB have the same joint effect (B). The samples of the validation set can be 22135321.2 5 classified with a sensitivity of 66.7% (exact 95% confidence interval, 34.9-90.1%) and specificity of 100% for the combination of CCND2 and PCSK2 (A). Using PLAB

and PCSK2 combined, 91.7%(exact 95% confidence interval, 61.5-99.8%) of all FTCs in the validation set can be correctly identified and 94.7% (exact 95%
confidence interval, 74.0-99.9%) of the benign thyroid nodules can be correctly classified as well (B). See also Table 3. The joint performance of all three genes is demonstrated in Fig. 3.
[015] Figure 3: ROC curve based on the joint performance of PCSK2, PLAB, and CCND2 in the classification of an independent validation set of 31 samples [12 FTCs, 12 nonfunctioning thyroid nodules (five FAs and seven adenomatous nodules), two normal tissue, and five autonomous adenomas] by linear discriminant analysis. The linear combination of gene expression levels of PCSK2, CCND2, and PLAB with the coefficients -0.2763, -0.1896, and 0.3666, respectively, is used for classification. The arrow indicates that when three genes are used together in this linear combination with a cutpoint of 2.0, a sensitivity of 100%, or 12 of 12, specificity of 94.7, or 18 of 19 (exact 95% confidence interval, 74.0-99.9%) and accuracy of 96.7, or 30 of 31 (exact 95% confidence interval, 83.3-99.9%) are reached. See also Table 3.
[016] Figure 4: ROC curve showing the perfornlance of using antibodies against PCSK2 and CCND2 together in a second independent validation series of 83 samples. Each sample was assigned to one of five classes, according to the pattern of NC-derived expression (Table 4). When categories 3, 4, and 5 are considered to represent test positive cases (FTC), the classification of follicular 4135321,2 8 neoplasias based on the protein expression of CCND2 and PCSK2 shows a sensitivity of 89.5% (exact 95% confidence interval, 78.5-96.0%), a specificity of 80.8% (exact 95% confidence interval, 60.6-93.4%) and accuracy of 86.7% (exact 95% confidence interval, 77.5-93.2%; indicated by arrow in the curve), thus supporting the data derived from the more quantitative gene expression analysis (Fig. 3).
[017] Figure 6: CCND2 (cyclin D2) mRNA sequence. Other aliases for CCND2 include KIAK0002 and fidIGC102758.
[018] Figure 6: CCND2 (cyclin D2) amino acid sequence. Other aliases for CCND2 include KIAK0002 and MGC102758.
10191 Figure 7: PCSK2 (proprotein convertase subtilisinikexin type 2) mRNA
sequence. Other aliases for PCSK2 include NEC2 (neuroendoerine convertase 2), PC2 (prohorrnone convertase 2), and SPC2 (subtilisin-like prohorrnone convertase 2).
[020] Figure 8: PCSK2 (proprotein convertase subtilisinikexin type 2) amino acid sequence. Other aliases for PCSK2 include NEC2 (neuroendocrine convertase 2), PC2 (prohormone convertase 2), and SPC2 (subtilisin-like prehorrrione convertase 2).
[021] Figure 9: PLAB mRNA sequence. Other aliases for PLAB include GDF-15 (growth differentiation factor 15), GDF15, MIC-1, MIC1, NAG-1, PDF
(prostate differentiation factor), NSAID (non-steroidal anti-inflammatory drug)-activated protein, cornl, and PTGFB (P OF-beta)...
22135321.2 7 [022] Figure 10: PLAB amino acid sequence. Other aliases for NAB
include GDF-15 (growth differentiation factor 15), GDF15, MICA , tvliC1, NAG-1, PDF (prostate differentiation fador), NSA ID (non-steroidal anti-Inflammatory drug)-activated protein, , and PTGFB (PTGF-beta)õ.
[023] Figure 11: hTERT (human telomerase reverse transcriptase) mRNA
sequence of transcript variant #1. Variant #1 represents the longest transcript. Other aliases for hTERT include TERT (telomerase reverse transcriptase), TP2, TRT
(telomerase reverse transcriptase), EST2, TCS1 (telornerase catalytic subunit), and hEST2.
[024] Figure 12: hTERT (human telomerase reverse transcriptase) amino acid sequence of lisoform 1. Other aliases for hTERT include TERT (telomerase reverse transcriptase), TP2, TRT (telomerase reverse transcriptase), EST2.

(telomerase catalytic subunit), and hEST2.
[0251 Figure 13: hTERT (human telomerase reverse transcriptase) mRNA
sequence of transcript variant #2, Variant #2, also called alpha, uses an in-frame alternate splice site in the coding region, compared to variant #1. lsoform 2 is shorter than isoform 1 and lacks part of reverse transcriptase (RI) motif 3. Isofoan 2 is a dominant-negative inhibitor of telomerase activity. Other aliases for hTERT
include TERT (telomerase reverse transcriptase), TP2, TRT (telomerase reverse transcriptase), EST2, TCS1 (telomerase catalytic subunit), and hEST2.
[026] Figure 14: hTERT (human telomerase reverse transcriptase) amino acid sequence of isoform 2. Other aliases for hTERT indude TERT (telomerase 22135321,2 8 reverse transcriptase), TP2, TRT (telornerase reverse transcriptase), EST2, (telomerase catalytic subunit), and hEST2.
10271 Figure 15: hTERT (human telomerase reverse transcriptase) mRNA
sequence of transcript variant #3. Variant #3 lacks two axons in its coding region, resulting in a frameshift and early termination compared to variant #1.
Isoform 3 has a shorter and distinct C-terminus compared to Isofomi 1. Other aliases for hTERT
include TERT (telomerase reverse transcriptase), TP2, TRT (telomerase reverse transcriptase), EST2, TCS1 (telomerase catalytic subunit), and hEST2.
10281 Figure 16: hTERT (human telomerase reverse transcriptase) amino acid sequence of isoform 3. Other aliases for hTERT include TERT (telomerase reverse transcriptase), TP2, TRT telomerase reverse transcriptase), EST2, TCS1 (telomerase catalytic subunit), and hEST2.
10291 Figure 17: hTERT (human telomerase reverse transcriptase) mRNA
sequence of transcript variant #4. Variant #4 has multiple differences in the coding region, resulting in a frameshift and early termination compared to variant #1.
lsoform 4 has a shorter and distinct C-terminus, compared to variant #1. Other aliases for hTERT include TERT (telomerase reverse transcriptase), TP2, TRT
(telomerase reverse transcriptase), EST2, TCS1 (telomerase catalytic subunit), and hEST2.
10301 Figure 18: hTERT (human telomerase reverse transcriptase) amino acid sequence of isoform 4. Other aliases for hTERT include TERT (telomerase reverse transcriptase), TP2, TRT (telomerase reverse transcriptase), EST2, (telomerase catalytic subunit), and hEST2.
22135321.2 9 [031] Figure 19 C044 niRNA sequence of transcript variant 01. Variant 01 represents the longest transcript. It encodes the longest isoform 1. Other aliases for CD44 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-111, and MGC10468.
[032] Figure 20: CD44 amino acid sequence of isoform 1. Other aliases for CD44 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-HI, and MGC10468.
[033] Figure VI: CD44 mRNA sequence of transcript variant 02. Variant 02 Jacks an in-frame coding exon compared to variant 01. The resulting isoform 2 lacks an internal region, as compared to Isoform 1. Other aliases for C044 'include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, C0VV44, MUTCH-I, ECMR-III, and MGC10468.
[034] Figure 22: CD44 amino acid sequence of isoform 2. Other aliases for CD44 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-III, and MGC10468.
[035] Figure 23: C044 mRNA sequence of transcript variant 03. Variant 03, also known as CD44R, lacks multiple coding-exons compared to variant 01. The translation remains in-frame. The resulting isoform 3 lacks an internal segment, as compared to isoform 1. Other aliases for CD44 include IN, LHR, MC56, MDU2, IVIDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-III, and MGC10468.
[036] Figure 24: CD44 amino acid sequence of isoforrn 3. Other aliases for C044 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-III, and MGC10468.
0i353212 10 [037) Figure 25: 0044 mRNA sequence of transcript variant #4. Variant #4 lacks multiple coding-exons compared to variant #1. The translation remains in-frame. The resulting isoforrn 4 lacks an internal segment, as compared to isoform 1.
Other aliases for 0044 include IN, LHR, MC56, MDU21 MDU3, MIC4, Pgpl, CDW44, MUTCH-I, ECMR-III, and tincio46e.
[038] Figure 26: 0044 amino acid sequence of isoform 4. Other aliases for CD44 include IN, LHR, MC56, MDU2, M003, MIC4, Pgpl, C0W44, MUTCH-I, ECMR-Ill, and MGC10468.
[0391 Figure 27: 01344 mRNA sequence of transcript variant #5. Variant #5 lacks multiple coding-exons compared to variant #1. The translation frame is changed. The resulting isoform 5, also known as 0044 isoform RC, has a distinct and shorter C-terminus, as compared to isoform 1. Other aliases for 0D44 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDVV44, IVIUTCH-I, ECMR-III, and IVIGC10468.
[040] Figure 28: 0044 amino acid sequence of isoform 5. Other aliases for 0044 include IN, LHR, MC56, MDU2, MDU3, MIC4, Pgpl, CDVV44, MUTCH-I, ECMR-III, and MGC10468.
[041] Figure 29: Frizzled-1 mRNA sequence. Other aliases for Frizzled-1 include FZD1, VVnt receptor, frizzled (Drosophila) homolog 1, frizzled 1, and frizzled, Drosophila, homolog of, 1.
[0421 Figure 30: Frizzled-1 amino acid sequence. Other aliases for Frizzled-1 include FZD1, VVnt receptor. frizzled (Drosophila) homolog 1, frizzled 1, and frizzled, Drosophila, homolog of, 1.
2213521.2 ii [043] Figure 31: MEDI mRNA sequence. Other aliases for =EDI
include IVISG1.
[044] Figure 32: MEDI amino acid sequence. Other aliases for CITED1 include MSG1 [045] Figure 33: ARHI mRNA sequence. Other aliases for ARHI include DIRAS3 and NOEY2.
[046] Figure 34; ARHI amino acid sequence. Other aliases for ARHI include DIRAS3 and NOEY2.
[047] Figure 35: Primer sets for glyceraldehyde-3-phosphate dehydrogenase, f3-actin, CCNID2, PLAB, and PCSK2.
DESCRIPTION OF THE EMBODIMENTS
[048] The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. However, before the present methods and compositions are disclosed and described, it is to be understood that this invention is not limited to specific methods, specific nucleic acids, specific polypeptides, specific cell types, specific host cells or specific conditions, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
[049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the 22135321.2 12 invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
[0501 Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being moded in all instances by the term "about.' Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
1051] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, hovvever, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[052] As used herein, "cDNA" means a DNA prepared using messenger RNA (mRINIA) as template. In contrast to genomic DNA and DNA polymerized from a genomic, non- or partially-processed RNA template, cDNA contains coding sequences of the corresponding protein in the absence of introns and other non-translated nucleic acids.
[053] "Gene" refers broadly to any region or segment of DNA associated with a biological molecule or function. Thus, genes include coding sequence, and may further include regulatory regions or segments required for their expression.
Genes may also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest, or synthesizing from known or predicted sequence information, and may include sequences encoding desired parameters.
p354] "Isolated," when used herein in the context of a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. it is preferably in a homogeneous state although it can be in either dry form or an aqueous solution.
Purity and homogeneity are typically determined using analytical chemistry techniques such as polyactylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant molecular species present in a preparation is substantially purified. An isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of Interest [055] "Malignant" or "cancerous" or 'cancer" refers to the properties of cells or tissue that distinguish them from benign or normal cells. Malignant, cancerous, and cancer cells invade, grow and destroy adjacent tissue, metastasize, and usually grow more rapidly than benign cells.
[056] "Normal cell" means a non-cancerous or non-malignant cell.
10571 "Nucleic acid" and "poly-nucleotide refer to deoxyribonucleotides or ribonucleotides, nucleotides, oligonucleotides, polynucleotide polymers and fragments thereof in either single- or double-stranded form. A nucleic acid may be of natural or synthetic origin, double-stranded or single-stranded, and separate from or combined with carbohydrate, lipids, protein, other nucleic acids, or other materials, and may perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (P: )Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and may be metabolized in a manner similar to naturally-occurring nucleotides.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608;
Cassol et al. (1992); Rossolini et al. (1994) Mal. Cell. Probes 8: 91-98). The term 22435321,2 15 nucleic acid is used interchangeably with gene, cDNik, and mRNA encoded by a gene.
[058] "Sample" refers to an isolated sample of material, such as material obtained from an organism, containing nucleic acid molecules. A sample may comprise a bodily fluid; a cell; an extract from a cell, chromosome, organelle, or Membrane isolated from a cell; genornic ON, RNA, or cDNA in solution or bound to a substrate; or a biological tissue or biopsy thereof. A sample may generally be obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
[059] "Stringent hybridization conditions" and 'stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. Nucleic acids having longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology¨Hybridization with Nucleic Acid Pmbes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be 5 C lower than the thermal melting point go for the specific sequence at a defined lianic strength and H. Typically, under "stringent conditions"
a probe will hybridize to its target subsequence, but to no other sequences.
The Tin is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are 22136321,2 16 selected to be equal to the Tr, for a particular probe. An example of stringent hybridization conditions for hybridization of cornplementani nucleic acids that have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCI
at 72 C for 15 minutes. An example of stringent wash conditions is a 0.2x SSC
wash at 65 C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40 C for 15 minutes. For short probes (e.g., 10 to 50 nucleotides), stringent conditkm typically involve salt concentrations of less than 1.0 M Na ion, typically 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least 30 C. Stringent conditions can also be achieved with the addition of destabilizing agents such as fonnamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially similar if the polypeptides that they encode are substantially similar.
This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[060] Identification of Thyroid Carcinoma 22135321.2 17 [061) 'Thyroid carcinoma is a common endocrine cancer with a favorable prognosis if subjected to timely treatment. However, the clinical identification of follicular thyroid carcinoma (FTC) among patients with benign thyroid nodules is still a challenge. Preoperative fine needle aspiration-based cytology cannot always differentiate follicular carcinoma as from benign follicular neoplasias.
Because current methods fail to improve preoperative diagnosis of thyroid nodules, we explored new molecular-based diagnoses, [0621 Briefly, we conducted a microarray-based study to reveal the genetic profiles unique to FTC and follicular adenomas (FM), to identify the most parsimonious number of genes that could accurately differentiate between benign and malignant follicular thyroid neoplasia. We confirmed our data by quantitative RT-PCR and immunohistochernistry in two independent validation sets with a total of 114 samples. We were able to identify three genes, cyclin P2 (CCAID2) (mf2NA
shown in Figure 5), protein convertase 2 (PCSK2) (rnRNA shown in Figure 7), and prostate differentiation factor (PLAB) (mRNA shown in Figure 9), that allow the accurate molecular classification of FTC and FA. Two independent validation sets revealed that the combination of these three genes could differentiate FTC
from FA
with a sensitivity of 100%, specificity of 94.7%, and accuracy of 96.7%. in addition, our model allowed the identification of follicular variants of papillary thyroid carcinoma with an accuracy of 85.7%. Three-gene profiling of thyroid nodules can accurately predict the diagnosis of FTC and FA with high sensitivity and specificity, thus identifying promising targets for Maher investigation to ultimately improve preoperative diagnosis.

[063] The invention provides methods of identifying malignant thyroid tissue, and for differentiating between non-malignant and malignant neoplasms.
According to the methods, in some embodiments a thyroid tissue sample is evaluated for the expression of at least two genes chosen from CCND2, PCSK2, and PLAB.
Evaluation of expression of any two of the three can be combined in the test.
Thus, in some embodiments, the thyroid tissue sample is tested for the expression of CCND2 and PCSK2, or alternatively for CCND2 and PLAB, or alternatively for PCSK2 and PLAB.
[064] Of course, the assay can test for the presence of all three of the genes.
Thus, in some embodiments, the thyroid tissue sample is tested for the expression of CCND2, PLAB, and PCSK2. Still further, the expression of additional genes may also be included, which may even further evidence the existence of malignant cells, or otherwise characterize a carcinoma. For example, in addition to testing for CCND2, PLAB, and PCSK2, one may also test for the expression of hTERT (rnRNA
sequences of hTERT variants are shown in Figures 11,13, 15, and 17).
[065] The invention also provides kits for identifying malignant thyroid tissue comprising means for assaying a thyroid tissue sample for the expression of at least two genes chosen from CCND2, PCSK2, and PLAB. In some embodiments, the kits comprise at least two of the following: (a) a container containing at least one CCND2 primer; (b) a container containing at least one PCSK2 primer; and (c) a container containing at least one PLAB primer. The kits may also include a container containing at least one hTERT primer. Kits according to the invention may also 22136321.2 19 include additional molecular biology reagents for PCR reactions, including control primer sequences.
[066] EXAMPLES
[067] Materials and Methods [068] Tissue specimens [069] In total, 55 samples (24 FTC and 31 benign thyroid samples) were independently acquired for gene expression analysis in our training and validation set mentioned below. All tissue specimens were snap frozen in liquid nitrogen after Surgical removal and stored at -80 C. IFinal histological classification for these Samples was obtained from paraffin-embedded tissue. In addition, sections from each snap-frozen tumor sample were independently subjected to hematoxylin and eosin stain and evaluated by a pathologist. A panel (training set) of 12 FTes and 12 FAs were accrued for microarray (GeneChipTm) analysis (Table 1).
TABLE 1. Histopathological classification of 12 FTC samples used for microarray analysis Sample ID Sex/age Pathologic diagnosis ThIM
02E187 n/a FTC¨Hurthle cell type; capsular Invasion pT2 03E139 F/61 FTC¨Hurthle cell type; widely Invasive pT2 03E077 F/48 FTC¨Hurthle cell type; minimal Invasive pT2 03E193 F/82 FTC¨Hurthie cell type; minimal Invasive pT3 03E041 F/72 FTC¨Hurthie cell type; hepatic Metastases 408 F/71 FTC¨Hurthle cell type; recurrence 95 F/69 FTC; recurrence 22 F/67 FTC pT4 177 F/78 FTC; widely invasive pT3 52 M/40 FTC; recurrence 03E191 F/62 FTC; minimal invasive pT2 03E192 F/25 FTC; minimal., angioinvasive pT2 tits, Not available; M, male; E, female.
22135321.2 20 [070] No atypical variant or Hurthle cell adenoma was included in our set of 12 FAs. RNA extraction of these 24 samples was performed for GeneChip analysis and quantitative RT-PCR. Furthermore, seven follicular variants of PTCs (FV-PTCs) and additional tissue samples from five normal thyroids have been obtained from unrelated patients and RNA was extracted for quantitative RT-PCR. To validate our findings from the training set, two independent validation sets were also obtained as follows. The first validation set comprised in total 31 samples among which were 12 FTCs, 12 nonfunctioning thyroid nodules (five FAs and seven adenomatous nodules), five autonomous adenomas (hot nodules), and two normal thyroid tissues.
The first validation series was subjected to quantitative RT-PCR. The second independent validation set comprised paraffin-embedded archival material from patients with FTC [including 14 minimally invasive FTC and seven minimally invasive Hurthle cell carcinomas (HCC)] and 26 patients with benign thyroid nodules (17FA
and nine follicular hyperplasia) was subjected to immunohistochemistry (IHC).
These samples were obtained through the Department of Pathology, The Ohio State University (Columbus, OH) and independently analyzed for histological diagnosis by the collaborating pathologist. All samples were obtained as anonymized materials without linked identifiers, with the approval of The Ohio State University's Institutional Review Board for Human Subjects.' Protection.
[0711 RNA extraction [0721 Total RNA was isolated from 0.2 g of snap-frozen tissue using the TRIzol Reagent (Invitrogen, Carlsbad, CA) and purified with the RNeasyKit (OIAGEN, Valencia, CA). Aliquots of 1 jig of total RNA were pretreated with DNase 22136321.2 21 I (Invitrogen), after which 500 ng were reverse transcribed into cDNA using the SuperScript')." II System (Invitrogen) and a random hexarner anchored primer (Roche, Indianapolis, IN) according to the manufacturers' recommendations.
[0731 Oligonucleotide expression miariarray analysis [074] Sample preparation, hybridization, and analysis were performed as described previously, except that version U133A GeneChips were used, which contain 22283 probe sets (17). In addition RNA quality was assured by using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) in accordance to the standards described by Auer at al. (18). The cell intensity fries (.CEL) were interrogated using the AffymetrixTm Microarray Suite 5.0 software. The percentage of probe-sets called present, the ratio of 3'-signal to 5'-signal of two housekeeping genes, the intensity of four hybridization controls, the scale factor between arrays and signal-to-background ratio were used for quality control assessment and to validate the in vitro transcription procedure. Furthermore, each array was cross-referenced to other arrays to identify array or single outliers by the method described by Li and Wong (19). All arrays passed these quality control steps. The DNA-Chip Analyzer Software (dChip) developed by Li and VVOng was used to no all arrays to a common array having a median overall brightness by using an invariant set of probes (19). A perfect match/mismatch difference model of the dChip software developed by Li and Wong was used to compute the model-based expression index (MBEI) (19). A summary table of the 80 differentially expressed genes is described by Weber et at (55).
[0751 Quantitative RT-PCIR
22135221.2 22 [076] Quantitative RT-PCR was performed using the primers noted below and the iQ SYBR Green RT-PCR system (Bio-Rad, Hercules, CA) on an iCycler Instrument (Bio-Rad) using the comparative threshold cycle (Ct) method (20).
Equal efficiency of the reference and target amplification was determined by a validation experiment for all reference and target genet. Samples were analyzed in triplicate for the target gene and normalized to the average Ct value of the two reference genes, p-actin and glyceraidehyde-3-phosphate dehydrogenase (primers listed in Figure 35), the latter two of which were analyzed as duplicate. AACt was determined by normalizing to the average ACt of five normal thyroid samples, indicating the relative difference in the expression level of the target gene between neoplasia and normal sample. The fold difference between FTC and FA is calculated by two to the power of the absolute difference in AACt between the two groups. All values are given as means and 95% confidence intervals of each group. Primer sequences were as follows: glyceraklehyde-a-phosphate dehydrogenase, GGGCTGC _____ 11 I IAACTCTGGTAA-3'(SEQ ID NO; 31) and 6-ATGGGTGGAATCATA1TGGAAC-3'(SEQ NO:32): 8-actin, 5%
CGTCATACTCCTGCTTGCTG-T(SEQ ID NO:33) and 5'-CCAGATCATTGCTCCTCCTGA-3'(SE0 ID NO:34); cyan D2 (CCND2), 5'-CACTrGTGATGCCCTGACTG-31(SEQ ID NO:35) and 5'-ACGGTACTGCTGCAGGCTAT-3' (SEC/ ID NO:36); prostate differentiation factor (FLAB), F-CAACCAGAGCTGGGAAGATT-T(SEC) ID NO:37) and 5'-AGAGATACGCAGGTGCAGGT (SEO ID NO: 38); and protein convertase 2 (PCSK2), 5'-GCCATGGTGAAAATGGCTAA-3'(SEQ ID NO:39) and 5-GAGTGTCAGCACCAACITGC-31(SEQ ID NO:40) (Figure 35). Primer sequence for ARHI and CITED1 have been described previously (21). One embodiment of the mRNA sequences for ARHI and CITECil are shown in Figures 33 and 31 respectively.
[0771 Primers for quantitative RT-PCR were designed to span an exon-exon boundary or an intronic sequence, to avoid amplification of any genomic DNA.
All quantitative RT-PCR products were initially visualized on a 2% agarose gel to ensure the presence of only a single amplicon product. The average sd between replicates was 0.15 and the average interassay sd for control genes was 0.32.
[0781 /HO
(079] IHC was performed as described previously (22). Antibodies against CCND2 (Santa Cruz Biotechnology, Santa Cruz, GA) were used ate dilution 1:150 and against PCSK2A (US Biological, Swampscott, MA) were used at a dilution of 1:100. A total of 83 sections were analyzed, consisting of 57FTCs and 28 benign thyroid nodules (17FA and rune follicular hyperplasia), Additional sections from five normal thyroid glands and adjacent normal thyroid tissue were used for comparison.
All slides were scored in a blinded fashion, and a second Individual randomly validated the results. We regarded cells as irnmunoreactive when an obvious nuclear (CCND2) or cytoplasmic (PCSK2) expression was seen_ We scored immunoreactivity as follows: retained (++) when more than 50% of nuclei/cytoplasm were strongly immunoreactive, reduced (+) when 10-50% of the nuclei/cytoplasm 22135321.2 24 were immunoreacfive, and absent (-) when less than 10% of the nuclei/cytoplasm were immunoreactive or all cells' nuclei showed no immunoreactivity at all (55). The absence of a commercially available antibody that could reliable allow staining of thyroid tissue led to refine the IHC analysis to COND2 and PCSK2.
[080] Statistical methods [081] Two-tailed Student's t test for independent samples, assuming equal variance, was used to determine difference between mean gene expression determined by RT-PCR of the three selected genes with 22 degrees of freedom (Table 2).
TABLE 2. Summary of quantitative RT-PCR data obtained for three genes, CCNDZ
PCSKZ and PLAB
Gene Affymetrix ID NMI public AnCt FTCaa AilCt FAasi Fold change FTC FA
ID vs .FA
4.03 -0.68 Down (-5.19 to -CCND2 200952_s_at AVV026491 (-1.18 to2.87) 10.2-fold 0.00001 0.18) -7.46 0.58 Down (-9.17 to 79 PCSK2 204870_s_at AL031664 - (-1.to 263-fold .=0 5 75) 2,95) 4.12 1.67 Up RAS 221577 x at AF003934 (2.9 to 5.34) (0.63 to 6.5-fold 0,0037 2.81) The approved gene symbol for PtAB by the Human Genome Organization Nomenclature Committee is GDF45 (growth differentiation factor 15).
a Given are ii,6Ct as mean of each group and exact 95% confidence intervals in parentheses.
P values are calculated with two-tailed Student's t-test for independent samples with 22 degrees of freedom.
1082] The hierarchical cluster analysis we used to present our data are based on 96 probe sets that we fiftered from the 22283 probe sets present on the FIG-U133A chip by setting the thresholds to 2-fold expressional changes at the lower 90% confidence bound in either direction, a P value less than 0.05 for the difference 22135321,2 25 in expression and no less than 50% present call for each gene in all 24 arrays. For our cluster analysis we choose the commonly used average linkage method. The distance measure in the clustering analysis is 1 minus the correlation coefficient (23).
[083] When the expression of a single gene is used for diagnosis, it becomes necessary to find a desirable threshold value that is used to distinguish the two groups. We obtained for each possible threshold value the sensitivity and specificity of diagnoses, which are percentages of FTC ("test positive") and FA ("test negative", i.e., not FTC) samples correctly identified, respectively. The best threshold value is the one that maximizes an appropriate combination of the two. To use multiple genes in combination for the purpose of diagnosis, we applied linear discriminant analysis, which is based on the assumption of multivariate normal distributions of the joint expressions, and finds the best linear combination of the expression values that discriminates the two groups. In a first round, we applied the technique of cross-validation to the training set to assess the performances of the diagnostic tests, in which each sample is in turn left out of the data, a test developed based on the remaining samples and then applied to the sample being left out.
The diagnoses can be compared with the true classes of the samples to indicate the performance of the method leading to the diagnostic test. In a second round, we applied the same technique of linear discriminant analysis, but this time using our validation set, to independently confirm our findings from the first round.
[084] RESULTS

CA, 2,534,871 Agent Ref. 70619/00002 [085] To dissect out the most parsimonious gene expressional differences that accurately classify FTC from benign follicular neoplasias, in particular FAs, we used a global expression array approach on 12 FTCs and 12 FAs ("training set").
So that we could also differentiate the earliest signs of malignancy from benign neoplasia, we included two minimally invasive FTCs and two minimally invasive HCC within our set of FTCs (Table 1). Using the dChip compare sample function, we used, as a first step, a straight forward but conservative approach to identify those genes that could reliably differentiate between FTC and FA. Using these criteria defined in the Materials and Methods section, we identified 96 probe sets, which represent 80 genes. To statistically validate these finding, we performed a random permutation analysis, in which we randomly permuted the labels of FTCs and FAs a large number of times, repeated the gene selection procedure using the same criteria, and recorded the number of genes identified (24). It demonstrated that these 80 genes were uncovered due to biological relevance and not by random coincidence (i.e. chance). Hierarchical cluster analysis showed that based on this set of 80 genes, FTCs and FAs could be accurately classified according to their histological group (Fig. 1). Notably, three of four minimally invasive carcinomas and all HCC clustered within the FTC group. Only sample 03E192, a minimally invasive FTC, clustered with the FA group. From this set of 80 genes, we set out to find the smallest number of genes that could reliably classify FTC from FA in an independent validation set. After ranking the probe sets based on their fold change and significance (P value and t statistics), we identified those genes that also showed the highest difference in expression levels between 22135321.2 27 minimally invasive FTCrs and FA and we exduded expressed sequence tags and hypothetical proteins. Based on these criteria, we identified a list of 11 genes, and we focused, in the first instance, on the two highest ranking genes CCND2 (fold change -11.72; Pvalue0.0025), and PLAB (fold change 7.86; P value 0.0039; this gene has been annotated under different names, such as GDF-15, MIC-1, or coml) (25). Besides CCND2, we also found CD44 (mRNA sequences of CD44 variants are shown in Figures 19, 21, 23, 25, and 27), a gene targeted by the Wnt signaling pathway, markedly under-expressed in FTC (fold change -4.5; P value 0.0016).
In addition, Frizzled-1 (one embodiment of Ftizzled4 rnRNA is shown in Figure 29), the membranous receptor for Wnt ligands, is also dysregulated (fold change -4.39; P
value 0.0081). Neither CCND2 nor PLAB have been previously associated with thyroid carcinogenesis.
10861 As a second step, we analyzed our gene expression data for probe sets with very high absent calls in only one group, either FTC or FA but not both, expecting that this approach will identify strongly under-expressed or silenced genes, which would in theory reliably differentiate these two histologies.
Such high absent calls can lead to high P values, and consequently, the gene will not be detected by standard selection process. This approach revealed the gene encoding PCSK2 [present call 7% (IVIBEI 12.05) in FTC vs .75% (MBEI 1743.51) in FA;
fold change 144.7, P value 0.011] on further analysis. Expressional differences of each of the three genes between FTC vs. FA in the training set was confirmed using quantitative RT-PCR (summarized in Table 2).
(0871 Genetic classification of FTC and FA
22135321.2 28 [0881 Based on our micro array data from the training set of 12 FTCs and 12 FAs, we then employed different statistical methods to predict the performance of our selected three genes in the accurate and reliable classification of Flt and FA.
We employed receiver-operated characteristics (ROC) curve analysis to evaluate the performance of our genetic classification using the expression of each of the three genes (CCND2, PCSK2, and PLAB) individually. The ROC curves shows the sensitivity (proportion of FTC samples correctly classified) and one minus the specificity (where specificity is defined as proportion of FA samples correctly classified, i.e. not carcinoma) from using all possible threshold values of expression in the classification (graph not shown). Because a very low false-negative rate is desired, and we note that to perfecfiy identify all FTC samples (12 of 12), the minimum proportions of misclassified FA samples based on our data are 33%
(four of 12), 16.7% (two of 12), and 75% (nine of 12) when the expression values of CCND2, PCSK2, and PLAB are used separately. Of significance, when expression values of CCND2 and PCSK2 were used jointly in the classification by applying the method of linear discriminant analysis, the two groups of samples, FTC and FA, can be distinguished perfectly (24 of 24) (Fig. 2A). PCSK2 and PLAB have the same joint effect (Fig. 28). To validate this microarray-based classification, we blindly analyzed the expression levels of CCND2, PCSK2, and PLAB in an independent validation set of 12 FTCs, 12 nonfunctioning thyroid nodules (five FAs and seven follicular hyperplasia), two normal thyroids and five autonomous adenomas (hot nodules). Linear discriminant analysis of this in dependent validation series confirmed that dual combinations of CCND2 and PCSK2 or PCSK2 and PLAB were 22435321.2 29 CA, 2,534,871 Agent Ref. 70619/00002 able to distinguish between FTCs and FAs with an accuracy of 87.1% (exact 95%
confidence interval 70.2-96.4%) (27 of 31 samples) and 93.5% (exact 95%
confidence interval 78.6-99.2%) (29 of 31 samples), respectively (Fig. 2 and Table 3). Furthermore, because both hot as well as cold nodules could be accurately identified, we showed that the differences between the two groups are in dependent from functional status of the thyroid nodule but due to malignant transformation. For an honest estimate of the clinical performance using all three genes together, i.e.
CCND2, PCSK2, and PLAB jointly, we applied the classifier from linear discriminant analysis, which correctly identified all 12 FTC samples from the validation set, and we estimated a false-positive rate of 5.3% (exact 95% confidence interval 0.13-26.03%) (1 of 19 samples) allowing an accuracy of 96.7% (exact 95% confidence interval 83.3-99.9%) (30 of 31 samples) (Fig. 3 and Table 3).
TABLE 3. The performance of classifiers in terms of sensitivity and specificity in the validation set (see also Figs. 2 and 3) Sensitivity in Specificity in Genes used in classification validation set validation set PCSK2, CCND2 66.7% (8of12) 100% (19of19) PCSK2, PLAB 91.7% (11of12) 94.7% (18of19) PCSK2, CCND2, PLAB 100% (12of12) 94.7% (18of19) [089] Furthermore, we validated our data by means of IHC for the most promising combination of two genes, CCND2 and PCSK2, in a second independent validation set of 57 FTCs and 26 benign thyroid nodules. Using PCSK2 and CCND2 jointly (ROC curve in Fig. 4), we observed a sensitivity of 89.5% (exact 95%
confidence interval 78.5-96.0%), specificity of 80.8% (exact 95% confidence interval 60.6-93.4%) and accuracy of this test of 86.7% (exact 95% confidence interval 77.5-93.2%) when we chose the cutoff value for identifying FTC
22135321.2 30 to be category 3 or larger (Table 4). Of note, complete absence of expression of PCSK2 and/or CCND2 was only seen in FTCs but never in benign thyroid nodules (Table 4). Furthermore, only 1 of 14 minimally invasive FTCs was misclassified due to retained immunostain for both antibodies, PCSK2 and CCND2. These observations affirm the accuracy of our genes to identify even minimally invasive neoplasias.
TABLE 4. Distribution of CCND2 and PCSK2 expression by immunohistochemistrya in 83 total follicular neoplasia samples Category .1 (+404) 2(+44+) 3(44+) 4(4+) 5(4-) Benign nodule 13 (50%) 8 (30.8%) 5(19.2%) 0 0 FTC 3 (5_3%) 3 (5.3%) 9 (15.8%) 06 (10.5%) 36 (63.1%) 'Images of samples are published as supplemental data on The Endocrine Society's Journals.
[090] Genetic classification of FV-PTC
[091] About 10% of suspicious FNA biopsies will be classified as FV-PTC in final histology. Therefore, we employed our three-gene based classifier system on a set of seven FV-PTC (Table 5). Six of seven FV-PTC samples analyzed were correctly identified as a malignant thyroid neoplasia (85.7%). In addition, we used CITED1 and ARHI, two other markers previously described by us, to further characterize these samples. It is of note that one sample (FV-PTC_269) does not show expression of CITED1, a predictive marker for FV-PTC and PTCs.
Interestingly, only in this sample we see a clear under-expression of CCND2 as seen in all other FTCs analyzed. Furthermore, sample FV-PTC_345 shows expression of CITED1, but was not identified by our three-gene profile as a malignancy. It is note worthy that we found strong expression of the imprinted tumor suppressor gene ARHI in this sample. As we showed previously, silencing of this 2213$321.2 31 gene is associated with FTC carcinogenesis (21). These data might indicate that histological diagnosis of FV-PTC addresses a heterogeneous group of follicular neoplasia-an aspect that needs further elucidation. We note, by including the seven FV-PTC in our validation set, we can accurately identify 94.7% of all malignant samples (18 of 19) and 94.7% of all benign samples (18 of 19) as well.
TABLE 6. The performance of classifiers in a series of seven FV-PTCs Sample AA Ct CCA102 M Ct PLAB AA Ct PCSK2 WA value Malignant CITEDI ARH1 FV-PTC_348 -0.92 5.25 -2.75 2.859 True +
FV-PTC_158 -0.04 6.38 -t08 2.645 True +
FV-PTC 243 -0.55 5.08 4.93 3.329 True +
FV-PTC_86 -0.84 5.98 -6.98 4.28 True +
FV-PTC_61 0.83 8.4 -5.45 4.428 True +
FV-PTC 345 0.82 0.1 -6.9 2.099 False +
FV-PTC _269 -3.35 3.78 -10.73 4.986 True -Values for the three-gene classifiers CCND2, PLAB, and PCSK2 are given in MCI
(see also Table 2). Six of seven FV-PTC
(85.7%) have been correctly identified as malignant. Sample FV-PTC 345 was not identified as malignant. LOA value is the value of the linear combination of the three genes used to discriminate malignantand nonmalignant samples. Expressions of the two genes CITED1 and ARH1 are marked with +, where as no detectable expression is labeled -.
[092] DISCUSSION
[093] Currently, the diagnosis of thyroid nodules relies primarily on cytology (4, 8). For the majority of patients with PTC, non-FTC, or inflammatory lesions, FNA-based cytology can make a diagnosis with high accuracy (4). However, there is a significant proportion of follicular neoplasias in which this FNA-based preoperative cytologic diagnosis fails (4 - 6, 8- 10). Several reports show that individual skill and experience largely affect the sensitivity of this diagnostic test, ranging from as low as 57% to as excellent as 98% (10). However, an estimated 20% (ranging from 9.2-42%) of all performed FNA-based cytologies will describe a suspicious follicular neoplasia, but only 10-20% of the patients that undergo surgery based on this diagnosis will actually have a malignant thyroid nodule (4, 5, 8). Based on investigative studies, immunohistochemical analysis has been proposed as a 22135321.2 32 reliable marker for differentiating between FTC and FA (25). However, most of these markers showed their limitations in clinical practice and failed to become established (4, 27). One underlying reason might be that neoplasias do not show their distinct malignant phenotype and therefore cannot be diagnosed by these methods.
[0943 Different global gene expression studies have been conducted over the last years to identify novel targets. A recent study employing serial analysis of gene expression proposed a four-gene profile to improve preoperative diagnosis of FTC, but the accuracy of 80% for the gene expression based model is not superior to other algorithms (28). In addition other microarray-based studies, that allowed the highly accurate differentiation between FTC and FA by employing a 106-genes profile, still failed to identify minimally invasive FTCs, which comprise a large proportion of all FTCs (5,14). Our approach overcame this problem by including diverse phenotypes of follicular thyroid malignancies, especially minimally invasive variants, in the microarray-based training set The inclusion of oncocytic variants of FTC (HCC) might appear distracting at first, because they are considered by some as a distinct clinicopathological entity and display unique molecular alterations (12, 29). Other groups have identified molecular alterations such as RET/PTC
translocations or BRAF mutations in a subset of oncocytic thyroid cancer (29-31).
Both these somatic alterations are common in PTC (15, 29). However, it is acknowledged that morphological features defining PTC and FV-PTC can be found in Huerthle cell carcinoma as well (29). Therefore, other reports endorse the idea of Huerthle cell PTC or FV of Huerthle cell PTC (29). Unsupervised cluster analysis and multidimensional scaling failed to differentiate FTC and HCC into two distinct classes, indicating that in our sample set, the similarities in gene expression out-weigh in FTC and HCC the differences. These findings and other reports support our hypothesis that FTC and some HCC may result from shared molecular alterations (21). Nonetheless, this area requires further clarification and it remains important to identify HCC separately.
(095] Our approach has allowed us to identify genetic nuances in the initiation of follicular carcinogenesis. The dysregulation of CCND2, the first gene we identified as being an indictor of thyroid malignancies, and a cell cycle regulator, is intriguing because over-expression is associated with cancer progression and malignant transformation (32, 33). However, there are emerging data that CCND2 may act in different ways beyond cell cycle control. Other reports showed that CCND2 is under-expressed in various cancers due to hypermethylation of its promoter (34, 35). Our findings might provide further insight into the biological mechanism of CCND2 inactivation. Previous reports indicated that the dysregulation of the Wnt signaling pathway might play an important role in thyroid carcinogenesis (36). The membranous Frizzled receptors serve as binding targets for the Wnt proteins and subsequent activation of its intracellular Dishevelled proteins lead to transcription of targets genes such as CCND2 and CD44 (36, 37). Our data demonstrated dysregulation of this pathway from the receptor to the target genes in FTC. Corroborating our findings, a previous report identified 11 genes of the Wnt pathway, including CCND2 and CD44, under-expressed in prostate cancer (37).
This seeming paradox that both over- and under expression of the same gene can 22135321.2 34 result in carcinogenesis is being explained by accumulating data showing that different signaling pathways and its downstream targets may act as oncogenes in some neoplasms and tumor suppressors in others (38, 39). Thus, further investigation would be required to determine how a profile of concurrent signaling pathways feed into directly opposed phenotypes.
[096] The second gene we identified, PLAB, encodes a member of the TGF-superfamily that is known to prevent apoptosis by activating the Akt pathway (25).
The importance of Akt activation in follicular thyroid carcinogenesis has been previously shown by us (40). Therefore, PLAB might provide an upstream target of this pathway. Furthermore, an estimated 10% of all FNA do not result in sufficient material for a cytological diagnosis (4). Due to the lack of serum biomarkers that could identify FICs, no preoperative noninvasive diagnosis is currently available for these patients. In this context, PLAB, a secreted protein, should be considered for further investigation to determine its feasibility as a diagnostic tool to identify thyroid malignancies from a simple blood test (41).
[097] The third gene identified in our analysis is PCSK2. The members of this family process latent precursor proteins into their biologically active products.
The mechanism by which the disruption of proprotein processing can promote tumorigenesis in thyroid tissue remains unknown. However, it has been shown that the inhibition of proprotein convertases enhances cell migration and metastases development of human colon carcinoma cells (42). Such a mechanism is plausible as well in thyroid carcinogenesis.

[0981 Even when we used only a combination of two of the three identified genes (CCND2 and PCSK2 or PLAB and PCSK2) we were still able to correctly classify 100% of the FTCs, including four minimally invasive ones, and all FAs.
Indeed, using an independent validation series of 31 samples, we demonstrated that the combination of all three genes CCND2, PCSK2, and PLAB performed well in differentiating FTC from FA, resulting in an accuracy of 96.7% (exact 95%
confidence interval of 83.3-99.9%). Furthermore, we were able to use a second validation series and a different technique, 1HC, to examine a combination of only CCND2 and PCSK2, which resulted in an accuracy of 86.7%. Thus, our results appear to be superior to those reported using RT-PCR methods to detect gene expression of telomerase, galectin-3, or a number of other markers to discriminate benign from malignant follicular thyroid tumors (4, 13, 43, 44), The employment of galectin-3111C has been reported to reliably identify malignant thyroid lesions (26, 45). However, we and others have shown previously that this method does not succeed in improving the differentiation between FTCs and FAs in all cases (27, 43).
Furthermore, analysis by means of 1HC often has its limitations, not only due to variability of antibodies or Interinstitutional variation (artifact) but also because of nonuniform classification and interpretation. In contrast, the gene expression analysis described here, in a total of 24 FTCs and 31 benign thyroid nodules, using the combination of three genes, resulted in 100% of FTCs being identified and 30 of 31 of benign thyroid nodules definitively identified as well. A very recent FNA-based study employing hTERT as a molecular differentiator succeeded with recognizable sensitivity and specificity (46). However, the data indicate that this test performs 22135321.2 36 much better in the identification of PTC and FV-PTC compared with FTC. Indeed, a full 20% of FTCs were missed. In addition, the performance of this test in identifying minimally invasive FTCs is unclear, and the authors conclude that additional molecular-based markers need to be explored (46). The robust results from our initial testing/training set confirmed by two independent validation sets have lent confidence that the invention as disclosed in its various embodiments herein might help to establish a new and reliable molecular adjunct for diagnosis of follicular thyroid nodules in the near future.
[099] There exist other studies that reported accurate differentiation of thyroid carcinomas, but notably, all these models were either based on high-density gene profiles (100 or more genes), which would not work in a presurgical diagnostic setting due to limited tissue and RNA available in such a setting, or do riot provide the accuracy needed (13, 14, 28, 47). Our classification model based on the limited number of genes, only three, provides the basis to pursue further evaluation.
Whereas the technique to perform gene expression analysis in limited cell material has been well established (48), it needs to be shown how in adequate and/or contaminated FNA will affect the accuracy of the methods of the instant invention.
[0100] FV-PTC will be found in about 10% (range 0-22%) of inconclusive FNA cytologies (5, 6, 49, 50) and it is of note that when we employed our three-gene profile, we were able to identify FV-PfiCs with an accuracy of 85.7%.
Still, we need to acknowledge that FV-PTC might pose a special challenge when employing the three-gene predictor model into an FNA based setting. Our data indicate that the histological diagnosis of FV-PTC might describe a heterogeneous group of thyroid 22136321.2 137 neoplasias. In this regard, it is of note that in a recent study by Lloyd et at a concordant diagnosis of FV-PTC among 10 pathologists was made only in 39% of all cases (51). This high degree of observer variation can lead toe considerable bias of data if analysis is based on the unreviewed diagnosis of RI-PIG.
[0101] However, considering the recent studies that reported the differentiation between FV-PTC and FA using hTERT or CITED1, it may be plausible to use a four-gene test comprising CCND2, PCSK2, and PLAB plus hTERT (46,52).
Therefore, there is accumulating molecular evidence that suggest that, in the near future, the majority of, if not all, thyroid malignancies can be targeted for definitive surgery, abolishing the requirement of a completion surgery (46,53,54). More importantly, most of the FAs that currently would have gone to unnecessary surgery would have been spared an extensive operation.
101021ln summary, we have demonstrated that genetic classification of follicular thyroid neoplasia with a minimal number of three genes is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of follicular malignancies. It is hoped that the quantkative nature of such a test will be a useful gene-based objective adjunct to the preoperative diagnosis of a disease that currently relies solely on cytology.
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22135Z21.2 46 CA 2,534,871 Agent Ref. 70619/00002 [0159] 55. "Weber F, Shen L, Aldred MA, Morrison CD, Frilling A, Saji M, Schuppert F, Broelsch CE, Ringel MD, Eng C 2005 Genetic classification of benign and malignant thyroid follicular neoplasia based on a three-gene combination.
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[0160] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only. The scope of the claims should not be limited by the preferred embodiments, but should be given the broadest interpretation consistent with the description as a whole.
22135321.2 47 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
The Ohio State Research Foundation (ii) TITLE OF INVENTION:
Three-Gene Test to Differentiate Malignant from Benign Thyroid Nodules (iii) NUMBER OF SEQUENCES:
(iv) CORRESPONDENCE ADDRESS:
Blake, Cassels & Graydon LLP
199 Bay Street Box 15, Connerce COurt West Toronto Ontario M5L 1A9 (v) COMPUTER READABLE FORM:
a)COMPUTER: IBM compatible b)OPERATING SYSTEM: Windows XP
c)SOFTWARE: Notepad (vi) CURRENT APPLICATION DATA:
(A)APPLICATION NUMBER: CA 2,534,871 (B)FILING DATE: 2006-02-15 (viii) PATENT AGENT INFORMATION:
a) NAME:BLAKE, CASSELS & GRAYDON LLP
b) REFERENCE NUMBER: 70619/00002 (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 6480 b) TYPE: nucleotide (ii) MOLECULE TYPE: COMA to MINA
(vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: I
=
agagcgagca ggggagagcg agaccagttt taaggggagg accggtgcga gtgaggcagc 60 cccgaggctc tgctcgccca ccacccaatc ctcgcctccc ttctgctcca ccttctctct 110 ctgccctcac ctctcccccg aaaaccccct atttagccaa aggaaggagg tcaggggaac 180 gctctcccct ccccttccaa aaaacaaaaa cagaaaaacc cttttccagg ccggggaaag 240 caggagggag aggggccgcc gggctggcca tggagctgct gtgccacgag gtggacccgg 300 tCcgCagggC Cgtgcgggac CgCaaCCtgc tCcgagaCga Ccgcgtcctg cagaacctgc 360 tcaccattga ggagcgctac ettccgtagt gctcctactt caagtgcgtg cagaaggata 420 tccaacccta catgcgcaga atggtggcca CCtggatgct ggaggtctgt gaggaacaga 480 agtgcgaaga agaggtcttc cctctggcca tgaattacct ggaccgtttc ttggctgggg 540 tCccgaCtcc gaagtcccat ctgcaactcc tgggtgctgt Ctgcatgttc ctggcctcca 600 aactcaaaga gaccagcccg ctgaccgcgg agaagctgtg catttaCacc gacaactcCa 640 tcaagcctca ggagctgctg gagtgggaac tggtggtgct ggggaagttg aagtggaacc 720 tggCagCtgt cactcctcat gacttcattg agcacatat gCgcaagCtg ccCcagcagc 780 gggagaagct gtctctgatc cgcaagcatg CtcagacCtt cattgctCto totocca(cg 840 6t 08917 16p6 6666 616eazeDDI. 3111111161 116211PDD1 23t111D163 111P1D2126 Oat 1613.631)3e 1)14424,z6 61Daanaee E)146666e eelDeDDDla 1E21314441 09SV alanDx1ID 1161262162 2621111)1D 6z4Dee6114 P6611.D DD alD6e66e6 MSt r',116aall 6e66m26612 26ta1D 2D6ez1DDI.D 1DD)62111E 661Dp2pp66 oytt pp6ePpeDee p24D62DD2D 611a63.1121 2D66p22611 66666D16pP p6662e6DD6 02EV 1D66662616 66 666 2612Do6666 11,11E/Dal 1111D1111) ftzDz1Do16 OZEV 662DDDD66e 6DDD16e461 DD11D1DpD, 122DeelbaD 26tDelazel 111E6111E1 ogzt 6eeYe66 6et66reure Pav66DDpv6 eee6zeee6 eD6eeopee p22D6e644D
00Zt Dat.6e64.6 6412,4aleal zevea646v 6e562 ppIDD6e662. 1Dlee66621, otn1126e662DD D? 656e p66663re66 peDDD16p66 PeD D6662. >71111EDED
090' 6PD1D6212D 6vDD6e6vtD 1)12426p6p DD6e2D6pel 6erD6rDD6e a6!6626ev5 OZO* 66p6e66111 a6PP61212e P616616662 peDe666266 2D214e6666 0966 DDDr61pD6r 116E61614a DE66e)1166 fte6v41361 61.6ev66614 1,D1.6v3a6e 006i 62p6v3paze pp6166v6eD PaD1D11.161 D66e6ep616 6e666ee622 6P2p6ae666 Q%i1vlbeelete al66nverp e6ele6623 6614et5lae t)1EED111 1666D2.66De ogLE pelDez65D6 p61,51DIDD )66eDDD6ep D6lezDeD6P rD66eD6D62 DveeD1.1462 OZLE D1D)1DDlp 21D311616 61e11166e D6peD6pD61 DDre63,p6p2 6e6D62D2re 099E 61D266e1DD 22266eD621 iDlelle66P 21261612aD pDp661.eD66 21DDD1DDDI
009E 2.113167,3- Da3Z14116e p6a2D1eDil 2DDD666epp 161D2PD11) 6666D6peee OPSE Da241iD6e6 b41)1.EnDD 20061116 1.366pDpell llpeopppep ZDP6PDPEP6 olivE 6rD26266ea De266e2aDe D6zaleez6D z64e6eDI.D6 66D64 11EDEEEDel ozvE 6eDz16126t D66peD3.2Dp DD2DDDaDD1 p666prDpee 621v-selevP Inv661v6zr 09Ef Dlpalnua 662DDDe6'ra 26PDDD1)66 6D2eDD66e6 62eD166212 12DDe666eD
00ff DD1DeD=6 6D666eD66p P663.65E)11 1D)D1DEB;) 666,216p616 16PD6yr6eD
00ZE elPe656e D6D22PDDD6 e66622.D64D 222.6eDp463 DEEDED1DDE lee4e)121a 08E 61111D5DeD 1.1v6leeD66 veve3snee lveDwelreeD p664)g1146 61n1.)Delz2 ozTE Dllel6v66, e6reeDlaDa arDeDletaD In1122211 13.11166ala al166113.12 490 1)1111'1111 111611661E 111E1E36E1 6611111111 161116E161 1113316EEE
000E E6611E1611 2D126e62e2 p6p61z662e 6E26rp62DD 26pPDDllee el1D61DP12 0/76z 21p23x2e2m eze21214eD PEE1El3.11e 464,64616 PEI'nED116 lee2.62D6t1 wom pael6pDp63 16aellIEll DDlellleD2 6pep2D6pe6 pelD61D6D6 122222211a oz 2111166112 6e34.16zall 63a4let141 1111ED1)13 1666evaDle papeep66De 09LZ 6ee611656e blv16R1e31 13641D61D 21e6eDeDel P2112ellve 6pe6222v1D
666eDtp6pa 4eeept6pee epelfte442 22DD6224p1 pere126122. p2D6puOvee Otr9Z 166pop611P E1612V23ED 6211122211 1141432Dep DpvD6u6623. 666e6e ogsz 226PDixeD6 6E6E111111 V111311111 111111EE61 zDel6D6pD2 ap26116pee ozsz D611vallze P126116126 31e1111211 11111216DD 1111311116 a4aze564.6 OW 1566rplepp 281315baebe lefterp6a0 Nabla.e446 eeee6veeN5 410.06114, OW D411)rzea Pelanr66e p6e)eD66ar 61.)1zeaDel 1.62D2reee2 OVEZ nImP161D06 Pe,e6r3113 lleopeDell ED3311PDP6 13513D113D ODP3P6P6EP
08ZZ oloe66eD3e 1DD1162661 D1.16PDeDel. 66112D116D 662.1664 D11D6D2.1a2.
OZZZ De11666aDa 6PDDDD161) al13313D, D061111)1D 21D2DDDD1D D1Devepea 091Z 62.eal6peD6 2e111662p6 661e6e1e6D 6r6DDDz262 leeppe6111 DeD6erre6D
OOTZ aTV014R31 Pre4P21611 pD6e4116eD pD21661D16 1pD6641p6a D616611111 OVOZ PP1Depeppe r6e22,1112 alvD6611D3 56161m66 6reenuel 611P1163.)1 or az1D16e661 pe6eDDIT66 62666mDp 66e56 606Deep61 eDeD1Depep 0?61DDreleaD66 64e2D6p264. teE66661Ee 1.DIV1)611E ZEDDtD3tDe 6e)641e)41 0981 6PAltee3D 21D13.322D6 laaleDr616 IDI161.rDza 26661DD2D6 62.1p6a66eD
008T pat66116eD 6uueiabaeberree pexaxelD61 16y16vpr1D
Ota 3161RrlleD e4,43u662 36e161606 enfteloel 16=e)D6v D1DzD62113 0891 DuDbee6e66 le666111ED Dee612e6le DeeD1D1D1r eDepepar16 v66E06662 0Z91 P61eDe666p 66eplere61 .pee6te6pe Dzaee6lrep 6rerD621T6 Plar64PD66 09ST 1E111E1161 P2.61.61DPe6 6peDllavel E16E16113p DD3PDP1PDV D6e1D6p6lp 00ST 1161eD1412. 344422Dp6e 66e6ellpeu Dee6661plp eD52DD62e2 61ezet6r6P
OW 126124686pp 61,DDeepe66 Ple41D64pr eez6e2ep26 peev162E22 e62.12621e2 OVET 46pDee6)6a e424642DD) 2xe666te4e D6eDtl6e6 111P23D616 6211vD6rep OZET pelppzre66 Dveve6Dv2D D166epppp6 p6e626ep6p 3414,1p6ep vllveveDli 09ZT 5E1DDP3D3D D61l1eree preeeenTe pv6a6o6v11 za615111)1 16z1111611 GON 6610111)11 plaozne61 DlEE1E1/16 Deov6p6p6e 6ePp6DD666 116eDD61v6 own 6e6161D,p6 Daple666D6 16De6vDelD 3E16E116 ee)De662De e8le66e66D
0801 16peDDae66 Dr646DeeDD p66eaa6DDE 26eD5pD64D D6eleepza a6166D66r OZOT 611P6PD6P6 6P3,611D6P PPD2D2621P 6616ap6pDp DeeDDeD2p6 peaD66aD6a 096 D6p62Dp61D D16201621 DeD3,D6D2a6 p626pe66e6 le66eD6eDD 3)666261oz 006 eDD6eD6e66 6162fte562 aeeD6D1e6a E6316DDEDD De261eDD61 1.16EE17.1DE
LZ-LO-TTOZ TLEIVESZO YD

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0909 OP3133B161 e'reaealp DE)ze3326a enx)3144Z lueleilzvaD 6663ae3are 0009 eD6e)p)aae aelaDapee 66 pp azDzabtlal 5614m114 Otr6S aal6ee,m/2 eee6aa3D18 13)61.662 pppaea6eae ale3D6P16a 0885 Dv6apPlaDD 616eeexeee 6666all'Dea n6e,6=6 Diappple)6 1.336e361D6 0Z85 ea6aaflae36 16Dea6161e6xxftebe61111 66m1663a 311E1333E6 ows E31-eaD6661 366113reva laapbaDa6a 6a625Dale, eaDeae6Dee De66e)aaa 00L5 eD16bledre )31e1.3eeea azehlfrala p6u6112z) 561.eez3e4 ea4pDazaez opgs aralarae66 ebenalebe eftea666e1 errea6161.1 amaeDD5e D6lepaa6a 0855 6E6m2111 eeD6P6tD1, e66161561a raD1.1156e) babefteeft aeaAe16aD
OZSS 2333emee 16teevell rEfe5labaeE lave0556te rea4116rae 094,5 r6e6IaDDre ))26re6r6 l'aDaelaa6 aaa'aD3lIe 655e ea.palo66a1 OOPS 6e36eel6e 6x,a6ee66e 6666ee66ea 66e6611veb 662616e166 aleaveze6e ovfs efteplabel allalefizle emelbzep a6analptp 61.adiplee r6er6e6664 ons f065rDeb66 fieDe33666z epal,laaal lee6666eD6 6br6erpal aDapebarbe OZZS Dae56e)66e eaeareftel 160eeefia,e 606aDa36e6 66=D6162 091S ,665eaDa e,66eo 56 61o6e66De6 e'llaa3eln 661ae86e6 Oars ea6aan)3 1t61361t3 1)6ne6a3 Dpenefte6 e66e6eD6e ene6a5eeel ovos 1)651.-epeap W3566e3266 uD,6ezEll aDDB13415 1DI.D156e661 Del61D1)51 026t, fizna2bbla anellftba DP13211335 l'ebeDeofte WiEpaDupp DB06E31335 0z6v aDeefilaaar 1.1366E66e pa33)116a) 6666ee6ee6 alaaa6e63 66e5e6a,6 099v 6Alimaav ,ee6e16Dpa DD3116PD66 ETBDD6BBB5 266eD6l3D6 ee6eelp6e) erle,121556 D61e)lalee elzee2eD5e eaD6av1vae ereeD)61eI 6elplere6 otLt ee66666ezD xPPEDD66a all)a)ana apDalebaala eeeD361n6 P333334162 Lys Glu Thr Ser Pro Leu Thr Ala Glu Lys Leu Cys Ile Tyr Thr Asp Asn Ser Ile Lys Pro Gin Glu Leu Leu Glu Trp Glu Leu Val Val Leu Gly Lys Leu Lys Trp Asn Leu Ala Ala val Thr Pro His Asp Phe Ile Glu His Ile Leu Arg Lys Leu Pro Gin Gin Arg Glu Lys Leu ser Leu Ile Arg Lys His Ala Gin Thr Phe Ile Ala Leu Cys Ala Thr Asp Phe Lys Phe Ala met Tyr Pro Pro Ser met Ile Ala Thr Gly Ser val Gly Ala Ala Ile Cys Gly Leu Gin Gin AS Glu Glu val Ser Ser Leu Thr Cys AS Ala Leu Thr Glu Leu Leu Ala Lys Ile Thr Asn Thr Asp val Asp Cys Leu Lys Ala Cys Gin Glu Gin Ile Glu Ala val Leu Leu Asn Ser Leu Gin Gin Tyr Arg Gin Asp Gin Arg Asp Gly Sec Lys Sec Glu Asp Glu Leu AS Gin Ala Ser Thr Pro Thr Asp val Arg Asp Ile Asp Leu (4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 4745 b) TYPE: nucleic acid (ii) MOLECULE TYPE: cONA to mRNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPT/ON: SEQ ID NO: 3 gagCtgagga ggagctgaaa atgcagattt agcatCaagc acagacctac actcgctctt 60 tCtCtccggt acacacagct ccccacattc gcacccctgc ccgcgcgccg ggccgcctga 120 ctgcacggct tcccctccav ccagatgctg gagaacacac actgattcgc tgctttccaa 180 gaCcctgttc agtctctttc tctatacaaa gattttttta aaaactatat ataagaattc 240 tttatttgca ccctccctcc gagtcccctg ctccgccagc ctgcgcgcct cctagcacca 300 cttttcactc ccaaagaagg atgaagggtg gttgtgtctc ccagtggaag gcggccgccg 360 ggttcctctt ctgtgtcatg gtttttgcat ctgctgagcg accggtcttc acgaatcatt 420 ttcttgtgga gttgcataaa gggggagagg acaaagctcg ccaagttgca gcagaacacg 480 gctttggagt ccgaaagctt ccctttgctg aaggtctgta ccacttttat cacaatggcc 540 ttgcaaaggc caagagaaga cgcagcctac accacaagca gcagctggag agagacccca 600 gggtaaagat ggctttgcag caggaaggat ttgaccgaaa aaagcgaggt tacagagaca 660 tcaatgagat cgacatcaac atgaacgatc ctctttttac aaagcagtgg tatctgatca 720 atactgggca agctgatggc actcctggcc ttgatttgaa tgtggctgaa gcctgggagc 780 tgggatacac agggaaaggt gttaccattg gaattatgga tgatgggatt gactatctcc 840 acccggacct ggcctccaac tataatgccg aagcaaotta coacttcagc agcaacgacc 900 ota. .331D6lellp ppeeelene e31366e66e liere1611-5 zear6eveep 3a1ernflee 099t 1P11,11eeee D9636133)z D611611ept P1156elaPr leelre616z neure6ler OZ9t Pree1141e1 ne346evel lana6e3e6 efte61DEpl D71e5=D1 nelle1111 09st 6-4vOlaela 6a4316aeu eee43e4er3 ZEILVDU35 3.1121144 00St nelea3616 elelyleive eeelneave 3Pael6e6le le614446ee ovvv D66areeke6 PR11DE4)5B D1P11DEeD6 eep6raleve ezellaeel6 16616166eD
08Et P31111e2e, D16416r1er Pelelveeye D6ep6elele 11D161perl lepprele36 out, 1.)4)1Dem 11.4verab4 =meafil 4raa46eEe v6a66eaaar 09Zt re566e36 6ee66 62)11r6ae6 repinee6e 6e'lleaev6 Deeelo=e 00Zt 31DP1PD1v1 v5v3ptp6ry 6ivellamb 1.61p1616a6 1PP1P4DDE1 133VDEV1V1 Obit eleareem 6113epplva rlelelp163. 6aelelp161 6z6lexelva ea3r6e16ze 080t Mpleaelr lere6e)yDe pftbeele16 16461e616 161elle)e6 oveeneeel aeop e6 11e3lappre

6)1enrer6 6zerrlrere ee6erplaar 096E 6v1116ime rella11116 6peg161.162 alpalplilp pp6eveplal lale6aelee 006E lee1616eee 111161eDae le1)2,11aD e61611D3e) PP131D1131 51D111e661 ot,gE e,664xelva nx11411D1 13611aelp ppeeer6131 epeft6ebee 113)mb62 08L 15Bee61D)Da lee31166e2 De)6e6e-)eD epElvDCiet 344D)41aD) r)6r664D36 e66e6e 6e5661.64, D44.3,34)1, DDED66VDD1 34306e311) 16111161yr 099E efte,31ae 3,1e3e)61e ED3D16M] D1E31.311)6 11e6o5e elpDe646er 009f emlavDe6 elDepp)D41 r3DzeDye) 2beete)331 ee3a661ea1 66re6nDee OtSE 361elpelal E.DefieEt1DD 310eDftela Be766D4)14 DeJlEIRDED e6566 08VE 16134Dae6e ree6e6466e 66 5I66 e6 e66 6v)3azeoll epfizr,66 ozvE 666 1111)x335v 661E16116e lplollbele 32)2411B4] 636e6361z6 09EE Pe61116arD 6666 603561e316 r6re66D)3a 6114,)66re m6661461 00EE Bleellepab 101z16ne 3D7D)1DP11 p3pleevEle6 veffiletTel relETEDDll ova ze03.34)666 464e6re,e Demax)6e 7E4342.141 11411e16e6 55555 09TE 126161161,1e 6331e1n1)z 5165v6111D epabeelln 111D3)a5le e6p3vee31 on 13136ezxel 146elerefte 6a661D3e3r ate664D6 eeDe6ee4le emerreT5 090E VRPeDDE13 6e1rDeD1Da 136666aDll 11VR11)Dbe pppleplabe 6p1u461416 000E eD1,4141Te reaD3Oe6e 6e4AD,lee eea164r216 e61)1a64a6 4Ae)3ela ow 4)61.61,653. 6,5e6e,e44 ayevelplee e)8646,66D )6EDIDelD1 yeDllelne6 088Z Pe3361peDD ea)E111212 26616160 aeD6er66pD avEveriee6 5663.e36aDe OZ8Z ;2622)M5e 6e6v1zolax D61e6eEee pl1p6164r6 166ee2ve6e a61066eD) 09LZ Q4110,146 e414e01.41. 2010)6e05 66314)Dlee elela6151D ear6056r)e 36ree61) zteDD6,6e 626z666eDe 66r61,6ep leree6tpl relpDp1262 otw D6666pe3Re ppl.1661!) D1611penep 6elD2DR51 6YEME3) 6epeellne 0852 163ppftere W63311163. 4123.144111 V3l337)441. 36e,e6661) eveeeeme ozsz seerepvere efi4D31P10 15561veeD4 le31613111 eallplzree 131oe51616 09tZ 1e1371151x, 34eDee3erD a)z.4636e4e 1.Vee2D764 el'llellaa 41,Davezeve 00tZ 66werltalv 21m7Z61DeD
13V11D6Re 61)112e11,1 116,111re )66embn )e31tD3Ive e35TIDDR76 6e366e)114 63en4)614 3x154.1,36 1)6PD)3D 1,DD33D153 lE)33112.33 6DameD61 66 e6 eelte11,,2 ouz e)6eeerelip )fte6e5e156 16D6ep6oe 661p6z6ue6 Cie6646e56 e6eee6eep 091Z z61.e6644 6vealbeDa r44:2666D61 66665 3423216)DD D546e6eDa OOTZ Pa661e351, 61e611D,e6 61606pe61 36166666ee 6e36)D)6D 6e366)1611 0170z 1v6661D6e6 61D33e65zD DeD66p6D 6De6p.e6666 6616)e)TD1 peppe6M1 0861 lappbelbee 7011436466 1661pD11.3e 63'0310156e Eppleo66DD tieOz361411 0Z61 eDlEfee)Dr 36661p23), 311)W5me e)le.Dee643 De6eNe6et 6t)ee2(be oggl eD156DeDve 31.61D66e), 161vD6p661 lel36,316 211Tever66 6666e616 0081 1.D6,e6,Dr epeolyvv6 1361661162 eD661)y),1 VDDV/PEPPE, e613Dle66E
Ota 3516331,66 et55616154D e3)11P6e6e 61)516DDE etebtaorbe zelab64vee 0991 r6166u336 1615t)61e64 1))16556Jv 1)56114 DeDltelale r661366D1 0Z9T 6666lee6a 66D66161n1 en266r6oe 53P31.1)6eD Dep66yervD D1DDe1161 0951 64DV6133ED 6666 6a661ple6a )1666mee ap66e661)4 p661DE3611 oosT M51618635 n66e6n3) fta6e)62 1r,e6661D3 4eD65e6131. DeD64Dee6 ow 63E163.41e6 v,e33m66 163.56n6De 153DDIee6OE vrel6e6663 teDbel,a) 08ET T3D41,5811. ,DeD141D 64.,6e6e67e 66 ape662)653 v6Dee3leDD
OZET 6ED1DVIMV 1D1E385 6164eD6pD3 lpn6)elD66 nr6n61pep 61D6oe6le on 1D6eD66.366 )t66661,3a )65626463e llve,6e366 rPe366)663 6D666ee3e GOZT 026,664r6 ,3661,366 Pa516YeD4 D6ebbb,333 6b6aebb1bp Defteattou OKI Rae6PDBD) 36666a36e) DeoBRDE131 epe611P61, 6eprpo62ra ral6raaepp 0901 3.331)366e6 )4VD1T3V5P D251E041F D6 m66 6ap66oDyeD 66v064365p OZOT P,17VVDP2 e6v26,66-1 z6e663.61.1 eletpee3re D161D61.361 )1116ee6v6 096 5v)6164e6) a3e6663en 6em11466 lae64e5exe DP166D1,33 El1D33V107 LZ-LO-TTOZ 1L81'ESZO YD

agaaa 4745 (5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 638 b) TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SD) ID NO: 4 Met Lys Gly Gly Cys Val Ser Gin Trp Lys Ala Ala Ala Gly Phe Leu Phe Cys Val Met Val Phe Ala Ser Ala Glu Arg Pro Val Phe Thr Asn His Phe Leu val Glu Leu His Lys Gly Gly Glu Asp Lys Ala Arg Gin val Ala Ala Glu His Gly Phe Gly val Arg Lys Leu Pro Phe Ala Glu Gly Leu Tyr His Phe Tyr His Asn Gly Leu Ala Lys Ala Lys Arg Arg Arg Ser Leu His His Lys Gin Gin Leu Glu Arg Asp Pro Arg val Lys Mt Ala Leu Gin Gin Glu Gly Phe Asp Arg Lys Lys Arg Gly Tyr Arg Asp Ile Asn Glu Ile Asp Ile Asn Met Asn Asp Pro Leu Phe Thr Lys Gin Trp Tyr Leu lie Asn Thr Gly Gin Ala Asp Gly Thr Pro Gly Leu Asp Leu Asn val Ala Glu Ala Trp Glu Leu Gly Tyr Thr Gly lyS Gly Val Thr Ile Gly Ile Met Asp Asp Gly Ile Asp Tyr Leu His Pro Asp Leu Ala Ser Asn Tyr Asn Ala Glu Ala Ser Tyr Asp Phe Ser Ser Asn Asp Pro Tyr Pro Tyr Pro Arg Tyr Thr Asp Asp Trp 71:1); Asn Ser His Gly Thr Arg cys Ala Gly Glu Val Ser Ala Ala Ala Asn Asn Asn Ile cys Gly val Gly Val Ala Tyr Asn ser Lys Val Ala Gly Ile Arg Met Leu Asp Gin Pro Phe met Thr ASp Ile Ile Glu Ala Ser Ser Ile ser His Met Pro Gin Leu Ile Asp Ile Tyr Ser Ala Ser Trp Gly Pro Thr Asp Mn Gly Lys Thr val Asp Gly Pro Arg Glu Leu Thr Leu Gin Ala Met Ala Asp Gly Val Asn Lys Gly Arg Gly Gly Lys Gly Ser Ile Tyr val Trp Ala Ser Gly Asp Gly Gly Ser Tyr Asp Asp Cys Asn Cys Asp Gly Tyr Ala Ser ser met Trp Thr Ile Ser Ile Mn Ser Ala Ile Asn Asp Gly Arg Thr Ala Leu Tyr Asp Glu Ser Cys Ser Ser Thr Leu Ala Ser Thr Phe Ser Asn Gly Arg Lys Arg Asn Pro Glu Ala Gly val Ala Thr Thr Asp Leu Tyr Gly Asn Cys Thr Leu Arg His Ser Gly Thr ser Ala Ala Ala Pro Glu Ala Ala Gly Val Phe Ala Leu Ala Leu Glu Ala Asn Leu Gly Leu Thr Trp Arg Asp met Gin HIS Leu Thr val Leu Thr Ser Lys Arg Mn Gin Leu His Asp Glu Val His Gin Trp Arg Arg Asn Gly Val Gly Leu Glu Phe Mn His Leu Phe Gly Tyr Gly val Leu Asp Ala Gly Ala Met Val Lys Met Ala Lys Asp Trp Lys Thr Val Pro Glu Arg Phe His Cys val Gly Gly Ser val Gin Asp Pro Glu Lys Ile Pro ser Thr Gly Lys Leu val Leu Thr Leu Thr Thr Asp Ala cys Glu Gly Lys Glu Mn Phe val Arg Tyr Leu Glu His val Gin Ala Val Ile Thr Val Asn Ala Thr Arg Arg Gly Asp Leu Asn Ile Mn met Thr ser Pro Met Gly Thr Lys ser Ile Leu Lev Ser Arg Arg Pro Arg Asp ASp Asp Ser Lys Val Gly Phe Asp Lys Trp Pro Phe Met Thr Thr His Thr Trp Gly Glu AS Ala Arg Gly Thr Trp Thr Leu Glu Leu Gly Phe val Gly Ser Ala Pro Gin Lys Gly val Leu Lys Glu Trp Thr Leu Met Leu His Gly Thr Gin Ser Ala Pro Tyr Ile Asp Gin Val Val Arg Asp Tyr Gln Ser Lys Leu Ala Met ser Lys Lys Glu Glu Leu Glu Glu Glu Leu Asp Glu Ala Val Glu Arg ser Leu Lys Ser Ile LeU Asn Lys Asn (6) INFORMATION FOR. SEQ ID MO: 5 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 1204 b) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 cggaacgagg gcaacctgca cagccatgcc cgggcaagaa ctcaggacgg tgaatggctc 60 tcagatgctc ctggtgttgc togtgctctc gtggctgccg catgaggcg ccctgtctct 120 ggccgaggcg agccgcgcaa gtttcccggg accctcagag ttgcactccg aagactccag 180 attccgagag ttgcggaaac gctacgagga cctgctaacc aggctgcggg ccaaccagag 240 ctgggaagat tcgaacaccg acctcgtccc ggcccctgca gtccggatac tcaogcCaga 300 agtgcggctg ggatccggcg gccacctgca cctgcgtatc tctcgggccg cccttcccga 360 ggggctcccc gaggcctccc gccttcaccg ggctctgttc cggctgtccc cgacggcgtc 420 aaggtcgtgg gacgtgacac gaccgctgcg gcgtcagctc agccttgcaa gaccccaagc 480 gcccgcgctg cacctgcgac tgtcgccgcc gccgtcgcag tcggaccaac tgctggcaga 540 atcttcgtcc gcacggcccc agctggagtt gcacttgcgg ccgcaagccg ccagggggcg 600 ccgcagagcg cgtgcgcgca acggggacga ctgtccgctc gggcccgggc gttgctgccg 660 tctgcacacg gtccgcgcgt cgctggaaga cctgggctgg gccgattggg tgctgtcgcc 720 acgggaggtg caagtgacca tgtgcatcgg cgcgtgcccg agccagttcc gggcggcaaa 780 catgcacgcg cagatcaaga cgagcctgca ccgcctgaag cccgacacgg agccagcgcc 840 ctgctgcgtg cccgccagct ataatcccat ggtgctcatt caaaagaccg acaccggggt 900 gtcgctccag acctatgatg acttgttagc caaagactgc cactgcatat gagcagtcct 960 ggtccttcca ctgtgcacct gcgcggggga ggcgacctca gttgtcctgc cctgtggaat 1020 gggctcaagg ttcctgagac acccgattcc tgcccaaaCa gCtgtattta tataagtctg 1080 ttatttatta ttaatttatt ggggtgacct tcttggggac tcgggggctg gtctgatgga 1140 actgtgtatt tatttaaaac tctggtgata aaaataaagc tgtctgaact gttaaaaaaa 1200 aaaa 1204

(7) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 308 b) TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 Met Pro Gly Gin Glu Leu Arg Thr val Asn Gly Ser Gin Net Leu Leu val Leu Leu val Leu ser Trp ieu pro His Gly Gly Ala Leu ser Lett Ala Glu Ala Ser Arg Ala Ser Phe PrO Gly Pro Ser Glu Leu His ser Glu ASO Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu Thr Arg Leu Arg Ala Asn Gin Ser Trp Glu Asp Ser Asn Thr Asp Leu Val Pro Ala Pro Ala Val Arg Ile Leu Thr pro Glu val Arg Leu Gly Ser Gly Gly HiS Leu His Leu Arg Ile Ser Arg Ala Ala LeU Pro Glu Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser Pro Thr Ala Ser Arg Ser Trp Asp val Thr Arg Pro Leu Arg Arg Gin Leu Ser Leu Ala Arg Pro Gin Ala Pro Ala Leu His Leu Arg Leu ser Pro Pro Pro Ser Gin Ser Asp Gin Leu Leu Ala Glu ser Ser Ser Ala Arg Pro Gin Leu Glu Leu His Leu Arg Pro Gin Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn Gly ASP Asp cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu val Gin val Thr Met Cys Ile Gly Ala Cys Pro ser Glm Phe Arg Ala Ala Asn Met His Ala Gin Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Glu Pro Ala Pro Cys Cys val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gin Lys Thr Asp Thr Gly val Ser Leu Gin Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile

(8) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 4015 b) TYPE: nucleic acid (ii) MOLECULE TYPE: cONA to mRNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens Lg ozLF VD7D3PD3a3 6631763bbl De6eDED7D3 1172311)15 )361DDVDED 6E331516E6 099E 136E62E336 E331646E53 6E613366E6 1366311646 E61366tE61 36633161E3 009E 613366E633 65444616E6 16043366E 61316E6661 ADDE36DD) 66E333E3E3 opsE 336636666e 666E666E33 34631E13136 663363e346 13336e16e3 3e3e6e36e6 02VE E63366E336 EDED))673 e33661e613 e661331.E33 E6ee3413e6 ED1D3)61DE
ozvE D66)3DEP33 6E353356E6 61313613E6 1363E63E66 66333136EE 66316E6136 ogEE PDbDetreD73 6e3E66e313 e316666133 13e336463e 133E316163 pe3E6313E6 00EE zpecee31364 3321e36ee3 3E33615136 616E361633 66E6311333 6131331663 ovu 363363666e v336666611 63161E666e 16Dpp6PP31 6ppr5lnl1V 3311E13611 ogTE 1p3D4D)66) EDE633131E 3163636i33 21111eDEDD 33EE6Ee661 146ee36E31.
OZTE ED141e3DD1 36e3613616 161E363E3z 4166E)V163 66E3613613 34331e6ee3 090E el31PDPEOD P36161661e 6Eppa336e3 EE6465e361 lae6613111 54336e3E32 000E 546ee64366 3644346666 414.343pee3 6316364eae 06e666136 61EE3413663 Ob6z 6leeD113 e3a116e336 r6r3ae3323 pe6637361P 1D6P7D1Del 3e636e6E36 oz 166E661333 E66333e3E6 6136436apa 6636166133 3311P13366 3E33366336 02Z4E6E311611 1.13563E366 1666133D56 E63E6EE6E4 64333113EE 616646E3E6 ogLz ee6636143e r62661636z 3661'n6E64 33316466e6 7)4.661D)7e 66e3433113 OOLZ peeee6363e DD)ED1DDeD z33E3E6166 1161131a1E 61E6616611 1635131136 ogz 436663E666 366341e666 6361116136 eepee6e661 epe6366ael 361613)6PD
08SZ 6131361363 eDnD1D14e )713666PD6 333105666e 3D626e)D16 3e1)31fte, onz 6666 D6 36163363E3 3E336161E3 41363E1.331 13163e63a4 3433661.6E3 09tZ 6666,e P61331D31. )6Q6P36P63 len6D161) 6117665p601) 6D316P3DP5 00tz tE0661Dpe alD6616,11 6e3e6AleD EI6DAE)34 33eerne6al Dpel)1)46D
OtEZ rn6e6eeD1 13D66E,E5D )16ye36664 eaD3633ftie eempDatiblb 3364e16631 08ZZ 63616)61ae 16or)ey6to Dp3eM4e) 1E36E33631 E3466E663e 34366E3E65 OZZZ VDDDDDleDD vng631,4W 3666)0461 E66166ee31 61.113E1643 6-01336336 09TZ 1DE66E733 65636164E0 61361631a3 DED6)661.D) 666E3E331E 1E63E66133 OOTZ 6661D6141) ZDADE0661) 317D66)DD3 6366353666 36E63e33ve 31361636e) OVOZ 11613E365e E64566E631. 33E3431636 E633556E6e EEE6e6e363 31163ee6e3 086T 36e6661631 6arlpe664r 32E61614E6 3366361366 63E64336ve )3334E3aap 0Z61 6331De6r33 16DP61D613 )3677D66E1 35e66631e 36E366e3a6 60e36et.65 0921 D161D6066 364)6e3616 66e6e6113 rD6Re6E,D1 pv66112D6p PP36116PED
ONT 6e66a31.516 eftp6633e4 3111113436 ETDEP6reep pa1163E3DE 6e661e3161 ova e1311D444D 166E3436a3 6E63163163 e464646e61 e6136613e3 61D)13.6veD
099T 3661Dalvft 6601663) 163).eD5p62 36DD663D11 616'1D66116 566e3336E5 ozgT 6E16361366 a1,6361.30 663616)60 leftE66163 E6136e66E1 6136313135 09ST re3161e36E E6656z1334 34E3116ee6 reppe3ee66 E313311363 36yee63ve3 OOST E366e33135 556z333366 e333 6466 4.166J36)64 3364335663 5463113663 otte16166e366 133336E36e 3e36e3a633 136136e364 6613361161 D.,7126VDR3 ogET e66E66e66e 6333336636 6161D13666 EDDDD6E6P 666333646a 316i66336E
OZET pfteDODOtl 66)61)50D 6136)D361) v363e6ee)1 Dalp616666 Delp,D3516 09ZT ED6D6DP3De P65641,61D 5E66333446 1.DDDDE45361 eve36613E1 3636E33336 00ZI 1DDEODD)61 166e,6 , 13E666E336 1e66133366 E331166613 1.17DarDpe6 OKI P661633.366 r66,136D156 1De61336pD 3366e61323 136E313E13 D11D11DD36 HOT 6161D6e36e 66ene6D66 PD13313P1) 133113E36e E33E6E6336 pe461663aD
OZOT )1.611Da6) EDP66647,17 ltbeD)V3D6 6)folepep) 1U))))))66 EID5)EDDE6 096 e33633666 63)1E333E3 3313E33636 3E35643131 36361666E6 64z13133E3 006 35e0er633 6DD)R6ED36 133e346166 4616434116 64633e616E 65666 OS 3E66E36663 33e3336661 33z6666E36 6611633363 E6636e6633 6E5433336a 02L 3635616366 E33366r6ee 3336116336 a346E26336 e33616e366 66536366E6 OZ/ 6E63616663 3335E33643 3666133333 466663366e 666e34636E 4e)Dee6643 099 DE,66yer636 4e6661316D 66ee6Dpu 66;6e4D6DE )eD7E0D)D 66DD355vD4 009 3E33613636 63).36E33E1 63.36336336 663616166E DDE1D76)61 D6VDDD1.D66 (WS 166116a611 1313636362 353E366136 133E341661 36163E63E6 3666363633 o9t, 6364361364 3655663,636 66536E6666 6361.3e363e 633e64663e pre3336133 OZt e46eD6o64 636E33E33E 33366E63 1333)36665 6363336666 3e662D61D6 09( D6a1,6631 a336613616 lee6ee6363 663636E636 161366E6E3 613646E633 00E D65 6D6 66 61D361 3D16166en 6D321)D1)) )))6336)DD DDD63366DE
ovz 6py66613 )616D61616 6433616en )661651D63 636n211D6 6D683nr66 091 66D5)5tDba 651360D661 )6156m)36 666666 )63.6341,6)e a366a.DODD6 OZT ap61.65eob DDE1DED)6E D6753361)7 D3.36D616D0 6E63364363 3334363636 09 3361E63633 DDDED3663) 3)661DD36e P66616DED6 )61D5a333.6 D61D6DE0E76 L :ON OI 035 :NOIldInS30 3DN3IO3S (!x) gggccagctt ttcctcacca ggagcccggc ttccactccc cacataggaa tagtccatcc 3780 Ccagattcgc cattgttcac ccctcgccct gccctccttt gccttccacc cccaccatcc 3840 aggtggagac cctgagaagg accctgggag ctctgggaat ttggagtgac caaaggtgtg 3900 ccctgtacac aggcgaggaC cctgcacctg gatgggggtc cctgtgggtc aaattggggg 3960 gaggtgctgt gggagtaaaa tactgaatat atgagttttt cagttttgaa aaaaa 4015

(9) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 1132 b) TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly Pro Gin Gly Trp Arg Lets val Gin Arg Gly Asp Pro Ala Ala Phe Arg Ala Leu val Ala Gin Cys Leu val Cys val Pro Trp As Ala Arg Pro Pro Pro Ala Ala Pro Ser Phe Arg Gin val Ser Cys Leu Lys Glu Leu val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala LyS Asn val Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Glu Ala Phe Thr Thr Ser val Arg Ser Tyr Leu Pro Asn Thr Val Thr Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg val Gly Asp Asp val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val Leu Val Ala Pro Ser Cys Ala Tyr Gin val Cys Gly Pro Pro Leu Tyr Gin Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly Pro Arg Arg Arg Leu Gly cys Glu Arg Ala Trp Asn His ser val Arg Glu Ala Gly val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gin Gly Ser Trp Ala HiS Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys val Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr ser Leu Glu Gly Ala Leu Ser Gly Thr Arg Hisser His Pro Ser Val Gly Arg Gin His His Ala Gly Pro Pro Ser Thr ser Arg Pro Pro Arg Pro Trp Asp Thr Pro Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr ser Ser Gly Asp Lys Glu Gin Leu Arg Pro Ser Phe Leu Lim Set" Ser Leu Arg Pro Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser Arg Pro Trp Met PrO Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gin Arg Tyr Trp Gin met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His Ala Gin Cys Pro Tyr Gly val Leu Leu Lys Thr His Cys Pro Leu Arg Ala Ala val Thr Pro Ala Ala Gly val Cys Ala Arg Glu Lys PrO Gin Gly Ser Val Ala Ala Pro Giti Glu Glu Asp Thr Asp Pro Arg Arg Leu val Gin Leu Leu Aeg Gin His Ser Ser Pro Trp Gill, Val Tyr Gly Phe Val Arg Ala Cys Leu Aro Arg Leu val Pro Pro Gly Leu Trp Gly Ser Arg His Asn Glu Arg Arg Phe Leu Arg Mn Thr Lys Lys Phe Ile Ser Leu Gly Lys HiS Ala Lys Leu Ser Leu Gin Glu Leu ihr Trp Lys Met Ser val Arg Asp Cys Ala Trp Leu Arg Arg ser Pro Gly val My Cys val Pro Ala Ala Glu His Arg Leo Arg GU 610 Ile LOU Al4 Lys Phe Leu His Trp Leu met ser val Tyr val Val, Glu Lep Leu Arg Ser Phe Phe Tyr val rhr Gip rhr 1hr Phe Gin LyS Asn Arg Leu Phe Phe Tyr Arg Lys Ser val Trp ser Lys Leu Gin Ser Ile Gly Ile Arg Gin His Leu Lys Arg Val Gin Leu Arg m Leu ser Glu Ala m Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile val Asn met Asp Tyr Val Val Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg Pro Gly Leu Leu Gly Ala ser val Leu Gly Leu Asp Asp Ile His Arg Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gin Asp Pro Pro Pro Glu Leu Tyr Phe val Lys Val Asp Val Thr Gly Ala Tyr ASP Thr Ile Pro Gin Asp Arg Leu Thr Glu val lie Ala Ser Ile Ile Lys Pro Gin Asn Thr Tyr cys val Arg Arg Tyr Ala val val Gin Lys Ala Ala HiS

Gly His Val Arg Lys Ala Phe Lys Ser HiS Val Sir Thr tell Thr Asp Leu Gln Pro Tyr met Arg Gin Phe Val Ala His LOU Gln Glu Thr ser Pro Leu Arg Asp Ala val val lie Glu Gin ser ser ser Leu Asn Glu Ala Ser Ser Gly Leu Phe AS val Phe Leu Arg Phe Met Cys His His Ala val Arg Ile Arg Gly Lys Ser Tyr val Gin cys Gin Gly Ile Pro Gin Gly Ser Ile Leu ser Thr LOU Leu cys ser Leu Cys Tyr tly Asp Met Glu Asn LyS Leu Phe Ala Gly Ile Arg Arg AS Gly Leu Leu LOU

Arg Leu Val AS ASP Phe Leu Leu val Thr Pro His Leu Thr His Ala Lys Thr Phe LOU Arg Thr Leu val Arg Gly val Pro Glu Tyr Gly cys Val val Asn Leu Arg Lys Thr val val Asn Phe Pro val Glu Asp Glu Ala Leu Gly Gly Thr Ala Phe Val Gin met Pro Ala His Gly Leu Phe Pro Trp Cys Gly Leo Leu Leu Asp Thr Arg Thr Leu Giu vai Gin Ser Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr *le Asn Arg Gly Phe Lys Ala Gly Arg Asn met Arg Arg Lys teu Phe Gly Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gin Val Asn Ser Leu Gin Thr val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gin Ala Tyr Arg Phe His Ala Cys Val Leu Gin Leu Pro Phe His Gin Gin Val Trp Lys Asn Pro Thr Phe Phe teu Arg Val Ile Ser Asp Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Giu Ala val Gin Trp Leu Cys His Gin Ala Phe Leu Leu Lys Leu Thr Arg His Arg val Thr Tyr val Pro Leu Ley Gly Ser Leu Arg Thr Ala Gin Thr Gin Leu Ser Arg Lys Leu Pro Gly Thr Thr Leo Thr Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp

(10) INFORMATION FOR SEQ I0 NO: 9 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 3979 b) TYPE: nucleic acid (ii) MOLECULE TYPE: cDNA to mRNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: hOmo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 geagcgctgC gtcctOctgc gcacgtggga agctaggcc etogccactc ccgtgatgcc 60 gcgcgctccc cgctgccgag ccgtgcgctc cctgctgcgc agccactacc gcgaggtgCt 120 gccgctggcc acgttcgtgc ggcgcctggg gccccagggc tggcggctgg tgcagcgcgg 180 ggacccggcg gctttccgcg cgctggtggc Ccagtgcctg gtgtgcgtgc cctgggacgc 240 acggccgccc cccgccgtcc cctccttccg ccaggtgtcc tgcctgaagg agttgOtggc 300 ccgagtgctg cagaggctgt gcgagcgcgg cgcgaagaac gtgctggcct trim-mac 360 gctgctggac ggggcccgcg ggggcccccc cgaggccttc accaccagcg tgcgcagcta 420 cctgcCcaac acggtgaccg acgcactgcg ggggagcggg gcgtgggggc tgctgagcg 480 ccgcgtgggc gacgacgtgc tggttcacct gctggcacgc tgcgcgCtct ttgtgctggt 540 ggctcccagc tgcgcctacc aggtgtgcgg gccoccacta taccagctcg gcgctgccac 600 ppe wpm :34A1 OZTI :H15,131 (e S)IISIII31.7µmvi0 3DN3003S (0 OT :ON OT bS W04 NOTIVW403NT (TT) 6L6E reeeeee5 3.116E3341a 0961 1664elear 64Delteee 166661613 616566666 55 6161)=66 006f 6661e66= r361=e56 6365e3ear 16=36161 66eer3304 666113e6 otrff 5513435e66 6333366ee 6e5a)33r5t 65166e3)4e nE)33D3e3 31a316111) 09LE 71)7,61733 6)133)3E74 4611e31531 16e3=le 3346elev65 eleae33331 0u1 3e3=366) 3)666e= D10)1411,6 r))666e= DeDD436531 363661)56e 099E DTDDiD11TEt 321D16))53. D3V]e,6VD3 16)661366 6eeDD6eD31 61663661 0091 D356261)56 =66eft) 6666) 3162e36133 66e6336611 164663.66 OVSE 13)656131 6066a3633 p3633366e) 13r3r31)66 35666666e 656e=163 ogyE e1)13666)3 63VD154]] 6t76t÷e7V 5e)566)) 56e))6e)r3 ))6=r336 0nq. 61613e661 DD1r3De6re 31136e3a3 33613e3663 neE3D6ED6 D166V6611 09E1 361Dy613tb 63e666633 Dla6re5631 60336r351 6e)336ene 66e3132316 0011 66613)13e) 36163e=e DI:616)7E7P 634)e6136e e)136 44 P)62e),P)) oyzE 6643664.6e 36463)656 334=6= 333663D6D3 6)666re))6 66664)6345 09TE 1P666e36e lidetDaftwe 64331v3313 ElD6131373 4)35631.70 13431e3163 OZT 636=1311 le3e))33ee ber661136e e36e)1r312. ITDDDZ36V) 643616461e 0901 363e334166 e)r45)66e3 6136133133 16er3r1D2 epreple561 61661e5e33 0001 4)36e3er04 60,351440 6134145333 6e3e34646e e543663644 D1665014D
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09V 6161e31136 3e4. 44316 36=3133 6616eD6eD3 6661e633 DD17)4.)5E6 0017? eD6631e31 631633516 666136= 61)36e662 36133e3136 6463415eDe 011? 6361r3e163 a6e331310 VDP614D3r2 3131.6,e1D6 e6e311136 6re36))15) 08ZZ P6661e)n 53)66Pr6e) pl&616)D61 el663z6361 61611plbe yer6e3e OZZZ eeDar3arD5 to3631e316 6e65De3136 5e366teD1 54113e163) 5e543)0336 091Z 33De66e D 666364.6163 61.)646=3 )eD5D66133 666e3e)34e 46)6643D
ouu 65f1D61611 1116166613 3.331563DO1 6365361666 3563eiDee 11.351536ep OtOZ 1151DED66e 64666631 )3e3131635 e6 6666e ree66235) 3115Dee6e3 0261 36e66646)1 barlar664e Dee64614e6 3366361366 6361336'er )31D1E)11 OZ6T 633336r33 36361)63D D3533)66rD 35te66631r D6rD66r31.6 66e566 099T 316136656 36136,23636 666e01.13 e36eDe6e31 ve6611e36r eu35115eeD
008T 6e661)4626 efte6601e1 31114=36 6eDes6ever 311163me 6663e3161 (*LT ezllapal÷ 156r313613 5e6)161153 e161616e61 e613661)rp nalalaBetl 0891 36643346r 66e646)643 4632r366e 363)663344 6461)661.16 566e a6e6 onT 6ra6D61365 alD6D6436 66365366 e6 665 e6a3666eD 6136)4)436 09ST re3)61e35e 0666a=1 71E)17,5EVEI eP3DEDPE66 P71)711363 363e6pee3 loosT ea56e=35 65643=66 e33 36166 1366336361 3361336563 616=3663 e16166e355 133336r36e 1e35e336)3 a35136e362 6617)6)16D 3D3DE6tDED
08Ei 66e6666e 5)33336636 6151)13666 r33)36t6e 666=6161 316165135e OZET 76VDDDDP?1 66361.36e63 6135333613 eD63e6ee31 =3616666 DP111DD616 09Z1 E)6DoDEDDE 66631)6a) 6661)2126 1333366363 reeD6643e1. 3636e33336 00ZT 13363=64 366e3633)3 13066e3D6 3'66133366 1.33116653.3 11131230125 e6616)1366 e663136366 1301336e3 6664343 436e343E4) 741.71,D)5 0901 6)61)6e35 66re)6D66 VD33,IE3.) 1D)34,VD6E vD1e6e5)36 3e1646E0)D
OZOT D31611D363 P,P661313DD 161e1)en6 8D6D4P3E3D lEDDD3)366 636)e)3rD6 096 e)3633666a 6=e=r3 DD43E?D676 3e356131)1 )6361655e6 6a1a3233e3 006 35e6e633 6333e6e336 133e)16466 3.615131116 616D31416e 5e666 08 e66.n5,56 n)a6664 ,34.6666e36 bea466, e66,5e55a 6e6,64.
OW )636616156 e)))66r6ee ,6116318 1D16E286 e1)616e366 66635)666 OZL 563616663 336E7D51.) D65483DDD3 3.55663365e 666k &e6 aPpet66ap 099 )66EIDYP6D6 465613163 66e03333e 6616e436)r DEDD6a) 66=66eD3 (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 Met Pro Arg Ala Pro Arg Cys Arg Ala val Arg Ser Leu Leu Arg Ser His Tyr Arg Glu val Leu Pro Leu Ala Thr Phe val Arg Arg Leu Gly Pro Gin Gly Trp Arg Leu Val Gin Arg Gly Asp Pro Ala Ala Phe Arg Ala Leu val Ala Gin Cys Leu val Cys val Pro Trp z Asp Ala Arg Pro Pro Pro Ala Ala Pro Ser Phe Arg Gin Val Sec Cys Leu Lys Glu Leu Val Ala Arg Val Leu Gin Arg Leu Cys Glu Arg Gly Ala Lys Asn Val Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly PrO Pro Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr val Thr AS Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg val Gly Asp Asp val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe val Leu val Ala Pro Ser Cys Ala Tyr Gin val Cys Gly Pro Pro Leu Tyr Gin Leu Gly Ala Ala Thr Gin Ala Arg Pro Pro Pro His Ala Ser Gly Pro Aril Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser val Arg Glu Ala Gly val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly Gly ser Ala sec Arg Ser Leu Pro Leu Pro Ly$ Arg Pro Arg i2,14.8 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gin Gly Ser Trp Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val val ser pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala Leu Ser Gly Thr Arg HiS Ser HiS Pro Ser Val Gly Arg Gin His HiS

Ala Gly Pro Pro ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro Cys Pro Pro val Tyr Ala Glu Thr Lys HiS Phe Leu Tyr Ser Ser Gly Asp Lys Glu Gin Leu Arg Pro Ser PheLOU Leu Ser Ser Leu Arg Pro Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gin IR Tyr Trp Gin met Ta Pro Leu he Leu Glu Leu Leu Gly Asn His Ala Gin Cys Pro Tyr Gly val Lew Leu Lys Thr His Cys Pro Leu Arg 405 410 41$
Ala Ala Val Thr Pro Ala Ala Gly val Cys Ala Arg Glu Lys Pro Gin Gly Ser val Ala Ala Pro Glu Glu Glu Asp Thr ASp Pro Arg Arg Leu Val Gin Leu Leu Arg Gin His Ser ser Pro Trp Gin val Tyr Gly Phe val Arg Ala Cys Leu Arg Arg Leu val Pro Pro Gly Leo Trp Gly ser Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Ly$ Lys Phe Ile ser Leu Gly Lys His Ala Lys Lem Ser Leu Gin Glu Leu Thr Trp Lys met Ser val Arg Asp Cys Ala Try Lev Arg Arg Ser Pro Gly val Gly Cys val Pro Ala Ala Glu His Arg Leu Arg GU" Glu Tie Leu Ala Lys Phe Leu His Trp Leu Met Ser val Tyr Val val Glu LOU Leu Arg ser Phe Phe Tyr val Thr Glu Thr Thr Phe Gin Lys ASh Arg Leu Phe Phe Tyr Arg Lys Ser Val Trp Ser Lys Leu Gin Ser Ile Gly Ile Arg Gin His Leu Lys Arg Val Gln Leu Arg Glu Leu ser Glu Ala Glu val Arg Gin His Arg Glu Ala Arg Pro Ala Leu Leu Thr Sitr Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile val Asn met Asp Tyr val val Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gin Asp Pro Pro Pro Glu Leu Tyr Phe val Lys Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gin Asn Thr Tyr Cys val Arg Arg Tyr Ala val val Gin Lys Ala Ala His Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp Leu Gin Pro Tyr Met Arg Gin Phe val Ala His Leu Gin Glu Thr Ser Pro Leu Arg As Ala Val val Ile Glu Gin Ser Ser Ser Leu Asn Glu Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe met Cys His His Ala Val Arg Ile Arg Gly Lys Ser Tyr val Gin Cys Gin Giy Ile Pro Gin Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu cys Tyr Gly Asp met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu Arg Leu Val Asp AS Phe Leu Leu val Thr Pro His Leu Thr His Ala Lys Thr Phe Leu Arg Thr Leu val Arg Gly val Pro Glu Tyr Gly Cys Val val Asn Leu Arg Lys Thr val val Asn Phe Pro val Glu Asp Glu Ala Leu Gly Gly Thr Ala Phe val Gin Met Pro Ala His Gly Leu Phe Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gin Ser Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe Mn Arg Gly Phe tys Ala Gly Arg Mn Met Arg Arg Lys Leu Phe Gly val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gin val Asn Ser Leu Gin Thr val cys Thr Asn Ile Tyr Lys Ile out v66e66e66r 8,Dpa36E06 6a61D1D566 mnbeeft f65,a36464 D26466D6r ozET bea3)3Eal 66D ) 64AD)54 eD6Dtkeal Dp64.6866 ae4÷)6446 ogzT PD6D6De3DT e6661aD643 6056a3;146 TDupp66364 eeeD6633ea D6)5p37)-36 0021 za6a3D61 166eD6',3) 4De66612aA le664a66 eaa44666a) ala3leuae6 OTT e65a6D1D56 r56Da6366 1pp6a3D6p) op56.61.D4D aa6e1peap 1.1))1'DDD6 OSOT (3)616eD6e 6Otear6366 PDZn4W13 17311DRAP epae6e6n6 3e16168apa OZOT 3Dabllapb) e3v6E161)3 1632DDeDD6 6D6DaRDVD3 1E3n)D)66 63EbepueD6 096 e6D)666a 6aDlle)3,eD 31DE36,6 Dea661Dla )6)64666v6 6141a1Dep 006 D6e,6r26DD 63Dpv6e136 arD161.66 16161,1116 61633e6a6a 6D3P661.616 ovg De66e)6557 ,)v..33)5661 )a16656e8 561463D36a t6W,e66D3 6e613)54 08L D6)6615)66 eD3u66e6re na6a263D5 1Dafte6D)6 Dp6a6p)66 666)6D66p6 OZL Be061666D 3D36z3.)643 )66613Daup 16666aD60e 666eal6D6r xeDaee66au 099 D666D2e6)6 le6664D463 66u26De 66a6v167a 7M15DD3D, 6666eD4 009 DvD61,66 6a1D6eDav1 61,6136,D8 66)64631362 ),n336A4 DeleD,D1D66 OVS 465ZD61611 ;,1D6D6361 DtorD661p6 aaDepaa66a )546Dt6Dt6 D66646D6ap 091' ba6aD6aD53 )6666016D6 6666p6666 6D613eD6De 6a7e6166De DEP,3)61)) on7 p4D6eD6D61 6)6tnrDDe DalaD66r63 DDD3D6656 663,D6666 )r661.61.6 09E 5D11D66)a app562.)6a6 eu6E,T.M51 66D6165D5 in 666 62D6161.63o 00E E,61.564)6e 56 6.6I 3)16z66rD3 6 1=aDD ap6Da63D Dp36)663r D63V6661.)1 D625,61636 b1D1626eD1 166z6ba,6) 6)6)Da1136 6,66D),e55 081 66D6D5eD5a 664.66D6i54 Da5eDDD8 6561.3,6D68 D646116De DA61,6D5 OZT z38166R6,6 Dava3e3315e D6D6aAiD) 11)61616m 6p6336163 DDD1D5D636 og 364e6D6) aqeDfiCtaa b64),Dbe e66516DeD6 a62,64)Dab )62D5)6e36 TT ;ON OI b3S ;N0Il4IWDS3O 3Dm3nb3S
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Pro Pro Ala Ala Pro ser Phe Arg Gin Val Ser Cys Leu Lys Glu lieu Val Ala Arg val Leu Gin Arg Leu Cys Glu Arg Gly Ala Lys Asn Val Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val Gly AS Asp val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe val Leu val Ala Pro Ser Cys Ala Tyr Gin Val Cys Gly Pro Pro Leu Tyr Gin Leu Gly Ala Ala Thr Gin Ala Arg Pro Pro PrO His Ala Ser Gly Pro Arg tp Arg Leu Gly Cys Glu Arg Ala Trp Asn HiS Ser Val Arg , Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg PrO Arg Arg Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro val Gly Gin Gly Ser Trp Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys val Val Ser Pro Ala Arg PrO Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280. 285 Leu ser Gly Thr Arg HiS Ser His Pro ser val Gly Arg Gin HIS His Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro TrO Asp Thr Pro Cys Pro Pro val Tyr Ala Glu Thr LyS His Phe Leu Tyr Ser ser Gly ASP Lys Glu Gin Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro Ser Leu Thr Gly Ala Arg Arg Leu val Glu Thr Ile Phe Leu Gly Ser Arg Pro Trp Het Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro GlA

Arg Tyr Trp Gin met Arg Pro Leu Phe LeU 1;14 LeU Leu Gly,Asn :4;

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Leu Lys Arg val Gin Leu Arg Glu ieu Ser Glu Ala Glu val Arg Gin His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Lea Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg Val Lys Ala Leta Phe Ser Val Leu Asn Tyr 610 Arg Ala Arg Arg Pro Gly Leu Leu Gly Ala Ser val Leu Gly Leu AS Asp Ile His Ar0 Ala Trp Arg Thr Phe val Leu Arg val Ar9 Ola Gin Asp Pro Pro Pro 01u Leu Tyr Phe Val Lys Val Asp val Thr Gly Ala Tyr Asp Thr Ile Pro Gin ASP Arq Leu Thr Glu val Ile Ala Ser Ile Ile Lys Pro Gin Asti Thr Tyr Cys Val Arg Arg Tyr Ala Val val Gin Lys Ala Ala HIS

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a) LENGTH: 795 b) TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: home sapiens (xi) SEQuENcE DESCRIPTION: SEG TO NO: 14 met Pro Arg Ala Pro Arg Cys Arg Ala Val Aria Ser Lep Leu Arg Ser His Tye Arg Glo val Leu Pnia Leg Ala Thr Phe Val Arg Arg Leo Gly Pro Gln Gly Trp Arg Lem VAT Gin Arg Gly ASO Pro Ala Ala'Phe Arg Ala Lep val Ala Gin Cys Leo Val Cy$ Val Pro rep Asp Ala Arg Pro SO: 55 60 PrO PrO Ala Ala: Pro .Ser Phe Arg Gln val ser cys Leu LS Glg tag val Ala Arg val Leo Gin Arg Lev cys Glo Arg Gly Ala Lys Mn Val Lag Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Glv Ala Phe Thy- Thr Ser VA1 Arg Ser Tyr Leu Pro Asa Thr Val Thr Asp Ala Lett Arg Gly Ser Gly Ala Trp Gly Leo Lew Leu Arg Arg val Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val Leu Val Ala Pro Ser Cys Ala Tyr Gin val Cys Gly Pro Pro Leu Tyr Gin Leu Gly Ala Ala Thr Gin Ala Arg Pro Pro ProC His Ala Ser Gly Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro val Gly Gin Gly Ser Trp Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr ser Ley Glu Gly Ala Leu Ser Gly Thr Arg HIS ser His Pro Ser val Gly Arg Gin His His Ala Gly Pro Pro Ser Thr ser Arg Pro Pro Arg Pro Trp Asp Thr Pro Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly Asp Lys Gill Gln ieu Arg Pro ser Phe Leo Leu Ser Ser Leu Ary Pro Ser Leu Thr Gly Ala Arg Arg Leu val Glu Thr Ile Phe Leo Gly Ser Arg Pro Trp Met Pro Gly thy PrO Arg Are Leu Pro Arg Leu Pro Gin .
Arg Tyr Trp Gin Met Arg Pro Leu Phe Leu 3u Leu LeU Gly Asn Ala Gin Cys Pro Tyr Gly val Leu Leu Lys Thr His Cy5 Pro Leu Arg Ala Ala val Thr Pro Ala Ala Gly Val cys Ala Are Glu Lys Pro Gin Gly Ser Val Ala Ala Pro Glu Glu Glu ASp Thr Asp Pro Arg Arg Leu 435 440 445, Val Gln Leu Leu Arg Gin His Ser ser pro Trp Gin val Tyr,Gly Phe Val Arg Ala cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser Arg His Ash Glu Arg Arg Phe Leu Arg Asn Ti- Lys Lys Phe Ile Ser Leu Gly Lys His Ala Lys Leu Ser Leu Gin Glu Leu Thr Trp Lys met Ser val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Tie Leu Ala Lys Phe Leu His Trp Leu Met Ser val Tyr val Val Glu Leu Leu Arg Ser Phe Phe Tyr val Thr Glu Thr Thr Pile Gin Lys Mn Arg Leu Phe Phe Tyr Arg Lys Ser val Trp Ser Lys Leu Gin Ser Ile Gly Ile Arg Gin His Leu Lys Arg val Gin Leu Arg Glu Leu ser Glu Ala Glu val Arg Gin HiS Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile val Aso Met Asp Tyr val Val 41Y Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg Pro Gly Leu Leu Gly Ala Ser val Leu Gly Leu Asp Asp Ile His Arg Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gin Asp Pro Pro Pro Glu Leu Tyr Phe val LyS Asp Arg Leu Thr Glu val Ile Ala Ser Xle Ile Lys Pro Gin Asn Thr Tyr Cys Val Arg Arg Tyr Ala val val Gin Lys Ala Ala His Gly His val Arg Lys Ala Phe Lys Ser His Val Leu Arg Pro val Pro Gly Asp Pro Ala Gly Leu His Pro Leu His Ala Ala Leu Gin Pro Val Leu Arg Arg His Gly Glu Gin Ala val Cys Gly Asp Ser Ala Gly Arg Ala Ala Pro Ala Phe Gly Gly (16) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS

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val Phe His val Glu Lys Asn Gly Arg Tyr Ser tie Ser Arg Thr Glu Ala Ala Asp Leu Cys Lys Ala Phe Asn ser Thr Leu Pro Thr Met Ala Gin Met Glu Lys Ala Leu Ser Ile Gly Phe Mu Thr Cys Arg Tyr Gly Phe Ile Glu Gly His val val Ile Pro Arg Iie His Pro Asn Ser Ile Cys Ala Ala Mn Asn Thr Gly val Tyr Ile Leu Thr Ser Asn Thr Ser Gin Tyr Asp Thr Tyr Cys Phe Mn Ala Ser Ala Pro Pro Glu Glu Asp Cys Thr Ser Val Thr Asp Lei Pro Asn Ala Phe Asp Gly Pro Ile Thr Ile Thr Ile val Asti Arg Asp Gly Thr Arg Tyr val Gin Lys Gly Glu Tyr Arg Thr Mn Pro Glu Asp Ile Tyr Pro Ser Mn Pro Thr Asp Asp Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr ser Gly Gly Tyr lie Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp Ser Pro Trp tie Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Arg Asp Gin Asp Thr Phe His Pro Ser Gly Gly Ser His Thr Thr His Gly Ser Glu ser Asp Gly His Ser His Gly Ser Gin Glu Gly Gly Ala Asn Thr Thr Ser Gly Pro Ile Arg Thr Pro Gin Ile Pro Giu Trp Leu Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala val cYs Ile Ala Val Asn ser Arg Arg Arg Cys Gly Gin Lys Lys LyS Leu val Ile Mn ser Gly Asn Gly Ala val Glu Asp Arg Lys Pro ser Gly Leu Asn Gly Glu Ala Ser Lys Ser Gin Glu Met val His Leu Vel Mn Lys glu Ser Ser Glu Thr Pro Asp Gin Phe met Thr Ala Asp Glu Thr Arg Mn Leta Gin Mn Val Asp met Lys Ile Gly val (24) INFORMATION FOR SEQ ID NO: 23 OZTE 44641.37e4 ele6622)rp 1)DDa6r161 1613t11e11 1,3414144 3eleetalee 090E 664444e444 leeD4444e1 166rane6 e6466e6re peppo44e4 r6r1,31.D0a 000E DeD41.1466 1p6p6na6r leD1D16rep r1r646r466 66pre6e666 r3DDD6D161 e1,1116r ,Prlp11661 PPPP1366e P3E66113313 P336Pneu) 6reettapee 088Z 14434D6rea lapaz366ra e6r,66e6 EDD6a4556 433rEfReo rD3D41a16a OZ8Z 1313616te D4a616elle D3611162 16161r13rD 166elveDe6 613ret6wer 09ZZ 6113eea412 1D1141.e114 443141e663 )16646r)44 Ilr÷ae6r) 6.641r)4r., 00a e0464.344) 4)4143413 eeeee611D1 EEDE)÷Dep 11)eDD1Da 4)1)1))1) Ot9Z DDD11I3nE 1)1.613D317 3135111n6 eD1111e1DP 361)113DEe ,16ar6rOt6 pesz 6err11=e 2er6r261)2 e6416e6eDr 6r4116661D 666eparen De61DD2)6e OZSZ ere6ee6er6 ere6eA5ere apprtieD4 4.4.264eta66 eare44646 4e41Druel3 0917Z 3E116441r) 16rerely4 D)4366e61D 1113a1D1v1. 66evez164 1)66Dler 004'Z 6rer1e1,lr D6a1e3D61!r 6e1rD161al 61366PEPD) 113PP6e1D) 16611e6))1 OVEZ 6666e D4r33.4e111 r36646a11.3 al6e36641 Depa)1)411 4elena)re 08ZZ 4n134e111 3661Delbeb Ebeepeem eble4116te oftelperp) e)leaz)4re OZZZ 6611e2e)3 D42066ree 4466reera ,1.6e6666 4,)646re44 6666e11 09TZ 2e4)6146R3 64r6a13116 3)1616r-31r learerllar re46111r46 1A64e1114 OOTZ 2e3r644461 D34e6e1.61e DD6101r66 re6666pre) 664ar6e6er r6lere66ze 0170Z rp6er6re6 r6prler,ra 1,2620111 41)fil5ee66 1P1MVPET e3)112p5115 el4r4))706 4365e6elep 443,Eq33)66 662)DD4eal 64)Deble)6 14454T21e3 0z61 vD16eve)64 11D66pre61 61,131D14 641rD3661 prlI1661ra 111.13,612a or 1.236e661151 )15re6643.6e 6enve6623 )161),1eD1 allIbErepto )161Dne51 0081 1111444111 1641244164 alla3.62131 363)eD6vD6 66e66eene 6ee6614a OtLI verr)D61)4 )137)P6E31 pe33D146e6 1.1.-6e6r) lale)14)40 erzplzpl)r 089T 6r66erz664 er6r64,6e6 er66731e36 6661)161Dr D6rDDerel) 0z9T 1416e3lial n6261,625 11113)P3a1 34e6111314 3e46134e4e re4re4rall 09si 164yerr14; 666vvreyea b6e6e)1641 larzlberrl 11341erlr2 6r)405111) 00sT DlOrD1r661 7),VPP11E1 ree6111116 46111T112r 17E2E16E16 2)1P124e2) 6rr64666re p De'rbe6 66 36e 6e)414z1er r6e4.11331 le6eper3z1 08E1 lepepr146 piDr161666 6446666632 66r613e111 113611e366 611nnleell OZET D6e1eDD1.1 Dp61111r1D 4661er641 lere66e666 1664) 666e4re146 09ZI 6e1131142 3e66erap6r 33e),D)311 13De3631 e613plaelb lele3e3e1) 00ZI veeeeDeeaa 11.)664e661 e436164666 71)))1tDal 4DD66e6611 Dr333146e, Olat D146,4044 lepEere6e) leeieel4re )666)112e pr61D111.)6 43ea6eDeeg 0801 e16111en D166r326er e441n4614 1264644114 1611.111)4) r43241.1ver OZOI elea6ella4 1434re6)61 reol44641e 64)elD6161 ev361e6epe r143epe666 096 4)6e66fteDe 14muelel EPP66116D1 pepeppftep 661171E11P D3E3,43)E) 006 m6466661 le6re64r)e 66.161er6r) 61Drr66er 3r6e64e643 6m)r6ar444 008 6epar6r331 Derrem346, 16r66re3Er 5656 166aper66e Da316tE06e 0v, D'A6e6r66, erDle6616 erl3tiver6e pe65e66161 36P661rPD6 626rDerole 0u, 616r136Pre re6re6t)66 616166er6r r6,16nrr, 16eD614rD6 441beD6a)) 099 r6e5114r66 )1r)be614 6ere6e664 e6rD3664e eYmp6;lo peD6e1eeD1 009 11,66m61, D1D)r64,63 )66r66De66 D1D131P36e 3e1.6)1661 gerre6e661 0ts 6)÷,11r26 166r,6414D 5o)61,prel elvv6111e6 ne6v)6)66 a 6e61Dfo 081' 5lba23,64 31)r666610 36e6)2)66 4664114E0u )061env) 06))z)633 zOnln)63 613)63141D Dz36P)313) 4r666raDD6 6aDa)61D, 66 6D3z 09( 1E1)31.16) 4D71.67 466 ftra 3,61,6e3 3,1166611, Da644rDaD
00E 6e)66e632 4repp6eD6 IDeereelee Eolperlal 13.61eD166 P11)16)D1D
OVZ D31DDD1)6) 1)6)113)16 16166611)6 14))))))6P pe315e11D6 10656A133, 081 )3PEVP11)1 1Dr1D64,66 6r666DD,6) aa61566e6n 66666D6666 Dea6r6vD6r OZT )4)a6e)T11 4E21e630)3 6e,661÷)4 6aelpe6) aD44.661ma )eDe6D6)]
09 1z366r,rD6 6e66nD666 616r1n6666 p1636661D1 116D616e3D 6ree6re6r6 EZ :ON CI b3S :NOLLAIU353G 33N3n03S (!.x) suWes atuog :WSINVDVO (V
:3311110S lvNI9I80 (LA) ',WNW ca vim 31n310W (H.) pp r DialDnu :adAl (q S86E :RIDN31 (e SDIISIV31IVWVH)3)N311b3S
LZ-LO-TTOZ TLEIVESZO YD

ttcacgatag aaataaggga ggtctagagc ttctattcct tggccattgt caacggagag 3180 ctggccaagt cttcacaaac ccttgcaaca ttgcctgaag tttatggaat aagatgtatt 3240 ctcactccct tgatctcaag ggcgtaactc tggaagcaca gcttgactac acgtcatttt 3300 taccaatgat tttcaggtga cctgggctaa gtcatttaaa ctgggtcttt ataaaagtaa 3360 aaggccaaca tttaattatt ttgcaaagca acctaagagc taaagatgta atttttcttg 3420 caattgtaaa tcttttgtgt ctcctgaaga cttcccttaa aattagctct gagtgaaaaa 3480 tcaaaagaga caaaagacat cttcgaatcc atatttcaag cctggtagaa ttggcttttc 1540 tagcagaacc tttccaaaag ttttatattg agattcataa caacaccaag aattgatttt 3600 gtagccaaca ttcattcaat actgttatat cagaggagta ggagagagga aacatttgac 3660 ttatctggaa aagcaaaatg tacttaagaa taagaataac atggtccatt cacctttatg 3720 ttatagatat gtctttgtgt aaatcatttg ttttgagttt tcaaagaata gcccattgtt 3780 cattcttgtg ctgtacaatg accactgtta ttgttacttt gacttttcag agcacaccct 3840 tcctctggtt tttgtatatt tattgatgga tcaataataa tgaggaaagc atgatatgta 3900 tat tgctgag ttgaaagcac ttattggaaa atattaaaag gctaacatta aaagactaaa 3960 ggaaacagaa aaaaaaaaaa aaaaa 3985 (25) INFORMATION FOR SEQ ID NO: 24 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 139 b) TYPE: amino acid (ii) MOLECULE TYPE- protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: Sal ID NO: 24 Net Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro Leu Ser Leu Ala Gin Ile Asp Leu Mn Ile Thr Cis Arg he Ala Gly val Phe His val Glu Lys Asn Gly Arg Tyr Ser Xle Ser Arg Thr.Glu Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala Gin met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Ser Leu His Cys ser Gin Gin ser Lys Lys Val Trp Ala Glu Glu Lys Ala Ser Asp Gin Gin Trp Gin Trp Ser Cys Gly Gly Gin Lys Ala Lys Trp Thr Gln 100 10$ 110 Arg Arg Gly Gin Gin Val Ser Gly Asn Gly Ala Phe Gly Glu Gin Gly 11$ 120 125 Val val Arg Asn ser Arg Pro val Tyr Asp ser (26) WORMATION FOR SEQ ID NW 25 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 4350 b) TYPE nucleic acid (ii) MOLECULE TYPE: cONA to mRNA

otsE 16epR66a33 ea166E6116 aeeP66Iree va6432563) z1111e6v11 DE051)36e3 ogyE e6e6ltz6F6 111))6a13) re35613361 e66Dele)61 19Z1D417T4 364eeepul ozyE 46664e1e6r le64De6e6ee 66146664z 6a4436ev64 eAlre6ee66 6all,r6e6a3 ogEE e,6556 vve661e)16 AvD65414 8661v1661 DPP D16 6ea31,11t) 00EE 114e)4)6e6 a6666p6111 livez316ee 631131DEUP DA1VEDDEP ae6e616r61 on 665 i6 (5v31))e) )31.31.133)1 DP1V6P3DD6 3eepalne3 1663e331)3 OSTE 313)6e)laa 6r366136)6 662 55 )3)666661e )1.1DVDD-4) 31.11Dee6e) OZTE ,361)2la 43r66136pe 61D)1330) 11D)415)7 66 6D6;? 664860,6634 ogof 3641333133 aleepeaie) e1ea)3166 61.66063,31 eee3)3a))e ee3e))13er 000E 6v3e1.66ee4 zpa)6666e4 66v61.2)661 ))411)1413 DDI.DDDD15.3. Dala31.33) 0t6Z 1D33)644. 21)33)34).3 6))311DRD1 666e)D6aD6 )62,666eza '05)626666a oggz 63.3e36eala 6e6)66662, 46e)36)66 611663r666 D1D61666DE )3E1.137131 029z ',6D6aft14, oz6,3664,1 236,61mD6 1,p,P15,616 61YeeprlD6 p66pppD6la oga pl6e663143, 4.0362133.1 3)=1366e 6111e)peel 1166D666e1 zael4eellz OOLZ albrela344.24434.4113.6 DI5a1400) 1.63e6zeen 44.t4-6124x4 tabteztql1 Ot9Z 11e6vE1646 11D6tleer4 ale64e411 1.)6D4.162e 6e661 XIDEDenD3 0135? ftz,1, De6 r)v663,e66r 666t6v)36z ,P6,666v 6orDp66wee eD65e61D3 OZSZ 16,a12,6vp veD6a3D2)6 P,1)PE3f0 114771766P D6D141D1D1 ppepr6e3)E.
ogyz 313342166r 63132e3e61 ))1eT)361.6 )116e66163 3636e))6ve e3)03)1e6) 00tZ 6eD636666) )664.3366e 3÷62e))36 epl.)66663) pe6e64)16e at4)e6e666 oyEz 6merept5e DeVDDE32.6 6V6DVDR4D4 16e06e661 av6,m66)) ogzz 166111t662 3113666326 pepi236661 5)1v6a163e 61t1lapv16 emE61e31 OZZZ 21z663ea12 3e663)36e6 le))3E0DDE )6136)3)36 )66e663666 )66e331)3e ogTz 32333613)3 ple1363e1) 6e6reD61)6 0E3336646 66116e363e e666z6e3)e OOTZ 66633113)6 6e36E63eza 44,e1)62)2 6>m163ze )3e,D6e3D6 1,643e3ex61 oyoz 364636R)1.1 )46)66a1v) 6)61662t)1 aftp62661) Bee6e533E6 pe33e3661t 0961 6)ea5e6le D1V3DEJ5D3 le,63D24D4 )6n625311 D56)D561A 2D144D346.) 0Z61 v356319'111 61D3e16161 11)13)3363 661161631a 3661616136 361e6616)e 0981 elera23666 616)14)61.6 1,6E66)6,64 A464r6166 )06166e3) 6662363661 OOST 3)ap33e)lv 3).e6veple) 3663364613 6661 6336 64)311.41 ea6e3e3lav OtLT ea36pe6)1E ))66e63e3) 666616n61 e)661)6636 63,3)146643 ap31363a61 089T Dne616661 661,TeD)43 6e1366 2t36 p3113113v1 31361e61e3 413z)34e33 ozgT b2 6666 6pp6tn)v) 666p363661 61)v363v36 6663e66e6) 363146ee)e 0951 6verz64616 645e6DDr6r e563.D3.31 3.666,4E. ,e 666 66463,66 OOST v3v3a63,66 ,)1611111) 1p)m))66 )6e66133e1 )6P311)636 6661 oyyi 6613an63e 11)6466)e) 1111)63e3) 1336361)61 643616e,45 6114e3661a NEI e6613Da363 631311.366 za6T66e633 ,666n43ez 64r3136661 el6166m) O?Lt63)6044 4)'m6)663 541e66Re6e 666661,aal De 3e 1,7e1D34JD
09ZT 361.66ee)43 1)6,6)6)31 61)alla6e s366e636e6 611516D55) )61665666D
002T 3)12366)66 1533e3666e 66356)661e )6e31333ev 36P330621 za6t6ran4a OKI )61)6)4)33 6)E(W3P3 666erae6)) z6mee6r) 3666463646 436e0366)) MOT 635631e3616 65 e6464.6ET)4 363e3e6ea) 6646e3)44) 66a446mt ozot P63 5b 60,61.3666 e))636)5)6 )6e6)646z) )31.D6336z, ))6))61D6D
096 66ea6e6e13 616))e)616 16131636)r 1612331)61 6337410115 re)136E64) 006 63311616e3 61.6eFt626p 1D1D1D9lna 15e),93626 696613D666 )6)061.66p (Nig pEeb,e)123 )6661:361)a ee )64r)a E,Ptin3VP Yel6D6D1ED e60aeD6261 OSL 6312334 31e÷,kt33 641e4,66)e 33e66)3346 paa.aae3666 6)5e6366De OZL v)t16e36e) 66636e6e)e rp6r36e313 D6D36n537 65 666336663) ogg )5666e3366 e))6666e)) 666)66)66r )3)6663)16 66661361)6 1)5)31366v oog 55 655 al361361)6 1361361,316 )536623263 663333)r61 lfreD63)D6 3)15D6Z)66 4563)6)R65 55 666 66p66)664) 663336631 4)636666)) ogy 616213,3ee6 664641636) 66166a66)) 6D666 )1 6ev6re2336 )66p66v66p OZt 61366m6e pe6e63)6e6 6u6e6pe 6 )66)6u66)6 P3DD36ED66 ogE 65DA036D) 5656556 37561DD)1? 1.66e6eD6D6 3)3v3666e6 63E6)13666 00E 41)6636vet 633566)6P6 6666)3606 tpre6e)6)e 1.66361)66) 66)66)6636 OtZ 265 neeftr3a4 )6)633)6)1 63)3)1)116 66)33v)36e 133)636)66 31)303p3e 666)661m66 e)1566e63) 6e636p)16p 6663)6e3)6 3361663636 OZT P13e336606 n66663,23 13136a6z16 p6r66361)6 661361)36e 61a6e66686 og 6e36366e66 366e66ee6p 66366366)6 6366E33.651 erepe3r6la e66.6e6116e sz :ON CI 035 :NOIldIlDS30 3DNWIWS (Lx) suo0es mot{ :WSINVD110 :3DIMOS 1YNI9M0 (L) tttgttaagg agttgatgtt tuctucctt aacaagacag caaaacgtaa acagaaattg 3600 aaaacttgaa ggatatttca gtgtcatgga cttcctcaaa atgaagtgct attttcttat 3660 ttttaatcaa ataactagac atatatcaga aactttaaaa tgtaaaagtt gtacactttc 3720 aacattttat tacgattatt attcagcagc acattctgag gggggaacaa ttcacacCac 3780 caataataac ctggtaagat ttcaggaggt aaagagggtg gaataattga cggggagata 3840 gcgcctgaaa taaacaaaat atgggcatgc atgctaaagg gaaaatgtgt gcaggtctac 3900 tgcattaaat cctgtgtgct cctcttttgg atttacagaa atgtgtcaaa tgtaaatctt 3960 tcaaagccat ttaaaaatat tcactttagt tctctgtgaa gaagaggaga aaagcaatcc 4020 tcttgattgt attgttttaa actttaagaa tttatcaaaa tgccggtact taggacctaa 4080 atttatctat gtctgtcata cgCtaaaatg atattggtct ttgaatttgg tatacattta 4140 ttctgttcac tatcacaaaa tcatctatat ttatagagga atagaagttt atatatatat 4200 aataccatat ttttaatttc acaaataaaa aattaaagt tmtacaaa attatatgga 4260 .
ttttgtgcct gaaaataata gagcttgagc tgtctgaact attttacatt ttatggtgtc 4320 tcatagccaa tcccacagtg taaaaattca 4350 (27) INFORMATION FOR SEQ 1D NO: 26 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 647 b) TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26 Met Ala Glu Glu Glu Ala Pro LyS Lys Ser Arg Ala Ala Gly Gly Gly Ala Ser Trp Glu Leu cys Ala Gly Ma Lett Ser Ala Arg Lou Ala Glu Glu Gly Ser Gly Asp Ala Gly Gly Arg Arg Arg Pro Pro Val Asp Pro ÷ 40 45 Arg An Leu Ala Arg Gln Leu Leu Leu Leu Leu Trp Leu Leu Glu Ala Pro Leu Leu Leu Gly val Arg Ala Gin Ala Ala Gly Gin Gly Pro Cl clo Cly pro Gly pro Gly Gin Gln Pro Pro Pro Pro Pro On Gin Gin Gin Ser Gly G3n Gin Tyr Mn Gly Glu Arg Gly Ile Ser val pro Asp His Gly Tyr Cys Gin Pro Ile Ser Ile Pro Leu Cys Thr Asp tie Ala Tyr Asn Gin Thr Tie met Pro Asn Leu Leu Gly His Thr Mn Gin Glu Asp Ala Gly Leu Glu Val His Gin Phe Tyr Pro Leu Val Lys Val Gin Cys Ser Ala Glu Leu Lys Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val Leu Glu Gin Ala Leu Pro Pro Cys Arg Ser ieu Cys Glu Arg Ala Arg Gin Gly Cys Glu Ala Leu Net Asn Lys Phe Gly Phe Gin Trp Pro ASp Thr Leu Lys Cys Glu Lys Phe Pro val His Gly Ala Gly Glu Leu Cys Val Gly Gln Asn Thr Ser Asp Lys Giy Thr Pro Thr Pro Ser Leu Leu Pro Glu Phe Trp Thr Ser Asn Pro Gin HiS Gly 41y Gly Gly His Arg Gly Gly Phe Pro Gly Gly Ala Gly Ala Ser Glu Arg Gly Lys Phe Ser Cys Pro Arg Ala Leu Lys val Pro Ser Tyr Leu Asn Tyr HIS Phe Leu Gly Glu Lys Asp cys Gly Ala Pro Cys Giu Pro Thr Lys val Tyr Gly Leu met Tyr Phe Gly Pro Glu Glu Leu Arg Phe Ser Arg Thr Trp Ile Gly Ile Trp Ser val Leu cYs Cys Ala Ser Thr Leu Phe Thr val Leu Thr Tyr Leu val Asp met Arg Arg Phe Ser Tyr Pro Glu Arg Pro fie Ile Phe Leu ser Gly Cys Tyr Thr Ala val Ala val Ala Tyr Ile Ala Gly Phe Leu Leu Glu Asp Arg val val cys Asn ASP Lys Phe Ala Glu Asp Gly Ala Arg Thr val Ala Gin Gly Thr Lys Lys Glu Gly Cys Thr Ile Lau Phe Net met Lou Tyr Phe Phe ser met Ala ser Ser lie Trp Trp val Ile Leu Ser Leu Thr Trp Phe Leti Ala Ala 611 Met Lys Trp Gly His Glu Ala Ile Glu Ala Asn Ser Gin Tyr Phe HiS

Leu Ala Ala Trp Ala val Pro Ala Ile Lys Thr Ile Thr Ile Leu Ala Leu Gly Gln val Asp Gly Asp val Leu ser Gly val cys Phe val Gly Leu Asn Asn val Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe val Tyr Leu Phe Ile Gly Thr ser Phe Leu Leu Ala Gly Phe Val ser Leu Phe Arg Ile Arg Thr Ile met Lys His Asp Gly Thr Lys Thr Glu Lys Leu Glu Lys Leu met val Arg Ile Gly val Phe ser WI Leu Tyr ppe outse :3dAl (q E6T :H/0N31 SMISIN3131#WW0 3DN3nb3S (!) gz :044 CI 03S 110d NOIIVWVWNI (6Z) EOZT vele oozT neer/peel! reeeereee3 ell3e216er merrelre v251163122 2r33e3,622 Ott222e65e564 v5v1V13an 6115VP,Au elp61A5ell 2ea34.11De6 6363.3e341D
000T .0410e0les e5,3666023 6626236256 334235e52e eaa5v633e6 624665043e 0z0I v662.561551 353162e613 6ee5501 v60202337e 56,12032e 3a21354337 096 6e5r333516 61o63.31133 131)11e,le 25e36666r5 66666 5562213r56 006 513evee364 ,373016,V35 5,5555333e e3,56v3323 v336236136 5126655134 OV9 225v235v1e 222.3ere523 6435e354e1 5e23654301 13e3233336 ap,)e)01al Ott EvD3.1,3a440 )3,3vev23e 33323333r, 3,Varlaal3 2766e2ve33 2722665323 cou 3226,27,73 323556625e 3366ee>3er 561ree3273 6e25666273 )e2,e35232 099 2e316626v3 52ev3634e5 e6 66b 2.33re3317e 2335525131 36r6ze5e6e 009 vmer33542 0606ve536 23313233e3 6626662e32 61e52.13e36 42366e6316 0ps 3er3ve3362 ere6234356 Vea351,14) ee334ezue r776373a13 r32e762355 OF0 236233236e 3ve3e3625e 323223666 6v3666e334 7434333243 6666 cap 3323235676 111231336) 3623230665 6236666231 5z53626366 1553651563 09E 0215635712 663763636 01367065) 6336636366 re 55166 6345566333 00E 36136e3,33 411)600W51 6536356253 5535535e36 15535161,r 35535,662a Otz 35236665v3 2.653235rap e366676653 656365064 25e033636 6356633 08T d1352DVDD5, Den62D662 663636,650 ev55533636 e713326663 36t5e62236 OZT 665521e366 765033z31) 665e346351 0263516261 352361345e 3355e6336r 09 23333e656z 3,111,013 1605erretv 055evetee ve2625566e 03leee5625 LZ :ON oi bBS :NOIldIVDSBO 30,43nMS (Lx) suds owoy :wSINVDVO (e :3)Wn0S 1VNI9ISO (LA) VNWOJ 01 Vt401 :um. Tirol-10w (R) = pm !.apnu (q EOZT :HIDN31 (V
SDUSIIMIDVIIVHD 3)N3nO3S
LZ :0N 01. Ws WA NOILVWWWNI (R) St9 len J4I J4I no ALD U9 sAl SE9, QE9 S49 Jos us y Jgl nal DJV J111 JAI a4d ski 6.1v du. JoS uSti nal JgI SA1 AL9 'la ail dii.a4d AID as J41 atI Alp LEA all nal Jg1 IOW

no, JAI ski au aw au d J41 a4d dsv cmd Jas IOW 0Jd 0Jd SLH
06S SOS, OVS
oJd eve, Alp Alp riy U. nal SIM
0Jd SA3 0Jd au I elv JAI
54S OLS gs JOS SA l SA 3 Jas Up 81.W IBA 041 JOS 6Jv no 0.41 ULD dSV 5JV Ogd 09S SSS OSS Sec elv ul9 ni9 JAL Ogd JAI SA3 elV alI LeA 0LI J4I etv 0Jd leA J41 SES OES
LZ-LO-TTOZ TLEItESZo vo (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28 Met Pro Thr Thr Ser Arg Pro Ala Leu Asp Val Lys Gly Gly Thr Ser Pro Ala Lys Glu Asp Ala ASM Gin Glu Met Ser Ser Val Ala Tyr Ser Asn Leu Ala val Lys Asp Arg Lys Ala val Ala Ile Leu His Tyr Pro Gly Val Ala Ser Asn Gly Thr Lys Ala Ser Gly Ala Pro Thr Ser Ser Ser Gly Ser Pro Ile Gly Ser Pro Thr Thr Thr Pro Pro Thr Lys Pro Pro Ser Phe Asn Leu HIS Pro Ala Pro His Leu LeU Ala Ser Met Gin Leu Gin Lys Leu Asn Ser Gin Tyr Gin Gly Met Ala Ala Ala Thr Pro Gly Gin Pro Gly Glu Ala Gly Pro Leu Gin Asn Trp Asp Phe Gly Ala Gin Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly Ala Gin Ser Pro Ala Ile Ile Asp Ser Asp Pro val ASO Glu Glu val Leu Met ser Leu val val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gin Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (30) INFORMATION FOR SEQ ID NO: 29 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 1642 b) TYPE: nucleic acid (ii) MOLECULE TYPE: a:NA to mRNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29 gctctgcgtt gggccagccc ctcacagctg gtttcttacc acgtattgcg caagcggaat 60 ctatgcctgt tacccacact ccctgcgccc ccgcaccccg ctcctgtgcg caagtcggaa 120 tataaaaccg cggaggagtg agctcttggg gtgtccagtt ggttgccgcg gcagtctctc 180 cgagcagcgc atttgtcttc taggctgctt ggttcgtgcc tccgagaaag gggtctcctg 240 ctgccagcta agtgtgggag aacttgtgca cgtatctccc ctccgaatcc caacgatggg 300 taacgccagc tttggctcca aggaacagaa gctgctgaag cggttgcggc ttctgcccgc 360 cctgcttatc ctccgcgcct tcaagcccca caggaagatc agagattacc gcgtcgtggt 420 agtcggcacc gctggtgtgg ggaaaagtac gctgctOcat aagtgggcga gcggcaactt 480 ccgtcatgag tacctgCcga ccattgaaaa tacctactgc cagttgctgg gctgcagcca 540 cggtgtgctt tccctgctca tcaccgacag caagagtggc gacggcaacc gcgctctgta 600 gcgccacgtt atagcccggg gccacgcttt cgtcctggtc tactcagtca ccaagaagga 660 aaccctggaa gagctgaagg ccuctatga gctgatctgc aagatcaaag gtaataacct 720 gcataagttc cccatcgtgc tggtgggcaa taaaagtgat gatacccacc gggaggtggc 790 cctgaatgat ggtgccacct gtgcgatgga gtggaattgc gccttcatgg agatttcagc 840 caagaccgat gtgaatgtgt aggagctOtt ccacatgctg ctgatttaca agalaaagcc 900 caccaccggc ctccaggagc ccgagaagaa atcccagatg cccaacacca ctgagaagct 960 gcttgacaag tgcataatca tgtgagctct gggccttaag agccagctct tcctatcttg 1020 tagcgtgtag aaaacgtgga ctcatttcac tatgttatat gtatatggtt gattttgtgc 1080 tgttgtttgg actgtaacat ccatgttgtc aatacgtata ccttgtaagt ggataacttt 1140 tctttttccc aggccagaga attcaaattg ttaaaacatt ggcatttgaa gaggagaaca 1200 aaatgtagca tgatgtattt aaagtaaggc ctttagtaat gaatgtattg agagaaaatg 1260 ttttgaaaag aacaaaacat caaaatgaat agaaagaaaa attggaaggt gtccttttgg 1320 taacccgatt attgtgtatt acctttaaat atttcacatc ctgtaagtgc ttaatcatat 1380 cttttaattg tgtatttaag aaaagtgttt tcacaacaaa agcttttgat aaattgctgc 1440 gtgacatata ctaaataaaa aaatgaatat gttgatcatt aggggtotgg gagcagagaa 1.500 aattgtgaaa gtgactctca ctaaagatgt tagtagtttc tcatgtcatt taaaaatgtt 1560 tgagtattct gcatagcagt ttgtaaaagt gtaacagctt attgacttaa taaagctttt 1620 cctgcatgca aaaaaaaaaa aa 1642 (31) INFORMATION FOR SEQ ID NO: 30 (i) SEQUENCE CHARACTERISTICS
a3 LENGTH: 229 b TYPE: amino acid (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE:
A) ORGANISM: how sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30 Met Gly Asn Ala Ser Phe 41y Ser Lys Glu Gin Lys Lev Leu L/t Arg Lou Arg Lev Leu Pro Ala Leu Leu Ile Leu Arg Ala Phe LyS Pro His Arg Lys Ile Arg Asp Tyr Arg Val Val val val Gly Thr Ala Gly Yal Gly Lys n Ser Thr Leu Leu His Lys Trp Ala Ser Gly Asn Phe Arg His Glu Tyr Leu Pro Thr /16 Glu Asn Thr Tyr cys Gin Leu Leu Gly Cys Ser His Gly Val Leu Ser Leu His Ile Thr Asp ser Lys Set Gly Asp Gly Asn Arg Ala Leu Gin Arg His val Ile Ala Arg Gly His Ala Phe 100 105, 110 val Leu Val Tyr Ser val Thr Lys Lys Glu Thr Leu Glu Glu Leu Lys Ala Phe Tyr Glu Leu Ile Cys Lys Ile Lys Gly Asn Asn Leu His Lys Phe Pro Ile val Leu Val Gly Mn Lys Ser Asp Asp Thr His Arg Glu val Ala Leu Asn Asp Gly Ala Thr Cys Ala met Glu Trp Asn cys Ala Phe met Glu Ile Ser Ala Lys Thr Asp Val Asn Val Gin Glu Leu Phe His Met Leu Leu Asn Tyr Lys Lys Lys Pro Thr Thr Gly Leu Gin Glu Pro Glu Lys Lys Ser Gin Met PrO Mn Thr Thr Glu Lys Leu Leu asp Lys CS Ile Ile Met (32) INFORMATION FOR SEQ ID NO: 31 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 22 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31 ggigctgcttt taactctgqt aa 22 (33) INFORMATION FOR SEQ ID NO: 32 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 22 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32 atgggtggaa tcatattgga ac 22 (34) INFORMATION FOR SEQ ID NO: 33 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 =

cgtcatactc ctgcttgctg 20 (35) INFORMATION FOR SEQ ID NO: 34 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 21 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 ccagatcatt gctcctcctg a 21 (36) INFORMATION FOR SEQ ID NO: 35 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35 cacttgtgat gccctgactg 20 (37) INFORMATION FOR SEQ ID NO: 36 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36 acggtactgc tgcaggctat 20 (38) INFORMATION FOR SEQ ID NO: 37 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (Xi) SEQUENCE DESCRIPTION: SEQ zo NO: 37 caaccagagc Cgggaagatt 20 (39) INFORMATION FOR SEQ ID NO: 38 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE! nucleic acid (ii) MOLECULE TYPE: DNA
(v1) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence syntbetiCDrinier (Xi) SEQUENCE DESCRIPTION: SEQ TD NO: i8 agagatacgc, aggtgcaggt 20 (40) INFORMATION FOR SEQ ID NO: 39 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid " MOLECULE TYPE: DNA, (0) ORIGINAL SOURCE!
A) ORGANISM: artificial sequence synthetic: primer .(A:i) SEQUENCE DESCRIPTION! SEQ ID NO! 39 gccatggtga aaatggctaa 20 (41) 114P0RmATIuN FOR sEqt ID NO: 40 (i) SEQUENCE CHARACTERISTICS
a) LENGTH: 20 b) TYPE: nucleic acid (ii) MOLECULE TYPE: DNA
,(vi) ORIGINAL SOURCE:
A) ORGANISM: artificial sequence synthetic primer (4i) SEQUENCE DESCRIPTION! SEQ ID NO; AO
gagtgtcagc accaacttgt ZO' 98:

Claims (12)

We Claim:

1. A method of differentially diagnosing non-malignant thyroid tissue from malignant thyroid tissue comprising testing a thyroid tissue sample for the expression of at least two genes chosen from CCND2, PCSK2, and PLAB, wherein a decreased level of expression of as compared to a control, a decreased level of expression of PCSK2 as compared to a control, or an increased level of expression of PLAB as compared to a control indicates the presence of malignant thyroid tissue in the sample.

2. The method according to claim 1, wherein the thyroid tissue sample is tested for the expression of CCND2 and PCSK2.

3. The method according to claim 1, wherein the thyroid tissue sample is tested for the expression of CCND2 and PLAB.

4. The method according to claim 1, wherein the thyroid tissue sample is tested for the expression of PCSK2 and PLAB.

5. The method according to claim 1, wherein the thyroid tissue sample is tested for the expression of CCND2, PLAB, and PCSK2.

6. The method according to claim 1, further comprising testing a thyroid tissue sample for the expression of at least one gene chosen from hTERT, CD44, CITED1, ARHI, and Frizzled-1, wherein an increased level of expression of hTERT as compared to a control, a decreased level of expression of CD44 as compared to a control, an increased level of expression of CITED1 as compared to a control, a decreased level of expression of ARHI as compared to a control, or a decreased level of expression of Frizzled-1 as compared to a control indicates the presence of malignant thyroid tissue in the sample.

7. A kit for differentially diagnosing non-malignant thyroid tissue from malignant thyroid tissue, comprising:
primers for detecting at least two genes selected from CCND2, PCSK2, and PLAB, wherein the primers comprise at least two of the following primers:

(a) at least one CCND2 primer, wherein the at least one CCND2 primer is chosen from 5'-CACTTGTGATGCCCTGACTG-3' (SEQ ID NO: 35) and 5'-ACGGTACTGCTGCAGGCTAT-3' (SEQ ID NO: 36);
(b) at least one PCSK2 primer, wherein the at least one PCSK2 primer is chosen from 5'-GCCATGGTGAAAATGGCTAA-3' (SEQ ID NO: 39) and 5'-GAGTGTCAGCACCAACTTGC-3' (SEQ ID NO: 40); and (c) at least one PLAB primer, wherein the at least one PLAB primer is chosen from 5'-CAACCAGAGCTGGGAAGATT (SEQ ID NO: 37) and 5'-AGAGATACGCAGGTGCAGGT-3' (SEQ ID NO: 38).

8. The kit according to claim 7, wherein the kit further comprises at least one primer for hTERT, at least one primer for CD44, at least one primer for CITED1, at least one primer for ARHI, or at least one primer for Frizzled-1.

9. The kit according to claim 7, wherein the primers comprise at least one CCND2 primer chosen from 5'-CACTTGTGATGCCCTGACTG-3' (SEQ ID NO: 35) and 5'-ACGGTACTGCTGCAGGCTAT-5' (SEQ ID NO: 36), and at least one PCSK2 primer chosen from 5'-GCCATGGTGAAAATGGCTAA-3' (SEQ ID NO: 39) and 5'-GAGTGTCAGCACCAACTTGC-3' (SEQ ID NO: 40).

10. The kit according to claim 7, wherein the primers comprise at least one CCND2 primer chosen from 5'-CACTTGTGATGCCCTGACTG-3' (SEQ ID NO: 35) and 5'-ACGGTACTGCTGCAGGCTAT-5' (SEQ ID NO: 36), and at least one PLAB primer chosen from 5'-CAACCAGAGCTGGGAAGATT-3' (SEQ ID NO: 37) and 5'-AGAGATACGCAGGTGCAGGT-3' (SEQ ID NO: 38).

11. The kit according to claim 7, wherein the primers comprise at least one primer chosen from 5'-GCCATGGTGAAAATGGCTAA-3' (SEQ ID NO: 39) and 5'-GAGTGTCAGCACCAACTTGC-3' (SEQ ID NO: 40), and at least one PLAB primer chosen from 5'-CAACCAGAGCTGGGAAGATT-3' (SEQ ID NO: 37) and 5'-AGAGATACGCAGGTGCAGGT-3' (SEQ ID NO: 38).

12. The kit according to claim 7, wherein the primers comprise at least one CCND2 primer chosen from 5'-CACTTGTGATGCCCTGACTG-3' (SEQ ID NO: 35) and 5'-ACGGTACTGCTGCAGGCTAT-5' (SEQ ID NO: 36), at least one PCSK2 primer chosen from 5'-GCCATGGTGAAAATGGCTAA-3' (SEQ ID NO: 39) and 5'-GAGTGTCAGCACCAACTTGC-3' (SEQ ID NO: 40), and at least one PLAB primer chosen from 5'-CAACCAGAGCTGGGAAGATT-3' (SEQ ID NO: 37) and 5'-AGAGATACGCAGGTGCAGGT-3' (SEQ ID NO: 38).