WO2008085007A1

WO2008085007A1 - Novel proliferation marker in papillary thyroid carcinoma and novel method of diagnosis detecting the marker

Info

Publication number: WO2008085007A1
Application number: PCT/KR2008/000190
Authority: WO
Inventors: Hyun-Kee Kim; Jin-Woo Kim
Original assignee: Hyun-Kee Kim; Jin-Woo Kim
Priority date: 2007-01-11
Filing date: 2008-01-11
Publication date: 2008-07-17
Also published as: KR20080066252A; KR100886670B1

Abstract

The present invention relates to the use of minichromosome maintenance protein 3 (MCM3) as a diagnostic marker for papillary thyroid carcinoma (PTC). In particular, the present invention relates to a diagnostic marker for papillary thyroid carcinoma consisting of MCM3, a diagnostic kit for papillary thyroid carcinoma comprising an MCM3 -specific antibody, and a method for diagnosing papillary thyroid carcinoma using the MCM3-specific antibody.

Description

NOVEL PROLIFERATION MARKER IN PAPILLARY THYROID CARCINOMA AND NOVEL METHOD OF DIAGNOSIS DETECTING THE MARKER

TECHNICAL FIELD

The present invention relates to the use of minichromosome maintenance protein 3 (MCM3) as a diagnostic marker for papillary thyroid carcinoma (PTC) .

BACKGROUND ART

Thyroid carcinoma is the most common malignancy of the endocrine system worldwide, and the most common type of thyroid carcinoma is papillary thyroid carcinoma (PTC) . Further, the incidence of papillary thyroid carcinoma (PTC) has most rapidly increased among other cancers , from 7.5 in 1991 to 8.4 in 2004 per 100 thousand persons . In particular, the main increase in incidence rate has occurred in mid-aged women (source: Ministry of Health and Welfare, Annual Report of the Korea Central Cancer Registry, Seoul, 2004) . As a consequence, there has been increasing interest on the tumorigenesis and prognosis of thyroid carcinoma.

Furthermore, papillary thyroid carcinoma (PTC) does not have the remarkable proliferation marker such as conventional proliferation markers suchKi-67 and PCNA. Theproliferative capacity of tumor cells is a characteristic feature of all the growing tumors. Assessment of cell proliferation may provide both pathologists and clinicians with more objective prognostic information. Ki-67 immunoreactivity has been widely used as a standard proliferation marker in several human cancers including lung cancer, soft tissue sarcoma, meningioma, prostate cancer, and non-Hodgkin' s lymphoma (Jansen RL, et al . "MIB-I labeling index is an independent prognostic marker in primary breast cancer", Br J Cancer 1998, 78:460-465; Perry A, et al . "The prognostic significance of MIB-I, p53, and DNA flow cytometry in completely resected primary meningiomas", Cancer (Phila.) 1998, 82:2262-2269; Mashal RD, et al . "Expression of cell cycle-regulated proteins in prostate cancer", Cancer Res 1996, 56:4159-4163; and Dallenbach F, et al . "Growth fractions in malignant non-Hodgkin' s lymphomas (NHL) as determined in situ with the monoclonal antibody Ki-67", Hematol Oncol 1984, 2:365-371) . Despite of its usefulness, the function of Ki-67 remains unclear although some reports suggests that Ki-67 plays a role in ribosome biosynthesis during cell proliferation, rather than being directly associated with cell cycle (MacCallum DE, et al . "The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle", J Pathol 2000, 190:537-544). Furthermore in PTC, it is controversial whether Ki-67 is a reliable prognostic marker of this tumor (Erickson LA, et al . "Expression of p27kipl and Ki-67 in benign and malignant thyroid tumors" , Mod Pathol 1998 , 11:169-174; and Katoh R, et al . "Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure usingmonoclonal antibodyMIB-I ", Hum Pathol 1995, 26:139-146) . Thus, another proliferation marker directly involving DNA replication is needed to more precisely assess the proliferative activity of PTC. It has been known that the minichromosome maintenance (MCM) protein family MCM2-7, which consists of six members, is the highest conserved group among DNA-binding proteins, and is required for DNA replication in eukaryotic cells (Tye BK, et al . "MCM proteins in DNA replication" Annu Rev Biochem 1999, 68:649-686) . The MCM proteins are involved in the initiation and regulation of DNA replication. The MCMprotein binds to the origin of DNA replication, and subsequently interacts with an origin recognition complex (ORC) and Cdc 6 protein to form a prereplicative complex. Upon the initiation of S-phase, the Cdc6 protein is released from the prereplicative complex and this regulation of DNA synthesis ensures that DNA replicates only once during each cell cycle (Chevalier S, et al . "Cell cycle control of replication initiation in eukaryotes" Curr Opin Cell Biol 1996, 8:815-821) . It has been reported that the expression of MCM proteins is observed cycling cells, and no expression is observed in quiescent and differentiating cells (Madine MA, et al . "The roles of the MCM, ORC, and Cdcβ proteins in determining the replication competence of chromatin in quiescent cells" J^" Struct Biol 2000, 129:198-210). Antibodies against MCM proteins may be used for defining the proliferative compartments in both normal and neoplastic tissues. Immunostaining assessment of all six MCM proteins has been observed to produce similar results in most of studies (Hiraiwa A, et al . " Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth" Int J Cancer 1997, 74:180-184; and Freeman A, et al . "Minichromosome maintenance proteins as biological markers of dysplasia and malignancy" Clin Cancer Res 1999, 5:2121-2132) .

However, with regard to the MCM protein expression, most studies have been conducted on MCM2 and MCM5 in various tumors (Ramnath N, et al . "MCM2 is an independent predictor of survival in patients with nonsmall-cell lung cancer", J Clin Oncol 2001, 19:4259-4266; Stoeber K, et al . "Diagnosis of genito-urinary tract cancer by detection of minichromosome maintenance 5 protein in urine sediments " JNatl Cancer Inst (Bethesda) 2002, 94:1071-1079; Gonzalez MA, et al . "Minichromosome maintenance protein 2 is a strong independent prognostic marker in breast cancer" J Clin Oncol 2003, 21:4306-4313; and Kato H, et al . "A new proliferation marker, minichromosome maintenance protein 2, is associated with tumor aggressiveness in esophageal squamous cell carcinoma" J Surg Oncol 2003, 84:24-30). Many studies have reported that MCM immunoreactivity can be used as a proliferation marker (Kato H, et al . "A new proliferation marker, minichromosome maintenance protein 2, is associated with tumor aggressiveness in esophageal squamous cell carcinoma" J^" Surg Oncol 2003, 84:24-30; and Ha SA, et al . "Cancer-associated expression of minichromosome maintenance 3 gene in several human cancers and its involvement in tumorigenesis " Clin Cancer Res 2004, 10:8386-8395) . Prior studies have reported that MCM2 may be a superior marker to Ki-67 in the assessment of cell-cycle entry using cadaver specimens (Rodins K, et al . "Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: relationship to tumor dormancy and potential clinical utility" Clin Cancer Res 2002, 8:1075-1081) . However, there is no report about the availability of MCM3 , in particular, relationship between the expression level of MCM protein and PTC. In the present invention, the availability of MCM3 is assessed as a novel and reliable diagnostic marker for PTC, thereby meeting the requirement of effective diagnostic marker for PTC diagnosis.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an effective diagnostic marker for papillary thyroid carcinoma.

It is another object of the present invention to provide a novel use of minichromosome maintenance protein 3 (MCM3) as a diagnostic marker for papillary thyroid carcinoma.

It is still another object of the present invention to provide a method for diagnosing papillary thyroid carcinoma using MCM3-specific antibodies.

Further, it is an object of the present invention to provide a diagnostic kit for papillary thyroid carcinoma comprising MCM3-specific antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a photograph showing the intrinsic characteristic of PTC and immunoreactivity of MCM3 in PTC, in which the left photograph shows the image of papillary fronds with fibrovascular stalks of PTC characteristic (100X) , and the right photograph shows the negative staining result of normal thyroid follicular cells (200X);

Fig. 2 is a photograph showing the immunoreactivity of MCM3 in PTC, in which the nuclear staining pattern in PTC is shown as a result of immunohistochemical staining of MCM3 (100X);

Fig. 3 is a photograph showing the result of immunostaining of MCM3 in proliferating lymphocytes, in which the immunostaining of MCM3 is shown in the proliferating lymphocytes in lymphocyte masses around PTC tissues (200X); and

Fig.4 is a photograph showing the result of Western blot analysis on the expression level of MCM3 protein in human thyroid tissues, in which the expression levels of MCM3 protein in human PTC tissues (c) are compared with the expression levels in the corresponding normal tissues (N) , and MCM3-specific antibody and β-actin-specific antibody are used as a probe for each blot of the same samples .

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel and effective diagnostic marker for papillary thyroid carcinoma (PTC) . In particular, the present invention provides a minichromosome maintenance protein 3 (MCM3) as a diagnostic marker for PTC, which is clarified by investigating the relationship between theminichromosomemaintenance protein 3 (MCM3) and clinicopathologic parameters of PTC.

The present inventors found that the expression level of MCM3 protein is remarkably high in the papillary thyroid carcinoma tissue, whereas remarkably low or undetectable in the normal thyroid tissue. Accordingly, diagnosis of papillary thyroid carcinoma can be achieved by detecting the expression level of MCM3 protein. The diagnostic method of the present invention comprises the steps of a) providing a tissue sample obtained from an individual, b) contacting the sample with a diagnostic agent that specifically binds to MCM3 , and c) detecting a complex formation of MCM3 with the diagnostic agent so as to diagnose papillary thyroid carcinoma (PTC) .

The diagnostic agent used in the diagnostic method may include any one selected from proteins and antigens that are capable of forming a specific bond with MCM3 , preferably MCM3-specific antibody. The antibody may be a monoclonal or polyclonal antibody. Further, the diagnostic agent may include a partial fragment of the antibody or a fragment that essentially contains a domain for binding with MCM3.

The MCM3-specific antibody can be prepared by any method known in the art, for example, a method descried in "The human cervical cancer oncogene protein is a biomarker for human hepatocellular carcinoma" Seung Kew Yoon et al , Cancer Research, 2004; 64: 5434-5441.

In the present invention, a monoclonal antibody produced in a rabbitmaybe used, and furthermore monoclonal orpolyclonal antibodies that are derived from other species may be used.

The complex formation of MCM3 and MCM-specific antibodies can be detected by using a detection label. As the detection label, any label known to those skilled in the art , including staining chromogens , fluorescent compounds, enzymes, ligands, luminescent compounds, and radioactive isotopes, may be employed. In a specific embodiment of the present invention, a biotin-labeled anti-mouse immunoglobulin G is used as a secondary antibody and 3 , 3-diaminobenzidine-hydrogen peroxide is used as a chromogen, so as to detect the complex formation of MCM3 and MCM-specific antibodies.

According to the diagnostic method of the present invention, the expression level of MCM3 protein in the sample obtained from an individual is determined to diagnose PTC by using the above described antibody according to any method known in the art such as immunohistochemical assay, Enzyme-Linked ImmunoSobent Assay (ELISA) , RadioImmunoAssay (RIA) , sandwich assay, Western blot or immunoblot on a polyacrylamide gel .

The diagnostic kit of the present invention comprises an MCM-specific antibody and a detection label. The diagnostic kit of the present invention may further comprise a secondary antibody. The antibody and detection label are the same as the above described, and the secondary antibody is preferably an immunoglobulin G (Ig G) .

To confirm the availability of MCM3 as a diagnostic marker through the relationship between MCM3 and various histopathologic parameters of PTC, Western blot analysis may be performed for immunohistochemical analysis onMCM3 in the PTC tissue andcomparisonbetweenMCM3 expression levels in PTC tissues and normal tissues. As a result of immunohistochemical analysis, it was found that the expression level of MCM3 was very high in PTC patients. In particular, it was found that the MCM3 expression level had been significantly associated with tumor size and extracapsular extension. Accordingly, it can be seen that the MCM3 expression level is a more sensitive and reliable marker to detect the fast proliferative compartment in the cells.

The availability of anti-MCM antibody as a proliferation marker for carcinoma cells induced in PTC was confirmed in the paraffin-embedded section using affinity-purified polyclonal antibodies. Papillary fronds with characteristic fibrovascular stalks were observed in the PTC tissue (Fig.1, left) , whereas a positive reaction with anti-MCM3 antibodywas not observed in the normal thyroid tissue (Fig. 1, right) . As a result of immunohistochemical staining based on immunoreactivity of anti-MCM3 antibody, the staining pattern corresponding to the distribution of MCM3 protein in nucleus was observed around the cancer nest (Fig. 2) . MCM3 overexpression was also observed in the proliferating lymphocytes in lymphocyte masses around PTC tissues (Fig. 3) .

As a result of Western blot analysis on the MCM3 expression level , it was found that the MCM3 overexpression was observed in all of the tissue samples from PTC patients (see Fig. 4) . In contrast, it was found that the MCM3 expression level was very low or undetectable in all of normal tissues.

Consequently, the MCM3 protein can be a reliable marker for PTC diagnosis. In particular, the MCM3 protein is useful for evaluating tumor growth, invasion and prognostic factors in PTC patients . Accordingly, it can be seen that the MCM3 protein is a useful proliferation marker for diagnosing tumor growth and invasion in PTC.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for the illustrative purpose only, and the invention is not intended to be limited by these Examples.

Example 1 : Collection of tissue sample from PTC patient Tissue samples and relevant clinical data that had been collected from total 30 patients with papillary thyroid carcinoma (PTC) were analyzed, which are shown in Table 1. The clinical data included age, gender, and tumor size. The average age of patient was 45 years (range: 20 to 67) .

Histopathologic features including lymph node metastases, lymphatic invasion, extrathyroidal extension, muscle extension, and tumor multiplicity were evaluated on routine hematoxylin-eosin (H-E) stained sections using generally agreed and accepted diagnostic criteria. Among 30 patients, 16 were patients with

early-stage (stage I ) PTC and 14 were patients with late-stage (stage

El ) PTC .

[Table 1]

MCM3 and Ki-67 labeling indices and clinicopathological features in

30 patients with PTC.

Size MCM3

No Age Sex multi ET ext LN mt Lym Inv M Inv MCM3 ' Ki-67

(cm) Grade

1 64 F 1.5 + + - - - 30 2 2

2 18 M 1.5 + + + + - 30 2 5

3 45 F 1.6 + - - - - 40 2 5

4 43 F 2.5 - + + + - 80 4 5

5 30 F 0.7 - - - - - 60 3 1

6 38 F 0.4 - - - - - 10 1 2

7 51 F 1.5 - + - - - 60 3 5

8 44 F 0.7 - + - - 10 1 1

9 55 M 3 - + + + + 70 3 3

10 37 F 0.8 - - - - - 80 4 1

11 63 F 2.5 + + - - + 70 3 3

12 53 F 1.5 + + - - + 50 2 3

13 55 F 1.2 - + - - - 80 4 3

14 45 F 1.5 - - - - - 80 4 3

15 40 F 1.8 - - - - - 10 1 1

16 64 M 1.8 - + - - - 20 1 2

17 56 F 0.6 - - - - - 10 1 1

18 28 F 0.8 - + - - - 10 1 3

19 33 M 4 - + - - - 80 4 3

20 56 M 1 - - - + - 20 1 2

21 54 M 4 - + - - + 20 1 5

22 55 F 1 - - - + - 70 3 2

23 64 M 2.8 - + + + + 30 2 3

24 45 F 2.5 + + - + + 40 2 1

25 50 F 1 - + - - - 5 1 2

26 47 F 1 - + - - + 40 2 3

27 47 F 1 + + 30 2 1 28 24 F 1 . 3 - + 30 2 1

29 67 F 0 . 8 - 10 1 10

3 0 57 F 1 . 8 + + - - - 10 1 2

1) Ratio of benign tumor cells to total 100 malignant tumor cells

Example 2: Production of antibodies against MCM3 protein Rabbit polyclonal antibodies were raised against the product of MCM3 gene. A plasmid expressing a whole MCM3 fused to a 6 x His tag was constructed by ligating a PCR product of MCM3 into a pBAD/TOPO expression vector (pBAD/TOPO ThioFusion Expression Kit , Invitrogen, USA) . The resulting plasmid was introduced into E.coli, and then the transformed E.coli was induced to produce fusion proteins in the presence of arabinose. The fusion proteins were purified using a Ni-nitrilotetraacetic acid-agarose column (ProBond purification system, invitrogen, USA) . The antibodies against thepurifiedprotein were raised in New Zealand White rabbits. The resulting sera from the rabbits were subjected to purification using affinity with MCM3 protein that was covalently linked to CNBr-activated Sepharose bead (Amersham Biosciences, USA).

Example 3 : Immunohistochemical staining

Immunohistochemical staining was performed using the streptavidin/biotin/peroxidase complex method (Dako, Denmark). For antigen retrieval, microwave pretreatment was performed in 10 rnM citrate buffer for 9 minutes. Endogenous peroxidase activity was blocked by 0.3% H₂O₂ in methanol for 30 minutes. Antibodies against MCM3 (Dako, Denmark) were diluted to a ratio of 1:50, and applied to the tissue samples fromPTCpatient andnormal thyroid tissue samples , followed by incubation at 4⁰C overnight. Biotin-labeled anti-mouse immunoglobulin (Ig) G was used as a secondary antibody, and 3 , 3-diaminobenzidine-hydrogen peroxide was used as chromogen. Sections were counter stained with hematoxylin, dehydrated, cleared, and then mounted. A primary antibody was substituted with a Tris-buffered saline solution to prepare a negative control.

Comparative Example: Immunohistochemical staining using Ki-67 antibody

Immunohistochemical staining was performed using the streptavidin/biotin/peroxidase complex method (Dako, Denmark). For antigen retrieval, microwave pretreatment was performed in 10 mM citrate buffer for 9 minutes. Endogenous peroxidase activity was blocked by 0.3% H₂O₂ in methanol for 30 minutes. Antibodies against Ki-67 (Dako, Denmark) were diluted to a ratio of 1:100, and applied, followed by incubation at 4°C overnight. Biotin-labeled anti-mouse immunoglobulin (Ig) G was used as a secondary antibody, and 3 , 3-diaminobenzidine-hydrogen peroxide was used as chromogen. Sections were stained with hematoxylin, dehydrated, cleared, and then mounted. A primary antibody was substituted with a Tris-buffered saline solution to prepare a negative control.

Result analysis

The immunohistochemical staining results from Example 3 and Comparative Example were analyzed. The sections were observed with high resolution (400X). Apparent nuclear staining was recognized as a positive reaction. MCM3 and Ki-67 labeling indices were assessed as the percentage of 1000 tumor cells in most activation area. The labeling indices of tumor (percentage of benign tumor cells positive for MCM3 andKi-67) were classified into four groups : group 1 (0-25%), group 2 (26-50%), group 3 (51-75%) and group 4 (76-100%) (Table 1; MCM3 grade) .

As a result of anti-MCM3 antibody reaction and immunohistochemical staining of PTC tissue, characteristic papillary fronds with fibrovascular stalks were observed (Fig. IA, left) . In respect to the immunoreactivity with anti-MCM3 antibody, the tumor cells having these papillary structure showed a nuclear staining pattern, mainly at the periphery of cancer nests, which is consistent with nuclear localization of MCM3 protein (Fig. 2) . In contrast to PTC tissue, any positive reaction with anti-MCM3 antibody was not observed in the normal thyroid tissue (Fig. 1, right panel), which

implicates that MCM expression is tightly associated with the PTC.

Further, Fig. 3 shows the result of immunostaining of MCM3 in

proliferating lymphocytes in the lymphocytic aggregates near the PTC.

Meanwhile, Analysis of Variance was performed to determine the

correlation of MCM3 andKi-67 expression with clinical variables (age,

gender, tumor size) and histopathologic parameters (lymph node

metastases, lymphatic invasion, extracapsular extension, muscle

invasion, tumor multiplicity) . A simple regression analysis was

performed to determine the relationship among MCM3 and Ki-67 labeling

indices .

The relationship between clinicopathologic parameters and MCM3

and Ki-67 labeling indices of tissues from 30 PTC patients were

summarized in Table 2.

[Table 2]

The correlation between MCM3 and Ki-67 Labelling index and

clinicopathologic factors.

Parameter Number of MCM3 LI Ki-67 LI cases

P-value P-value

Gender

Male 7 0 . 939 0 . 454

Female 23

Age > 45 20 0.776 0.265 <45 10

Tumor size <1.0cm 7 0.031 0.271 1.0-2.0cm 16 2.0-4.0cm 5 >4.0cm 2

Multiplicity

Presence 7 0.071 0.759

Absence 23

Node metastasis

Presence 4 0.702 0.184

Absence 26

Extrathyroidal extension Presence 20 0.037 1.000

Absence 10

Lymphatic invasion Presence 7 0.590 0.759

Absence 23

Muscle invasion

Presence 0.134 0.933

Absence 22

In the immunohistochemical analysis, Ki-67 that is generally

used in the related art as a tumor marker was immunohistochemicalIy

analyzed as a control group, and compared with the analysis result

of MCM3 , which is shown in Table 2. As a result, the MCM labeling

index (LI) shows a significant statistical correlation with both tumor

size (P=O.031) and presence of extrathyroidal extension (P=O.037),

whereas Ki-67 LI did not show any significant statistical correlation with 8 clinicopathologic parameters in 30 PTC patients. Therefore, it is recognized that Ki-67 does not appear to be a good candidate marker for assessment of PTC proliferation.

In the primary tumor, the significant correlation between MCM3 andKi-67 labeling indices was not observed, andMCM3 LI was considerably higher.

As a result of analyzing the expression levels of MCM3 and Ki-67 protein in PTC, it was found that Ki-67 LI was low in PTC . In agreement with the analysis result, most studies demonstrated low Ki-67 LI in well differentiated thyroid carcinoma (Timler D, et al . , "Expression of proteins: Dl cyclin and Ki-67 in papillary thyroid carcinomas" Folia Histochem Cytobiol 2001, 39:201-202 Kjellman P, et al . "MIB-I index in thyroid tumors : apredictor of the clinical course inpapillary thyroid carcinoma" Thyroid 2003, 13:371-380 Siironen, et al . "Prognostic factors in papillary thyroid cancer: an evaluation of 601 consecutive patients" Tumour Biol 2005, 26:57-64). The present inventor confirmed that Ki-67 LI has no significant correlation with clinicopathologic parameters of tumor.

As mentioned above, MCM3 LI is much higher than Ki-67 LI, which implicates that Ki-67 may be expressed during a shorter interval of the cell cycle than MCM3. Specifically, MCM3 is present throughout the cell cycle, and the mRNA expression level of MCM3 dramatically increases in Gl-S transition phase. Ki-67 is mainly expressed during S, G2, and M phases, whereas MCM3 is expressed during early Gl phase in normal and neoplasm cells, and Ki-67 expression is not detected at this stage.

Further, as a result of analyzing the correlation between clinicopathologic parameters , age and tumor multiplicity did not show notable relationship with other parameters among 8 clinicopathologic parameters of Table 2. On the other hand, tumor size was significantly associated with extrathyroidal extension (P-O.016). Further, the present inventors found that there was a significant correlation between extrathyroidal extension and muscle invasion (P=O.019). Additionally, there was a significant relationship between lymphatic invasion and metastasis to regional lymph node (P=O.000) . Finally, it was found that gender (male) was considerably correlated with tumor size (P=O.015), lymph node metastases (P=O.007), and lymphatic invasion (P=O.015).

Example 4: Western blot analysis

For Western blot analysis, tissue samples were homogenized in a lysis buffer (2OmM Tris (pH7.4), 1% NP40, 5mM EDTA, 10% glycol, 0.1% SDS, andl50mMNaCl) containingprotease inhibitormixture (Sigma, USA) . The homogenized samples were clarified by centrifugation at 5, 000 G for 10 minutes and cleared. Equivalent volumes of cell lysates containing 20 μg of total proteins were loaded onto a 10% SDS-polyacrylamide gel, followed by electrophoresis for separation. The protein samples were then transferred to nitrocellulose membrane. The blots were incubatedwithpolyclonal anti-MCM3 serum, and developed using an enhanced chemiluminescence detection kit (Pierce, USA) .

The MCM3 expression levels in six PTC and normal thyroid tissues were compared and shown in Fig. 4. In consistent with Figs. 1 to 3, it was found that the expression level of MCM3 protein were higher in PTC than in normal tissue. The MCM3 expression level was found to be very low or undetectable in the normal thyroid tissues.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing papillary thyroid carcinoma (PTC) , comprising the steps of a) providing a tissue sample obtained from an individual, b) contacting the sample with a diagnostic agent that specifically binds to MCM3 (minichromosome maintenance protein 3 ) , and c) detecting a complex formation of MCM3 and the diagnostic agent in step b) to diagnose papillary thyroid carcinoma (PTC) .

2. The method according to claim 1, wherein the diagnostic agent comprises any one selected from proteins or antigens capable of specifically binding to MCM3.

3. The method according to claim 2, wherein the diagnostic agent comprises an MCM3-specific antibody.

4. Themethodaccording to claim 3, wherein the antibody is amonoclonal or polyclonal antibody, or a partial fragment of antibody that essentially contains a domain for binding to MCM3.

5. The method according to claim 1, wherein the complex formation in step (c) is detected by using any one detection label selected from the group consisting of chromogens , fluorescent compounds, enzymes, ligands, luminescent compounds, and radioactive isotopes.

6. The method according to claim 1, wherein the complex formation in step (c) is detected by any one method selected from the group consisting of immunohistochemical assay, Enzyme-Linked ImmunoSobent Assay (ELISA) , RadioImmunoAssay (RIA) , sandwich assay, Western blot or immunoblot on polyacrylamide gel .

7. A kit for diagnosing PTC, comprising a diagnostic agent that specifically binds to MCM3 and a detection label.

8. The kit according to claim 7 , wherein the diagnostic agent comprises an MCM3-specific antibody.

9. The kit according to claim 8, wherein the antibody is a monoclonal or polyclonal antibody, or a partial fragment of antibody that essentially contains a domain for binding to MCM3.

10. The kit according to claim 7, wherein the detection label is any one selected from the group consisting of chromogens, fluorescent compounds, enzymes, ligands, luminescent compounds, and radioactive isotopes.

11. The kit according to claim 7, further comprising a secondary antibody.

12. The kit according to claim 11, wherein the secondary antibody is immunoglobulin G.

13. A diagnostic marker for papillary thyroid carcinoma, consisting of MCM3.