DE102005011003B9 - Method and means for the differential diagnosis of thyroid tumors - Google Patents

Abstract

Method for differential diagnosis of thyroid tumors for distinguishing benign tumors from malignant tumors in which:
1.) RNA is isolated from a tissue sample of the thyroid gland,
2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of genes selected from the genes listed in Table 1, Table 2, Table 3 and Table 4 with the gene numbers 1 to 153, wherein a quantitative determination of the mRNA Transcripts of at least three genes from the genes listed in Table 3 and a quantitative determination of the mRNA transcripts of at least four genes from the genes listed in Table 4, wherein
a.) an increased amount of transcripts of genes Nos. 1 to 3, 5 to 8, 11, 13 to 15 and 19 to 22 and genes 23 to 27 and 33 provides an indication of the presence of a benign tumor,
b.) a reduced amount of transcripts of genes Nos. 4, 9, ...

Description

The The invention relates to methods and means for differential diagnosis of thyroid tumors, in particular for distinguishing benign nodular thyroid changes (so-called hot and cold knots) of malignant thyroid carcinomas (papillary and follicular Carcinoma). The procedure and the means come in clinical Tumor diagnostics for use.

The frequency nodular Thyroid abnormalities, in the following text as a thyroid nodule is located in iodine deficient areas, such as Germany, at approx. 30 percent of the adult population (1). The diagnosis of a thyroid nodule is often a random one Findings in the survey of clinical status, or a secondary finding in sonographic examinations (2; 3).

The Classification of thyroid nodules is based on both morphological (4) and functional Criteria ("hot" or "cold" node), about 85% all thyroid nodules scintigraphically represent themselves as "cold" nodes (5) .This group comes in the differential diagnosis of nodular thyroid disease a special meaning, since thyroid carcinomas usually impress as "cold" knots cold node is a node that is functionally inactive, d. H. less thyroid hormones produced as healthy thyroid tissue. If there is a cold knot, it must be clarified whether it is a benign Tumor is or suspected to be a thyroid carcinoma. The average Risk for a thyroid carcinoma is very low (1-5% the thyroid nodule). The prevalence of thyroid carcinomas is also not generally elevated in iodine deficient areas.

About five percent of all thyroid nodules are scintigraphically called "hot" nodules (autonomous thyroid adenomas) (5) due to constitutively activating mutations of the TSH receptor (TSHR), (up to 82%) and the G _s α protein (bis 35%) The hot knot is a benign tumor that produces more thyroid hormone than healthy thyroid tissue, creating "latent" or "overt hyperfunction." At this node, unlike the cold knot, there is no suspicion of carcinoma, it is almost always benign, there may be a single (solitary) autonomic nodule, or the thyroid gland may be interspersed with many hot knots.

The most important diagnostic procedure for the differentiation of benign (benign) and more vicious (malignant) thyroid disease is the fine needle aspiration cytology (FNAZ). By means of FNAZ can in usually a classic papillary, anaplastic and medullary thyroid carcinomas as well as metastases of other primary tumors, if necessary in addition with appropriate immunocytological markers, preoperatively diagnosed become (6; 7). quality or expressiveness of the FNAZ are decisive of the Experience of the puncturer and the evaluating cytopathologist dependent: In a very experienced team can by means of FNAZ a very high precision (85 to 100%), specificity (72-100%) and sensitivity (55-98%) the differential diagnosis of thyroid nodules (nodular thyroid disease) be achieved. However, results show the most extensive so far FNAZ study in 15,000 patients over a period of 16 years at the Mayo Clinic because of nodular thyroid disease were treated that too at experienced Investigators in up to 32% of punctured thyroid nodules sufficient cytological evaluation is not possible (7). For this purpose, on the one hand non-exploitable (up to 21%), on the other hand, "suspicious" points (about 10%) responsible (6-11). In the latter group is usually the problematic findings of a follicular Neoplasia before. Under this, currently not further differentiable, Cytological group diagnosis are follicular adenomas, follicular carcinomas as well as the follicular and oxyphilic variant of the papillary thyroid carcinoma summarized. In the absence of appropriate differential diagnostic markers for follicular neoplasia this is currently considered an indication for nodal resection, although only in a maximum of 20 to 25% of cases indeed a carcinoma is present (7).

So far are for differentiated thyroid carcinomas The following molecular causes are known: Ret rearrangements in about 20% of the papillary thyroid carcinomas found (16). PAX8-PPAR rearrangements were recently in follicular thyroid carcinomas demonstrated. They do not occur in papillary thyroid carcinomas However, they were also found in benign thyroid adenomas (12, 18).

The previous preoperative Lead selection process to a histological carcinoma rate in thyroid surgery because of thyroid nodules from 5 to a maximum of 30%. As a result, over 70% of those are currently due Carcinoma suspected in thyroid nodules conducted Operations actually unnecessary.

The invention has for its object to provide methods and means for the diagnosis of nodular thyroid disease, in particular for the differential diagnosis of benign thyroid keno ("hot" and "cold" nodes) against malignant thyroid carcinomas, especially against the follicular and papillary thyroid carcinomas.

The The invention is based on scientific investigations of the differential Gene expression in benign cold and hot thyroid nodules in comparison to the respective corresponding, healthy environment tissues and the subsequent comparison of these data with the corresponding ones Gene expression data from thyroid carcinomas. The invention in benign and malicious thyroid tumors differently expressed genes are used as markers for the differential diagnosis used by thyroid tumors.

Of the Invention is based on the object, a method and means for differential diagnosis of thyroid tumors, in particular to Differentiation of benign thyroid nodules from thyroid carcinomas specify. Malignant Tumors to be diagnosed with the invention are especially follicular and papillary Thyroid carcinoma.

The The invention is based on scientific investigations of the differential Gene expression in benign cold and hot thyroid nodules in comparison to the respective corresponding, healthy environment tissues and the subsequent comparison of these data with expression data in papillary and follicular Thyroid cancer. The study of differential gene expression of approximately 10,000 genes was performed using high density gene expression arrays (Affymetrix GeneChips U95Av2, Affymetrix; Santa Clara, CA, USA). By comparison the expression data in the thyroid nodules or carcinomas with the Data of healthy surrounding tissues was normalized, which made it possible Expression differences of genes between the different ones Determine pathologies.

The according to the invention in benign and malicious thyroid tumors differently expressed genes are used as markers for the differential Diagnosis of thyroid tumors used.

• 1.) RNA aus einer Gewebeprobe der Schilddrüse isoliert wird,
• 2.) in der isolierten RNA eine quantitative Bestimmung der mRNA-Transkripte mindestens eines Genes ausgewählt aus den in Tabelle 1, Tabelle 2, Tabelle 3 und Tabelle 4 aufgeführten Genen mit den Gen-Nummern 1 bis 153 erfolgt, wobei eine quantitative Bestimmung der mRNA-Transkripte von mindestens drei Genen aus den in Tabelle 3 aufgeführten Genen und eine quantitative Bestimmung der mRNA-Transkripte von mindestens vier Genen aus den in Tabelle 4 aufgeführten Genen erfolgt, wobei a.) eine erhöhte Menge an Transkripten der Gene Nr. 1 bis 3, 5 bis 8, 11, 13 bis 15 und 19 bis 22 sowie der Gene 23 bis 27 und 33 einen Hinweis auf das Vorliegen eines gutartigen Tumors liefert, b.) eine erniedrigte Menge an Transkripten der Gene Nr. 4, 9, 10, 12, 16 bis 18 sowie der Gene 28 bis 32, 34 und 35 einen Hinweis auf das Vorliegen eines gutartigen Tumors liefert, c.) eine erhöhte Menge an Transkripten der Gene Nr. 16, 32, 36 bis 41, 43 bis 45, 47 bis 52, 54 bis 62, 64 bis 68, 70, 71, 74, 75, 77 bis 85, 87, 89, 91 bis 94, 101 bis 107, 110, 112 bis 115, 118, 119, 121 bis 125, 130, 131 sowie der Gene Nr. 133 und 134 einen Hinweis auf das Vorliegen eines bösartigen Tumors liefert, d.) eine erniedrigte Menge an Transkripten der Gene Nr. 2, 13, 15, 22, 33, 42, 46, 53, 63, 69, 72, 73, 76, 86, 88, 90, 95 bis 100, 108, 109, 111, 116, 117, 120, 126 bis 129, 132 sowie der Gene Nr. 8 und 135 bis 153 einen Hinweis auf das Vorliegen eines bösartigen Tumors liefert.

According to the invention the object is achieved by a method for the differential diagnosis of thyroid tumors for the differentiation of benign tumors against malignant tumors in which:

• 1.) RNA is isolated from a tissue sample of the thyroid gland,
• 2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of at least one gene selected from the genes listed in Table 1, Table 2, Table 3 and Table 4 with the gene numbers 1 to 153, wherein a quantitative determination of the mRNA Transcripts of at least three genes from the genes listed in Table 3 and a quantitative determination of the mRNA transcripts of at least four genes from the genes listed in Table 4, where a.) An increased amount of transcripts of genes Nos. 1 to 3 , 5 to 8, 11, 13 to 15 and 19 to 22 and genes 23 to 27 and 33 provides an indication of the presence of a benign tumor, b.) A reduced amount of transcripts of genes Nos. 4, 9, 10, 12, 16 to 18 and genes 28 to 32, 34 and 35 provides an indication of the presence of a benign tumor, c.) An increased amount of transcripts of genes Nos. 16, 32, 36 to 41, 43 to 45, 47 to 52, 54 to 62, 64 to 68, 70, 71, 74, 75, 77 bi s 85, 87, 89, 91 to 94, 101 to 107, 110, 112 to 115, 118, 119, 121 to 125, 130, 131 and genes Nos. 133 and 134 provides an indication of the presence of a malignant tumor, d.) a reduced amount of transcripts of genes Nos. 2, 13, 15, 22, 33, 42, 46, 53, 63, 69, 72, 73, 76, 86, 88, 90, 95 to 100, 108, 109, 111, 116, 117, 120, 126 to 129, 132 and genes Nos. 8 and 135 to 153 provides an indication of the presence of a malignant tumor.

In preferred embodiments the method is a quantitative determination of mRNA transcripts of at least five Genes selected from the genes listed in Table 1, Table 2, Table 3 and Table 4, in further preferred embodiments the mRNA transcripts of at least 10 or at least 15 Genes analyzed to further improve the informative value.

The The genes listed in Tables 1 to 4 are continuous numbered (gene number). To uniquely identify the genes are each the publication number of the associated Genbank entry in the Entrez database of the National Center for Biotechnology Information (NCBI), Bethesda, USA (http://www.ncbi.nih.gov/entrez/query.fcgi?db=gene) and the identification number of the Affymetrix chip (ProbeSet-ID) specify. Under "ProbeSet-ID" is in the tables the term standardized by Affymetrix of a group of 16-20 Specimens immobilized on the Affymetrix chip are indicated, with which hybridizes the respective gene and the proof of a serve specific transcript. The most common name of each Gene is also indicated.

Tables 1 to 4 show the SLR values of the genes in the respective tissues (columns "expression in HK" (hot node), "expression in KK" (cold node), "expression in PTC" (papillary carcinoma), " Expression in FTC "(follicular carcinoma)). The SLR (signal log ratio) was determined as follows from the expression data on which the invention is based:

One Signal log ratio (SLR) value of 1 thus corresponds to a 2-fold higher Expression of the gene in the respective tumor tissue in comparison to the normal Surrounding tissue, a SLR value 2 equals 4 times higher Expression compared to the surrounding tissue.

One negative SLR value corresponds to a reduced expression in the respective Tumor tissue compared to the normal surrounding tissue. So corresponds an SLR value of -1 one in tumor tissue reduced by half compared to normal tissue Expression. An SLR value of -2 corresponds to one quarter of the expression in normal tissue reduced expression.

The SLR values are in the 1 to 4 shown in bar charts. In the 1 to 4 the mean value of the signal log ratio for hot nodes is given as "mean HK" (HK as tumor tissue, UG _HK as surrounding tissue in formula 1). Correspondingly obtained values for the benign cold thyroid nodules (CC), papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are given as "mean KK", "mean PTC" or "mean FTC." The numbers above and below the bars, respectively correspond to the gene numbers from Tables 1 to 4.

In Table 1 lists the genes whose expression is in benign be called thyroid nodules on average their expression in normal surrounding tissue, opposite theirs Expression in cold nodules and their expression in thyroid carcinomas (papillary and follicular Thyroid carcinoma) at least twice as high or at least reduced by half is.

• 1.) RNA aus einer Gewebeprobe der Schilddrüse isoliert wird,
• 2.) in der isolierten RNA eine quantitative Bestimmung der mRNA-Transkripte von zusätzlich mindestens drei Genen ausgewählt aus den in Tabelle 1 aufgeführten Genen mit den Gen-Nummern 1 bis 22 erfolgt,
• 3.) eine erhöhte Menge an Transkripten der Gene Nr. 1 bis 3, 5 bis 8, 11, 13 bis 15 und 19 bis 22 sowie eine erniedrigte Menge an Transkripten der Gene Nr. 4, 9, 10, 12 und 16 bis 18 einen Hinweis auf das Vorliegen eines gutartigen heißen Knotens liefert.

A preferred aspect of the invention is therefore a method for the differential diagnosis of thyroid tumors for distinguishing benign, hot nodules from benign cold nodules, malignant follicular carcinomas, and malignant papillary carcinomas, in which:

• 1.) RNA is isolated from a tissue sample of the thyroid gland,
• 2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of additionally at least three genes selected from the genes listed in Table 1 with the gene numbers 1 to 22,
• 3.) an increased amount of transcripts of genes Nos. 1 to 3, 5 to 8, 11, 13 to 15 and 19 to 22 and a reduced amount of transcripts of genes Nos. 4, 9, 10, 12 and 16 to 18 provides an indication of the presence of a benign hot nodule.

In preferred embodiments the method is a quantitative determination of mRNA transcripts at least four or five Genes selected from those listed in Table 1 Genes, in further preferred embodiments, the mRNA transcripts of at least 10 or at least 15 genes analyzed to improve the validity.

The differential expression values summarized in Table 1 are explained in more detail below with reference to gene gene no. 2 (type I 5 iodothyronine deiodinase) and gene no. 4 (single stranded DNA binding protein):
Gen No. 2 (type I 5 iodothyronine deiodinase, NM_000792, sample set ID 31966_at) has an SLR of 2.0 in hot nodes (HK) compared to the normal hot node surrounding tissues, which is 4-fold higher in expression the HK compared to the surrounding tissue hot knot (UG _HK ). In cold knots (CC) the SLR 0.8 is cold knot (UG _KK ) compared to the normal surrounding tissues. This corresponds to a 1.2-fold higher expression in the KK compared to UG _KK . In comparison of the papillary carcinomas (PTC) to their surrounding tissue, the SLR is -3.8, which corresponds to a 0.07-fold expression, ie a reduced by about a factor of 14 expression in the PTCs compared to their surrounding tissues (UG _PTC ). In follicular carcinomas (FTC), the SLR is -0.8 compared to the normal surrounding tissues of follicular carcinomas (UG _FTC ). This corresponds to a 0.6-fold expression, ie a reduced by approximately factor 1.7 expression in the FTCs compared to their surrounding tissues.

consequently is gene # 2 (type I 5 iodothyronine deiodinase) in hot nodes across from Cold knots and opposite follicular and papillary thyroid carcinomas specifically overexpressed and therefore provides a specific marker for diagnosis of hot thyroid nodules represents.

In PTC is the type I 5 iodothyronine deiodinase specifically diminished expressed. It therefore also provides a specific marker Diagnosis of thyroid carcinoma, in particular for the diagnosis of papillary thyroid carcinomas and is shown again in Table 3.

Gene No. 4 (single stranded DNA binding protein, NM_012446, Sample Set ID 32668_at) has an SLR of -0.9 in HK compared to its surrounding tissues (UG _HK ), corresponding to 0.53-fold expression in the HK, ie a reduced by a factor of 1.9 expression in the HK compared to their surrounding tissues (UG _HK ). In cold knots (CC), the SLR is 0.3 compared to its surrounding tissue, which corresponds to a 1.23-fold expression in the CC compared to the corresponding surrounding tissues. In comparison of the papillary carcinomas to their surrounding tissue, the SLR is 0.8, which corresponds to a 1.74-fold expression in the PTCs compared to their surrounding tissues. In comparison of the follicular carcinomas to their surrounding tissue, the SLR is 0.0, which corresponds to an unchanged expression.

consequently is gene No. 4 in HK expressed specifically reduced and presents therefore a specific marker for the diagnosis of hot thyroid nodules represents.

1 Figure 3 shows in a bar graph the differential expression of genes 1 to 22 of Table 1, which act as specific markers for hot thyroid nodules. The numbers above or below the bars correspond to the gene numbers from Table 1.

In Table 2 lists the genes whose expression is in benign cold thyroid nodules on average their expression in normal surrounding tissue, opposite theirs Expression in hot knots and opposite their expression in thyroid carcinomas (papillary and follicular Thyroid carcinoma) at least twice as high or at least reduced by half is.

• 1.) RNA aus einer Gewebeprobe der Schilddrüse isoliert wird,
• 2.) in der isolierten RNA zusätzlich eine quantitative Bestimmung der mRNA-Transkripte von mindestens drei Genen ausgewählt aus den in Tabelle 2 aufgeführten Genen mit den Gen-Nummern 23 bis 35 erfolgt,
• 3.) eine erhöhte Menge an Transkripten der Gene Nr. 23 bis 27 und 33 sowie eine erniedrigte Menge an Transkripten der Gene 28 bis 32, 34 und 35 einen Hinweis auf das Vorliegen eines gutartigen kalten Knotens liefert.

A preferred component of the invention is therefore also a method for the differential diagnosis of thyroid tumors for distinguishing benign cold nodules from benign, hot nodules, malignant follicular carcinomas and malignant papillary carcinomas in which:

• 1.) RNA is isolated from a tissue sample of the thyroid gland,
• 2.) in the isolated RNA additionally a quantitative determination of the mRNA transcripts of at least three genes selected from the genes listed in Table 2 with the gene numbers 23 to 35,
• 3.) an increased amount of transcripts of genes Nos. 23 to 27 and 33 and a decreased amount of transcripts of genes 28 to 32, 34 and 35 provides an indication of the presence of a benign cold nodule.

In preferred embodiments the method is a quantitative determination of mRNA transcripts at least four or five Genes selected from those listed in Table 2 Genes, in further preferred embodiments, the mRNA transcripts of at least 10 or at least 15 genes analyzed to improve the validity.

The differential expression values summarized in Table 2 are explained in more detail below with reference to genes No. 23 (fibroblast growth factor 7) and No. 29 (chromosomes 21 open reading frame 5):
Gene No. 23 (fibroblast growth factor 7, NM_002009, Sample Set ID 1466_s_at) has an SLR of -1.2 in HK compared to its surrounding tissues, which corresponds to 0.44-fold expression in HD. In comparison, cold nodes to their surrounding tissue is the SLR 1.3, which corresponds to a 2.46-fold expression in the CC compared to the corresponding surrounding tissues. In comparison of the papillary carcinomas to their surrounding tissue, the SLR is -0.8, which corresponds to a 0.57-fold expression in the PTCs compared to their surrounding tissues. In comparison of follicular carcinomas to their surrounding tissue, the SLR is -3.0, which corresponds to a 0.125-fold expression in the FTCs compared to their surrounding tissues.

consequently is gene # 23 (fibroblast growth factor 7) in KK versus HK, FTC and PTC specifically overexpressed and therefore provides a specific marker for the diagnosis of cold thyroid nodules represents.

Gene No. 29 (chromosome 21 open reading frame 5, NM_005128, Sample Set ID 37827_r_at) has an SLR of 0.5 in HK compared to its surrounding tissues (U _HK ), which corresponds to 1.41-fold expression in HD. In comparison cold knots to their surrounding tissue is the SLR -0.9, which corresponds to a 0.54-fold expression in the CC compared to the corresponding surrounding tissues. In comparison of papillary carcinomas to their surrounding tissue, the SLR is 0.8, which corresponds to one 1.74-fold expression in the PTCs compared to their surrounding tissues. In comparison of the follicular carcinomas to their surrounding tissue, the SLR is -0.1, which corresponds to a 0.93-fold expression in the FTCs compared to their surrounding tissues.

consequently is gene number 29 (chromosome 21 open reading frame 5) in KK versus HK, FTC and PTC are expressed less expressed and therefore provides a specific Marker for the diagnosis of cold thyroid nodules.

2 Figure 3 shows in a bar graph the differential expression of genes 23 to 35 of Table 2, which act as specific markers for cold thyroid nodules. The numbers above or below the bars correspond to the gene numbers from Table 2.

In Table 3 lists the genes whose expression is malignant papillary thyroid carcinomas on average their expression in normal surrounding tissue, opposite theirs Expression in follicular thyroid carcinomas and opposite their expression in benign thyroid tumors (hot knots and cold knots) at least twice as high or at least around the half is reduced.

• 1.) RNA aus einer Gewebeprobe der Schilddrüse isoliert wird,
• 2.) in der isolierten RNA eine quantitative Bestimmung der mRNA-Transkripte von mindestens drei Genen ausgewählt aus den in Tabelle 3 aufgeführten Genen erfolgt,
• 3.) eine erhöhte Menge an Transkripten der Gene Nr. 16, 32, 36 bis 41, 43 bis 45, 47 bis 52, 54 bis 62, 64 bis 68, 70, 71, 74, 75, 77 bis 83, 85, 87, 89, 91 bis 94, 101 bis 107, 110, 112 bis 115, 118, 119, 121 bis 125, 130, 131 sowie eine erniedrigte Menge an Transkripten der Gene Nr. 2, 15, 46, 63, 69, 88, 95 bis 97, 108, 109, 111, 120, 126 bis 129 und 132 einen Hinweis auf das Vorliegen eines bösartigen papillären Schilddrüsenkarzinoms liefert.

A preferred component of the invention is therefore also a method for the differential diagnosis of thyroid tumors for distinguishing malignant papillary carcinomas from benign hot nodules, benign cold nodules and malignant follicular carcinomas, in which:

• 1.) RNA is isolated from a tissue sample of the thyroid gland,
• 2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of at least three genes selected from the genes listed in Table 3,
• 3.) an increased amount of transcripts of genes Nos. 16, 32, 36 to 41, 43 to 45, 47 to 52, 54 to 62, 64 to 68, 70, 71, 74, 75, 77 to 83, 85, 87, 89, 91 to 94, 101 to 107, 110, 112 to 115, 118, 119, 121 to 125, 130, 131 and a reduced amount of transcripts of genes Nos. 2, 15, 46, 63, 69, 88 , 95 to 97, 108, 109, 111, 120, 126 to 129 and 132 provides an indication of the presence of a malignant papillary thyroid carcinoma.

In preferred embodiments the method is a quantitative determination of mRNA transcripts at least four or five Genes selected from those listed in Table 3 Genes, in further preferred embodiments, the mRNA transcripts of at least 10 or at least 15 genes analyzed to improve the validity.

The differential expression values summarized in Table 3 are explained in more detail below with reference to genes No. 36 (tissue inhibitor of metalloproteinase 1) and No. 42 (homogentisate 1 2-dioxygenase):
Gene No. 36 (tissue inhibitor of metalloproteinase 1, NM_003254, Sample Set ID 1693_s_at) has an SLR of 0.0 in HK compared to its surrounding tissues, corresponding to unaltered expression in HD. In comparison, cold nodes to their surrounding tissue is the SLR -0.7, which corresponds to a 0.62-fold expression in the CC compared to the corresponding surrounding tissues. In comparison of papillary carcinomas to their surrounding tissue, the SLR is 2.9, which corresponds to a 7.46-fold expression in the PTCs compared to their surrounding tissues. In comparison of the follicular carcinomas to their surrounding tissue, the SLR is 0.0, which corresponds to an unchanged expression in the FTCs compared to their surrounding tissues.

consequently is gene No. 36 (tissue inhibitor of metalloproteinase 1) in PTC across from HK, KK and FTC specifically overexpressed and therefore provides a specific marker for the diagnosis of thyroid carcinomas, in particular for the diagnosis of papillary thyroid carcinomas represents.

gene No. 42 (homogentisate 1 2-dioxygenase, NM_000187, Sample Set ID 31844_at) has an SLR of 1.3 in HK compared to its surrounding tissues on, which corresponds to a 2.46-fold expression in the HK. Compared cold knots to their surrounding tissue is the SLR 0.8, which corresponds a 1.74-fold expression in the CC compared to the corresponding Surrounding tissues. In comparison of papillary carcinomas to their surrounding tissue is the SLR -3.4, this corresponds to a 0.09-fold expression d. H. one by about factor 10.5 reduced expression, in the PTCs compared to their Surrounding tissues. In comparison of follicular carcinomas to their surrounding tissue is the SLR -3.3, this corresponds to a 0.1-fold expression d. H. one by about factor 10 reduced expression in the FTCs compared to their surrounding tissues.

consequently is gene no. 42 (homogentisate 1 2-dioxygenase) in PTC and FTC (see p. Table 4) HK and KK expressed specifically reduced and therefore provides a specific marker for the diagnosis of thyroid carcinomas.

3 Figure 3 shows in a bar graph the differential expression of genes 2, 13, 15, 16, 22, 32, 33 and 36 to 132 of Table 3, which act as specific markers for papillary thyroid carcinoma. The numbers above or below the bars correspond to the gene numbers from Table 3.

In Table 4 lists the genes whose expression is in malignant follicular thyroid carcinomas on average their expression in normal surrounding tissue, opposite theirs Expression in papillary thyroid carcinomas and opposite their expression in benign thyroid tumors (hot knots and cold knots) at least twice as high or at least around the half is reduced.

• 1.) RNA aus einer Gewebeprobe der Schilddrüse isoliert wird,
• 2.) in der isolierten RNA eine quantitative Bestimmung der mRNA-Transkripte von mindestens vier Genen ausgewählt aus den in Tabelle 4 aufgeführten Genen erfolgt,
• 3.) eine erhöhte Menge an Transkripten der Gene Nr. 133 und 134 sowie eine erniedrigte Menge an Transkripten der Gene Nr. 8 und 135 bis 153 einen Hinweis auf das Vorliegen eines bösartigen follikulären Schilddrüsenkarzinoms liefert.

A preferred component of the invention is therefore also a method for the differential diagnosis of thyroid tumors for distinguishing malignant follicular carcinomas from benign hot nodules, benign cold nodules and malignant papillary carcinomas, in which:

• 1.) RNA is isolated from a tissue sample of the thyroid gland,
• 2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of at least four genes selected from the genes listed in Table 4,
• 3.) an increased amount of transcripts of genes Nos. 133 and 134 as well as a decreased amount of transcripts of genes Nos. 8 and 135 to 153 provides an indication of the presence of a malignant follicular thyroid carcinoma.

In preferred embodiments the method is a quantitative determination of mRNA transcripts of at least five Genes selected from those listed in Table 4 Genes, in further preferred embodiments, the mRNA transcripts of at least 10 or at least 15 genes analyzed to determine the significance to improve.

The differential expression values summarized in Table 4 are explained in more detail below with reference to genes No. 133 (UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase) and No. 138 (BENE protein):
Gen No. 133 (UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, NM_001497, Sample Set ID 40960_at) has an SLR of 0.1 in HK compared to the surrounding tissues, which corresponds to a 1.1-fold expression in the HK. In comparison, cold knots to the surrounding tissues is SLR-0.3, which corresponds to a 0.8-fold expression in the CC compared to the surrounding tissues. In comparison of the follicular carcinomas to the surrounding tissues, the SLR is 2.0, which is a fourfold expression in the FTC compared to the surrounding tissues, and in comparison of the papillary carcinomas to the surrounding tissues, the SLR is 0.0, which corresponds to an unchanged expression in the PTC compared to the surrounding tissues.

consequently Gen. No. 133 in FTC is overexpressed specifically against HK, KK and PTC and therefore provides a specific marker for the diagnosis of follicular thyroid carcinomas represents.

gene No. 138 (BENE protein, NM_005434, Sample Set ID 33331_at) an SLR of 0.7 in HK compared to the surrounding tissues on, this corresponds to a 1.62-fold expression in HD. Compared cold knot to the surrounding tissues is the SLR 1.0, which corresponds a 2-fold expression in the CC compared to the surrounding tissues. In comparison, the follicular Carcinomas to the surrounding tissues is the SLR -1.6, which corresponds to one 0.33-fold expression in the FTC compared to the surrounding tissues and in comparison of the papillary Carcinomas to the surrounding tissues is the SLR 1.3, which corresponds 2.46-fold expression in the PTC compared to the surrounding tissues.

consequently is opposite Gen. No. 138 in FTC HK, KK and PTC are specifically reduced and therefore expressed a specific marker for the diagnosis of follicular thyroid carcinomas represents.

4 Figure 4 shows the differential expression of genes 8, 13, 22, 33, 42, 53, 72, 73, 76, 84, 86, 90, 98, 99, 100, 116, 117 and 133 to 153 from Table 4, which are specific Markers for follicular thyroid carcinoma act, in a bar chart. The numbers above or below the bars correspond to the gene numbers from Table 4.

The distinction between benign tumors and malignant tumors is preferably in addition, a quantitative determination of the transcripts of a reference gene is carried out in the isolated RNA. Expression of the reference gene serves as an internal standard. The determined value for the mRNA expression of the tumor-specific gene is preferably divided by the determined value for the expression of the reference gene. From the height of the quotient thus obtained, conclusions can then be made about the degree of malignancy of the tumor.

The Reference gene is a gene found in healthy thyroid tissue as well as in benign and malignant Tumors of the thyroid gland at the same height is expressed. Preferred reference genes are shown in Table No. 5 specified.

Advantageous is with the inventive method an improved distinction of benign cold thyroid nodules from malicious thyroid carcinomas by quantifying the above genes in a very small amount Sample of thyroid tissue, especially in FNAZ material, possible. By quantifying these genes, the percentage of thyroid punctures, in those according to the prior art, a sufficient assessment possible is to be minimized. Thus, you can unnecessary thyroid surgery be avoided.

Of the Comparison of the measured values with reference ranges for the different benign and malignant thyroid diseases thus allows a distinction between benign (benign) and malignant (malignant) thyroid nodules.

• a.) eine erhöhte Menge an Transkripten der Gene Nr. 23 bis 27 und 33 einen Hinweis auf das Vorliegen eines gutartigen Tumors liefert,
• b.) eine erniedrigte Menge an Transkripten der Gene Nr. 28 bis 32, 34 und 35 einen Hinweis auf das Vorliegen eines gutartigen Tumors liefert,
• c.) eine erhöhte Menge an Transkripten der Gene Nr. 16, 32, 36 bis 41, 43 bis 45, 47 bis 52, 54 bis 62, 64 bis 68, 70, 71, 74, 75, 77 bis 85, 87, 89, 91 bis 94, 101 bis 107, 110, 112 bis 115, 118, 119, 121 bis 125, 130, 131 sowie der Gene Nr. 133 und 134 einen Hinweis auf das Vorliegen eines bösartigen Tumors liefert,
• d.) eine erniedrigte Menge an Transkripten der Gene Nr. 2, 13, 15, 22, 33, 42, 46, 53, 63, 69, 72, 73, 76, 86, 88, 90, 95 bis 100, 108, 109, 111, 116, 117, 120, 126 bis 129, 132 sowie der Gene Nr. 8 und 135 bis 153 einen Hinweis auf das Vorliegen eines bösartigen Tumors liefert
• e.) und wobei zusätzlich ein Referenzgen in der Gewebeprobe gemessen wird.

In a preferred embodiment of the method, in the isolated RNA, a quantitative determination of the mRNA transcripts of at least three genes selected from the genes listed in Table 2 (specific genes for benign cold nodules) and at least three genes selected from the in Table 3 and at least four genes selected from the genes listed in Table 4, wherein

• a.) an increased amount of transcripts of genes Nos. 23 to 27 and 33 provides an indication of the presence of a benign tumor,
• b.) a reduced amount of transcripts of genes Nos. 28 to 32, 34 and 35 provides an indication of the presence of a benign tumor,
• c.) an increased amount of transcripts of genes Nos. 16, 32, 36 to 41, 43 to 45, 47 to 52, 54 to 62, 64 to 68, 70, 71, 74, 75, 77 to 85, 87, 89, 91 to 94, 101 to 107, 110, 112 to 115, 118, 119, 121 to 125, 130, 131 and genes Nos. 133 and 134 provides an indication of the presence of a malignant tumor,
• d.) a reduced amount of transcripts of genes Nos. 2, 13, 15, 22, 33, 42, 46, 53, 63, 69, 72, 73, 76, 86, 88, 90, 95 to 100, 108, 109, 111, 116, 117, 120, 126 to 129, 132 and genes Nos. 8 and 135 to 153 provides an indication of the presence of a malignant tumor
• e.) and additionally measuring a reference gene in the tissue sample.

In alternative embodiments the procedure will continue to make the case more meaningful to improve, in addition the expression of at least one gene from the group of genes specific for hot knots from Table 1 and / or further genes from Tables 2, 3 and 4 measured.

The Removal of the tissue sample is preferably carried out by puncture of the thyroid or Fine needle aspiration cytology (FNAZ). From the tissue sample is in well-known RNA isolated (14).

Prefers the quantitative measurement of mRNA gene expression by means of quantitative RT-PCR. In the known process of RT-PCR is first with Help the enzyme reverse transcriptase (RT) based on the isolated RNA complementary DNA (cDNA) synthesized. As a primer for the RT can in principle a Oligo-dT can be used. The use of random hexamer primers for the RT led but according to the invention to a significant increase the sensitivity (at least factor 4). The random hexamer oligonucleotide each contains a mixture of A, G, T, C at all six positions and hybrizes nonspecifically mRNA sequences.

Separate cDNAs are used in the subsequent PCR amplified with a sequence-specific primer pair. The primers for the PCR preferably have a length from 15 to 30 base pairs and are designed to be intron-overlapping in the In case of contamination of the RNA with genomic DNA only cDNA to amplify.

The PCR is particularly preferably carried out as a real-time PCR. Known real-time PCR methods are z. B. TaqMan ^®, FRET (fluorescence resonance energy transfer), LightCycler ^® and Beacon Ver drive. By using fluorescent-labeled primers or the use of gene-specific oligonucleotides as hybridization probes with different dye or fluorescent marker-binding (TaqMan ^®, FRET, Beacon) can advantageously the PCR product be specifically quantified during the PCR in these methods.

In a particular embodiment the method according to the invention additionally a proof of Pax8 / PPARγ- and / or Ret / PTC rearrangements and / or BRAF mutations likewise by means of PCR, particularly preferably according to the method described by Cheung CC et al (17) for the Pax8 / PPARγ- and from Nikiforova MN (18) for the Ret / PTC rearrangements and Salvatore G et al. (19) for the BRAF mutations Method.

At the Screening for the Pax8-PPARγ rearrangement (17) the occurrence of a rearrangement between the Pax8 and the PPARγ gene, that in part of the follicular thyroid carcinomas was detected, analyzed. This rearrangement is therefore An additional diagnostic marker for follicular thyroid carcinomas represents.

At the Screening for the Ret PTC Rearrangement (18) will occur the most common Rearrangements between the cytoplasmic domain of the ret proto-oncogene with ubiquitous in the thyroid gland expressed promoters (Ret / PTC1: H4 and Ret / PTC3: ele1). These rearrangements were in a part of the papillary thyroid carcinomas detected and therefore provide an additional diagnostic marker for papillary thyroid carcinomas represents.

At the BRAF mutation screening (19) will be the base sequence of 45% of the papillary thyroid carcinomas mutant gene BRAF (V600E) (Homo sapiens BRAF) marker analyzed. A BRAF mutation therefore provides an additional diagnostic marker for papillary thyroid carcinomas represents.

By the analysis of these additional diagnostic markers, the validity of the method according to the invention on the Differentiation between benign and malignant thyroid tumors even further increased become.

By the measurement of such an additional Markers can the validity of the method according to the invention on the Differentiation between benign and malignant thyroid tumors even further increased become.

• 1) für die cDNA-Synthese: a) Random-Hexamer-Primer, b) Reverse Transkriptase;
• 2) für die PCR: c) pro nachzuweisendem mRNA-Transkript mindestens zwei Primer, die mit der cDNA eines der Gene ausgewählt aus den Gruppen der tumorspezifischen Gene aus den Tabellen 1, 2, 3 und 4 hybridisieren; d) mindestens zwei Primer, die mit der cDNA eines Referenzgens, bevorzugt ausgewählt aus der in der Tabelle 5 angegebenen Gruppe der Referenzgene, hybridisieren, e) eine über 80°C beständige DNA-Polymerase, wie z. B. taq-Polymerase;

sowie dNTP-Mix (bevorzugt 10 mM), DDT, RNase-Inhibitor und entsprechende Reaktionspuffer.To carry out the method according to the invention for differential diagnosis by means of RT-PCR, a diagnostic kit is preferably used which contains the following components in each case:

• 1) for cDNA synthesis: a) random hexamer primer, b) reverse transcriptase;
• 2) for the PCR: c) for each mRNA transcript to be detected, at least two primers which hybridize with the cDNA of one of the genes selected from the groups of the tumor-specific genes from Tables 1, 2, 3 and 4; d) at least two primers which hybridize with the cDNA of a reference gene, preferably selected from the group of reference genes given in Table 5, e) a DNA polymerase which is stable above 80 ° C, such as e.g. B. taq polymerase;

and dNTP mix (preferably 10 mM), DDT, RNase inhibitor and corresponding reaction buffer.

In a less preferred embodiment contains the kit for the cDNA synthesis oligo-dT instead of the random hexamer.

Examples for primer pairs, which the specific amplification of genes Nos. 66 and 67 as well Allow 154 to 157 are given in the SEQ ID no 1 to 12.

In an embodiment of the kit is in each case one of the primers from 2) c) (for the tumor-specific Gene) and 2) d) (for the reference gene) fluorescence-labeled.

• f) eine Fluoreszenz-markierte Hybridisierungssonde oder ein Paar Fluoreszenz-markierter Hybridisierungssonden, die spezifisch mit dem tumorspezifischen Gen aus Tabelle 1, 2, 3 oder 4 hybridisieren, und
• g) eine Fluoreszenz-markierte Hybridisierungssonde oder ein Paar Fluoreszenz-markierter Hybridisierungssonden, die spezifisch mit dem Referenzgen aus Tabelle 5 hybridisieren.

In a particularly preferred embodiment, the diagnostic kit for carrying out a real-time PCR preferably contains as additional constituents:

• f) a fluorescence-labeled hybridization probe or a pair of fluorescently-labeled hybridization probes which hybridize specifically with the tumor-specific gene from Table 1, 2, 3 or 4, and
• g) a fluorescently labeled hybridization probe or a pair of fluorescently labeled hybridization probes that specifically hybridize to the reference gene of Table 5.

Either as primers, as well as hybridization probes are preferred DNA oligonucleotides with a length chosen from 10 to 30 nucleotides.

In an embodiment of the kit the hybridization probe next to the fluorescent dye another Dye that quenches the fluorescence by a fluorescence resonance energy transfer (FRET), as long as the sample does not hybridize with the amplified cDNA.

In an alternative embodiment The kit uses two hybridization probes in which one probe each is labeled with a dye. Such Probe pair is chosen that they are at a small distance, preferably at a distance of 1-5 bp, on hybridize to the amplified cDNA. By the hybridization The fluorescent dyes are brought so close to each other, that FRET takes place and the cDNA can be quantified. Then be (in the extension phase of the PCR), by the exonuclease activity of the polymerase, the probes are separated from the cDNA. In a preferred variant is in a pair of probes, the first probe at the 3 'end with fluorescein (FL) and the other at its 5'-end marked with LightCycler-Red-640.

Fluorescence-labeled hybridization probes are e.g. B. as LightCycler ^®, TaqMan ^® or "molecular beacons" in the market and enable quantification during PCR.

Examples for hybridization probes, which the specific recognition of cDNA of genes No. 67, 154, 155 and 157 allow are given in SEQ ID no 13 to 20. The sequences according to SEQ ID no 13 and 14, 15 and 16, and 17 and 18 may each be used as a pair of probes be used as they are at a small distance, preferably in 1 to 5 bp, hybridize to the amplified cDNA.

For this purpose, the SEQ ID no. 13, 15 and 17 preferably at the 3 'end with fluorescein (FL) and SEQ ID NO. 14, 16 and 18 labeled with LightCycler-Red 640 at its 5 'end and allow the quantification by the LightCycler ^® process.

The 3'-hydroxyl group SEQ ID no. 14, 16 and 18 is for this purpose preferably with a phosphate group against the unwanted Extension blocked by the polymerase. After hybridization remains between the two probes a distance of one to five bases, so that the dyes do not collide.

The probe for gene no. 67 (COX-2) according to SEQ ID no. 19 and for gene no. 155 (β-actin) according to SEQ ID no. 20 is preferably labeled at the 5 'end with VIC and at the 3' end with TAMRA-3 'and allows for the quantification according to the TaqMan ^® method.

For a multiplex PCR differ the fluorescent dyes with which the primer or hybridization probes are labeled, in their absorption and / or emission spectra. This allows a parallel quantification the amplified cDNA of the tumor specific gene and the cDNA of the reference gene.

By following figures and embodiments The invention and the invention underlying scientific Knowledge closer explains:

there demonstrate

1 Mean ± SEM of the signal log ratio of genes 1 to 22 from Table 1 in hot thyroid nodules, benign cold thyroid nodules, and papillary and follicular thyroid carcinomas, respectively, relative to expression in the corresponding surrounding tissue.

2 Mean ± SEM (SEM = standard mean error) of the signal log ratio of genes 23 to 35 from Table 2 in hot thyroid nodules, benign cold thyroid nodules, and papillary and follicular thyroid carcinomas, respectively, relative to expression in the corresponding surrounding tissue.

3 Mean ± SEM (SEM = standard error of the mean) of the "signal log ratio" of genes 2, 13, 15, 16, 22, 32, 33, and 36 to 132 of Table 3 in hot thyroid nodules, benign cold thyroid nodules, and papillary and follicular thyroid carcinomas, respectively, relative to expression in the corresponding surrounding tissue.

4 Mean ± SEM (SEM = standard error of the mean) of the signal log ratio of genes 8, 13, 22, 33, 42, 53, 72, 73, 76, 84, 86, 90, 98, 99, 100, 116, 117 and 133-153 of Table 4 in hot thyroid nodules, benign cold thyroid nodules and papillary and follicular thyroid carcinomas, respectively, relative to expression in the corresponding surrounding tissue.

embodiment 1:

Investigation of differential gene expression with Affymetrix GeneChips U95Av2 (Affymetrix, Santa Clara, CA, USA):

At first was to total RNA from 15 hot Thyroid nodules, their surrounding tissue, 22 cold thyroid nodules, and their surrounding tissue using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) isolated according to the manufacturer's instructions. Subsequently was total RNA using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quality and quantity of RNA were used on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6,000 LabChip Kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer's instructions determined.

production, Purification and analysis of the cRNA was carried out according to the supplied Logs of Affymetrix. The synthesis of double-stranded cDNA took place from 10 μg Total RNA. there was the Superscript II reverse transcriptase (Superscript II, Life Technologies, Gaithersburg, MD USA) and an oligo dT primer with a T7 RNA polymerase promoter sequence (Genset SA, Paris, France). The cDNA was then analyzed by means of Phenol-chloroform extraction purified. This was followed by cRNA synthesis in one in vitro transcription using ENZO BioArray RNA Labeling Kits (Affymetrix, Santa Clara, CA, USA). Unincorporated nucleotides were purified by RNeasy kit purification (QIAGEN, Hilden, Germany) away. The cRNA was then used according to the Affymetrix protocol fragmented and over Night on the Affymetrix GeneChips U95Av2 hybridized. To wash, To dye and scanning of the GeneChips was also done according to the instructions of the Affymetrix protocol.

Of the Comparison of the expression data of these 15 hot thyroid nodules, 22 cold thyroid nodules and their normal surrounding tissue was with expression data of 8 follicular Carcinomas and 8 normal tissues and 8 papillary carcinomas and their surrounding tissues.

The Data analysis was carried out in a first step with the "Significance analysis of microarray "(SAM) method. It is an algorithm with the help of which differentially expressed genes with statistically adjusted significance (in terms of on the big one Number of genes calculated in parallel) can be identified. there The settings of the SAM analysis were chosen so that a maximum of 1% false positives Genes were detected. In a second step of data analysis From the resulting gene set of the SAM analysis, the genes became whose absolute signal values were greater than 200, which guarantees that these Genes can be detected by real-time RT-PCR and additionally at least two-fold differences in expression between the investigated entities (hot nodes, cold nodules, papillary carcinomas and follicular Carcinomas), which should ensure that quantitative differences in expression can be detected by real time RT-PCR. The resulting gene sets were finally according to her specificity divided into genes that have a specific expression pattern for hot thyroid nodules exhibit; Genes that have a specific expression pattern for cold thyroid nodules exhibit; Genes that have a specific expression pattern for papillary carcinomas and genes that have a specific expression pattern for follicular thyroid carcinomas exhibit.

embodiment 2:

to execution the method according to the invention For the differential diagnosis of thyroid tumors is first a Tissue sample taken by means of fine needle aspiration cytology (FNAZ).

Fine needle aspiration is carried out on a lying patient after skin disinfection without local anesthesia using disposable cannulas with an outer diameter of 0.6 to 0.7 mm, if possible under sonographic control. At least two punctures should be performed fan-like into different nodal areas per node become. Cyst punctates should be centrifuged and heparin should be added when shipping the cyst fluid. After emptying cysts, a new puncture of solid nodal parts should also be sought.

embodiment 3:

to Extraction of RNA will make the disposable cannulae with the tissue sample embodiment 2 in 1 ml TRIzol (in 1.5 ml Eppendorf tubes) rinsed several times thoroughly. Subsequently, will vortex the homogenate and incubate for 5 min at room temperature. This is followed by an addition of 200 .mu.l of chloroform. Before another Incubation at room temperature (2-3 min) becomes the Eppendorf tube strong for about 15 seconds shaken. After the incubation step, centrifugation is carried out for 15 minutes at 4 ° C. and 15,000 rpm. The upper phase will follow transferred to a new tube and after addition of 500 μl Isopropanol slightly swirled the tube and again for 10 min incubated at room temperature. (The interphase and the phenolic Phase are used to extract the DNA (see embodiment 8th)). This is followed by another centrifugation for 15 min at 4 ° C and 15,000 rpm. After the supernatant was removed, the RNA pellet is washed with 150 ul of 70% ethanol and again for 5 min at 4 ° C and centrifuged at 15,000 rpm. The supernatant is removed again and the RNA pellet dried briefly at room temperature. Subsequently, will it in 12 μl Water added.

The synthesis of the cDNA was carried out in a reaction mixture consisting of 11.5 μl of RNA (see above), 0.5 μg of random hexamer primer, 5 × first-strand buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl ₂ ) (GibcoBRL, Karlsruhe, Germany), 0.5 mM dNTPs, 5 mM DTT (GibcoBRL), 15 U Prime RNase Inhibitor (PeqLab, Erlangen, Germany), and 200 U Moloney murine leukemia virus reverse transcriptase (GibcoBRL, Karlsruhe, Germany). Germany) for 1 hour at 37 ° C with a final heat step at 94 ° C for 5 min.

embodiment 4:

The cDNA obtained in accordance with Example 3 is analyzed quantitatively on a quantitative PCR as follows on a LightCycler (Roche, Mannheim, Germany):
The expression of the overexpressed in papillary thyroid carcinoma gene cyclooxygenase-2 (Homo sapiens COX-2, gene no. 67 from Table 3) was analyzed as a diagnostic marker. As an endogenous (internal) control, the expression of the reference gene β-actin (gene No. 155 from Table 5) was quantitatively analyzed in a parallel PCR approach.

First were optimal PCR conditions, according to the manufacturer's specifications established (14). The amplicons (COX-2 and β-actin) were subsequently in cloned the pGEM-T vector (Promega, Madison, WI, USA) and calibration curves, by means of plasmid dilution series, using the LightCycler DNA Master SYBR Green I kit (Roche), created.

• – 2 μl LightCycler DNA Master SYBR Green I,
• – 5 mM zusätzliches MgCl₂,
• – je 0,5 μM Forward- und Reverse-Primer gemäß SEQ ID No. 3 und 4,
• – 2 μl cDNA Template aus Ausführungsbeispiel 3.

The 20 μl reaction batch for the detection of COX-2 is composed as follows:

• - 2 μl LightCycler DNA Master SYBR Green I,
• 5 mM additional MgCl ₂ ,
• - 0.5 μM forward and reverse primer according to SEQ ID no. 3 and 4,
• 2 μl of cDNA template from Example 3.

The PCR is carried out with the following temperature profile:
95 ° C 1 min, 40 cycles: 95 ° C 0 sec, 64 ° C 7 sec, 72 ° C 13 sec.

• – 2 μl LightCycler DNA Master SYBR Green I,
• – 4 mM zusätzliches MgCl₂,
• – je 0,5 μM Forward- und Reverse-Primer gemäß SEQ ID No. 7 und 8,

und 2 μl cDNA Template aus Ausführungsbeispiel 3.The 20 μl reaction batch for the detection of the reference gene β-actin is composed as follows:

• - 2 μl LightCycler DNA Master SYBR Green I,
• 4 mM additional MgCl ₂ ,
• - 0.5 μM forward and reverse primer according to SEQ ID no. 7 and 8,

and 2 μl cDNA template from embodiment 3.

The PCR is carried out with the following temperature profile:
95 ° C 1 min, 40 cycles: 95 ° C 0 sec, 55 ° C 7 sec, 72 ° C 9 sec.

The Primers were synthesized by MWG (Ebersberg, Germany).

embodiment 5:

The cDNA obtained in accordance with Example 3 is differentially analyzed by quantitative PCR on a LightCycler (Roche, Mannheim, Germany) as follows:
The expression of the overexpressed in papillary thyroid carcinoma gene cyclooxygenase-2 (Homo sapiens COX-2, gene no. 67 from Table 3) was analyzed as a diagnostic marker. As an endogenous control, the expression of the reference gene β-actin (gene No. 155 from Table 5) was quantitatively analyzed in a parallel PCR approach.

First were optimal PCR conditions, according to the manufacturer's specifications established (14). The amplicons (COX-2 and β-actin) were subsequently in cloned the pGEM-T vector (Promega, Madison, WI, USA) and calibration curves, by means of plasmid dilution series, using the LightCycler DNA Master Hybridization Probe Kits (Roche), created.

• – 2 μl LightCycler DNA Master Hybridization Probes,
• – 5 mM zusätzliches MgCl₂,
• – je 0,5 μM Forward- und Reverse-Primer gemäß SEQ ID No. 3 und 4,
• – 0,15 μM der 5'-VIC- und 3' TAMRA markierten TaqMan-Sonde mit einer DNA Sequenz gemäß SEQ ID No. 19

und 2 μl cDNA Template aus Ausführungsbeispiel 3.The 20 μl reaction batch for the detection of COX-2 is composed as follows:

• - 2 μl LightCycler DNA Master Hybridization Probes,
• 5 mM additional MgCl ₂ ,
• - 0.5 μM forward and reverse primer according to SEQ ID no. 3 and 4,
• 0.15 μM of the 5'-VIC and 3'TAMRA-labeled TaqMan probe with a DNA sequence according to SEQ ID no. 19

and 2 μl cDNA template from embodiment 3.

The PCR is carried out with the following temperature profile:
95 ° C 1 min, 40 cycles: 95 ° C 0 sec, 64 ° C 7 sec, 72 ° C 13 sec.

• – 2 μl LightCycler DNA Master Hybridization Probes,
• – 4 mM zusätzliches MgCl₂,
• – je 0,5 μM Forward- und Reverse-Primer gemäß SEQ ID No. 7 und 8,
• – 0,15 μM der 5'-VIC- und 3' TAMRA markierten TaqMan-Sonde mit einer DNA Sequenz gemäß SEQ ID No 20

und 2 μl cDNA Template aus Ausführungsbeispiel 3.The 20 μl reaction batch for the detection of the reference gene β-actin is composed as follows:

• - 2 μl LightCycler DNA Master Hybridization Probes,
• 4 mM additional MgCl ₂ ,
• - 0.5 μM forward and reverse primer according to SEQ ID no. 7 and 8,
• 0.15 μM of the 5'-VIC and 3'TAMRA-labeled TaqMan probe with a DNA sequence according to SEQ ID No 20

and 2 μl cDNA template from embodiment 3.

The PCR is carried out with the following temperature profile:
95 ° C 1 min, 40 cycles: 95 ° C 0 sec, 55 ° C 7 sec, 72 ° C 9 sec.

The Primers were synthesized by MWG (Ebersberg, Germany). The 5'-VIC and 3 'TAMRA marked TaqMan probes are available from Applied Biosystems (Darmstadt, Germany) available.

embodiment 6:

For other markers or reference genes, a PCR is carried out according to the conditions given in Example 4. The additionally added MgCl ₂ , the annealing temperature and the elongation time are dependent on the marker or reference gene to be amplified and are optimized so that a melting curve with a single maximum value is detected. In addition, the amplicons were analyzed by agarose gel electrophoresis for correct size of the band.

The amplifications are generally carried out with the following temperature profile:
95 ° C 1 min, 40 cycles: 95 ° C 0 sec, x ° C 7 sec, 72 ° C y sec.

Where x is the optimized annealing temperature for the specific gene and y is the elongation time of the specific amplicon. The PCR conditions for the genes Nos. 66 and 67 from Table 3 and the genes 154 to 157 from Table 5 are summarized in the following overview: Gene no. Gene Symbol MgCl ₂ Conc. Annealing temperature x Elongation time y 66 ETS 1 5mM 64 ° C 13 sec 67 COX 2 5mM 64 ° C 13 sec 154 GAPDH 4mM 57 ° C 8 sec 155 β-Actin 4mM 55 ° C 9 sec 156 18S rRNA 4mM 64 ° C 15 sec 157 TG 4mM 62 ° C 18 sec

embodiment 7:

• 1. Für die cDNA-Synthese: – 20 μg Random-Hexamer-Primer – 0,1 M DTT – 10 mM dNTP-Mix (je 10 mM dATP, dCTP, dGTP und dTTP) – 10.000 U Reverse Transkriptase – 10.000 U RNase Inhibitor – 5 × Erststrang-Puffer
• 2. Für die real time PCR – 5 nmol Forward-Primer COX-2 (Nr. 96; Markergen) gemäß SEQ ID No. 3, – 5 nmol Reverse-Primer COX-2 (Nr. 96; Markergen) gemäß SEQ ID No. 4, – 1 nmol einer 5'-VIC- und 3' TAMRA markierten TaqMan-Sonde für COX-2 (Nr. 67; Markergen) mit einer DNA Sequenz gemäß SEQ ID No. 19, – 5 nmol Forward-Primer β-Aktin (Nr. 155; Referenzgen) gemäß SEQ ID No. 7, – 5 nmol Reverse-Primer β-Aktin (Nr. 155; Referenzgen) gemäß SEQ ID No. 8, – 1 nmol einer 5'-VIC- und 3' TAMRA markierten TaqMan-Sonde für β-Aktin (Nr. 155; Referenzgen) mit einer DNA Sequenz gemäß SEQ ID No. 20, – 5 × PCR-Puffer – 100 U Taq-Polymerase.

A kit for carrying out the method according to the invention by real time PCR contains, by way of example, the following components for detecting the gene overexpressed in papillary thyroid carcinoma Homo sapiens cyclooxygenase-2 (COX-2, gene no. 67 from Table 3) as a diagnostic marker and the reference gene β-actin (Gene No. 155 from Table 5):

• 1. For the cDNA synthesis: - 20 μg random hexamer primer - 0.1 M DTT - 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP and dTTP) - 10,000 U reverse transcriptase - 10,000 U RNase inhibitor - 5 × first strand buffer
• 2. For the real-time PCR - 5 nmol forward primer COX-2 (No. 96, marker gene) according to SEQ ID no. 3, - 5 nmol reverse primer COX-2 (No. 96, marker gene) according to SEQ ID NO. 4, - 1 nmol of a 5'-VIC and 3 'TAMRA-labeled TaqMan probe for COX-2 (No. 67, marker gene) with a DNA sequence according to SEQ ID NO. 19, - 5 nmol forward primer β-actin (No. 155, reference gene) according to SEQ ID no. 7, - 5 nmol reverse primer β-actin (No. 155, reference gene) according to SEQ ID no. 8, - 1 nmol of a 5'-VIC and 3 'TAMRA-labeled TaqMan probe for β-actin (No. 155, reference gene) with a DNA sequence according to SEQ ID NO. 20.5 PCR buffer - 100 U Taq polymerase.

embodiment 8th:

After this the aqueous phase for RNA extraction (embodiment 3) was removed, the DNA from the interphase and the organic Phase precipitated by ethanol. For this purpose, 0.3 ml of 100% ethanol for interphase and organic phase added and mixed by gentle panning. Then be the samples 2-3 stored at 15-30 ° C min and then the DNA at 2,000 x g for 5 min at 4 ° C centrifuged. After removal of the phenol / ethanol supernatant The DNA is washed with 1 ml of 0.1 M sodium citrate in 10% ethanol solution. The DNA pellet is stored for 30 minutes at room temperature in the solution and subsequently at 2,000 × g for 5 min at 4 ° C centrifuged. After this washing step has been repeated, is the DNA in 1.5 ml of 75% ethanol for 15-20 stored at room temperature and then at 2,000 × g for 5 min at 4 ° C centrifuged. The DNA pellet is air dried and incubated in 8 mM NaOH resuspended.

embodiment 9:

• – 10 mM Tris-HCl, 50 mM KCl, 1,5 mM MgCl₂ (pH 8,3),
• – 0,2 mM dNTP-Mix
• – je 0,8 pmol Forward-(5'-TCATAATGCTTGCTCTGATAGGA-3') und Reverse-(5'-GGCCAAAAATTTAATCAGTGGA-3')Primer,
• – 5 μl DNA Template aus Ausführungsbeispiel 9,
• – 1 U AmpliTaq DNA Polymerase (Applied Biosystems).

With the DNA obtained according to Embodiment 8, BRAF mutation screening (19) is carried out as follows:
The 25 μl reaction mixture for the amplification of BRAF is composed as follows:

• 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ (pH 8.3),
• - 0.2 mM dNTP mix
• 0.8 pmol each forward (5'-TCATAATGCTTGCTCTGATAGGA-3 ') and reverse (5'-GGCCAAAATTTAATCAGTGGA-3') primer,
• 5 μl of DNA template from embodiment 9,
• 1 U AmpliTaq DNA polymerase (Applied Biosystems).

The PCR is carried out with the following temperature profile:
94 ° C 5 min., 40 cycles: (94 ° C for 30 sec, 60 ° C for 30 sec, 72 ° C for 60 sec), 72 ° C for 5 min.

To followed by amplification and subsequent purification of the PCR product using QIAquick PCR Purification Kit (Qiagen) (according to the Manufacturer's instructions), a sequencing of the PCR product with the forward primer using the Big Dye Terminator Kit (Applied Biosystems) on an ABI Prism DNA sequencer.

The Primers were synthesized by MWG (Ebersberg, Germany).

embodiment 10:

• – 10 mM Tris-HCl, 50 mM KCl, 1,5 mM MgCl₂ (pH 8,3),
• – 0,2 mM dNTP-Mix
• – je 0,8 pmol Forward-(5'-AACCTCTCGACTCACCAGAC-3') und Reverse-(5'-AGAATGGCATCTCTGTGTCAAC-3')Primer,
• – 2 μl cDNA Template aus Ausführungsbeispiel 3,
• – 1 U AmpliTaq DNA Polymerase (Applied Biosystems).

The cDNA obtained according to Example 3 is screened for Pax8-PPARγ rearrangement (17) as follows:
The 30 μl reaction mixture for amplification of the rearrangement is composed as follows:

• 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ (pH 8.3),
• - 0.2 mM dNTP mix
• 0.8 pmol each forward (5'-AACCTCTCGACTCACCAGAC-3 ') and reverse (5'-AGAATGGCATCTCTGTGTCAAC-3') primer,
• 2 μl of cDNA template from embodiment 3,
• 1 U AmpliTaq DNA polymerase (Applied Biosystems).

The PCR is carried out with the following temperature profile:
94 ° C 5 min., 40 cycles: (94 ° C 40 sec, 60 ° C 1 min, 72 ° C 1 min), 72 ° C 5 min.

To After amplification, the PCR product becomes electrophoresed separated on a 1.5% agarose gel.

embodiment 11:

• – 10 mM Tris-HCl, 50 mM KCl, 1,5 mM MgCl₂ (pH 8,3),
• – 0,2 mM dNTP-Mix
• – je 1 mM Forward-(Ret/PTC1: 5'-GTCGGGGGGCATTGTCATCT-3', Ret/PTC3: 5'-TGGAGAAGAGGAGCTGTATC-3') und Reverse-(Ret/PTC1: 5'-AAGTTCTTCCGAGGGAATTC-3', Ret/PTC3: 5'-CTTTCAGCATCTTCACGG-3')Primer,
• – 2 μl cDNA Template aus Ausführungsbeispiel 3,
• – 1 U AmpliTaq DNA Polymerase (Applied Biosystems).

The cDNA obtained according to Example 3 is screened for Ret-PTC rearrangement (18) as follows:
The 30 μl reaction mixture for amplification of the rearrangement is composed as follows:

• 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ (pH 8.3),
• - 0.2 mM dNTP mix
• 1 mM forward (Ret / PTC1: 5'-GTCGGGGGGCATTGTCATCT-3 ', Ret / PTC3: 5'-TGGAGAAGAGGAGCTGTATC-3') and reverse (Ret / PTC1: 5'-AAGTTCTTCCGAGGGAATTC-3 ', Ret / PTC3 : 5'-CTTTCAGCATCTTCACGG-3 ') primer,
• 2 μl of cDNA template from embodiment 3,
• 1 U AmpliTaq DNA polymerase (Applied Biosystems).

The PCR is carried out with the following temperature profile:
95 ° C 4 min., 40 cycles: (95 ° C 30 sec, 55 ° C 30 sec, 72 ° C 30 sec), 72 ° C 5 min.

To After amplification, the PCR product becomes electrophoresed separated on a 1.5% agarose gel.

bp

Basenpaare

cDNA

zur mRNA komplementäre DNA

COX-2

Cyclooxygenase-2

cRNA

Kopie-RNA

DNA

Desoxyribonukleinsäure

DDT

Dithiothreitol

ETS-1

Human erythroblastosis virus oncogene homolog 1

FNAZ

Feinnadelaspirationszytologie

FRET

Fluoreszenz-Resonanz-Energie-Transfer

FTC

follikuläres Schilddrüsenkarzinom

GAPDH

Glyzerinaldehyd-Dehydrogenase

HK

heiße Schilddrüsenknoten

KK

kalte Schilddrüsenknoten

MEN

Multiple endokrine Neoplasie

μM

μmol/Liter

mM

mmol/Liter

mRNA

Messenger-RNA

NCBI

National Center for Biotechnology Information, Bethesda, USA

PBS

Phosphatsalzpufferlösung

PCR

Polymerasekettenreaktion

PTC

papillären Schilddrüsenkarzinomen

RT-PCR

Reverse Transkriptase PCR

RNA

Ribonukleinsäure

mRNA

Messenger-RNA

rRNA

Ribosomale Ribonukleinsäure

SEM

Standardfehler des Mittelwertes

SLR

Signal log ratio

TG

Thyroglobulin

TSH

thyroid stimulating hormone, Thyrotropin

TSHR

Thyrotropinrezeptor

UG

Umgebungsgewebe

In the description of the invention the following abbreviations are used:

bp

base pairs

cDNA

DNA complementary to mRNA

COX-2

Cyclooxygenase-2

cRNA

Copy RNA

DNA

deoxyribonucleic acid

DDT

dithiothreitol

ETS-1

Human erythroblastosis virus oncogene homolog 1

FNAZ

fine needle aspiration cytology

FRET

Fluorescence resonance energy transfer

FTC

follicular thyroid carcinoma

GAPDH

Glyceraldehyde dehydrogenase

HK

hot thyroid nodules

KK

cold thyroid nodules

MEN

Multiple endocrine neoplasia

uM

mol / liter

mM

mmol / l

mRNA

Messenger RNA

NCBI

National Center for Biotechnology Information, Bethesda, USA

PBS

Phosphate salt buffer solution

PCR

Polymerase chain reaction

PTC

papillary thyroid carcinoma

RT-PCR

Reverse transcriptase PCR

RNA

ribonucleic acid

mRNA

Messenger RNA

rRNA

Ribosomal ribonucleic acid

SEM

Standard error of the mean value

SLR

Signal log ratio

TG

thyroglobulin

TSH

thyroid stimulating hormone, thyrotropin

TSHR

thyrotropin

UG

surrounding tissue

Bibliography:

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• 7. Gharib H. Changing concepts in the diagnosis and management of thyroid nodules. Endocrinol Metab Clin North Am 1997; 26: 777-800.
• 8. Fuhrer D, Warner J, Sequeira M, Paschke R, Gregory J, Ludgate M. Novel TSHR germline mutation (Met463Val) masquerading as Graves' disease in a large Welsh kindred with hyperthyroidism. Thyroid 2000; 10 (12): 1035-1041.
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• 13. Engelbach M, Kunt T, P et al. Predictive genetic studies. Individualization of diagnostics and therapy in families with multiple endocrine neoplasia type II. Dtsch Med Wochenschr 2000; 125 (3): 37-44.
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• 19. Salvatore, G. et al. Analysis of BRAF Point Mutation and Ret / PTC Rearrangement Refines the Fine-Needle Aspiration Diagnosis of Papillary Thyroid Carcinoma. J Clin Endocrinol Metab. 2004; 89 (10): 5175-5180.

SEQUENCE LISTING - SEQUENCE LISTING

Claims (9)

Method for the differential diagnosis of thyroid tumors for the differentiation of benign tumors against malignant tumors in which: 1.) RNA is isolated from a tissue sample of the thyroid gland, 2.) in the isolated RNA, a quantitative determination of the mRNA transcripts of genes selected from the in Table 1, Table 2, Table 3 and Table 4 listed genes with the gene numbers 1 to 153, wherein a quantitative determination of the mRNA transcripts of at least three genes from the genes listed in Table 3 and a quantitative determination of the mRNA transcripts of at least four genes from the genes listed in Table 4, wherein a.) an increased amount of transcripts of genes Nos. 1 to 3, 5 to 8, 11, 13 to 15 and 19 to 22 and genes 23 to 27 and 33 b.) a reduced amount of transcripts of genes Nos. 4, 9, 10, 12, 16 to 18 and genes 28 to 32, 34 and 35 a H c.) an increased amount of transcripts of genes Nos. 16, 32, 36 to 41, 43 to 45, 47 to 52, 54 to 62, 64 to 68, 70, 71, 74 , 75, 77 to 85, 87, 89, 91 to 94, 101 to 107, 110, 112 to 115, 118, 119, 121 to 125, 130, 131 as well as genes Nos. 133 and 134 an indication of the presence of a d.) provides a reduced amount of transcripts of genes Nos. 2, 13, 15, 22, 33, 42, 46, 53, 63, 69, 72, 73, 76, 86, 88, 90, 95 to 100, 108, 109, 111, 116, 117, 120, 126 to 129, 132 and genes Nos. 8 and 135 to 153 provides evidence of the presence of a malignant tumor. Method according to Claim 1 for distinguishing malicious papillary Carcinoma or malignant follicular carcinoma, from benign hot Knot, benign cold knot and the other mentioned malicious Carcinoma in which: a.) in the isolated RNA a quantitative Determination of mRNA transcripts selected from at least three genes those listed in Table 3 Genes are done and an increased Amount of transcripts of genes Nos. 16, 32, 36 to 41, 43 to 45, 47 to 52, 54 to 62, 64 to 68, 70, 71, 74, 75, 77 to 83, 85, 87, 89, 91 to 94, 101 to 107, 110, 112 to 115, 118, 119, 121 to 125, 130, 131 and a reduced amount of transcripts of Gene Nos. 2, 15, 46, 63, 69, 88, 95 to 97, 108, 109, 111, 120, 126 to 129 and 132 an indication of the existence of a malicious papillary thyroid carcinoma supplies; or b.) in the isolated RNA, a quantitative determination of the mRNA transcripts selected from at least four genes those listed in Table 4 Genes are done and an increased Amount of transcripts of genes Nos. 133 and 134 and a decreased Amount of transcripts of genes No. 8 and 135 to 153 an indication the presence of a malicious follicular thyroid carcinoma supplies. Method according to claim 1 or 2 for distinguishing from benign hot Nodules or benign cold nodules, from malignant follicular carcinomas, malicious papillary Carcinomas and from each other called benign cold Node in which: a.) in the isolated RNA a quantitative Determination of mRNA transcripts selected from at least three genes those listed in Table 1 Genes with the gene numbers 1 to 22 are carried out and an increased amount Transcripts of genes Nos. 1 to 3, 5 to 8, 11, 13 to 15 and 19 to 22 and a reduced amount of transcripts of the genes Nos 4, 9, 10, 12 and 16 to 18 indicate the existence of a benign hot knot supplies; or b.) in the isolated RNA, a quantitative determination of the mRNA transcripts selected from at least three genes those listed in Table 2 Genes with the gene numbers 23 to 35 are made and an increased amount Transcripts of genes Nos. 23 to 27 and 33 and a decreased Amount of transcripts of genes 28 to 32, 34 and 35 an indication to the presence of a benign cold knot supplies. Method according to one of claims 1 to 3, characterized that in the isolated RNA in addition a quantitative determination of the transcripts of a reference gene he follows. Method according to claim 4, characterized in that that the reference gene from the group of those given in Table 5 Genes with the gene numbers 154 to 157 are selected. Method according to one of claims 1 to 5, characterized as a tissue sample, a thyroid punctate or a Feinnadelaspirationszytologie (FNAZ) becomes. Method according to one of claims 1 to 6, characterized that in addition an analysis for the presence of Pax8 / PPARγ and / or Ret / PTC rearrangements and / or performed by BRAF mutations becomes. Diagnostic kit for differential diagnosis of thyroid tumors for the differentiation of benign tumors against malignant tumors by means of RT-PCR, to perform a Process according to claims 4 to 7, containing the following constituents: 1.) for the cDNA synthesis: a.) random hexamer primer or oligo dT, b.) Reverse transcriptase; 2.) for the PCR: a.) per proof mRNA transcript at least two primers specific to the cDNA one of the genes selected from the genes listed in Table 1, Table 2, Table 3 and Table 4 with the gene numbers 1 to 153 hybridize; b.) at least two primers specific with the cDNA of a reference gene selected from those given in Table 5 Hybridize genes with the gene numbers 154 to 157, c.) a DNA polymerase resistant to more than 80 ° C, such as corresponding reaction buffer. Diagnostic kit according to claim 8, characterized in that it contains as additional constituents for the PCR: a.) per mRNA transcript to be detected, a fluorescence-labeled hybridization probe or a pair of fluorescence-labeled hybridization probes which are specifically linked to the cDNA of one of the genes selected from the genes listed in Table 1, Table 2, Table 3 and Table 4 with the gene Hybridize numbers 1 to 153, and b.) A fluorescence-labeled hybridization probe or a pair of fluorescently labeled hybridization probes which specifically hybridize to the cDNA of a reference gene selected from the genes numbered 154 to 157 given in Table 5.