Langenbecks Arch Surg (2004) 389:193–197 DOI 10.1007/s00423-004-0479-6
W. Czyz˙ E. Balcerczak M. Jakubiak Z. Pasieka K. Kuzdak M. Mirowski
Received: 8 December 2003 Accepted: 5 March 2004 Published online: 24 April 2004 Springer-Verlag 2004
W. Czyz˙ · Z. Pasieka · K. Kuzdak Department of Endocrinological and General Surgery, Copernicus Memorial Hospital, Lodz, Poland E. Balcerczak · M. Mirowski ()) Molecular Biology Laboratory, Department of Pharmaceutical Biochemistry, Medical University, 1 Muszynskiego Street, 90-151 Lodz, Poland e-mail:
[email protected] Tel.: +48-42-6779130 Fax: +48-42-6779130 M. Jakubiak Department of Pathology, Copernicus Memorial Hospital, Lodz, Poland
ORIGINAL ARTICLE
HMGI(Y) gene expression as a potential marker of thyroid follicular carcinoma
Abstract Background: We assessed HMGI(Y) gene expression in thyroid tumors, control thyroid tissue and in the blood of patients diagnosed with papillary and follicular thyroid cancers to try to differentiate between malignant and benign disease. Methods: HMGI(Y) gene expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 cases of thyroid tumors. Among this number 11 were diagnosed as papillary carcinoma, 37 as follicular carcinoma, and 12 as follicular adenoma. All carcinoma cases selected for this study were classified according to the tumor, lymph node metastases, distant metastases (TNM) classification. Results: HMGI(Y) gene expression was detected only in follicular carcinomas, whereas in papillary carcinomas, follicular adenomas and control tissues there was no positive reaction. In follicular carcinomas the percent-
Introduction High mobility group (HMG) proteins are involved in the physiological regulation of chromatin function. They are an important part of the transcriptional apparatus and can interact with DNA by binding with regions rich in A/T pairs, as well as with some transcriptional factors, forming complexes that stimulate transcription [1–3]. They recognize architectural DNA minor groove domains, rather than those composed of bases. They play an important role in cell proliferation and differentiation. The HMG protein family comprises three separate particles:
age of positive cases (number of samples with presence of HMGI(Y) gene transcript) was the highest and reached approximately 84. There was no statistical dependence between the presence of HMGI(Y) gene expression and tumor size or the presence of lymph node and distant metastases. HMGI(Y) gene expression was also analyzed in whole blood taken from a selected group of patients diagnosed with papillary or follicular carcinomas. Among follicular carcinomas there were 83% of positive cases, whereas among papillary carcinomas there were only 6%. Conclusions: On the basis of our study, we conclude that HMGI(Y) gene expression analysis could be helpful in differentiation between follicular carcinoma and adenoma. Keywords HMGI(Y) gene · Thyroid neoplasms · Follicular carcinoma
HMGI, HMGY, and HMGI-C. The HMGI and HMGY proteins are splice variant products of the same gene. Each protein contains three DNA binding motifs, known as AT-hooks, that bind the minor groove of the AT tract in DNA. The third protein, HMGI-C, is encoded by a separate gene, HMGI-C. HMGI and HMGY are the substrates for cyclin-dependent kinase (cdc2), responsible for histon H1 phosphorylation when the cell enters the phase of mitosis, and, thus, is involved in the cellular cycle. The nucleotide sequence of the HMGI(Y) gene has been established, and in its structure there are regions rich in G/C pairs (approximately 201 bp), non-coding 50 (approxi-
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mately 288 bp), non-coding 30 (approximately 1,182 bp) and the sequence encoding 96 amino acid protein [1, 4, 5]. Tissues of healthy adults do not express HMGI(Y) protein, or express it at a very low level. This gene overexpression is observed in differentiating and proliferating tissues during the embryological period [6]. It has been proven that in malignant cell lines, including breast, colon, prostate or thyroid carcinomas, this gene is also activated [7, 8–14]. HMGI(Y) gene overexpression is probably connected with translocation t(1;6) (p35; p21). There are several molecular mechanisms engaged in HMGI(Y) gene expression, e.g., fusion with other genes, deletion of the 30 end, V-mos and V-Ki-ras oncogene activation [15–17]. Various studies conducted on a limited panel of thyroid carcinomas have described HMGI(Y) overexpression on RNA and protein levels. Patients with benign thyroid tumors showed the presence of HMGI(Y)/HMGI(Y) on a significantly lower level than in cancer [12–14]. Distinguishing between benign and malignant follicular thyroid neoplasms on the basis of fine needle aspiration biopsy (FNAB) and macroscopic evaluation before and during surgery still poses numerous problems. There is a constant need for the seeking of markers that would be helpful for the reliable differentiation between follicular carcinoma and adenoma in preoperative diagnoses [18]. In this study we assessed the usefulness of HMGI(Y) gene expression in tumors and in patients’ blood as a differentiating factor for benign and malignant thyroid neoplasms.
Materials and methods Patients All patients underwent surgery between 1994 and 2002 in the Department of Endocrinological Surgery at the Medical University of Lodz. The age of patients with thyroid follicular carcinoma ranged from 18 to 69 years (mean 39 years), and that of patients with thyroid follicular adenoma ranged from 33 to 61 years (mean 44 years). All patients were female, except one male in the follicular adenoma group. We also investigated a group of 11 patients with papillary thyroid carcinoma. The control group comprised 17 patients with benign, nodular, non-toxic goiter. Samples Tissue samples from 60 patients diagnosed with thyroid tumors were obtained by standard surgical procedures. Among this group 11 cases were classified as papillary carcinoma (eight classic and three solid, morphological, variants), 37 cases as follicular carcinoma and 12 as follicular adenoma. As a control, 17 samples of normal thyroid tissue (pyramidal lobe) of patients with simple nodular goiter were taken during surgery. In the case of follicular adenomas, partial thyroid resection was done, and in follicular carcinomas, total thyroidectomy was performed. Clinical stage of disease was evaluated according to the UICC TNM classification, 1997 edition. Distant metastases were detected in whole-body scan after radio-iodine ablation. The tissue samples for assessment of
gene expression after excision were instantly deep frozen at 80C. All specimens were sub-serially cut, and final diagnosis was performed and confirmed on the basis of hematoxylin and eosin staining. The blood samples from 17 patients with papillary carcinoma and six from follicular carcinoma were also taken for HMGI(Y) gene transcript detection. The control group for these patients constituted 15 blood samples taken from healthy donors. RNA extraction RNA from tissue and whole-blood samples was isolated by use of the Total RNA Prep Plus Minicolumn Kit (A&A Biotechnology, Poland) based on an RNA isolation method developed earlier [19]. The isolated RNA has an A260/280 ratio of 1.6–1.8. Reverse transcriptase-polymerase chain reaction cDNA was obtained by use of the RevertAid cDNA Synthesis Kit (Fermentas, Lithuania). The reaction mixture, containing total RNA (3 mg), 1 ml oligo(dT) 18 primer (0.5 mg/ml), and 6 ml deionized, nuclease-free water, was prepared. After being mixed and spun down, the mixture was incubated at 70C for 5 min, then chilled on ice. After incubation the following components were added: 4 ml 5 reaction buffer, 1 ml ribonuclease inhibitor (20 U/ml), 2 ml 10 mmol/l dNTP mix. This mixture was incubated at 37C for 5 min. After incubation 1 ml RevertAid M-MulV reverse transcriptase (200 U/ml) was added. Finally, the mixture was incubated at 42C for 60 min. The reaction was stopped by being heated at 70C for 10 min. Polymerase chain reaction cDNA was amplified in 35 cycles, under the cycles parameters: denaturation (94C; 60 s), annealing (53C; 60 s), extension (72C; 30 s). The polymerase chain reaction (PCR) mixture contained 1.2 ml of 1.5 mmol/l MgCl2, 0.4 ml of 10 mmol/l mixed dNTPs, 0.2 ml of 0.5 U Taq polymerase, 2 ml of 10 reaction buffer and 0.7 ml of 0.5 mmol/l of each primer. Sets of primers for HMGI(Y) were planned on the basis of the literature [13]. As a control, bactin gene was amplified [20]. The PCR products were separated by electrophoresis on 2% agarose gel. Statistics Statistical analysis was carried out with the chi-square test, chisquare test with Yates correction, and the V2 test.
Results The presence of HMGI(Y) gene expression in the tissue of 60 thyroid neoplasms was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). In all 11 tumors classified as papillary thyroid carcinoma (T1N0M0: eight cases; T2N0M0: three cases) no HMGI(Y) gene expression was detected. In 31 out of 37 tissue samples classified as carcinoma folliculare, HMGI(Y) gene product (93 bp) was observed, whereas in all 12 analyzed tissues samples classified as adenoma folliculare, its expression was not detectable. None of 17 control non-neoplastic thyroid tissues showed the presence of HMGI(Y) gene
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Table 1 Expression of HMGI(Y) gene in thyroid tumors and normal thyroid tissue (number of cases) Histological type
Papillary thyroid cancer Follicular thyroid cancer Follicular adenoma Control
HMGI(Y) Positive
Negative
0 31 0 0
11 6 12 17
Table 2 HMGI(Y) gene expression in follicular carcinomas according to the UICC tumor, lymph node metastases, distant metastases (TNM) classification (number of cases), 5th edition HMGI(Y) Positive Depth of tumor invasion T1 6 T2 18 T3 5 T4 1 Lymph node invasion N-negative 13 N-positive 18 Metastases M0 26 M1 5
Negative
Percentage of positive cases
2 3 1 1
75 86 83 50
5 1
72 95
5 1
84 83
Table 3 HMGI(Y) gene expression in peripheral blood of papillary carcinoma patients and follicular carcinoma patients Number of cases
Fig. 1 Example of RT-PCR analysis of the HMGI(Y) gene expression (98 bp) in a thyroid tumors: carcinoma papillare 2–5, carcinoma folliculare 7, 8 and 10–15, adenoma folliculare 6 and 9, control 16; b peripheral blood of follicular carcinoma patients: 2–5, 7 positive and 6 negative HMGI(Y) cases; c all analyzed cases were b-actin positive 1–7. Lanes a-1, b-1 and c-8 are molecular size markers
transcript (Table 1). There was statistical dependence between HMGI(Y) gene expression and histological types of thyroid carcinomas. Presence of its expression was observed only in carcinoma folliculare (P=0.0001). As a reference gene for RT-PCR analysis b-actine was used (Fig. 1). More detailed analysis of follicular thyroid carcinoma showed the presence of HMGI(Y) gene transcript intrathyroideally (T1, T2 and T3 categories, 1997 UICC classification). The values reached approximately 76% and 83%, respectively. Unfortunately, there were only two cases classified as extrathyroideal (T4) and one of them was positive, the other was negative. No statistical dependence was observed between HMGI(Y) gene expression and stage of disease (Table 2). In the group of 13 out of 18 patients without metastases to lymph nodes (N0) the presence of HMGI(Y) gene transcript was observed. All 19 patients except one who were classified as N positive, showed HMGI(Y) gene
Clinical stage
Papillary carcinoma patients 4 I (T1N0M0) 7 II (T2N0M0) 3 III (T2N1M0) 2 III (T3N1M0) 1 IV (T4N1M1) Follicular carcinoma patients 3 II (T2N0M0) 1 III (T2N1M0) 1 IV (T3N1M1) 1 IV (T4N1M1)
HMGI(Y) Positive
Negative
0 0 1 0 0
4 7 2 2 1
3 1 0 1
0 0 1 0
expression (Table 2). There was no statistical dependence between HMGI(Y) gene expression and lymph node invasion. The same group of patients was divided according to the presence and absence of distant metastases. Both groups showed high and very similar percentages of HMGI(Y) gene expression: 84 for M0 and 83 for M1 cases (Table 2). No statistical dependence was observed between HMGI(Y) gene expression and distant metastases. The HMGI(Y) gene expression was also checked in 23 peripheral blood samples. There were 17 samples obtained from patients who had been diagnosed with papillary carcinoma, and 16 of the samples were HMGI(Y) negative (Table 3). In the five out of six blood samples (83%) taken from patients who had been diagnosed with follicular carcinoma HMGI(Y) gene expression was detected (Table 3). No HMGI(Y) gene expression was noticed in the blood samples taken from healthy donors.
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Discussion If unnecessary surgery and complications after thyroidectomy are to be avoided, it is important that we search for new diagnostic and prognostic features of thyroid carcinoma. It is possible for cytological diagnosis to be established in FNAB of papillary, medullary and anaplastic thyroid carcinoma. However, follicular neoplasms are difficult to be distinguished, not only from FNABs but also from histological examination. Diagnosis by FNAB is extremely difficult, due to scanty cellular, not histological, material. One must remember that a negative result from a FNAB does not exclude the presence of malignant foci in the thyroid lobes, if one takes into account that follicular carcinoma can manifest with more than one nodule. The large majority of follicular thyroid neoplasms are adenomas; some of them are difficult to be classified as benign or malignant tumors. Although in most cases surgical resection is sufficient to protect the patient from recurrence of goiter, there is always a risk of misdiagnosis and too radical or too scanty a resection of thyroid tissue. This is a problem for both pathologists and surgeons, and affects the results of surgical procedures by inducing numerous complications. In almost all cases the diagnosis of follicular thyroid carcinoma is made after the operation. However, the diagnosis should always be verified and confirmed by assessment of selected proteins with the use of immunohistochemistry or DNA ploidy [21]. Most of them reveal some suspicious features common to malignant transformation. There is, however, no single reliable marker that differentiates benign thyroid follicular tumors from malignant ones. Recently, some authors have indicated the usefulness of HMGI(Y) and HMGI(C) gene expression analysis in various carcinomas, including those in the thyroid gland. They proved that HMGI(Y) gene expression is connected with malignant transformation [3, 8–13, 22, 23]. In the present study we tried to establish the possible usefulness for HMGI(Y) gene expression as a preoperative and peri-operative marker for better diagnosis of thyroid carcinoma. This study focuses on follicular tumors of the thyroid gland and papillary thyroid carcinomas. We decided to use RT-PCR for direct detection of HMGI(Y) expression in postoperative tissues as well as in peripheral blood taken from the patients before they underwent their surgical procedures. We have noticed that the presence of HMGI(Y) gene expression in postoperative histopathological diagnosis is connected with follicular carcinomas, but not with papillary carcinomas or follicular adenomas. Chiapetta et al. [13] found HMGI(Y) positivity in tissues as high as 100% by RT-PCR and 96% with the use of immunohistochemistry in papillary carcinomas. We did not achieve such results by studying tissue, although we observed one positive case of papillary carcinoma where gene transcript was detected. Detailed information about
morphology of papillary carcinoma subtypes is required if the results are to be discussed. Histopathological variants of papillary carcinoma analyzed by Chiapetta may probably differ from ours. We did not observe positivity in follicular benign lesions either. We assumed that it would be useful to classify histopathological subtypes of papillary carcinoma for better precision of analysis of our material according to current criteria. In accord with our knowledge, in this study we have shown for the first time the expression of HMGI(Y) gene in the peripheral blood of patients with follicular carcinoma. This is a preliminary study and should be confirmed in larger-scale research. Estimation of HMGI(Y) gene transcript in the peripheral blood samples seems to be a more effective and less time consuming procedure for the patients’ diagnoses. Currently, we are conducting further investigations to explain the diagnostic role of the determination of HMGI(Y) gene expression in FNAB material and peripheral blood. There are only limited data describing the analysis of HMGI(Y) gene expression in peripheral blood of patients with solid tumors. Sezer et al. [7], using a semi-nested RTPCR, investigated HMGI-C gene expression in the peripheral blood of breast-cancer patients. They noticed that no HMGI-C could be detected in any of the healthy donors’ samples, but in the peripheral blood of patients with breast cancer this expression was correlated with a poor prognosis. We believe that the analysis of HMGI(Y) gene expression may have an influence on the preoperative diagnostic algorithm and allow better planning of the surgical strategy. There is still no good screening parameter that could be helpful in widely used diagnostic procedures before surgical treatment. Recently, the term residual neoplasm disease has been introduced for patients after radical excision of a malignant tumor. This means that a number of malignant cells are still present in the body and are to be detected by some methods. We suggest that assessment of HMGI(Y) in FNAB and peripheral blood specimens before and after operation can be helpful in establishing diagnosis in follicular thyroid tumors.
Conclusion The conclusion of our study is that estimation of HMGI(Y) gene expression in tissue and blood samples can be helpful in the diagnosis of follicular thyroid carcinoma. Acknowledgments The work was supported by grant 502-13-844 from the Medical University of Lodz, Poland. We wish to thank Prof. Ryszard Wierzbicki for his critical reading of the text.
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