Detection of lymph node metastases of papillary thyroid cancer ...

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Abstract Background: The range of lymphadenectomy in differentiated thyroid cancer remains still a matter of controversy because of the lack of.
Langenbecks Arch Surg (2005) 390: 209–215 DOI 10.1007/s00423-004-0528-1

L. Pomorski Krzysztof Kaczka S. Piaskowski I. Wójcik P. Rieske M. Matejkowska K. Kuzdak

Received: 16 June 2004 Accepted: 31 August 2004 Published online: 8 January 2005 # Springer-Verlag 2005 L. Pomorski . K. Kaczka (*) . K. Kuzdak Department of Endocrinological and General Surgery, Medical University of Lodz, Pabianicka 62, 93-513 Lodz, Poland e-mail: [email protected] Tel.: +48-42-6895171 Fax: +48-42-6895172 S. Piaskowski . I. Wójcik . P. Rieske Department of Molecular Pathology and Neuropathology, Medical University, Lodz, Poland M. Matejkowska Department of Pathomorphology, The Memorial Copernicus Hospital, Lodz, Poland

ORIGINA L ARTI CLE

Detection of lymph node metastases of papillary thyroid cancer—comparison of the results of histopathology, immunohistochemistry and reverse transcription-polymerase chain reaction—a preliminary report

Abstract Background: The range of lymphadenectomy in differentiated thyroid cancer remains still a matter of controversy because of the lack of reliable diagnostic methods for nodal metastases, other than histopathology. Aim: To compare the results of detection of lymph node metastases of papillary thyroid cancer by conventional histopathology and immunohistochemistry with the results of reverse transcription–polymerase chain reaction for thyroglobulin mRNA. Patients and methods: Each of 166 cervical lymph nodes obtained from 21 patients was divided into two halves: one was used for conventional histopathology and immunohistochemistry, the other part was investigated by molecular examination. Results: We obtained different results from examination of the lymph nodes in six (28.6%) patients. In four pa-

Introduction Surgery is the treatment of choice for differentiated thyroid cancer. Total thyroidectomy is the most typical operation. Only in the case of papillary microcarcinoma is there a possibility to do a less extensive operation [1, 2]. In some American centres, when the diagnosis is low-risk differentiated thyroid cancer, a less radical operation is performed, based on prognostic scales such as AGES, AMES, DAMES [3–5]. Extent of lymph node resection still remains a matter of controversy. There are two main kinds of lymphadenectomy in cases of differentiated thyroid cancer. Currently, radical neck dissection (Crile’s operation) is not performed because it does not improve prognosis and

tients (19.1%) reverse transcription– polymerase chain reaction (RT-PCR) was more sensitive in detection of positive lymph nodes; in two patients (9.5%) it revealed fewer metastasised lymph nodes than did histopathology. The rest of the patients did not have any differences: 12 (57.1%) of them had negative lymph nodes and three (14.3%) had positive lymph nodes in all examinations. Conclusions: (1) Thyroglobulin (Tg) RT-PCR is an appropriate method of detection for thyroid cancer cells. (2) In combination with histopathology, it might help to qualify patients’ nodal status better. Keywords Papillary thyroid cancer . Lymph node metastases . Histopathology . Immunohistochemistry . Reverse transcriptase–polymerase chain reaction

decreases quality of life [6]. Surgical options include a limited “berry picking” operation or more aggressive modified radical neck dissection. During the first kind of operation only single, clinically or intraoperatively suspected lymph nodes are removed. In some publications it has been reported that treatment of lymph node metastases does not affect survival, so “berry picking” is operation enough [7–9]. Some surgeons suppose that many metastatic lymph nodes are clinically occult or could be destroyed during postoperative radioiodine therapy [10]. In cases of nodal relapse there is always the possibility to remove them [11, 12]. Other authors suggest more aggressive treatment. They notice that many patients with differentiated thyroid cancer

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have involved lymph nodes: in the ipsilateral cervicocentral compartment (42%–86%), in the ipsilateral cervicolateral compartment (32%–68%), in the contralateral cervicolateral compartment (12%–24%) and in the mediastinal compartment (3%–20%) [13]. Most examinations from recent years have shown a prognostic impact of lymph node metastases [13–20]. Based on the above statement, to reach oncological radicality, a more radical operation is indicated. There are also other factors that suggest the performing of modified radical neck dissection. Recurrence—the worst prognostic factor—appears the most frequently in the lymph nodes [14, 21–23]. The nodal metastases often present no iodine uptake and they can be treated only by surgery [10–12, 24, 25]. Some surgeons obtain better results in treatment of distant metastases after dissection of the regional lymph nodes [10]. There is also the possibility of formation of further metastases from the lymph node metastases [23]. We suppose that the extent of lymph node dissection is controversial because of the lack of reliable diagnostic methods for nodal metastases, other than histopathology. Nowadays, the detection of disseminated tumour cells is one of the main topics in current oncology. Immunohistochemistry and polymerase chain reaction are new tools for finding a small number of neoplasmatic cells in the lymph nodes and peripheral blood. The results of these studies may help to develop more sensitive methods for the early detection of cell dissemination and may refine risk groups that might benefit from more extensive surgical procedures or adjuvant therapy.

Aim In the study our intention was to detect well-differentiated thyroid carcinoma cells in the cervical lymph nodes, by histopathology, immunohistology, and reverse transcription–polymerase chain reaction (RT-PCR), and compare the results obtained. In immunohistological and molecular examination the same marker—thyroglobulin—was researched. In this way mistakes with regard to different expressions of various markers are avoided.

Patients and methods Informed permission was acquired from 21 patients (five men, 16 women) with papillary thyroid cancer, which had been recognised preoperatively on the base of FNAB or intraoperatively according to histopathological examination. None of the patients had recognised toxic goitre and had been treated with radioiodine before the operation. The protocol study was approved by the Ethics Committee of the Medical University of Lodz, Poland. During the operation, total thyroidectomy with central lymph node dissection (level VI according to the WHO classification and

Robbins, level I according to Dralle and Gimm) and unilateral or bilateral modified, radical neck dissection (level II–V according to the WHO classification and Robbins, level II–III according to Dralle and Gimm) was performed [13, 26]. During the operation the suspected or enlarged lymph nodes from the mediastinal compartment (upper mediastinum) were dissected by cervical approach (without sternotomy). It was carried out in three of our patients (denoted as 1.SJ, 12.RK, 14.KZ in Table 1) who had the tumour located in the lower part of the thyroid gland. Type of operation conformed with the guidelines of the Polish Society of Surgeons and the Polish Society of Oncological Surgery [27]. Immediately, the removed lymph node samples were carried to the Department of Pathology and divided into two pieces. One half of each lymph node was used for conventional histopathology and immunohistochemistry; the other was snap-frozen in liquid nitrogen, stored at −70°C and used for RT-PCR. To avoid contamination, the lymph nodes were cut free from other tissue and a fresh scalpel was used each time. Histological and immunohistochemical examination The lymph nodes were fixed in formalin, embedded in paraffin and sectioned for histological studies. The sections were stained with haematoxylin and eosin and obTable 1 Results of the detection of tumour cells in lymph nodes by conventional histopathology/immunohistochemistry (CH/I) and thyroglobulin (Tg) RT-PCR (M male, F female) Patient Age/gender Number of lymph nodes CH/I 1.SJ 2.BS 3.MD 4.AJ 5.KW 6.AS 7.MS 8.MM 9.SS 10.KF 11.CZ 12.RK 13.AA 14.KZ 15.EM 16.SM 17.KK 18.EB 19.WS 20.KJ 21.SW Total

35/M 31/F 52/F 51/F 61/M 52/F 57/F 35/F 45/M 54/F 37/F 58/F 60/F 44/M 57/M 43/F 47/F 19/F 55/F 48/F 54/F –

15 3 3 4 14 7 6 11 5 8 4 12 10 12 10 12 7 9 5 4 5 166

0/15 0/3 0/3 0/4 5/14 0/7 0/6 5/11 0/5 1/8 0/4 0/12 3/10 0/12 5/10 0/12 0/7 3/9 0/5 0/4 3/5 25/166

Tg RT-PCR 0/15 0/3 0/3 0/4 5/14 0/7 0/6 5/11 0/5 2/8 0/4 0/12 3/10 1/12 3/10 5/12 0/7 5/9 0/5 0/4 2/5 31/166

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served under a light microscope. Immunohistochemical staining for Tg was performed on formalin-fixed, paraffinembedded sections with anti-Tg antibodies (Dako, Germany). Presence of Tg was visualised with alkaline phosphatase. Total RNA isolation and reverse transcriptase– polymerase chain reaction

AGAGGGAAACGGCC-3′; TG reverse 5′-TGTGTGGAC GCAGAGGGGATGGAGGTGTAT-3′ and β-actin forward 5′-GTGGGGCGCCCCAGGCACCA-3′; β-actin reverse 5′-CTCCTTAATGTCACGCACGATTTC-3′. After denaturation for 3 min at 94°C the total amount of reaction products was amplified for 35 cycles for TG (94°C, 30 s; 56°C, 30 s; 72°C, 60 s) and 35 cycles for β-actin (94°C, 30 s; 58°C, 30 s; 72°C, 30 s) on the Pelitier Thermal Cycler system (PTC-200, MJ Research). The PCR products were resolved on a 2% agarose gel and visualised by ethidium bromide staining on a UV table. In each experiment water was used as a negative control and sample with presence of Tg RNA as a positive control. The results of molecular studies were presented after computer image analysis by the system Gel Doc1000 Bio Rad Image with the program Molecular Analyst applied.

Total cellular RNA was isolated from frozen tissue samples with a commercial kit (A&A Biotechnology, Poland) according to the manufacturer’s protocol. Approximately 1–2 μg of total RNA was reverse transcribed into singlestrand cDNA in a final volume of 20 μl containing 4 μl 5×buffer (250 mmol Tris–HCl pH 8.3, 375 mmol KCl, 15 mmol MgCl2, 50 mmol DTT), 1.5 μg oligo(dT), 0.5 mmol dNTP, 20 U RNase inhibitor (Promega, Madison, Wis., USA) and 200 U M-MLV reverse transcriptase (Promega). First-strand cDNA synthesis reaction was performed at 37°C for 90 min. The following PCR reactions were performed in a final volume of 20 μl containing 4 μl of the RT solution, 5 μl 10×reaction buffer (10 mmol Tris–HCl pH 9.0, 50 mmol KCl), 0.5 μmol of each primer, 0.2 μmol of each dNTP, 1.5 mmol MgCl2 and 0.5 U Taq-DNA polymerase (Promega). A 348-bp fragment of transcript of TG gene and a 548-bp fragment of transcript of β-actin gene were amplified in separate PCR reactions using the following primers (Fig. 1): TG forward 5′-TGTGAGCTGC

Results

Fig. 1 RT-PCR analysis of TG transcript in representative samples of a patient with thyroid cancer. a β-actin mRNA expression. b TG mRNA expression. Lanes 1–10 samples of a patient with thyroid cancer (13 AA); lane 11 positive control (RNA from a patient with

papillary thyroid cancer confirmed by pathology); lane 12 negative control (water instead of RNA); lane 13 molecular weight marker (100 bp ladder). Presence of mRNA TG was detected in lanes 1, 2 and 3 (b)

Twenty-one patients cured at the Department of Endocrinological and General Surgery, the Medical University of Lodz, Poland (16 female, five male, aged 19–61 years, mean 47.4 years), were included. All the patients had histologically recognised papillary thyroid cancer. In our prospective study, we analysed 166 lymph nodes (from three to 15 per patient, mean 7.9). The size of the lymph nodes varied from 0.3 cm to 2.6 cm. A 100% correspon-

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dence was observed between conventional histopathology and immunohistochemistry, so we analysed these results as one group (pathological examination). In our study we obtained different results from the detection of metastasised lymph node in six (28.6%) patients. The remaining ones did not have any differences: 12 (57.1%) of them had all negative lymph nodes and three (14.3%) had positive, the same lymph nodes in all examinations. Of the 166 lymph nodes, 25 (15.1%) were positive by pathological examination; they were found in seven patients (33.3%) (Table 1). The pathologically positive lymph nodes were located in the cervicocentral compartment—10 (40%)—and in the cervicolateral compartment —15 (60%) (Table 2). There were no metastasised nodes in the mediastinal compartment. RT-PCR revealed Tg amplification products in 31 (18.7%) lymph nodes of nine patients (42.9%). Eleven (35.5%) of them were situated in the cervicocentral compartment, 19 (61.3%) in the cervicolateral compartment and one (3.2%) in the mediastinal compartment (Table 3). Among nine Tg RT-PCR positive patients three had the same results in the pathological and molecular examinations (patients denoted in Table 1 as 5. KW, 8.MM, 11.AA). According to RT-PCR, patient 16. SM had one positive lymph node in the cervicocentral compartment and four in the cervicolateral compartment. The pathological examination detected no metastasised lymph nodes in that patient. The molecular examination found two more positive lymph nodes in the cervicolateral compartment of patient 18.EB and one more in the same compartment of two patients (10.KF and 14.KZ). In two of our patients (15.EM and 21.SW) the molecular examination was less sensitive than histopathology was. By histopathology, patient 15. EM had five metastasised lymph nodes and patient 21.SW had three. Tg mRNA was revealed in three lymph nodes of patient 15.EM and in two lymph nodes of patient 21.SW. We observed an interesting phenomenon in patients 14.KZ and 10.KF. Our pathologists described, in one lymph node from each of those patients, the presence of thyroglobulin in macrophages, but they could not find neoplasmatic cells

Table 2 Localisation of histopathology/immunohistochemistry positive lymph nodes Patient

Compartment Cervicocentral

5.KW 8.MM 10.KF 13.AA 15.EM 18.EB 21.SW

4/10 5/11 1/1 0 0 0 0

Cervicolateral 1/4 0 0 3/10 5/10 3/9 3/5

Mediastinal 0 0 0 0 0 0 0

Table 3 Localisation of Tg RT-PCR positive lymph nodes Patient

Compartment Cervicocentral

5.KW 8.MM 10.KF 13.AA 14.KZ 15.EM 16.SM 18.EB 21.SW

4/10 5/11 1/1 0 0 0 1/2 0 0

Cervicolateral 1/4 0 1/7 3/10 0 3/10 4/9 5/9 2/5

Mediastinal 0 0 0 0 1/9 0 0 0 0

in these lymph nodes. The molecular examination showed a Tg mRNA band from these lymph nodes. We treated that as false positive results of the molecular examination. In another patient, 11.CZ, the pathologist also found thyroglobulin in macrophages in two nodes, but Tg RT-PCR was negative. In 12 other patients both the histopathology and the molecular examination did not find any metastases in the lymph nodes.

Discussion Recurrence (locoregional relapse or distant metastases) is the worst prognostic factor in thyroid cancer [14, 21–23]. It occurs even if the conventional histopathological examination after the initial surgery did not reveal lymph node involvement [28]. An explanation for this phenomenon might be micrometastases in the cervical lymph nodes, which were not detected by the conventional histopathology. It is supposed that lymphogenic dissemination of cancer cells is probably more important than haematogenic spread, especially in papillary thyroid cancer. The routine histopathological examination has limited sensitivity because only one or two sections of each lymph node are investigated. In addition, the detection of small clusters or single cancer cells is difficult for pathologists. The definition of sentinel lymph node in the case of thyroid cancer is not as precise as in breast cancer or malignant melanoma. Studies from recent years have shown no precise, predictable pattern of spread of lymph nodes metastases [29–32]. This fact also encourages us to look for a new methods of lymph node assessment. Burt and Goudie introduced the new technique—immunohistochemical examination—of detecting thyroid cells [33]. Thyroglobulin was found by the immunoperoxidase technique in routine histological sections in all recognised cases of well-differentiated thyroid cancer, in only one case of 20 anaplastic thyroid tumours and in none of other examined tumours. Later, Pisani et al. proved the value of the

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immunohistochemical examination in the detection of thyroid cancer cells in cervical lymph nodes [34, 35]. To increase the sensitivity and specificity of cancer cell detection in lymph nodes, various genetic methods were introduced. They have the advantage of a higher sensitivity and the possibility of researching not only single sections of a lymph node in a single examination. RT-PCR is a rapidly developing technique, which has been successfully applied to the investigation of micrometastases of many solid, malignant, tumours such as: gastric cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer [36–44]. In gastrointestinal cancers, detection of micrometastases by molecular methods, in histopathologically negative lymph nodes, was the most important factor indicating a possibility of local relapse and the need for more aggressive treatment [35–37]. Liefers et al., using CEA RT-PCR, divided 26 patients with stage II colorectal cancer into two groups. The 5year survival rate for patients with RT-PCR positive lymph nodes was 50%, compared to 91% in the group without metastases in the molecular examination [36]. In our study we researched detection of metastases by various methods in resected, cervical lymph nodes of patients with papillary thyroid cancer. For our investigation we chose, as a marker, thyroglobulin. It is one of the most specific thyroid antigens and normally presents only in thyroid cells and neoplasmatic cells derived from follicular cells. Others, besides thyroid peroxidase and iodine symporter, are not as specific for thyroid. For example, cytokeratin-20, used by Weber et al., is expressed in normal gastrointestinal epithelium, urothelium, Merkel cells and derived cancers [21, 37–40, 45]. In 15 (71.4%) of 21 patients we obtained the same results of lymph node investigation. Twelve of them had lymph nodes that were cancer-cell free both in histopathology and RT-PCR. In three patients we found 100% correspondence of metastasised lymph nodes. Our investigation revealed nine (5.4%) RT-PCR positive lymph nodes, although, according to the histopathology, they were classified as tumour free. Five of them were present in the patient who had no metastasised lymph nodes in the histopathological examination. One person had two more positive lymph nodes in the molecular examination. We think that two patients need more frequent follow-up so that the clinical value of these results can be estimated. Two patients had one more positive lymph nodes in RT-PCR. The histopathological examination revealed the presence of thyroglobulin in macrophages but not cancer cells in these lymph nodes. We treated RT-PCR results from these two nodes as false positive. According to us, thyroglobulin from macrophages gave a positive Tg signal. We suppose it is possible to obtain more PCR-positive, histopathologically negative lymph nodes by applying more cycles of PCR. In our study we did not do that because we

were afraid of illegitimate transcription and false positive results. In part, this phenomenon has been described by other investigators [46–48]. Bojunga et al. and Aust et al. proved that Tg mRNA expression, determined by RTPCR, is specific for thyroid cells when the normal assay sensitivity (30 cycles) is used but is not specific when the high assay sensitivity (40 cycles) is used [46, 47]. We received similar results from our material, which is why we did not perform the molecular examination with the high sensitivity. The reason for this high sensitivity of RTPCR with more cycles is unclear. In two patients RT-PCR failed to detect all histopathologically positive lymph nodes. Like Weber et al. we can think of only one explanation for these false negative results [21]. We may have made a mistake during the cutting of the lymph nodes—only one part of them could have been infiltrated by cancer cells and the other one, tumour-free, used for the molecular examination. We are fully aware that lymph nodes from only a small number of patients were analysed in this study, and further studies should be performed to support these results. The number of resected lymph nodes (average 7.9) was enough, according to the criteria of International Union Against Cancer [49]. According to them, during the operation, a minimum of six lymph nodes should be resected. From the technical point of view it is difficult to find, in fresh tissue samples and afterwards, precisely cut lymph nodes that are smaller than 3 mm. Other authors, performing similar studies, analysed even slightly lower numbers of resected lymph nodes [21]. As we mentioned above, the definition of sentinel lymph node in the case of thyroid cancer is not as useful as in breast cancer or malignant melanoma. Only in particular cases can the use of methylene blue or radioisotope help in finding a sentinel lymph node. Assuming that in 12% to 86% of cases some of the lymph nodes are involved in metastasis, we think that frozensection examination has limited value and does not change the surgeon’s operating tactic, including the performance of elective resection of regional lymph nodes. It can only suggest that a more extended lymphadenectomy is made also in the ipsilateral and contralateral compartments.

Conclusion Thyroglobulin mRNA RT-PCR might be applied for more accurate assessment of lymph node metastases (especially micrometastases) of papillary thyroid cancer. Acknowledgements This work was supported by grant 503-11016 Medical University, Łódź, Poland.

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