ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 1O, No. 1, 41-48, 1996 

The role of 201Tl scintigraphy in evaluating proliferative activity in thyroid neoplasms 

Kunihiro NAKADA, Chietsugu KATOH, Kakuko KANEGAE, Eriko TSUKAMOTO, Kazuo ITOH, Masayori FURUDATE and Nagara TAMAKI 

Department of Nuclear Medicine, Hokkaido University School of Medicine 

To identify the relationship between the uptake of 201Tl and the proliferative activity in thyroid neoplasms, 201Tl scintigraphy was performed in 57 patients with thyroid neoplasms. 201Tl uptake ratio was calculated in both the early and the delayed images and then compared with factors representing cellular or practical proliferative activity of the lesions. The labeling index (LI) for proliferating cell nuclear antigen (PCNA) was determined quantitatively by flow cytometry. There was a significant correlation between the uptake ratio and LI for PCNA. The correlation coefficient for the delayed ratio (DR) vs. LI was better than that for the early ratio (ER) vs. LI. As parameters for practical proliferation, the surgical stage in primary thyroid carcinoma or 131 I uptake in recurrent thyroid carcinoma was focused on. DR was strongly related to these parameters, regardless of the histopathological features or size of the lesions. Our results suggest that 201Tl uptake in delayed thyroid scan is useful in assessing proliferative activity in thyroid neoplasms. 

Key words: cell cycle, proliferating cell nuclear antigen, flow cytometry, 201Tl scintigraphy, thyroid neoplasm 

INTRODUCTION 

201Tl SCINTIGRAPHY has been used in diagnosing thyroid malignancies.1-8 The high radioactivity of 201Tl in the delayed scan seems to be a sign of malignant nodules,2-4,7 although positive uptake and retention of 201Tl have been described in benign nodules or chronic thyroiditis.3,6,8 Although some authors have insisted that the sensitivity of 201Tl scintigraphy is low,5,6 these results also suggest that 201Tl uptake is influenced by some factors which are independent of histopathological characteristics. Further-more, 201Tl uptake has been reported to indicate the biological or histological aggressiveness in brain tu-mor.9-13 Based on the above reports, we supposed that 201Tl scintigraphy has similar predictive value in assessing proliferative activity in thyroid tumor. Prospective studies were undertaken to identify the relationship between the 201Tl uptake and the proliferative activity in thyroid neoplasms. 

MATERIALS AND METHODS 

Patients 
For a period of 30 months between August, 1992 and January, 1995, 201Tl scintigraphy was performed on a total of 57 patients suspected of thyroid tumor. There were 44 females and 13 males with ages ranging from 17 to 84 years (mean 57.0 +- 14.1). All patients satisfied the following criteria: a) Ultrasonogram (US) measurement of the longest diameter of the tumor was > 2.0 cm long and <= 5.0 cm for the primary tumor, and > 1.5 cm long and <= 5.0 cm for the recurrent tumor; b) Nodules were not associated with dominant cystic changes or massive calcification. US-guided needle biopsy was done within 3 weeks after 201Tl scintigraphy. Thirty-five patients (group M) had malignant tumors, and 22 patients (group B) had benign neoplasms. Histopathological diagnosis is listed in Tables 1 and 2. All carcinomas were well-differentiated in the epidermal grade. Chronic thyroiditis was classified as a benign nodule. Nineteen patients with primary thyroid cancer underwent surgery within 2 months after the study. Pathological determination of the t and n factor was done according to General Rules for the Description of Thyroid Cancer.14 Five patients were classified as t4 because of positive extrathyroidal involvement (ETI), and the rest were classified into t2. Ten patients had positive lymph node (L/N) metastases. Twelve patients had recurrent thyroid cancer in the neck, the mediastinum, or the sternum. They received 131-I therapy within 2 months after the study. Whole body 131-I scintigram was obtained 4 to 7 days after the administration of the therapeutic dose (3.7 GBq-5.55 GBq). 131-I uptake in tumor was good in 5 patients, and little or nil in the others. 

201-Tl scintigraphy 
Static anterior images of the thyroid were obtained at 10 min (early scan) and 120 min (delayed scan) after intravenous administration of 3.7 MBq of 201-Tl with a gamma camera (Searle, USA) equipped with low-energy-all-purpose parallel-hole collimator. Data were stored in an on-line computer Scintipac 1200 (Shimadzu, Japan) for 10 minutes in a 64 x 64 matrix. The region of interest (ROI) 3 x 3 pixel in size was drawn both for the nodular lesion and background regions (BG) in the contralateral cervical area of the lesion. The nodular lesion to BG ratio (early ratio; ER, delayed ratio; DR) was calculated from mean counts for each ROI. When nodularity was uncertain on the scintigram, the nodular ROI was set up according to US findings. 

Flow cytometric analysis 
The labeling index (LI) of proliferating cell nuclear antigen (PCNA) was determined as described by Landberg et al.(15,16) with minor modifications. The specimen was di-vided into 2 samples. One was incubated with mouse fluoroscein isothiocyanate (FITC)-labeled PCNA antiserum (PC-10; DAKO Corporation, Glostrap, Denmark) before propidium iodide (PI) staining. The other was prepared as a negative control in incubation with mouse FITC-labeled IgG (Coulter Corporation, Hialeah, Fin-land). LI for PCNA in each specimen was calculated by subtracting the LI in negative control cells from that in PCNA-FITC-labeled cells. Flow cytometer FACS-CAN (Becton-Dickinson, USA) and its application software were used for the analysis. 

Comparative study 
 A) Relationship between 201-Tl uptake ratio and cellular proliferative activity: The correlation between ER vs. LI for PCNA and between DR vs. LI for PCNA was determined in group M and group B. 
 B) Relationship between 20]TI uptake ratio and practical proliferative activity in thyroid cancer: In patients with primary cancer, DR was compared with surgically confirmed t and n factor. In patients with recurrent cancer. DR was compared with 131-I uptake on the scintigram. In the text, figures are the means +-s.d. Student's t-test was employed for between-group comparison. The difference was regarded as significant when the p-value was less than 0.05. 

RESULTS 

201Tl uptake 
ER ranged from 1.16 to 3.67 (mean 2.24 +- 0.56). DR ranged from 1.10 to 3.51 (mean 1.78 +- 0.51). ER and DR in group M were 2.19 +- 0.53 and 1.87 +- 0.51, respec-tively. In group B, ER and DR were 2.27 +- 0.53 and 1.58 +- 0.34, respectively. There was no statistical difference between the two groups in ER, but DR was significantly higher in group M than that in group B (Fig. 1 ). 
Flow cytometric analysis 
LI for PCNA (%) was successfully determined in all cases, and ranged from 0.5 to 51.2% (mean 14.3 +- 12.1 ). LI was 17.6 +- 13.4% in group M, and 10.3 +- 6.1% in group B. Group M had significantly higher LI than group B (Fig. 2). 
Comparative study 
A) Relationship between uptake of 20]TI and cellular proliferative activity: The correlation coefficients for ER vs. LI in group M and group B were 0.576 and 0.650, respectively (Fig. 3A, 3C). The correlation coefficients for DR vs. LI in group M and group B were 0.817 and 0.838, respective]y (Fig. 3B, 3D). The correlation between 201-Tl uptake and LI for PCNA was significant in the two groups.
 B) Relationship between uptake of 201-Tl and practical proliferative activity in thyroid cancer: DR was used for this purpose because of better correlation with PCNA. In primary thyroid cancer, DR was 2.14 +- 0.53 in lesions with L/N metastases, and was 1.41 +- O.18 in those without L/N metastases (Fig. 4A). DR was 2.28 +- 0.62 in lesions with positive ETI and 1.62 +- 0.39 in those with negative ETI (Fig. 4B). DR was significantly higher in lesions which were accompanied by L/N metastases or ETI. Representative cases are presented in Figure 5. In recurrent carcinoma, DR was 2.34 +- 0.32 in patients with no or poor 131I uptake, and was 1.68 +- 0.36 in those with good uptake, respectively (Fig. 6). DR was noticeably higher in tumors which showed no or poor 131I uptake than in those with good 131I uptake. Representative cases are shown in Figure 7. 

DISCUSSION 

The assessment of proliferating activity in thyroid tumor is difficult by morphological methods, such as micros-copy, US, or CT scan. The possibility of 201Tl scintigraphy as a representative for 18F-FDG PET has been recently suggested for brain tumor.10-14 Tumor uptake of 201Tl is regulated by various factors,17-20 such as vascularity, activity of the Na+-K+-ATPase, Tl+-Na+-2Cl co-transport system, or development of cellular mitochondria. These factors are not direct predictors of proliferative activity and there has been no study on the relationship between 201Tl uptake and proliferative factors in thyroid tumors. We focused on PCNA as a predictor of proliferative activity. PCNA is a nuclear protein which is produced in the late G1 and S phases in the cell cycle and acts as a co-protein of DNA polymerase D.21,22 Like bromodeoxyuridine (BUdR), PCNA is regarded as a parameter for the cells in the S phase. One of the advantages of PCNA over BUdR is that there is no need of pre-administration at tissue sampling. PCNA is detectable by immunohistochemical staining,23-26 but the results have been strongly influenced by the conditions in fixation of the materials.24-26 The FCM technique is expected to make the results more reliable and objective with less artifacts. Biopsy specimens were fragmental but heterogeneity within the specimens may be less than with surgical ones. In our study, DR or LI for PCNA was higher in malignant tumors than in benign nodules. More importantly, there was a significant correlation between 201Tl uptake and LI for PCNA. This finding suggests that thyroid tumors which concentrate or retain more 201Tl include more cells in the S phase. The reason for the better correlation coefficient for DR vs. LI than for ER vs. LI is not clear. ER may be related to blood perfusion, but DR may represent a cellular mechanism to retain 201Tl longer. The results may indicate that the vascularity of the tumor and the number of proliferating cells within the tumor do not always correlate. DR is a better representative in predicting cellular proliferative activity, and a thyroid tumor with high DR has the potential for rapid growth regardless of its histopathological classification. This may support the suggestion by Harada et al.8 that the 201Tl scan is useful in determining the surgical indications for thyroid tumor rather than to differ-entiate between benign and malignant tumors, and that a case showing 201Tl accumulation on the scan has the possibility of further growth. The objective in the comparative study was to identify the value of 201Tl scanning in characterizing thyroid car-cinomas from the point of view of tumor proliferation. The surgical stage is a practical expression of the prolif-erative activity of thyroid carcinoma. Above all, the presence of ETI is one of the prognostic factors in thyroid carcinoma.27-29 In this study, DR was significantly higher when ETI or L/N metastases were observed. In contrast, DR in thyroid cancer without ETI or L/N metastases did not differ from that in benign neoplasm. Moreover, there is positive correlation between 201Tl uptake and PCNA in benign lesions as well as malignant tumors. These facts may help to resolve the controversy over the sensitivity and specificity of 201Tl scintigraphy.2-7 The uptake of radioiodine is closely related to the prognosis of patients with recurrent thyroid carcinoma.30 Some of the iodine-negative recurrent thyroid cancers rapidly grow in spite of thyroid hormone replacement. Our results show that although DR is higher in recurrent cancer with no or poor 131I uptake, it remains the same in the benign nodule in tumors with good 131I uptake. 201Tl scintigraphy has been reported to be useful in localizing metastatic tumors that do not concentrate 131I.31,32 Our results may be interpreted as indicating that DR demonstrates variations in the tendency for the tumor to concentrate 131I. Recurrent tumors with higher DR have more autonomously prolif-erating cells which may be independent of TSH regula-tion, resulting in less 131I uptake on the scintigram. 
 Differences among our subjects in the histological grade were small. Eighteen out of 19 primary lesions and all the recurrent lesions had "well differentiated" epidermology. The gratest diameter in lesions were some-how arranged to reduce the partial volume effect on the uptake ratio. Since DR indicates the potentiality of thy-roid cancer in invasive growth, in metastasizing, or in its resistance to radioiodine therapy, it provides important information for the management of thyroid cancer which cannot be assessed by other morphological studies. DR may also be helpful in determining indications for thyroid hormone administration in chronic thyroiditis or for surgery in benign nodules. In conclusion, this study strongly suggests that the delayed 201-Tl scan is useful in predicting proliferative activity in thyroid neoplasms. 

ACKNOWLEDGMENT 

We thank Mr. Hiroshi Matsumoto for his great assistance in FCM analysis. Part of this study was supported by a Grant-in-Aid form for the Cancer Research Fund from the Ministry of Education of Japan (Group leader: Mr. Akira Yokoyama). 

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