Radiol Oncol 1997; 31: 13-7. Relative DNA concentration in thyrocytes from scintigraphically hot nodi Natasa V. Budihna,1 Miran Zupanc,1 Ruda Zorc-Pleskovic,2 Miran Porenta,1 Olga Vraspir-Porenta2 1 Department of Nuclear Medicine University Medical Centre, Ljubljana, Slovenia, 2 Institute of Histology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia The aim of our study was to study the cytological appearance and the relative DNA content of scintigraphically hot thyroid nodi. Methods: Sixty-seven patients with hyperthyroidism due to hot nodi were treated. The relative DNA content of thyrocytes in hot nodi was determined by single cell cytophotometry and compared to results of cytology, and scintigraphy. T4, T3, TSH and thyroglobulin were measured in sera of the patients as well. Results: The modal value of the relative DNA concentration in thyrocytes was in 16 hot nodi diploid (Type 1), in 21 hyperdiploid (Type 2). The 12 nodi with diploid (Type 3) as well as 18 with hyperdiploid (Type 4) modal value of the relative DNA concentration had signs of increased proliferation. The thyrocytes of 4 normal controls were diploid. Cytomorphological signs of atypia and degenerative changes of thyrocytes were significantly more frequent in Types 3 and 4 than in Types 1 and 2. Conclusion: Dominant scintigraphically hot thyroid nodi are diploid or hyperdiploid. Some of them are in the state of proliferation. DNA cytophotometry can be useful as an additional diagnostic method in cases with thyroid (hot) nodi of uncertain cytology, especially when therapy with low dose of radioiodine is planned. Key words: hyperthyroidism - radionuclide imaging; DNA; cytophotometry Introduction Autonomous nodi are present in about 40% of patients with endemic goiter.1 Scintigraphy with technetium ("m Tc) or iodine (l31J) regularly shows an autonomous nodule as a hot spot with a more or less suppressed paranodal thyroid gland. A hot spot on the scan of the thyroid can present a true adenoma, a hyperplastic clone of hyperactive autonomous thyrocytes or a highly differentiated follicular Correspondence to: Nataša V. Budihna, M. D., Ph. D., Department of Nuclear Medicine, University Medical Centre, Zaloška 7, 1000 Ljubljana, Slovenia. UDC: 616.441-008.61-091.83 carcinoma. It is therefore desirable to differentiate among these conditions before the therapy is given, although in general the incidence of thyroid carcinoma is low.2-3 The aim of our study was to determine a pattern of DNA distribution in hyperactive thyroid nodi. Materials and methods Subjects Sixty-seven hyperthyrotic patients with autonomous goiter, sent to our department for routine investigations before the therapy with radioiodine, were in- 14 Budihna NV et al. eluded in the study. The inclusion criterion was the presence of a single hot node or utmost 3 hot nodi with a discernible dominant hot node. There were 57 females and 10 males, aged 43 to 89 years (mean 61.5 years). The normal control group consisted of 4 females, aged 33 to 37 years, sent to our department for suspected autoimmune thyroiditis. In each of them thyroid disease was excluded by hormonal tests, negative thyroid autoantibodies and by cytological examination of the thyroid. Methods The final clinical diagnosis of thyroid disease was based on the disease history, the physical examination of the patients, the results of thyroid cytology, ultrasonography, scintigraphy, serum T4, T3, TSH and thyroglobulin concentration. The results of cy-tophotometry were compared with cytomorphology (cytology) and final diagnosis. Scintigraphy and ultrasonography The planar scintigraphy of the thyroid was performed with Siemens Basicam gamma camera, 20 minutes after intravenous application of 80 MBq of 99mTc-pertechnetate. Scintigram with 2 MBq of l3IJ was accomplished 24 hours after the oral application of radiotracer. Ultrasonography (US) of the thyroid was performed by using high resolution transducer (10 MHz, Diasonics DRF 300). The diameter of dominant hot nodi was measured with US. Scintigraphically hot nodi were identified on the ultrasound scan with the help of scintigrams. Fine needle biopsy of a dominant hot node, guided by US, was done after scintigraphy. Smears for cytology and the single cell cytophotometry were prepared from each sample obtained with fine needle aspiration biopsy. Cytomorphology Smears were stained by the May-Gruenewald-Giem-sa method. The morphological changes in thyroid cells were grouped into 6 classes: 1 normal thyrocytes 2 hyperactive thyrocytes 3 thyrocytes with degenerative changes 4 proliferation or atypical thyrocytes 5 malignancy suspected 6 definite malignancy Cytochemical DNA assessment The single cell cytophotometry was performed after the Feulgen staining procedure, including acid hydrolysis in 4 N HC1 at 28°C for 60 min. DNA measurements were carried out on microspectro-photometer Opton USPM 30/50 at wave length of 580 nm and diaphragm 0.63. The objective's magnification was 25x or 40x. Processing was done by the computer. Hundred and fifty to 200 thyrocytes and 25-100 leucocytes were evaluated in each smear. The modal relative DNA concentration of leucocytes in the same thyroid biopsy smears served as a reference value for the normal diploid DNA concentration, the so called "L" value.4,5 The thyrocytes with the modal relative DNA concentration were considered to be in G1 (gapl) phase of cell division cycle, G2 (gap 2) phase was double modal value. The cells with the intermediate DNA concentration were considered to be in S (synthesis) phase. According to their relative DNA content, thyrocytes from hot nodi were classified as diploid (0.75G2 (%) Type 1 16 94 ±6 5 + 6 0.25 ±0.5 1 ± 1.7 Type 2 21 94 ±5 4 ± 4 0.7+1.4 0.1 ±0.4 Type 3 12 68 ± 19 5 ± 3 23.9 ± 17 2.7 ± 2.8 Type 4 18 78 ± 13 8 ± 8 14 ± 12 1.7 ±2.9 Controls 4 96 ± 3.6 1-8 0 0 Leucocytes 67 100 0 0 0 Legend: G1, G2, S (%) - percentage of thyrocytes in individual phases of cell division cycle >G2 - percentage of thyrocytes with more than tetraploid DNA concentration 16 Budihna NV et ai. Table 4. The comparison of cytomorphologic results in different types of DNA frequency distribution histograms in 67 hot nodi. Histogram Patients Active Aniso- Oncocytes Degenerated Microfollicles thyrocytes nucleosis thyrocytes (N) (% pts) (% pts) (% pts) (% pts) (% pts) Type 1 16 100 0 0 0 10 Type 2 21 80 5 0 10 15 Type 3 12 83 42 8 33 25 Type 4 18 94 17 6 39 11 cytomorphology was more frequent in nodi with the signs of proliferation in the DNA frequency distribution histogram. In a recent study the somatic mutation of TSH receptor gene was shown in the major part of autonomous nodi. The proof of this mutation might have an implication on the prognosis of thyroid adenoma.6 According to standards somatic mutation is present in 58 % of autonomous nodi in our study. It is apparently more frequent among solitary hot nodi where the average relative DNA concentration was slightly higher than in groups with 2 or 3 hot nodi. Several authors assume the aneuploidy characteristics of true adenomas.7"10 Also Lukasz found predominant hyperdiploidy in thyroid adenomas." According to these authors it is conceivable that among hot nodi in our patients those with the DNA frequency distribution histograms of Types 2 and 4 are true adenomas. In favour of this it is also the cytomorphology, which showed higher grades of atypia in these nodes. Among 67 hot nodi in our patients 2 (2.8%) were malignant. Citomorphologically one patient had follicular carcinoma (solitary hot node) and the other one (3 hot nodi) oncocytoma. In both cases the single cell cytophotometry showed the DNA distribution histograms reflecting proliferation and hyperdiploidy. Since similar changes were found in some benign follicular nodi as well, such changes can not be considered a proof of malignancy. Contrary to our experience, Bengtsson et al.12 considers the DNA cytophotometry convenient for the differentiation of malignant from benign lesions in the thyroid. Most other authors believe that DNA cytophotometry cannot reliably differentiate between thyroid cancer and benign thyroid adenoma,7"9'l3"14 The percentage of malignant hot nodi in our patients was small but significant. After our opinion cytomorphology is therefore mandatory in all dominant, especially solitary, hot nodi before the therapy, especially if the radioiodine therapy is consid- ered. 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