World J. Surg. 14, 425--430, 1990
World Journal of Surgery 9 1990 by the Soci6t6 lnternationale de Chirurgie
Prediction of Distant Metastasis in Follicular Adenocarcinoma of the Thyroid H i r o y u k i I w a s a k i , M . D . , A k i h i k o M a t s u m o t o , M . D . , K u n i h i k o Ito, M . D . , Y o s h i o K u r e , M . D . , A k i r a Suzuki, M . D . , K i m i n o r i Sugino, M . D . , O s a m u Ozaki, M . D . , and J a e d u k N o h , M . D . ito Hospital, Tokyo, Japan, and First Department of Surgery, Yokohama City University School of Medicine, Yokohama City, Japan Follicular adenocarcinoma of the thyroid sometimes displays distant metastasis in spite of its low histological grade of malignancy. We have studied whether distant metastasis can be detected by the presence of blood vessel invasion. We have also examined the problem from the Viewpoint of ceil.to:cell conjugation. This Study examined 160 cases of follicular adenocarcinoma of the thyroid. Histological specimens were made by sectioning tumors at their largest diameter. Blood vessel endothelia wer e specifically stained immunohistochemically with factor VIll-related antigen and Ulex europeaus agglutinin I (UEA-I). Then, the presence Of blood vessel invasion was examined in detail. Furthermore, the conjugation of lectin-derived wheat germ agg!utinin (WGA) to tumor cells was examined. Conjugation of tumor cells was also examined using sialic acid as a marker. Blood vessel invasion was recognizedin 95 (59.4%) of 160 cases. Distant metastases were identified in 25 cases (15.6%) in which blood vessel invasion was also found. Relative to the group without distant metastases, the group with distant metastases showed not only a significantly higher sialic acid content but also higher levels of WGA-binding protein in the tissues. The risk of distant metastasis in patients with follicular adenocarCinoma of the thyroid appears to be related both to the extent and to the frequency of blood vessel invasion. Furthermore, assessment of the conjugation of Cells appears to have predictive value for occurrence of distant metastases.
Among differentiated carcinomas, follicular carcinom a of the thyroid generally has a good prognosis; however, the determination Of clinical treatment is difficult because this type of carcinoma is frequently associated with distant metastasis. We suspected that it is possible tO predict the development of distant metastasis by looking for blood Vessel invasion in pathologic specimens [1, 2]. Furthermore, the conjugation of lectin-derived wheat germ agglutinin (WGA) to tissues as well as the sialic acid in tissues were both examined. In this report, we demonstrate that both WGA conjugation and sialic acid content correlate with occurrence of distant metastasis. Material
The subject s were 160 patients with the diagnosis of follicular carcinoma of the thyroid who recieved their initial operation Presented at the International Association of Endocrine Surgeons in Toronto, Ontario, Canada, September, 1989. Reprint requests: Hiroyuki Iwasaki, M.D., Ito Hospital, 4-3-6 Jingumae Shibuya-ku, Tokyo 150, Japan.
either at Ito Hospital or at the First Department of Surgery, Yokohama City University. All operations were performed during the 12-year period from 1976 to 1987. Methods
Sections were routinely cut with the primary pathological specimens cut at the largest diameter. The presence of blood vessel invasion was examined in detail by staining the specimens with Ulex europeaus agglutinin I (UEA-1) lectin and factor VIII-related antigen which are well known markers for blood vessel endothelial cells. Staining procedures are shown in Fig. 1. UEA-I lectin (E.Y Laboratory) and factor VIII-related antigen (Biomeda Corp.) were purchased commercially. As shown in Fig. 2, blood vessel invasion, recognized as tumor cell clusters inside vessels, was classified into 3 types. Type I blood vessel invasion was expansive and was found outside the capsule of the tumor. Type II was present near the capsule, and type III was either beneath the capsule or inside the tumor. Classification of invasion according to dimension seemed inaccurate because large blood vessels were often found near the capsule. Therefore, this classification is based on the position Of a blood vessel showing invasion (i.e., within the tumor, within the capsule, or outside the tumor). In cases where classifications overlapped, preference was given to the type with a lower number (Figs. 3-5). We examined the serum sialic acid in 32 cases and the tumor tissue sialic acid content in 10 cases. Serum sialic acid was examined when a case was diagnosed either as follicular carcinoma o r as carcinoma with distant metastases. The normal range of serum sialic acid is between 44 and 71 mg/dl. Determination of tissue sialic acid content was carried out as follows: first, tumor tissue was homogenized with phosphate buffered saline (pH, 7.0); after samples were centifuged at 3,000 rpm for 15 minutes, the upper portion was examined for sialic acid and protein content. We determined sialic acid per mg protein in tissues according to the method of Lowry. Normal tissues surrounding follicular adenomas were used as controls (10 cases). WGA lectin (purchased commercially from E~ Laboratory) was used in the examination of WGA-glycoprotein binding in
426
World J. Surg. Volo 14, No. 3, May/June 1990
Formalin paraffin sections (4 um) Step 1 I)eparaffinization
(xylen, I00~ ethanol)
Step 2 2~ Proteina~e for 30 min.
'
1
Step 3 Blocking the intrinsic peroxidase activity (methanol 0.3~ H202) Step 4 Lectin binding for 60 min. (biotinylated lectins: E-Y Lab.)
V~IR-Agbinding for 60 min. ( (BiomedaCorp.)
V
Step 5 ABC procedure for 30 min. (Avidin-Biotin-Peroxidase Complex) Step 6 Coloration (3,3"-diaminobenzidine) Step 7 Counterstaining (Hae~atoxylin)
Sections were washed in PBS (phosphate buffered salin) after each step.
Fig. 1. Staining procedures.
TYPE
I
~i:: ....
TYPE
~
TYPE
~I
::~.5:~
~
Fig. 2. Classification of blood vessel invasion. Dark circles indicate blood vessel invasion and dots show tumor cells. Type I, expansive invasion, is found outside the capsule of the tumor. Type II is present near the capsule, and type III is either beneath tile capsule or inside the tumor. tumor cells. Staining procedures were identical to those used for UEA-1 lectin. Results
Blood vessel invasion was observed in 95 (59.4%) of the 160 cases. Type I invasion was seen in 25 cases (26.3%), type II invasion in 47 (49.5%), and type III invasion was seen in 23 cases (24.2%). Distant metastases were present in 15 patients showing type I invasion, in 6 patients with type II invasion, and in 4 patients with type III invasion. It is evident that expansive blood vessel invasion was found mostly in cases with distant metastases (Table 1). Serum sialic acid was 58.7 + 9.4 mg/dl in the group without distant metastases, whereas in the group with distant metastases, the sialic acid value was 65.0 --- 9.2 mg/dl. There was no
Fig. 3. Type I blood vessel invasion stained with factor VIII-related antigen (• Arrows indicate endothelial cells of the blood vessel. Fig. 4. Type II blood vessel invasion stained with UEA-1 (x 100). Fig. 5. Type III blood vessel invasion stained with UEA-1 (x200).
significant difference between the 2 groups (Fig. 6). The sialic acid content of tumor tissues was significantly higher in the group with distant metastases (Fig. 7). WGA stain results are shown in Table 2. In the group without distant metastases, WGA binding occurred mainly on the apical surface of tumor cells; however, in the group with distant
H. Iwasaki et al.: Thyroid Carcinoma Metastases
427
Table 1. Types of blood vessel invasion.
- - p
World Journal of Surgery 9 1990 by the Soci6t6 lnternationale de Chirurgie
Prediction of Distant Metastasis in Follicular Adenocarcinoma of the Thyroid H i r o y u k i I w a s a k i , M . D . , A k i h i k o M a t s u m o t o , M . D . , K u n i h i k o Ito, M . D . , Y o s h i o K u r e , M . D . , A k i r a Suzuki, M . D . , K i m i n o r i Sugino, M . D . , O s a m u Ozaki, M . D . , and J a e d u k N o h , M . D . ito Hospital, Tokyo, Japan, and First Department of Surgery, Yokohama City University School of Medicine, Yokohama City, Japan Follicular adenocarcinoma of the thyroid sometimes displays distant metastasis in spite of its low histological grade of malignancy. We have studied whether distant metastasis can be detected by the presence of blood vessel invasion. We have also examined the problem from the Viewpoint of ceil.to:cell conjugation. This Study examined 160 cases of follicular adenocarcinoma of the thyroid. Histological specimens were made by sectioning tumors at their largest diameter. Blood vessel endothelia wer e specifically stained immunohistochemically with factor VIll-related antigen and Ulex europeaus agglutinin I (UEA-I). Then, the presence Of blood vessel invasion was examined in detail. Furthermore, the conjugation of lectin-derived wheat germ agg!utinin (WGA) to tumor cells was examined. Conjugation of tumor cells was also examined using sialic acid as a marker. Blood vessel invasion was recognizedin 95 (59.4%) of 160 cases. Distant metastases were identified in 25 cases (15.6%) in which blood vessel invasion was also found. Relative to the group without distant metastases, the group with distant metastases showed not only a significantly higher sialic acid content but also higher levels of WGA-binding protein in the tissues. The risk of distant metastasis in patients with follicular adenocarCinoma of the thyroid appears to be related both to the extent and to the frequency of blood vessel invasion. Furthermore, assessment of the conjugation of Cells appears to have predictive value for occurrence of distant metastases.
Among differentiated carcinomas, follicular carcinom a of the thyroid generally has a good prognosis; however, the determination Of clinical treatment is difficult because this type of carcinoma is frequently associated with distant metastasis. We suspected that it is possible tO predict the development of distant metastasis by looking for blood Vessel invasion in pathologic specimens [1, 2]. Furthermore, the conjugation of lectin-derived wheat germ agglutinin (WGA) to tissues as well as the sialic acid in tissues were both examined. In this report, we demonstrate that both WGA conjugation and sialic acid content correlate with occurrence of distant metastasis. Material
The subject s were 160 patients with the diagnosis of follicular carcinoma of the thyroid who recieved their initial operation Presented at the International Association of Endocrine Surgeons in Toronto, Ontario, Canada, September, 1989. Reprint requests: Hiroyuki Iwasaki, M.D., Ito Hospital, 4-3-6 Jingumae Shibuya-ku, Tokyo 150, Japan.
either at Ito Hospital or at the First Department of Surgery, Yokohama City University. All operations were performed during the 12-year period from 1976 to 1987. Methods
Sections were routinely cut with the primary pathological specimens cut at the largest diameter. The presence of blood vessel invasion was examined in detail by staining the specimens with Ulex europeaus agglutinin I (UEA-1) lectin and factor VIII-related antigen which are well known markers for blood vessel endothelial cells. Staining procedures are shown in Fig. 1. UEA-I lectin (E.Y Laboratory) and factor VIII-related antigen (Biomeda Corp.) were purchased commercially. As shown in Fig. 2, blood vessel invasion, recognized as tumor cell clusters inside vessels, was classified into 3 types. Type I blood vessel invasion was expansive and was found outside the capsule of the tumor. Type II was present near the capsule, and type III was either beneath the capsule or inside the tumor. Classification of invasion according to dimension seemed inaccurate because large blood vessels were often found near the capsule. Therefore, this classification is based on the position Of a blood vessel showing invasion (i.e., within the tumor, within the capsule, or outside the tumor). In cases where classifications overlapped, preference was given to the type with a lower number (Figs. 3-5). We examined the serum sialic acid in 32 cases and the tumor tissue sialic acid content in 10 cases. Serum sialic acid was examined when a case was diagnosed either as follicular carcinoma o r as carcinoma with distant metastases. The normal range of serum sialic acid is between 44 and 71 mg/dl. Determination of tissue sialic acid content was carried out as follows: first, tumor tissue was homogenized with phosphate buffered saline (pH, 7.0); after samples were centifuged at 3,000 rpm for 15 minutes, the upper portion was examined for sialic acid and protein content. We determined sialic acid per mg protein in tissues according to the method of Lowry. Normal tissues surrounding follicular adenomas were used as controls (10 cases). WGA lectin (purchased commercially from E~ Laboratory) was used in the examination of WGA-glycoprotein binding in
426
World J. Surg. Volo 14, No. 3, May/June 1990
Formalin paraffin sections (4 um) Step 1 I)eparaffinization
(xylen, I00~ ethanol)
Step 2 2~ Proteina~e for 30 min.
'
1
Step 3 Blocking the intrinsic peroxidase activity (methanol 0.3~ H202) Step 4 Lectin binding for 60 min. (biotinylated lectins: E-Y Lab.)
V~IR-Agbinding for 60 min. ( (BiomedaCorp.)
V
Step 5 ABC procedure for 30 min. (Avidin-Biotin-Peroxidase Complex) Step 6 Coloration (3,3"-diaminobenzidine) Step 7 Counterstaining (Hae~atoxylin)
Sections were washed in PBS (phosphate buffered salin) after each step.
Fig. 1. Staining procedures.
TYPE
I
~i:: ....
TYPE
~
TYPE
~I
::~.5:~
~
Fig. 2. Classification of blood vessel invasion. Dark circles indicate blood vessel invasion and dots show tumor cells. Type I, expansive invasion, is found outside the capsule of the tumor. Type II is present near the capsule, and type III is either beneath tile capsule or inside the tumor. tumor cells. Staining procedures were identical to those used for UEA-1 lectin. Results
Blood vessel invasion was observed in 95 (59.4%) of the 160 cases. Type I invasion was seen in 25 cases (26.3%), type II invasion in 47 (49.5%), and type III invasion was seen in 23 cases (24.2%). Distant metastases were present in 15 patients showing type I invasion, in 6 patients with type II invasion, and in 4 patients with type III invasion. It is evident that expansive blood vessel invasion was found mostly in cases with distant metastases (Table 1). Serum sialic acid was 58.7 + 9.4 mg/dl in the group without distant metastases, whereas in the group with distant metastases, the sialic acid value was 65.0 --- 9.2 mg/dl. There was no
Fig. 3. Type I blood vessel invasion stained with factor VIII-related antigen (• Arrows indicate endothelial cells of the blood vessel. Fig. 4. Type II blood vessel invasion stained with UEA-1 (x 100). Fig. 5. Type III blood vessel invasion stained with UEA-1 (x200).
significant difference between the 2 groups (Fig. 6). The sialic acid content of tumor tissues was significantly higher in the group with distant metastases (Fig. 7). WGA stain results are shown in Table 2. In the group without distant metastases, WGA binding occurred mainly on the apical surface of tumor cells; however, in the group with distant
H. Iwasaki et al.: Thyroid Carcinoma Metastases
427
Table 1. Types of blood vessel invasion.
- - p
