[Clinical study on prognostic factors in thyroid carcinoma].

We treated 227 patients, 45 men and 182 women, with thyroid carcinoma at our hospital from 1984 to 1998. Of these, 177 had papillary carcinoma and 50 follicular carcinoma. The extent of resection was based on tumor size in papillary carcinoma but not follicular carcinoma, and 70% of carcinoma patients underwent hemithyroidectomy. Neck lymph nodes were resected in 93.2% of papillary carcinoma patients, with D1 neck dissection in 45.7% and D2 or D3 neck dissection in 47.5%. In contrast, 70% of follicular carcinoma patients with lymph node resection had D1 dissection. Locoregional recurrence was noted in 22 patients and distant metastasis in 6 cases. Nonsurvivors numbered 17, 12 papillary and 5 follicular carcinoma patients, died of their primary disease. Almost all deaths were in patients with advanced disease, pT3 in 3, pT4 in 10, N1a in 3 and N1b in 8. The prognostic factors for papillary carcinoma were extracapsular spread, age, and distant metastasis, while the only factor for follicular carcinoma was distant metastasis. The 5-year survival for patients with papillary carcinoma was 93.0% and 10-year survival 88.8%, compared to 5-year survival for 93.5% of follicular carcinoma patients and 10-year survival for 93.5%.

A simple method to estimate the secretion of saliva from minor salivary glands using iodine-starch reaction.

OBJECTIVE/HYPOTHESIS: This study was undertaken to detect the faculty of secretion of saliva from minor salivary glands by analyzing a color reaction on a test tape containing iodine and starch that was applied on the lower lip. STUDY DESIGN: A study involving 63 patients with oral dryness, 7 patients with Sjogren syndrome, and 70 healthy individuals was performed. METHODS: A test tape (1 x 1 cm) containing iodine and starch was set on the mucosal area anterior to the labia frenulum for 30 seconds. Because the number of blue spots was considered to correspond to the number of ostia of the salivary gland on the lower lip that was examined, the number of blue spots occurring as a reaction of iodine and starch on the test tape was counted and was compared among three groups. In addition, the relationship between the histopathological findings and the number of spots was analyzed. RESULTS: The average number of spots in the patients with oral dryness (4.52+/-3.18 [mean +/- SD]) was lower than that in healthy individuals (9.49+/-2.52, P <.01), and that in the patients with Sjögren syndrome (2.14+/-1.35) was the lowest among all groups in the study. Moreover, this reduction in the number of spots in those patients was accompanied by histopathological changes of the minor salivary glands. CONCLUSION: These findings suggest that this simple, noninvasive method can be successfully used for the estimation of the faculty of secretion of saliva from the minor salivary glands.

Analysis of DNA hybridization in polyacrylamide gel.

We have made the first apparatus for fluorescent detection, monitoring the hybridization process of fluorescently labeled DNA fragments in a polyacrylamide gel. Using this, the analysis on the thermal denaturation/reassociation process of DNA fragments in the gel was employed, for improving the performance of In-Gel Competitive Reassociation (IGCR) technique, one of genome subtraction methods. We showed that Fluorescence Resonance Energy Transfer (FRET) in the gel occurred by positioning two fluorescent dyes at 3' and 5' ends of DNA fragments. The characterization of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.

Improvement of IGCR technique using FRET.

The fluorescent detection system has been introduced into the study on denaturation/reassociation process of DNA fragments in gel, for improving In-Gel Competitive Reassociation technique, one of genome subtraction methods. The annealing behaviour of the mixture of 3'-Fluorescein-labelled and 5'-Cy5-labelled DNA fragments was analysed by Fluorescence Resonance Energy Transfer (FRET) technique from donor Fluorescein to acceptor Cy5. We showed that two fluorescent dyes labelled at 3' and 5' ends of DNA fragments caused FRET in both the solution and the gel. The characterisation of fluorescence-labelled fragments in gel and the changes of their fluorescence intensity will be reported.

Laboratory maintenance of Eucyclops serrulatus (Copepoda: Cyclopoida).

A simple method for culturing a freshwater copepod Eucyclops serrulatus is described. A flagellate Chilomonas paramecium serves as the food organism. Starting from a single, egg-bearing female collected in a pond, the copepod proliferated for at least 10 generations apparently with no adverse changes. The stock culture has been maintained with minimal effort for at least 19 months. The method is expected to make the copepod useful in the laboratory as an intermediate host of various parasitic helminths.

[Evaluation of frozen section diagnosis of parotid gland tumors].

A retrospective study is presented comparing the results of 167 frozen section diagnoses of surgical extirpated parotid gland tumors with permanent-section diagnoses. Percentages of correct diagnosis for malignancy (cases correctly classified as benign or malignant tumors) and of correct diagnosis for histopathology (cases in which frozen section diagnosis and permanent-section diagnosis were identical) were calculated. Percentages of correct diagnosis for malignancy in all cases, benign cases, and malignant cases were 98.8%, 99.3%, and 95.8%, respectively. Percentages of correct diagnosis for histopathology in all cases, benign cases, and malignant cases were 94.0%, 97.2%, and 75.0%, respectively. These results are superior to the previous reports both of frozen section diagnosis and of fine-needle aspiration biopsy diagnosis although the data for histopathological diagnosis in malignant tumors are average compared to previous reports. We conclude that diagnoses of most parotid gland tumors based on frozen section examination are reliable and accurate, but caution should be exercised in malignant tumors diagnosis.

[Clinical study of parotid tumors: a 20 year statistical analysis of 633 cases].

During the 20 years from 1977 to 1996, 633 cases underwent excisions of parotid tumors (539 benign and 94 malignant) in the department of Otolaryngology of Kansai Medical University. The incidence of Warthin's tumors in the years from (1987 to 1996) was higher than that in the years from 1977 to 1986. On the other hand, the incidence of mucoepidermoid carcinomas was less than in previous reports. Tumors accompanied with spontaneous pain or facial nerve palsy and with invasion to both lobes frequently indicated malignancy. Facial nerve palsy caused by either benign or malignant tumors before operation showed no improvement after surgery. The incidences of facial nerve palsy (1.0% in benign, and 18.3% in malignant) and Frey's syndrome (17.8% in benign, and 18.3% in malignant) after surgery were lower than those in other reports of both benign and malignant tumors. Five-year mortality was 76.1%. All deaths that occurred five years after operation involved highly grade malignant tumors.

[Voice restoration and clinical outcome in patients undergoing laryngectomy with voice prostheses].

Since the introduction of the tracheoesophageal fistula (TEF) technique for restoration of voice in 1983, we performed voice restoration using primary tracheoesophageal fistula in 83 patients undergoing laryngectomy. The success rate for voice restoration with TEF was 80.6% (good voice; 69.8%, fair voice; 10.8%). Thirty-one patients out of 51 patients who have been followed for over 3 years had 34 problems with TEF (15 granulations of TEF, 7 difficulties in insertion of voice prostheses, 4 stenoses of the tracheal stoma, 5 other reasons). However, most patients could use TEF by following appropriate procedures. Our results suggest that the Blom Singer TEF procedure is a safe, simple and effective means of alaryngeal communication.

Intracellular calcium changes and chemosensitivities of human epidermoid carcinoma cell lines after exposure to cisplatin.

In order to elucidate the mechanisms of cisplatin (cis-diamminedichloroplatinum; CDDP)-resistant tumor cells, we previously established a CDDP-resistant KB cell line (KBrc cells) from a parental KB cell line derived from epidermoid carcinoma (KB cells). The KBrc cells were resistant to 5 kinds of platinum (Pt) drugs. Intracellular Pt concentrations in KBrc cells were lower than in KB cells. Decrease of intracellular Pt concentrations was one of the CDDP-resistant mechanisms. When we measured changes of intracellular calcium ion concentration ([Ca2+]i) during exposure to high-dose CDDP, a sustained elevation of the [Ca2+]i level was observed in the KB cells. These results suggest that the mechanisms underlying CDDP resistance involve changes in calcium channels and an alteration of calcium homeostasis in the tumor cell line.

Occult papillary thyroid carcinoma in Hashimoto's thyroiditis presenting as a metastatic bone tumor.

Some occult thyroid carcinomas are hypothesized to regress and be eventually obliterated. We report here a patient whose condition supports this hypothesis. A 51-year-old male with primary hypothyroidism due to Hashimoto's thyroiditis suffered from a rib bone tumor. He had a diffuse goiter with no nodular lesion. Serum FT4 and TSH concentrations were 0.8 ng/dl and 36.4 microU/ml on taking 100 microg/day of T4. Anti-Tg- and -TPO-Ab were strongly positive (99 and 1380 U/ml). The iodine 123 scintigraphy demonstrated clear accumulation in the rib tumor, whereas the thyroid was scarcely visible. Biopsy of the rib tumor showed papillary proliferation of large atypical cells, which were immunohistochemically positive for thyroglobulin. Metastatic bone tumor of papillary thyroid carcinoma was therefore strongly suspected. He underwent a total thyroidectomy and the thyroid was stepwise sectioned completely at 3 mm intervals. The thyroid condition was diagnosed as Hashimoto's thyroiditis demonstrating diffuse and dense fibrosis, lymphocyte infiltration with lymphoid follicles and flattened atrophied follicles, but no carcinomatous foci were found. He was treated with I-131 and scintigraphy after the ingestion showed distinct accumulation in the rib tumors similar to that before thyroidectomy. No other abnormal uptake was observed. It is suggested that the primary occult thyroid papillary carcinoma regressed and was obliterated possibly by some immunologic or other host-resistance factors after it metastasized to the distant bone.

An increase in laryngeal aerosol deposition by ultrasonic nebulizer therapy with intermittent vocalization.

In acute or chronic laryngitis as well as pharyngitis, nebulization with steroid and antibiotics is considered to be a safe and effective treatment. However, the optimal dose, frequency, duration, and method of administration by ultrasonic nebulization is not known. To evaluate whether there is continual improvement using ultrasonic nebulizer therapy, the deposition rate of aerosol particles into the upper airway was studied. We analyzed the deposition rate in the upper airway by a counting system of radioactive isotope (99mTc-diethylene triaminepentaacetic acid; 99mTc DTPA). Volunteers in good health inhaled an aerosol containing 99mTc DTPA under various conditions of inhalation. Deep and slow inhalation at a respiratory rate of 12 breaths/minute resulted in a high deposition rate of 99mTc DTPA in the lung and a low deposition rate in the larynx. In contrast, faster inhalation at a respiratory rate of 36 breaths/minute resulted in an increase in the isotope deposition rate in the larynx. Furthermore, when the volunteers vocalized intermittently during the fast inhalation at a respiratory rate of 36 breaths/minute, the isotope deposition rate in the volunteer's larynx was higher than in all other inhalation conditions. These results suggest that fast inhalation with intermittent vocalization is one of the important inhalation methods used to improve the deposition rate of aerosol particles into the upper airway with an ultrasonic nebulizer.

Simultaneous xenotransplantation of human Graves' thyroid tissue and autologous bone marrow cells in severe combined immunodeficient mice: successful reconstitution of human Graves' hyperthyroidism.

Human thyroid xenografts and the autologous bone marrow (BM) cells from five patients with Graves' disease (GD) were simultaneously xenografted into severe combined immunodeficient (SCID) mice to study the role of BM cells for the perpetuation of human GD autoimmunity and hyperthyroidism. All SCID mice engrafted with thyroid tissue (TH) alone, TH + autologous peripheral blood mononuclear cells, and TH + autologous BM cells produced similar amounts of human IgG; however, the production in TH + BM-engrafted mice peaked later than that of mice without BM. Production of thyroperoxidase antibody and thyroglobulin antibody in TH + BM-bearing SCID mice peaked in later weeks after xenografting than in those without BM. Moreover, human Graves' hyperthyroidism was actually reconstituted in TH + BM-transplanted mice; this was confirmed by (A) significantly higher levels and longer periods of secreting thyroid-stimulating antibody than those in mice without BM engraftment. (B) persistent hyperthyroxinemia up to the end of the experiment. (C) extremely high radioidine uptake of the xenografted thyroid tissue, and (D) histological findings of the maintenance of hyperplastic change of the xenografted thyroid epithelial cells. Human BM stem cells (CD34) were identified only in mice with TH + BM xenografts when analyzed by immunohistochemistry. In conclusion, (A) we have developed an animal model for human hyperthyroid GD by simultaneous xenotransplantation of GD thyroid tissue plus autologous BM cells into SCID mice, and (B) BM cells have a crucial role for perpetuating human GD autoimmunity and hyperthyroidism in this system.

High urinary hydroxyproline excretion in patients with advanced head and neck cancer.

We studied the urinary excretion of hydroxyproline, measured as the hydroxyproline-to-creatinine ratio (HOP/Cr), in sixty-six patients with head and neck disease including, 18 patients with head and neck cancer. Urinary hydroxyproline excretion was determined before treatment by the linear gradient elution method. Although the urinary excretion of hydroxyproline is considered to be useful in monitoring cancer tissue such as prostate carcinomas, no studies have been performed on the urinary excretion of hydroxyproline in patients with head and neck cancer. The HOP/Cr was significantly higher in cancer patients than in the healthy control. The ratio of patients with T3/T4 cancers was higher than in patients with benign tumors (p<0.05), chronic inflammatory diseases (p<0.01) or T1/T2 cancer (p<0.05). The high HOP/Cr with T3/T4 head and neck cancer corresponded to the extent of tumor invasion into the surrounding tissues such as muscles and bone. This suggests that HOP/Cr may be useful as a supplementary parameter for the assessment of tumor invasiveness which is masked in head and neck carcinomas such as skull base or maxillary carcinomas.

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma.

To evaluate the prognostic significance of cyclin D1 protein/gene expressions in human head-and-neck squamous-cell carcinoma (HNSCC), we examined amplification of the cyclin-D1 gene (CCND1) by the differential PCR method and over-expression of cyclin-D1 protein by immunohistochemistry in 45 paraffin-embedded sections from HNSCC. Amplification of CCND1 was found in 10 (22%) cases and over-expression of cyclin D1 was found in 24 (53%) cases. CCND1 amplification was also found in 3 (25%) of 12 cases of dysplastic lesions adjacent to HNSCC. The overall 5-year survival of patients with CCND1 amplification or with protein over-production was significantly lower than that of patients without (p < 0.0001 and p < 0.05, respectively). However, with multivariate analysis, only amplification of CCND1 retained an independent prognostic value (p = 0.0018). These suggest that CCND1 amplification occurs at early stages of HNSCC tumorigenesis and is a more useful prognostic factor than over-expression of cyclin D1 in HNSCC.

IFN-gamma has a protective role against thyroid-specific autoantibody production in severe combined immunodeficient (SCID) mice xenografted with Graves' thyroid tissue.

We studied the effects of exogenous human IFN-gamma or neutralizing monoclonal antibody (mAb) to IFN-gamma on xenografted human Graves' thyroid tissue in severe combined immunodeficient (SCID) mice to investigate a possible role of IFN-gamma in the pathogenesis of human Graves' disease. Human thyroid tissues from four patients with Graves' disease were xenografted into SCID mice. Two weeks after xenografting, mice were divided into three groups with human IgG levels similar to each other. Mice in the first group were treated with human IFN-gamma daily for 6 weeks; mice in the second (similar) group were treated with an mAb to IFN-gamma; mice in the third group were given mouse IgG only (control group). Blood samples were taken every 2 weeks for human IgG and thyroid-specific autoantibodies (Tg-Ab, TPO-Ab, and thyroid-stimulating antibody). After 6 weeks' treatment, mice were killed, and the thyroid xenograft was examined for thyrocyte HLA-DR expression. Human IgGs were produced equally in all three groups; mice treated with IFN-gamma showed significantly lower amounts of thyroid autoantibodies than those in the control group. Thyrocyte HLA-DR expression was markedly increased in xenografts from mice with IFN-gamma administration. On the other hand, anti-IFN-gamma mAb injection caused only slight suppression of HLA-DR expression on xenografted thyroid cells. In conclusion, IFN-gamma may down-regulate the production of thyroid-specific autoantibodies but not human IgG, at least under these circumstances; there thus may be specific inhibitory effects of IFN-gamma against thyroid-specific autoantibody production of intrathyroidal plasma cells, and this animal model may help to elucidate the possible role of cytokines in the pathogenesis of Graves' disease.

[Study of malignant lymphoma in the parotid gland region].

Malignant lymphomas arising in the salivary glands are very uncommon. The vast majority of these lesions are classified as extranodal non-Hodgkin's lymphoma. Clinical presentation, management, histopathology, and outcome in 11 cases of non-Hodgkin's lymphomas of the parotid gland region were reviewed. The ages of the patients ranged from 25 to 80 years (average 56.0 years) and the male to female ratio was 5:6. Presenting symptoms were painless masses (10 cases) and a painful mass (1 case) in the parotid gland region. None of the patients had facial nerve paralysis. It seems to be difficult to diagnose malignant lymphomas of the salivary glands preoperatively, because an open biopsy of the salivary glands is generally contraindicated. We failed to diagnose malignant lymphoma in the parotid gland preoperatively. The clinical stages were stage I in 5 cases, stage II in 5 cases, and stage III in 1 case. There was no patient with stage IV. Three of the stage I tumors were diagnosed as MALT (mucosa-associated lymphoid tissue) lymphomas. Eight of the tumors were treated surgically and an open biopsy of the parotid gland tumor was performed in 1 case and open biopsy of a neck lymph node in 2 cases for the purpose of diagnosis. After the diagnosis, these cases were followed up with or without radiotherapy and chemotherapy. Follow-up ranged from 6 to 109 months. The 5-year-survival rate was 60%. The outcome for this group was found to be as good as that reported by others.

[Mechanism of cisplatin and peplomycin therapy on head and neck carcinoma].

Results of chemotherapy for head and neck cancer are now improving owing to the development of concomitant use of chemotherapeutic agents such as cisplatin or peplomycin. Concomitant use of the two agents has been favored clinically. However, fundamental study on the combination therapy has not been carried out sufficiently. In the present study we studied the cell kinetics of tumor cells by the combination of cisplatin and peplomycin using flowcytometric analysis and electron microscopy. Survival of tumor cells was lowest, when peplomycin was administrated 3 days after cisplatin. Cell kinetics showed an accumulation at the S and G2M stage in this situation, By the electron microscopic study, microvilli of tumor cells disappeared and the blebbing of tumor cells was observed, when cisplatin alone was administrated. However, when the combination of cisplatin and peplomycin was administrated to the tumor cells, tumor cells enlarged. The results suggest that the mechanism of combination therapy differed from the mechanism of cisplatin alone.

Alteration of sensitivity to cis-diamine(glycolate)-platinum(II) (254-S) in oral tumor xenografts following multiple applications.

The emergence of resistance to platinum analogues is considered to be a major problem in the treatment of head and neck cancer. Therefore, it is important to clarify the mechanisms of resistance to these analogues and the mechanisms of the processes related to this resistance. The study of emergence of resistance in the solid tumors is particularly relevant. Ln the present study, the effect of a platinum analogue (254-S), on the response of an oral carcinoma cell line grown as a xenograft in nude mice, was studied. The effect of a full dose administered as a single intraperitoneal injection of 254-S (15 mg/kg X 1) on tumor growth was not significantly different from the effect of repeated intraperitoneal injections of 254-S, administered 3 times at 1/3 of this dose (5 mg/kg x3), or 5 times at 1/5 of this dose (3 mg/kg x5). However, when a single full-dose intraperitoneal injection of 254-S (15 mg/kg x1) was administered to each group of mice again at the 9th and 12th weeks after the initial treatment, different effects on tumor growth were observed among each group. The groups which received repeated treatment with 254-S (5 mg/kg, x3, or 3 mg/kg x5) showed a decrease in the inhibition of tumor growth, suggesting the emergence of resistance to 254-S. The study of platinum accumulation in the tumor tissues and a flow cytometric analysis of proliferating cell nuclear antigen (PCNA) supported the possibility that resistance to 254-S increases in tumor tissues treated repeatedly. These observations suggest that the potential use of this experimental assay as a model, may provide further insights into the therapeutic mechanisms of resistance to antineoplastic agents in the treatment of solid cancerous head and neck tumors.