Claudio Giani

Serum thyroglobulin in thyroid carcinoma and other thyroid disorders

Measurements of serum thyroglobulin (hTg) were performed using a specific radioimmunoassay. Sera with detectable anti-thyroglobulin (anti-Tg) antibody titers (>1∶10) as assessed by passive hemagglutination were discarded. Assays were carried out under conditions in which anti-Tg titers less than 1:10 produced no interference. The assay sensitivity was 1.25 ng/ml and the mean ± SE concentration of serum hTg in 58 control subjects was 9.5 ± 0.9 ng/ml (range< 1.25–27 ng/ml). A slight but significant (p<0.025) increase in the mean hTg level was observed in 12 pregnaint women at delivery (25.7 ± 5.2 ng/ml). Moderate to marked elevations of serum hTg were observed in patients with nontoxic goiter (61.4 ± 15 ng/ml; n = 23), subacute thyroiditis (138 ± 67 ng/ml; n =5), toxic adenoma (129 ±47 ng/ml; n =13), untreated (424 ± 101 ng/ml; n = 35) or treated (328 + 222 ng/ml; n =14) toxic diffuse goiter. 88 patients with thyroid carcinoma and 10 with nonthyroidal malignancies were studied. The mean level of serum hTg was increased in untreated differentiated thyroid carcinoma (89.5 ± 19 ng/ml; n = 13) but not in undifferentiated (10 ±2.9 ng/ml; n =6) or medullary (0.8 ±0.2 ng/ml; =3) carcinoma. In treated differentiated thyroid carcinoma the mean hTg levels were normal (8.2 ± 2.2 ng/ml) in patients (n = 24) with no evidence of either a thyroid residue or metastatic disease, moderately increased (56.6 ± 16 ng/ml) in patients (n =27) with residual thyroid tissue, markedly elevated in patients with lymph node metastases (199 ± 50 ng/ml; n = 15) and extremely elevated in those with bone (4004 ± 982 ng/ml; n = 8) or lung (2520 ± 620 ng/ml; n = 5) metastases. There was no significant difference in serum hTg between functioning (n =23) and nonfunctioning (n =5) metastases as assessed by 131| whole body scan. A slight but significant (p < 0.0005) increase in the mean concentration of hTg was observed in nonthyroidal malignancies (21.7 ±4.5 ng/ml; n = 10). Serial measurements showed a transient increase of serum hTg after131| therapy of differentiated thyroid carcinoma, toxic diffuse goiter or toxic adenoma, with peak values usually occurring within the first three days. A fall of serum hTg after administration of suppressive doses of thyroid hormone to patients with nontoxic goiter and a rise after discontinuation of thyroid suppressive therapy in patients with metastatic differentiated thyroid carcinoma was observed. The present data confirm and extend previous data indicating that serum hTg is frequently elevated in thyroid disease, and that the release of hTg from malignant and nonmalignant thyroid tissue is at least in part thyrotropin (TSH) dependent and it is enhanced by radioiodine therapy. Measurements of serum hTg do not differentiate from benign and malignant thyroid disease, but may be usefullly employed in the follow up of differentiated thyroid carcinoma. Of particular interest was the finding that nonfunctioning metastases may be detected by measurement of serum hTg and that bone or lung metastases are associated with much higher levels of serum hTg than lymph node metastases.

Serum thyroglobulin concentrations and 131I whole body scans in the diagnosis of metastases from differentiated thyroid carcinoma (after thyroidectomy)

SUMMARY. Measurements of circulating thyroglobulin (hTg) and 131I whole body scan were performed in 101 patients with differentiated thyroid carcinoma who had been subjected to surgical thyroidectomy and 131I ablation of remaining thyroid tissue. All 45 patients with positive scans (i.e. functioning metastases) had elevated hTg concentrations. Of fifty‐six patients with negative scans forty‐two had undetectable or very low hTg levels and were considered to be free of metastatic thyroid tissue, whereas fourteen showed the presence of non‐functioning metastases in the clinical and/or radiological examination. In this group of patients, eleven had elevated serum hTg levels while the other three patients had detectable hTg concentrations within the normal range. These results indicate that serum hTg measurements correlate very well with scan findings and have the added advantage of detecting non‐functioning metastases which would not be detected by scanning. We concluded that measurement of serum hTg may be used together with scanning, as the first step in the follow‐up of thyroidectomized patients with differentiated thyroid carcinoma.

IGF-II mRNA and protein are expressed in the stroma of invasive breast cancers: an in situ hybridization and immunohistochemistry study

Insulin-like growth factor-II (IGF-II) is a potent mitogen for a variety of cell types and is considered an important regulator of breast cancer growth. In this study, we analyzed IGF-II mRNA and protein expression in a series of 80 cases of invasive breast cancer. Seventy-five cases produced informative results for IGF-II mRNA expression, and were scored on an arbitrary scale. Two cases (2.6%) had no significant IGF-II mRNA expression. 35 cases (46.7%) expressed low levels of IGF-II mRNA, 20 cases (26.7%) moderate IGF-II mRNA, while 18 (24%) expressed high levels of IGF-II message. Generally, IGF-II mRNA was expressed in the smooth muscle walls of blood vessels and ducts, as well as in the stroma tightly adjacent to and surrounding tumor epithelium. IGF-II mRNA content was also directly related to the amount of the stroma within the tumor (p<0.05). In 10 cases (13.3%) IGF-II mRNA was detected in the stroma of normal lobules. Fifty-six out of 75 were positive for IGF-II immunostaining. Again, protein staining was generally observed in the smooth muscle of both blood vessels and ducts, as well as in the stroma surrounding tumor epithelium. In normal lobules and ducts the IGF-II protein was detected in the myoepithelium. Unequivocal IGF-II protein staining was seen in tumor epithelium in only three cases. The results of our study demonstrate that, in breast cancer, IGF-II mRNA is expressed in the smooth muscle and stromal components in the majority of invasive breast cancers. IGF-II expression correlates positively with the amount of stromal tissue present within a tumor. This suggests that IGF-II may have an important growth regulatory effect on breast tumor epithelium through paracrine pathways.

RECIPROCAL CHANGES OF SERUM THYROGLOBULIN AND TSH IN RESIDENTS OF A MODERATE ENDEMIC GOITRE AREA

Subjects living in iodine deficient areas were reported to have elevated serum thyroglobulin (Tg) concentrations. This finding was interpreted as related to thyroid stimulation. Discrepant results, however, were found when serum Tg concentrations were correlated either with serum TSH or with goitre size. In this study we investigated the relationships between goitre size, serum Tg and serum TSH in 488 unselected adult subjects living in an endemic area of North‐Western Tuscany (Garfagnana district). The control group comprised 352 subjects residing in a non‐endemic area. In the endemic area a high prevalence of goitre was found (80·1%), thyroid enlargement being slight to moderate in the majority of cases and very large only in six subjects. Serum Tg concentrations increased and serum TSH levels decreased with the size of goitre. Statistical analysis by the chi‐square cross correlation test showed that the converse changes of serum Tg and serum TSH in relation to goitre size were highly significant. These findings indicate that the increase of serum Tg occurring in endemic goitrous subjects may be related to factors other than TSH stimulation. Functional autonomy of the thyroid may account for the finding of low serum TSH and elevated serum Tg values in patients with large goitres. The present data do not exclude the possibility that the release of Tg is influenced by TSH stimulation, but indicate that other factors may be responsible for the increased levels of Tg found in endemic goitre.

Evaluation of a solid-phase immunoradiometric assay (IRMA) for serum thyroglobulin: effect of anti-thyroglobulin autoantibody

Determination of circulating thyroglobulin (Tg) is a useful tool in the diagnostic evaluation of several thyroid disorders [l--S]. Serum Tg is currently measured by a double antibody radioimmunoassay (RIA) technique [1,7,9]. This method is not suitable for Tg determination in sera containing anti-Tg autoantibody, since the latter interferes in RIA resulting in either falsely high or falsely low Tg concentrations [7,10]. Recently Bayer and Kriss [ 1 l] described a solid-phase immunoradiometric assay (IRMA) for Tg which avoids falsely elevated results due to anti-Tg autoantibody. However, the presence of anti-Tg autoantibody has been shown to produce falsely depressed Tg values [ll]. The object of the present study was to clarify the mechanism of this interference, and to compare results of assays by IRMA and RIA in samples with and without anti-Tg autoantibody.

Increased prevalence of primary hyperparathyroidism in treated breast cancer.

Hypercalcemia occurring in patients with advanced breast cancer (BC) is generally due to osteolytic metastases or to the activity of circulating tumor-derived products. In these conditions, the production of endogenous PTH is reduced. The frequency of hypercalcemia due to primary hyperparathyroidism in breast cancer is unknown. We examined the occurrence of primary hyperparathyroidism in a large group of women with treated BC. A total of 100 consecutive women aged 28–80 years with treated breast cancer were enrolled. One hundred and two healthy age-matched women and 60 age-matched female patients with differentiated thyroid carcinoma examined before thyroidectomy were used as controls. Intact serum PTH and serum calcium were measured in all patients and controls. Hypercalcemia associated with elevated serum PTH concentration indicating primary hyperparathyroidism was found in 7 BC patients (7%) and in none of healthy women or patients with thyroid cancer. The pre-operative staging of BC patients with primary hyperparathyroidism was I in six and II in one of them, and no patient had evidence of distant metastases. A parathyroid adenoma was found in all 6 BC patients submitted to neck exploration, one patient refused surgery. Serum calcium and PTH concentrations returned to normal levels after surgery. Two BC patients had increased serum PTH and normal calcium concentrations. One of them had low serum 25-hydroxyvitamin D [25(OH)D]. One patient with spread bone metastases had neoplastic hypercalcemia with undetectable serum PTH concentration. All remaining 90 BC patients had serum calcium and PTH concentrations within normal limits, but their mean (±SD) values (9.6±0.5 mg/dl for serum calcium, 38.0±16.4 mg/dl for serum PTH ) were slightly but significantly greater than in normal controls (9.3±0.5 mg/dl, p=0.003 and 27.9±10.6 pg/ml, p=0.0001, respectively) and in patients with thyroid cancer (9.2±0.6 mg/dl, p=0.001 and 26.2±11.0 pg/ml, p=0.001), with no relationship with clinical staging or anti-tumor therapy. In conclusion: 1) an increased frequency of parathyroid adenoma was found in BC patients with mildly aggressive neoplastic disease; 2) in BC patients with no evidence of primary hyperparathyroidism mean serum PTH and calcium concentrations were significantly greater than in healthy controls and in patients with thyroid carcinoma; and 3) this finding was unrelated to clinical staging or anti-tumor therapy. Thus, primary hyperparathyroidism should be considered as a possible cause of hypercalcemia in patients with non-aggressive breast cancer. We suggest that serum PTH should be determined in all BC patients with increased serum calcium concentration, especially in those with no evidence of metastatic disease.

Favorable predictive value of thyroid autoimmunity in high aggressive breast cancer

A high incidence of anti-thyroid antibodies (TAb) has been found in patients with breast cancer (BC). The aim of this study was to evaluate the prognostic value of TAb in a group of 47 women submitted to mastectomy for high malignancy degree BC. All patients were evaluated for thyroid disorders after breast surgery and before any anti-tumoral adjuvant therapy. Five yr after BC diagnosis 31/47 (65.9%) patients were alive (survivors group: SG) and 16/47 (34.1%) were dead (deaths group: DG). The overall prevalence of TAb was 15/47 (31.9%): 14/31 (45.1%) in SG and 1/16 (6.2%) in DG (p=0.008). Five-yr mortality was 15/32 (46.9%) in TAb- and 1/15 (6.7%) in TAb+ patients (p=0.01). Eight out of 47 (17.0%) patients had Hashimoto’s thyroiditis and 7 of them (87.5%) were in SG. Estrogen receptor (ER) was measured in 43/47 (91.5%) BC specimens. ER was detected in 19/30 (63.0%) patients in SG and 3/13 (23.1%) in DG (p=0.01). Five-yr mortality was 10/21 (47.6%) in ER- and 3/22 (13.6%) in ER+ patients (p=0.008). Absence of ER expression [odds ratio (OR) 6.54; p=0.006] and absence of TAb (OR 9.37; p=0.03) were related to a higher mortality rate. TAb were detected in 8/21 (38.1%) ER- and in 7/22 (31.8%) ER+ patients; no relation was found between ER expression and TAb positivity (p=ns). Patients with ER+ and TAb+ have a better prognosis and the absence of a significant relationship between these two parameters suggests an independent prognostic role in high malignancy degree BC women.

Relevance of estrogen and progesterone receptors enzyme immunoassay in malignant, benign and surrounding normal thyroid tissue

Several authors have demonstrated the presence of estrogen receptors (ER) and progesterone receptors (PR) in thyroid tissue, generally using dextran coated charcoal method (DCCA). The aim of this study was to measure ER and PR in thyroid specimens using an immunoenzymatic method, and to evaluate the meaning of different prevalence of ER and PR in malignant and benign thyroid disease, as compared with normal thyroid tissue. We have measured ER and PR in thyroid tissue from 28 benign and 20 malignant thyroid lesions, and in 38 samples of surrounding normal thyroid tissues. The sensitivity of ER-EIA and PR-EIA was 1.0 and 1.5 fmol/mg protein, respectively. In thyroid carcinoma the frequency of ER positivity (ER+) was 7/20 (35%); it was significantly higher in the surrounding normal tissue (15/20;71%) (p=0.03). In benign thyroid disease, the prevalence of ER+ was 11/28 (39%) and in the surrounding normal tissue it was 11/18 (61%) (p=not significant). PR+ was detected in 7/20 (35%) thyroid cancers and in 15/28 (53%) benign lesions without significant difference with the frequency detected in the surrounding normal tissues. ER and PR concentrations (mean±SD) in thyroid cancer was 2.2±2.2 and 2.2±2.9 respectively, similarly to that detected in benign thyroid disease and in normal tissue. The simultaneous presence of ER and PR (ER+PR+) was also evaluated. We have found that the frequency of ER+ PR+ was significantly higher in benign lesions (8/28; 28.6%) as compared with malignant samples (1/20; 5%) (p<0.05); the frequency of ER+PR+ was significantly higher in normal tissue surrounding the malignant lesions (9/20;45) (p=0.003). Our data indicate i) EIA method is appropriate to detect ER and PR in thyroid tissue. ii) The frequency of ER+ and ER+PR+ specimens is significantly higher in normal thyroid tissue than in pathologic tissues. This indicates that ER and PR immunoassays may be useful tools to evaluate the normal biological activity of thyroid cells.

High prevalence of breast cancer in patients with benign thyroid diseases.

Background: In patients with breast cancer (BC) a high prevalence of benign thyroid diseases (BTD) has been described, Hashimoto’s thyroiditis accounting to a large extent for this association. The aim of this study was to evaluate the prevalence of BC in a large group of patients with BTD. Patients: Clinical records of 622 consecutive patients with BTD were examined. BC prevalence in BTD patients was compared with BC frequency in general population living in the same country. Results: BC prevalence in patients with BTD (38/622; 6.11%) was significantly higher (p=0.0002) compared to BC frequency in general population (2.07%). When patients were divided according to the age of menopause, in females older than 49 yr BC frequency in BTD was significantly higher than in age-matched population (7.6 vs 3.3%; p=0.006), while in females aged 30–49 yr BC frequency in BTD was higher, but not statistically significantly, than in age-matched population (3.7 vs 0.5%; p=0.06). No significant difference in BC prevalence was found when patients were grouped according to the diagnosis of thyroid disorders: Graves’ disease, Hashimoto’s thyroiditis, nodular goiter associated or not associated with serum thyroid autoantibodies (TAb). No significant difference in BC frequency was observed between TAb+ (26/377; 6.9%) and TAb- (12/245; 4.9%) patients. The distribution of known risk factors for breast malignancies was similar in patients with or without BC. Conclusion: In patients with BTD the prevalence of BC is significantly higher than the expected, showing the usefulness of screening for breast malignancy of patients with BTD.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell‐mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross‐reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 104‐fold lower than in thyroid while in non‐BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 103‐fold lower than in thyroid. Full length TPO (Mr 105–110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC‐specific) identified.