An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

An Immunohistochemical Study of Leu 7 and PCNA Expression in Thyroid Neoplasms

Monoclonal Antibody (MoAb) HNK, or anti-leu-7, is reactive with several neuroendocrine and nonneuroendocrine tumors. The aim of this study is to examine anti-leu-7 reactivity in thyroid neoplasms and its relationship to cellular proliferation as determined by anti-PCNA reactivity. The expression of anti-leu-7 in 56 thyroid neoplasms (24 papillary carcinomas, 14 follicular carcinomas, two medullary carcinomas and 16 follicular adenomas) was examined immunohistochemically. Papillary and follicular thyroid carcinomas reacted with anti-leu-7 in a membranous and cytoplasmic pattern in 88% and 93% of cases, respectively. The adjacent benign tissues were nonreactive. Only eight cases diagnosed as follicular adenomas were reactive with anti-leu-7. Furthermore, the mean proliferative index (PI), as measured by the percentage of nuclei immunoreactive with anti-PCNA, was greater than 30% in all thyroid neoplasms reactive with anti-leu-7. The PI was 58% for papillary carcinomas and 68% and 48% for follicular carcinomas, and follicular adenomas, respectively. Lesions originally classified as follicular adenomas that were nonreactive with anti-leu-7 had a PI of 24% and were reclassified as hyperplastic nodules. These data suggest that anti-leu-7 may be useful for characterizing thyroid neoplasia.     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Deletions of the long arm of chromosome 10 in progression of follicular thyroid tumors

Previous studies of follicular thyroid tumors have shown loss of heterozygosity (LOH) on the short arm of chromosome 3 in carcinomas, and on chromosome 10 in atypical adenomas and carcinomas, but not in common adenomas. We studied LOH on these chromosomal arms in 15 follicular thyroid carcinomas, 19 atypical follicular adenomas and 6 anaplastic (undifferentiated) carcinomas. Deletion mapping of chromosome 10 using 15 polymorphic markers showed that 15 (37.5%) of the tumors displayed LOH somewhere along the long arm. Thirteen of these tumors showed deletions involving the telomeric part of chromosome 10q, distal to D1OS 187. LOH on chromosome 3p was found in 8 (20%) cases. Seven of these also showed LOH on chromosome 10q. In eight cases LOH was seen on chromosome 10q but not 3p. In comparison, the retinoblastoma gene locus at chromosome 13q showed LOH in 22% of the tumors. Most of these also had deletions on chromosome 10q. The results indicate that a region at the telomeric part of 10q may be involved in progression of follicular thyroid tumors.     

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.     

Primary unit for statistical analysis in morphometry: patient or cell?

In a series of 16 oxyphilic follicular neoplasms of the thyroid (8 adenomas and 8 carcinomas), three different approaches for the analysis of morphometric data were evaluated. It was shown that the statistical design of morphometric studies is by nature nested due to subsampling of cells within each patient. Therefore, the most appropriate analysis would be to account for this hierarchical structure. However, related statistical methods are not at present well established, especially as far as classification rules are concerned. Therefore, the nested design is converted into the simple factorial one by considering only one kind of statistical unit - either patients or cells. The results of the study presented indicate that ignoring the patient as unit of analysis leads to a substantial error in statistical output, regardless of the particular procedure applied. Moreover, the size of the error can be neither diminished nor controlled. Choosing patients as primary units assures accurate results and also has an advantage of gaining some additional information by calculating several distributional estimates in each patient. However, this approach often requires a reduction of dimensions and, furthermore, is not encouraged in certain fields of quantitative cytology. Advantages and disadvantages of all approaches have been summarized and practical recommendations for their use have been worked out.     

Neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical expression in follicular cell-derived thyroid tumors: a novel diagnostic tool?

Discrimination of follicular cell-derived benign and malignant tumors of the thyroid is one of the major problems encountered in surgical pathology. In the present study, we evaluated the immunohistochemical expression of NGAL, an iron-binding protein involved in the infiltrative potential of cancer cells, in a cohort of tumors including 8 follicular adenomas (FA), 2 Hurthle cell adenomas (HA), 2 atypical adenomas (AA), 8 minimally invasive follicular carcinomas (MIFC), 9 widely invasive follicular carcinomas (WIFC), 3 Hurthle cell carcinomas (HC) and 8 papillary carcinomas (PC) with 5 follicular-variant PC (FVPC) and 3 not otherwise specified (PC-NOS). Our goal was to test whether evaluation of NGAL immunoexpression may be of use in the differential diagnosis of benign and malignant thyroid neoplasias. 92% of benign tumors (specificity) were negative for NGAL, whereby NGAL immuno-expression was found in 82% (sensitivity) of malignant tumors, and, specifically, in 100% of MIFC, in 87% of WIFC, in 100% of HC, in 80% of FVPC. None of the PC-NOS displayed NGAL staining. When only tumors with a follicular architecture were considered, NGAL specificity for malignant lesions was 92%; sensitivity, positive predictive value and negative predictive value were 92%, 96% and 85%. Diagnostic accuracy of NGAL expression in the differential diagnosis between benign and malignant follicular tumors was 92%. In conclusion, NGAL protein seems to represent a marker of malignant follicular cell-derived thyroid tumors, and especially of those with follicular architecture. Hence assessment of its expression might be of use with respect to differential diagnosis from follicular benign neoplasias.     

Comparison of CD15, galectin-3 and HBME-1 expression in follicular thyroid neoplasms

INTRODUCTION: Estimation of malignancy in thyroid follicular neoplasms is a common diagnostic problem, thus revealing of differences in expression of some antigens in both benign and malignant lesions seems to be essential. The aim of this study is to evaluate the immunohistochemical expression of CD15, galectin-3 and HBME-1 in follicular adenomas and carcinomas. MATERIAL AND METHODS: Samples of 38 follicular adenomas (23 "classical", 5 with intracapsular invasion, 10 oncocytic) and 15 follicular carcinomas (9 "classic", 6 oncocytic) were stained immunohistochemically with anti-CD15, galectin-3 and HBME-1. RESULTS: In the whole group we found statistically significant differences in CD15 expression between follicular adenomas and carcinomas. "Classic" follicular carcinomas (without oncocytic tumors) showed stronger CD15 and HBME- 1 expression than "classic" adenomas. Adenomas with intracapsular invasion differed from "classic" adenomas only in HBME-1 expression. In oncocytic tumors the expression of examined antigens was similar. CONCLUSIONS: 1. In the group of nonoxyphilic tumors positive reaction with HBME-1 was more common in adenomas with intracapsular invasion and carcinomas, but positive reaction with anti-CD15--only in carcinomas. We suggest that reactivity with these antibodies could mark malignancy. 2. Oncocytic tumors had similar expression of CD15 and HBME-1 and galectin-3.     

Expression of human pendrin in diseased thyroids.

We examined pendrin expression in various diseased thyroid tissues by immunohistochemistry (IHC) using antiserum raised against human pendrin and by real-time quantitative RT-PCR. In normal thyroids the antiserum reacted with the apical membrane of follicular cells and its immunoreactivity was faint. In Graves' thyroids, the IHC expression of pendrin appeared in a pattern similar to that of normal thyroids but it was more extensive and stronger, especially in areas showing marked proliferation of follicular cells. The immunoreactivities of pendrin in nodular goiters varied from case to case. In follicular adenomas, pendrin was localized in the follicle-forming parts of the tumor but was negative in trabecular parts. Pendrin was negative in all follicular carcinomas, papillary carcinomas, and in one case of medullary carcinoma. In quantitive mRNA analysis, the relative values of pendrin mRNA were significantly low in papillary carcinoma (p<0.01), whereas the values in other diseased thyroids were not significantly different from those in normal thyroids. These results suggest that pendrin may play a role in thyroid hormone production as the apical porter of chloride/iodide and investigation of pendrin leads to a better understanding of functional aspects of the iodine transportation system in thyroid diseases.     

Necrotic Debris In Thyroid Aspirates: A Feature of Follicular Carcinoma of the Thyroid

Fine needle aspirates from 44 follicular thyroid tumours (30 adenomas, 14 carcinomas) have been studied. All aspirates contained neoplastic cells in follicular and trabecular arrangements. The individual tumour cells showed varying degrees of anisonucleosis and nuclear pleomorphism. Colloid was scanty or absent from all smears. Granular or filamentous necrotic material was observed in both biopsies and smears from one moderately and two poorly differentiated follicular carcinomas, but in none of the adenomas. This suggests that necrotic debris may be a feature of follicular carcinoma of the thyroid.     

Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly,but the difference is not useful in diagnosis of individual cases

T. Rozkos, A. Ryska, J. Cap, F. Sobande and J. Laco
Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases Introduction: The aim of our study was to search for new, readily available and statistically reliable cytological markers for differentiating benign and malignant follicular thyroid neoplasms pre‐operatively. Methods: Cohesiveness of tumour cells in cytology slides from a series of 58 follicular tumours diagnosed between 1998 and 2004 inclusive was studied, including 48 follicular adenomas, and eight minimally invasive and two widely invasive follicular carcinomas. Photomicrographs of the cytology slides were taken and the digital images were analysed using computer image analysis software. We evaluated the relative proportions of cells arranged in groups of various sizes. The cohesiveness of the cells in cytological smears was then correlated with the immunohistochemical expression of E‐cadherin in corresponding histological slides. Results: Cases from 15 men (26%) and 43 women (74%) with a mean age of 50 years (range, 19–79) were analysed. In follicular adenomas and carcinomas, respectively, isolated cells were seen in 16.8% and 24.7% (P = 0.028), groups of two to five cells in 9.7% and 11.5% (P = 0.145) and groups of more than five cells in 73.5% and 63.8% (P = 0.041). The mean cell count in groups with more than five cells was 46.5 and 27.0 in adenomas and carcinomas, respectively (P < 0.001). Cell cohesiveness, either as percentage of cells in groups of more than five (R² = 0.026) or as mean cell count per group of more than five (R² = 0.005), was not found to be dependent on the expression of E‐cadherin. Using a threshold of 13% isolated tumour cells in cytological smears, follicular adenomas and carcinomas could be distinguished with 90% sensitivity and 41% specificity. Conclusions: Although we demonstrated a statistically significant difference in cell cohesion between follicular adenomas and carcinomas, these could not be distinguished in the clinical setting by evaluation of the percentage of isolated cells in cytological smears because the specificity was too low. The absence of correlation of cellular cohesiveness with E‐cadherin expression indicates that other factors are probably responsible for the loss of cohesiveness observed in follicular thyroid malignancy.     

Effect of thyroid-stimulating hormone and norepinephrine on cyclic AMP levels in human normal thyroids and human thyroid tumors

The effect of TSH (100mU/ml) and norepinephrine (100 muM) on the cyclic AMP levels was studied in 10 human normal tissues, 10 thyroid adenomas and 4 thyroid carcinomas (3 papillary and 1 follicular). Normal tissues responded to TSH with a marked elevation of the cyclic AMP level. Response patterns of 10 thyroid adenomas to TSH were variable; the patterns of 6 cases resembled those of normal tissues, 3 responded mildly, and one had no response to TSH. Thyroid carcinomas had a higher basal level of cyclic AMP than those of normal tissues, although they responded only slightly to TSH. Two among 4 thyroid carcinomas had no response to TSH. Norepinephrine stimulated the accumulation of cyclic AMP in 4 thyroid adenomas and 3 thyroid carcinomas, while it had little effect on normal tissues. Responses to norepinephrine was observed only in thyroid tumors, although they had low response to TSH. It is suggested from these results that tumor cells originating from thyroid follicular cells have a modified response to hormones due to neoplastic alterations.     

Evaluation of HMFG2 and thyroglobulin in the diagnosis of thyroid fine needle aspiration (FNA)

In order to appraise the usefulness of HMFG₂ and thyroglobulin (Tg) as specific markers for the diagnosis of thyroid disease, we studied 63 FNA smears. Cases tested included 30 benign (nine colloid goitres, six cases of Hashimoto's thyroiditis, six Hürthle cell adenomas, nine follicular adenomas) and 33 malignant lesions (nine follicular carcinomas, 12 papillary carcinomas, nine anaplastic carcinomas, three medullary carcinomas). All cases with malignant lesions except the anaplastic carcinomas were positive for HMFG₂. Immunoreactive cells to HMFG₂ were also found in 15 adenomas out of 30 benign cases. Positive Tg reaction was found in benign and malignant thyroid lesions, except six cases of Hashimoto's thyroiditis, nine anaplastic and three medullary carcinomas. The results obtained indicate that morphology paired with immunocytochemistry can usually depict a more specific profile of thyroid lesions for better evaluation of the pathology.     

Computerized morphometric study on fine needle aspirates of cellular follicular lesions of the thyroid

OBJECTIVE: To investigate the value of computerized nuclear morphometry in the differential diagnosis of cellular follicular lesions of the thyroid cytologically diagnosed on fine needle aspiration (FNA) smears. STUDY DESIGN: Sixty cases of FNA thyroid smears were cytologically diagnosed and classified as follows: 30 cases of follicular carcinoma, 20 cases of cellular hyperplastic nodules and 10 cases of follicular adenoma. Using an image analysis system, two morphometric variables, nuclear area and major axis length of the nucleus, were measured for each case. RESULTS: For both nuclear morphometric variables, statistical differences were found between carcinomas and hyperplastic nodules as well as between carcinomas and adenomas. No statistical differences were found between the nuclear variables in either hyperplastic nodules or adenomas. CONCLUSION: The results confirm the aim of our study, to establish nuclear morphometry by computerized image analysis as an additional tool in the differential diagnosis of thyroid follicular lesions cytologically diagnosed on FNA smears.     

Fine needle aspiration diagnosis of hyperplastic and neoplastic follicular nodules of the thyroid. A morphometric study.

The differentiation of hyperplastic nodules, follicular adenomas and follicular carcinomas from fine needle aspiration (FNA) cytology smears may be difficult. To better define the diagnostic criteria, we studied the morphometric parameters of nuclear area (NA), nuclear:cytoplasmic ratio and nuclear roundness (NR) in single cells and cell aggregates. In addition, we quantitated the percentage of touching or overlapping nuclei (NO) and the percentage of extent of nuclear area of overlap (NAO) in cellular aggregates. We measured cellular samples from FNA aspirates obtained from 20 hyperplastic nodules, 21 follicular adenomas, 5 encapsulated follicular carcinomas and 22 invasive follicular carcinomas, all of which were subsequently confirmed by histologic examination. Cellular aggregates provided the maximum diagnostic information. Stepwise discriminant analysis revealed that nuclear size, nuclear roundness and the percentage NAO allow optimum differentiation of hyperplasia, adenomas and carcinomas. Clearly, all of the poorly differentiated carcinomas (large NA, low NR, high NO and NAO) could be reliably diagnosed. Discriminant analysis allowed the differentiation of carcinoma from adenoma in 20/22 carcinomas (91%) and all 21 adenomas (although 2 adenomas were called hyperplasias and 3 hyperplasias were called adenomas).     

Computer analysis of chromatin arrangement and nuclear texture in follicular thyroid tumours

The differentiation of the thyroid glands follicular neoplasias into adenomas and carcinomas is currently done using the histological criteria recommended by WHO. This pilot study of 10 human follicular carcinomas and 10 folliculars adenomas demonstrates the possibility of a cytological classification using digital picture processing of high resolution cell images. Giemsa stained paraplast sections were scanned with a Colour-TV-camera, different channels were used with respect to staining and analyzing methods and computed with an image processing system. The computer aided cytophotometric methods detected significant differences in the chromatin arrangement and structure.     

Mitochondrial D-Loop instability in thyroid tumours is not a marker of malignancy

Despite the numerous studies describing a high frequency of mitochondrial DNA (mtDNA) somatic mutations in many types of human primary tumors the mechanisms that generate such mutations and the role of mtDNA mutations in tumor development remain unclear. We present the results obtained in the study of mtDNA displacement-loop (D-Loop) region in a series of 66 thyroid tumors, and respective adjacent parenchyma, including benign (adenomas, n=30) and malignant tumors (follicular carcinomas, n=17 and papillary carcinomas, n=19). Three repetitive regions were analyzed [two mononucleotide repetitive (D310 and D568) and one dinucleotide repetitive (D514)]. Thirty-two (48.5%) of the 66 tumors [15/30 (50.0%) adenomas, 8/17 (47.1%) follicular carcinomas and 9/19 (47.4%) papillary carcinomas] harbored somatic insertions in D-Loop repetitive regions. Twenty (30.3%) of the 66 tumors [12/30 (40%) adenomas, 3/17 (17.6%) follicular carcinomas and 5/19 (26.3%) papillary carcinomas] harbored somatic insertions at the D310 mononucleotide repeat. Three (4.6%) of the 66 tumors [1/30 (3.3%) adenomas and 2/17 (11.8%) follicular carcinomas] harbored somatic insertions at the D568 mononucleotide repeat. Fifteen (22.7%) of the 66 tumors [3/30 (10.0%) adenomas, 5/17 (29.4%) follicular carcinomas and 7/19 (36.8%) papillary carcinomas] harbored somatic insertions at the D514 dinucleotide repeat. Five (7.6%) of the 66 tumors [1/30 (3.3%) adenomas, 1/17 (5.9%) follicular carcinomas and 2/19 (10.5%) papillary carcinomas] harbored somatic insertions in more than one region, and in one of them (a carcinoma) alterations were detected in the three regions. We conclude that mutations in the mtDNA D-Loop region are frequent in benign and malignant thyroid tumors and cannot be considered a marker of malignancy. Our study shows, furthermore, two repetitive regions (D310 and D514) that appear to be susceptible to mutation in thyroid tumors.