The Wnt/β-catenin pathway drives increased cyclin D1 levels in lymph node metastasis in papillary thyroid cancer

We examined the expression of cyclin D1 in conjunction with β-catenin and the phosphorylated inactive form of glycogen synthase kinase 3β (GSK-3β) in benign, nonneoplastic thyroid tissue as well as papillary thyroid carcinoma primary tumors and nodal metastases. We aim to unravel the regulation of cyclin D1 and determine if this cell cycle protein is a useful biomarker for metastatic disease. It is clear that expression of cyclin D1 (P < .0001), β-catenin (P < .0001), and inactive form of GSK-3β (P < .0001) are significantly higher in papillary thyroid carcinoma primary tumors than in corresponding benign, nonneoplastic tissue thyroid specimens. Interestingly, β-catenin and cyclin D1 expressions in papillary thyroid carcinoma are correlated (P = .025), implying that β-catenin is a factor driving higher levels of cyclin D1 consistent with previous cell models linking Wnt/β-catenin signaling and cyclin D1 expression. Conversely, inactive form of GSK-3β expression does not correlate with cyclin D1 (P = .52) or β-catenin expression (P = .54). We also did not observe any relationship between tumor size and marker expression. Comparing papillary thyroid carcinoma primary tumors with or without nodal metastases, we did not see any differences in expression of inactive form of GSK-3β (P = .95), β-catenin (P = .14), or cyclin D1 (P = .46). However, in papillary thyroid carcinoma lymph node specimens, the up-regulation of cyclin D1 (P = .0083) was highly significant compared with primary tumors. pGSK-3β and β-catenin expression did not vary between primary tumors and nodal specimens. In conclusion, we have demonstrated that expression of cyclin D1 is linked to nodal metastases and that cyclin D1 levels are regulated by Wnt/β-catenin signaling. GSK pathway-mediated regulation of β-catenin or cyclin D1 expression does not appear operative in papillary thyroid carcinoma.     

Altered expression of desmocollin 3, desmoglein 3, and β-catenin in oral squamous cell carcinoma: correlation with lymph node metastasis and cell proliferation

Desmocollin 3 (Dsc3) and desmoglein 3 (Dsg3) are both transmembrane glycoproteins that belong to the cadherin family of calcium-dependent cell adhesion molecules. β-Catenin is a member of the cadherin–catenin complex that mediates homotypic cell–cell adhesion and is also an important molecule in the wnt signaling pathway. In this study, we examined the simultaneous expression level of Dsc3, Dsg3, and β-catenin in oral squamous cell carcinomas (OSCCs) and normal oral epithelia using immunohistochemistry. There was a significant correlation (p < 0.05) among the following variables in OSCCs: reduced or loss of expression of Dsc3, Dsg3, and β-catenin compared to normal oral epithelium, reduced or loss of expression of Dsc3 and histological grade (moderately or poorly differentiated), and reduced or loss of expression of β-catenin and lymph node metastasis. Furthermore, a positive correlation was found between reduced or loss of β-catenin staining and reduced or loss of Dsc3 staining in lymph node metastatic cancer tissue (r = 0.734, p < 0.05). These results suggest an abnormal expression of Dsc3, Dsg3, and β-catenin induced in the progression of oral carcinomas and that the Dsc3 expression level might be related to the regulation of β-catenin in lymph node metastasis and cell proliferation in OSCCs.     

Reduced HBME-1 immunoreactivity of papillary thyroid carcinoma and papillary thyroid carcinoma-related neoplastic lesions with Hürthle cell and/or apocrine-like changes

BACKGROUND: We have recently observed that Hürthle cell tumours and papillary thyroid carcinoma with tumour cells showing decapitation of luminal portion of the cytoplasm (apocrine-like changes) display negative or decreased immunoreactivity for HBME. The purpose of this study is to correlate papillary thyroid carcinoma with positive and negative immunoreactivity for HBME with the histopathological features. METHODS AND RESULTS: Two hundred and five thyroid neoplasms including carcinoma and adenomas were grouped into Hürthle cell tumours, tumours with or without some features of Hürthle cells, tumours with apocrine-like changes and adenomas with or without limited nuclear features of papillary thyroid carcinoma but not diagnostic for papillary thyroid carcinoma. All neoplasms were submitted for immunostaining with cytokeratin 19 (CK19) and HBME. Papillary thyroid carcinoma, follicular carcinoma and follicular adenoma that have areas of limited nuclear features but not diagnostic for papillary thyroid carcinoma showed stronger immunostaining for HBME than their respective counterparts with Hürthle cell changes. All Hürthle cell tumours showed negative to focal reactivity. This decrease of reactivity for HBME was proportional to the levels of Hürthle cell changes. In addition, focal to extensive apocrine-like changes were seen in most Hürthle cell neoplasms and rarely seen in non-Hürthle cell neoplasms. Apocrine-like changes abolished or decreased HBME immunoreactivity of papillary thyroid carcinoma and tumours with limited nuclear features. Immunostaining for cytokeratin AE3 was not affected by Hürthle cell or apocrine-like changes. CONCLUSIONS: All papillary thyroid carcinomas without Hürthle cell or apocrine-like differentiation are reactive for HBME. Hürthle cell tumours and tumours with Hürthle cell or apocrine-like changes show negative or focal reactivity for HBME. Except for this limitation, HBME is a sensitive marker for papillary thyroid carcinoma and tumours with limited nuclear features.     

Altered expression of estrogen receptor β2 is associated with different biological markers and clinicopathological factors in papillary thyroid cancer

Estrogen and estrogen receptor (ER)-α and -β play a role in the development and progression of thyroid cancer. ERβ2 is one major splicing variant of ERβ. In this study, we investigated the clinical significance of ERβ2 protein expression in the papillary thyroid carcinoma (PTC) lesion. ERβ2 expression was immunohisto-chemically examined in formalin-fixed, paraffin-embedded thyroid tissues from 106 patients with PTC by Elivision™ plus two-step system as previously described. The relationships between ERβ2 expression and clinicopathological/biological factors were then analyzed. ERβ2 protein was expressed in all the PTC patients studied. It was positively associated with Ki-67 expression in female PTC patients with advanced reproductive age (>45 years, in low-estrogen status) and with VEGF expression in male PTC patients with reproductive age (18~45 years, in low-estrogen status) (P=0.005 and P=0.044, respectively). There was no association between ERβ2 expression and tumor size, extrathyroidal extension and tumor-node-metastasis stage in PTC patients. In addition, ERβ2 expression was lower in female patients of reproductive age (18~45 years, in relatively high-estrogen status) with lymph node metastasis than that in those patients without lymph node metastasis (P=0.035). The present results suggest that the expression of ERβ2 in PTC is associated with the progression of the disease. Its potential effect may vary with different estrogen status. Further study will assess the underlying molecular mechanisms of ERβ2 in PTC.     

Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients’ age but not with tumour aggressiveness

A high prevalence of the BRAF^V600E somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF^V600E mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF^K601E mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF^V600E (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF^V600E (29.5 years). The BRAF (BRAF^V600E) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.V. Trovisco and P. Soares contributed equally to this workFundação para a Ciência e Tecnologia POCTI/FEDER (POCTI/NSE/48171/2002)     

Frequency and Distribution of DNA fragmentation in Hashimoto’s thyroiditis and development of papillary thyroid carcinoma

Downregulation of apoptosis and high expression of bcl-2 play an important role in the development of follicular lymphoma. However, little is known about apoptosis in thyroid disease, particularly with respect to the development of papillary carcinoma from Hashimoto’s thyroiditis. To study the early stages of cell death in various types of thyroid disease, surgical specimens from 31 patients including Hashimoto’s thyroiditis (HT,n=7), papillary carcinoma (PC,n=12), Hashimoto’s thyroiditis with papillary carcinoma (HTPC,n=5), and Graves’ disease (GD,n=7) were examined by anin situ nucleotidyl transferase assay (ISNTA), which detects DNA fragmentation. Control normal thyroid tissue (NT,n=7) was obtained from surgically resected papillary thyroid carcinomas sampled away from the primary tumor. An immunohistochemical (IHC) method was used to detect bcl-2 expression. Positive ISNTA nuclei in thyroid follicular cells or tumor cells per section were counted in all parenchymal areas, excluding areas of lymphocyte aggregates. The intensity of bcl-2 staining was graded on a scale of 1+ to 3+. The number of ISNTA-positive thyroid follicular cells was a significantly higher in HT compared to GD. In addition, there was significantly lower number of ISNTA positive non-neoplastic thyroid follicular cells in HTPC compared to HT alone. Strong expression of bcl-2 was found in all cases of GD and NT, but much less bcl-2 staining was seen in HT. There was moderate expression of bcl-2 in HTPC and PC. These findings suggest that (1) DNA fragmentation of the thyroid follicular cells plays an important role in the thyroid injury in HT but not in GD, (2) expression of bcl-2 may overcome the apoptosis in GD but not in HT, and (3) downregulation of DNA fragmentation of the follicular cells in Hashimoto’s thyroiditis associated with papillary carcinoma may suggest an important mechanism for tumor pathogenesis.     

Differentiated thyroid carcinomas with vascular invasion: a comparative study of follicular,Hürthle cell and papillary thyroid carcinoma

AIM: Non-medullary thyroid carcinomas arise from follicular cells. The purpose of this study is to correlate clinical and pathological properties of these tumours with the rate of distant metastasis from a series of thyroid tumours excised at one institution. METHODS: A total of 311 non-medullary thyroid tumours were identified and divided into: 29 follicular carcinoma (FC), 12 Hürthle cell carcinoma (HC), 13 Hürthle cell papillary thyroid carcinoma (HPTC) with vascular invasion (VI), 32 papillary thyroid carcinoma (PTC) with VI and 225 PTC without VI. The mean follow-up was 6.5 years with a range of 1-17 years. The tumours were histologically subdivided into minimal or wide invasion for FC and HC and focal or extensive invasion for PTC and HPTC, and stratified according to status of VI. RESULTS: The rate of distant metastasis was similar for FC, malignant Hürthle cell tumours and PTC with VI, and increased with extent of invasion. VI was seen in 12% of all PTC and 0% of HPTC in this study. PTC without VI were associated with a much lower potential of distant metastasis, were smaller in size and occurred in patients of younger age than PTC with VI. In addition, there was a tendency for increased potential for distant metastases with increased tumour size and patient age for all groups of tumours in the study. Patient age and tumour size appeared to play a smaller role than that of VI in predicting distant metastasis. CONCLUSIONS: Our study suggests that the rate of distant metastasis relates to VI, patient age and tumour size, regardless of Hürthle cell, FC or PTC differentiation. PTC of large size, and in patients older than 45 years, have a high propensity for vascular invasion.     

HIF-1α up-regulation is associated with adverse clinicopathological and biological factors in neuroblastomas

Dungwa J V, Hunt L P & Ramani P (2012) Histopathology  61, 417–427 HIF‐1α up‐regulation is associated with adverse clinicopathological and biological factors in neuroblastomas Aims: To study the expression of hypoxia‐inducible factor 1α (HIF‐1α) in clinical neuroblastoma (NB) samples and its association with the clinicopathological features, biological features and survival of NB patients. Methods and results: Immunohistochemistry indicated that elevated HIF‐1α expression was present in 30 of 90 (33%) NBs. This expression was correlated significantly and positively with higher clinical stage (P = 0.002), ≥18 months of age at presentation (P = 0.020), high‐risk group (P = 0.005), unfavourable pathology (P = 0.002), MYCN amplification (P < 0.001), 1p deletion (P = 0.004) and 17q gain (P = 0.002). Enzyme‐linked immunosorbent assays showed that total HIF‐1α protein was significantly higher in NBs of patients with all examined adverse prognostic factors except for age. Univariate survival analysis revealed that higher‐than‐median HIF‐1α total protein levels were associated significantly with a decrease in event‐free survival (EFS) (P = 0.017), but not in overall survival (OS) (P = 0.12). HIF‐1α immunoexpression by ≥10% of tumour cells was associated significantly with decreased OS and EFS (P = 0.002 and P = 0.004, respectively), but not in multivariate analysis after adjusting for the high‐risk group (P = 0.16 and P = 0.19, respectively). Conclusions: HIF‐1α was increased significantly in patients with NB associated with unfavourable characteristics. HIF‐1α is a prognostic indicator of poor OS and EFS and defines subgroups of NBs with aggressive clinical behaviour.     

The role of cytokeratin 19 in the differential diagnosis of true papillary carcinoma of thyroid and papillary carcinoma-like changes in Graves’ disease

Graves’ disease is an autoimmune disease predominantly seen in females. All types of thyroid cancers may co-exist with Graves’ disease but papillary carcinoma is the most frequent. Vesicular nuclei, nuclear grooves, and papillary formations that may be seen in Graves’ disease may lead the pathologist to an overdiagnosis of papillary carcinoma. The differential diagnosis between a true papillary carcinoma and foci mimicking papillary carcinoma in Graves’ disease may be challenging by light microscopic features only. This study is designed to determine whether CK19 is effective in the discrimination between the true papillary carcinoma of thyroid and foci resembling papillary carcinoma in Graves’ disease. Twenty-five cases with papillary carcinoma and 25 cases with Graves’ disease containing foci resembling papillary carcinoma were included in the study. All 25 cases with papillary carcinoma stained positive with CK19, whereas only six of 25 cases with Graves’ disease showed weak staining, and the remaining 19 cases were completely negative. It is known that CK19 may show faint staining in benign thyroid lesions such as adenomas. Staining pattern with CK19 together with histopathological findings may be helpful in the differential diagnosis between foci mimicking papillary carcinoma and true papillary carcinoma in Graves’ disease.     

Expression of p63 in papillary thyroid carcinoma and in Hashimoto's thyroiditis: a pathobiologic link?

p63 proteins are p53 homologs that are postulated to regulate squamous stem cell commitment. An immunohistochemical survey of p63 expression in normal thyroid and in reactive, neoplastic, and inflammatory thyroid disorders was performed. Sections from routinely fixed and processed archival thyroidectomy specimens were pretreated with citric acid, pH 6.0, for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as a chromagen, and then were counterstained with hematoxylin. The results showed that p63 expression was negative in normal thyroid tissue, nodular goiters, and oncocytic follicular adenomas. Positivity was rare and weak in follicular adenomas. p63-positive foci were commonly found in Hashimoto's thyroiditis (1 or more foci in 78.8% of cases), but rare in Graves' disease. Twenty-seven of 33 papillary thyroid carcinomas (81.8%) displayed p63-positive foci. Staining was uncommon in follicular carcinomas and rare in medullary carcinomas. One case of insular carcinoma was p63-positive. All squamoid structures were p63-positive; p63-positive structures morphologically consistent with solid cell nests were also identified. Based on the results of this study, we conclude that p63 is commonly expressed in papillary thyroid carcinoma and in Hashimoto's thyroiditis. Given the debated association of papillary thyroid carcinoma with Hashimoto's thyroiditis, it is possible that p63 expression may be a potential pathobiologic link between the two disorders. The finding of p63 in benign squamoid nests supports a possible interrelationship between these structures and both Hashimoto's thyroiditis and papillary carcinoma. The high percentage of papillary carcinomas with p63-positive foci appears to distinguish papillary carcinoma from other neoplasms originating in the thyroid.     

High expression of REGγ is associated with metastasis and poor prognosis of patients with breast cancer

REGgamma (REGγ) has been recently found in several types of human cancer, however, its clinical significance in metastasis and prognosis of breast cancer remains unknown. In this study, immunohistochemical staining and western blot analysis were performed to evaluate REGγ expression in both mouse and human breast cancer specimens. We found that in MMTV-PyMT mice, 14 out of 20 (70%) mouse mammary carcinomas were REGγ positive, which was significantly higher than control (0/20, 0%, P < 0.001) and lower than metastatic lung tumour (20/20, 100%, P = 0.027). Further investigation for REGγ expression in 136 human breast cancer tissues with the paired peritumoural normal breast tissues and 140 breast benign disease tissue samples showed that REGγ was undetectable in normal breast tissues and nonmetastatic axillary lymph nodes (ALNs), whereas 111 out of 136 (81.6%) breast cancer tissue samples were REGγ positive, which was significantly higher than breast benign disease tissues (9/140, 6.4%, P < 0.001) and lower than metastatic ALNs (116/116, 100%, P < 0.001). The 5-year disease-free and overall survivals of patients with negative/low level of REGγ were significantly higher than those of patients with high level of REGγ (P < 0.05). Cox regression analyses further indicated that REGγ could serve as a novel independent prognostic factor for breast cancer (OR = 4.369, P = 0.008). Our results suggest that the high expression of REGγ might predict metastasis and poor prognosis in breast cancer.     

Prognostic value of c-FLIPL/s,HIF-1α, and NF-κβ in stage II and III rectal cancer

Locally advanced rectal cancer (LARC) is associated with a 25 % rate of metastases. The prognostic and predictive relevances of the expression of five proteins (c-FLIP_L/s, HIF-1α, β-catenin, p65, and p105/p50 NF-κβ) were assessed. This is a retrospective study. From 1998 to 2009, 152 patients with stage II/III rectal cancer were treated with radio-chemotherapy. TMAs constructed with tumor and normal tissue from the diagnostic endoscopic biopsy and the surgical specimen after chemoradiotherapy were subjected to immunohistochemical (IHC) analysis. Results were correlated with clinical and pathological data, including progression-free survival (PFS). Four different IHC conditions were independent prognostic parameters for PFS: (1) cytoplasmic c-FLIP_L/s (p?=?0.007), (2) nuclear HIF-1α (p?=?0.020), (3) a change in the cytoplasmic p65 between the diagnostic biopsy and the post-treatment specimen (p?=?0.004), and (4) a change in the cytoplasmic c-FLIP_L/s between the diagnostic biopsy and the post-treatment specimen (p?=?0.021). Three different protein expression profiles, combining biomarkers, showed prognostic significance. IHC evaluation of these biomarkers in our three protein expression profiles may help to identify patients with worse prognosis and design more effective therapeutic strategies to personalize the treatment of rectal cancer.     

Elevated expression of integrin αv and β5 subunit in laryngeal squamous-cell carcinoma associated with lymphatic metastasis and angiogenesis

In the past several years, the αv integrin subfamily has been repeatedly found to be involved in tumor progression and angiogenesis. The aim of this study was to investigate the expression of the integrin αv subfamily in laryngeal squamous cell carcinoma (LSCC), and to correlate the expression rate with tumor biological behavior and angiogenesis of the LSCC. The integrin αv subfamily, including αv, β1, β3, β5, β6 and β8 subunits, was immunohistochemically found to be expressed in 64 patients with LSCC, and we analyzed the relationship between the expression rate and the clinicopathological stage of this cancer. Immunohistochemical staining for CD105 was carried out in the same group of the patients. The intratumoral microvessel density (IMVD) of the LSCC was calculated by CD105 staining, and the correlation between the IMVD and αv subfamily expression was discussed. The results showed that all members of the integrin αv subfamily could be detected in the LSCC. The expression rate of integrin αv and β5 subunits in primary cancer was significantly higher than in normal tissue, and their expression rate in the group with lymphatic metastasis was significantly higher than in the group without metastasis. The IMVD of the group with positive expression of αv and β5 subunits was significantly higher than in the group with negative expression, but there were no significant effects on the β1, β3, β6 and β8 subunits in these biological processes. In conclusion, the expressions of integrin αv and β5 subunits were significantly associated with lymphatic metastasis and angiogenesis of the LSCC. Among the members of integrin αv subfamily, integrin αvβ5 might play an important role in invasion and metastases of the LSCC, and it may become a valuable marker for the evolution of the LSCC.