Overexpression of PDE5 is observed in certain human cancers, but PDE5 expression in well-differentiated thyroid carcinoma (WDTC) is unknown. We therefore examined PDE5 expression and its relationship with the clinicopathological features of WDTC. Real-time qPCR and Western blotting were performed to analyze the expression of PDE5 mRNA and protein in paired WDTC tumor and adjacent nontumor tissues. Immunohistochemistry was used to analyze the expression of PDE5 in paraffin-embedded tissues obtained from 103 cases of WDTC. Statistical analyses were performed to examine the correlation between PDE5 expression and clinicopathological features. The expression of PDE5 mRNA and protein was upregulated in WDTC lesions compared to their paired noncancerous tissues. The expression of PDE5 was significantly correlated with age (
Thyroid cancer (TC) is the most common endocrine malignancy; the incidence of which has been steadily increasing over the last three decades [
PDE5 is a cytoplasmic enzyme that regulates the levels of cGMP by hydrolyzing it [
Paraffin-embedded samples were obtained from 103 patients diagnosed with WDTC who underwent surgery between July 2009 and December 2015 at the Department of Breast and Thyroid Surgery and Department of Pathology, the First Affiliated Hospital of Sun Yat-sen University. All the patients’ ages ranged from 7 to 81 years (median = 50). The patient group included 19 and 57 patients who were diagnosed with WDTC with distant metastasis and lymph node metastasis, respectively, confirmed by imaging or pathological examination, and 27 patients with primary WDTC. Clinicopathological information such as age, tumor size, lymph node status, and histological type was obtained by reviewing their medical records and pathology reports.
Four matched pairs of fresh WDTC tumor and adjacent noncancerous tissue samples (at least 2 cm away from the margin of the tumor tissue) were also obtained for determining the mRNA and protein levels of PDE5. Histopathological analysis with hematoxylin-eosin staining of frozen sections confirmed that the tumor tissues comprised >70% cancer cells without necrosis and that no cancer lesions were present in the noncancerous tissues.
The study was approved by the Medical Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Informed consent was obtained from all the patients for the use of their clinical specimens.
We followed the methods of Zhang et al. [
Four matched pairs of WDTC tumor tissues and adjacent nontumor tissues were harvested and lysed in 50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mM sodium orthovanadate, 10
Immunohistochemical staining was carried out on formalin-fixed, paraffin-embedded sections (4
The extent of PDE5 immunostaining was evaluated independently by two pathologists who were blinded to the survival outcomes of the participants. They determined the proportion of positively stained tumor cells (staining area) and the intensity of staining. Staining intensity was scored as follows: 0, no staining; 1, weak staining (light yellow color); 2, moderate staining (yellow brown color); and 3, strong staining (brown color). The proportion of positively stained tumor cells was scored as follows: 0, no positively stained tumor cells; 1, <5% positively stained tumor cells; 2, 6–25% positively stained tumor cells; 3, 26–50% positively stained tumor cells; and 4, >50% positively stained tumor cells. A modified immunoreactivity scoring method was used to evaluate the immunostaining results by multiplying staining intensity with the staining area (staining index, SI) as previously described [
Chi-square test and logistic regression analysis were used for analysis of differences between the groups. Differences were considered significant when the
All cases were risk stratified on the basis of the clinical and pathological features in accordance with the 2015 American Thyroid Association Risk Stratification System Guidelines, TNM staging, GAMES, and Mayo Clinic Scoring System (metastases, age, complete resection, invasion, size, or MACIS).
Real-time qPCR and Western blot analysis showed that the expression levels of PDE5 mRNA and protein, respectively, were markedly higher in all four WDTC lesions than in their paired adjacent noncancerous tissues (Figures
Upregulation of PDE5 expression in well-differentiated thyroid carcinoma (WDTC). (a, b, c) Expression of PDE5 in WDTCs as determined by immunohistochemistry. Strong staining (A1, ×10; A2, ×40) and negative staining (B1, ×10; B2, ×40) can be observed. A WDTC sample (C1, ×10) with strong PDE5 staining in the carcinoma tissues (C2, ×40) and normal thyroid follicular epithelial cells with weak staining (C3, ×40). (d and e) Western blots show the expression levels of the PDE5 protein in four WDTC tumor tissues (T) and their paired adjacent noncancerous tissues (ANT);
Clinicopathological features of 4 thyroid papillary carcinoma cases.
Age | Tumor size | Lymph node status | Distant metastasis status | |
---|---|---|---|---|
Case 1 | 30 | 2 cm | Positive | Negative |
Case 2 | 5 | 4 cm | Positive | Negative |
Case 3 | 43 | 1.5 cm | Negative | Negative |
Case 4 | 54 | Multiple | Positive | Negative |
Furthermore, the immunohistochemical staining patterns revealed that PDE5 was mainly localized in the cytoplasm of the tumor cells. No staining or weak cytoplasmic staining was detectable in the follicular epithelial cells of the adjacent noncancerous thyroid tissue (Figure
The immunohistochemical staining results showed that 52 (50.5%) of the 103 WDTC patients had a high level of PDE5 expression (SI > 6), whereas 51 (49.5%) had a low level of PDE5 expression (SI ≤ 6).
The correlation between PDE5 protein expression and the clinicopathologic features of WDTC is shown in Table
Correlation between PDE5 expression and clinicopathological features.
Clinicopathological parameters | Total | The level of PDE5 expression |
| ||
---|---|---|---|---|---|
High (%) | Low (%) | ||||
Gender | Male | 36 | 19 (36.5) | 17 (33.3) | 0.733 |
Female | 67 | 33 (63.5) | 34 (66.7) | ||
Age (years) | <45 | 67 | 39 (75.0) | 28 (54.9) |
|
≥45 | 36 | 13 (25.0) | 23 (45.1) | ||
Histological type | PTC | 100 | 51 (98.1) | 49 (96.1) | 0.618 |
FTC | 3 | 1 (1.9) | 2 (3.9) | ||
Focus | Single | 58 | 30 (57.7) | 28 (54.9) | 0.774 |
Multiple | 43 | 21 (42.3) | 22 (45.1) | ||
Invasion of capsule or ETE | No | 89 | 45 (86.5) | 44 (86.3) | 0.969 |
Yes | 14 | 7 (13.5) | 7 (13.7) | ||
Distant metastasis | M0 | 84 | 47 (90.4) | 37 (72.5) |
|
M1 | 19 | 5 (9.6) | 14 (27.5) | ||
LN involvement | N0 | 46 | 16 (30.8) | 30 (58.8) |
|
N1 | 57 | 36 (69.2) | 21 (41.2) | OR = 2.79 |
ETE: extracapsular extension; PTC: papillary thyroid cancer; FTC: follicular thyroid cancer.
PDE5 expression and lymph node involvement in patients aged ≥45 years.
LN involvement | The level of PDE5 expression |
| |
---|---|---|---|
High (%) | Low (%) | ||
N0 | 1 (7.7) | 13 (56.5) |
|
N1 | 12 (92.3) | 10 (43.5) | OR = 15.60 |
This study found a significant relationship between PDE5 expression and lymph node involvement in WDTC. PDE5 may be a potential marker of regional lymph node involvement and disease prognosis in WDTC. The results would be valuable from the viewpoint of future diagnosis.
At variance with cAMP and its pathway, widely investigated as the main mediator of the TSH effects on thyroid cells, previous studies are widely focused on the TSH effect regulation of cAMP and its pathway on thyroid cells and involved in several thyroid diseases [
The role of PDE5 in cancer pathogenesis and prognosis is uncertain and complex. Peak et al. [
Cervical lymph node metastasis has been reported to be present in 20–90% of patients with WDTC [
In summary, our study investigated PDE5 expression in WDTC. We found high PDE5 expression in patients with lymph node metastasis; moreover, lymph node metastasis was associated with disease recurrence, treatment failure, and morbidity. Thus, the PDE5 expression level may in the future help identify patients who are required of aggressive treatment.
The authors declare that they have no potential conflicts of interest.
Ning Zhang and Zeng Fang contributed in conducting immunohistochemistry staining, clinical data collection, and statistical analysis. Qiufang Li and Kebing Wang contributed in conducting Western blot and real-time qPCR. Zeng Fang and Songqi Li contributed in collecting the fresh tissue samples. Wen Li and Shenming Wang supervised the project and carried out experimental design and preparation of the final manuscript. All authors read and approved the final manuscript. Ning Zhang and Zeng Fang contributed equally to this article.
This study was supported by grants from the National Natural Science Foundation of China (81172337, 81370368, and 81672417).