Periostin expression and its supposed roles in benign and malignant thyroid nodules: an immunohistochemical study of 105 cases

Background Thyroid tumors are often difficult to histopathologically diagnose, particularly follicular adenoma (FA) and follicular carcinoma (FC). Papillary carcinoma (PAC) has several histological subtypes. Periostin (PON), which is a non-collagenous extracellular matrix molecule, has been implicated in tumor invasiveness. We herein aimed to elucidate the expression status and localization of PON in thyroid tumors. Method We collected 105 cases of thyroid nodules, which included cases of adenomatous goiter, FA, microcarcinoma (MIC), PAC, FC, poorly differentiated carcinoma (PDCa), and undifferentiated carcinoma (UCa), and immunohistochemically examined the PON expression patterns of these lesions. Results Stromal PON deposition was detected in PAC and MIC, particularly in the solid/sclerosing subtype, whereas FA and FC showed weak deposition on the fibrous capsule. However, the invasive and/or extracapsular regions of microinvasive FC showed quite strong PON expression. Except for it, we could not find any significant histopathological differences between FA and FC. There were no other significant histopathological differences between FA and FC. Although PDCa showed a similar PON expression pattern to PAC, UCa exhibited stromal PON deposition in its invasive portions and cytoplasmic expression in its carcinoma cells. Although there was only one case of UCa, it showed strong PON immunopositivity. PAC and MIC showed similar patterns of stromal PON deposition, particularly at the invasive front. Conclusions PON may play a role in the invasion of thyroid carcinomas, particularly PAC and UCa, whereas it may act as a barrier to the growth of tumor cells in FA and minimally invasive FC. Supplementary Information The online version contains supplementary material available at 10.1186/s13000-021-01146-8.


Introduction
Papillary carcinoma (PAC) is the most common tumor in the thyroid gland [1]. Among follicular thyroid lesions, difficulties are associated with differentiating between follicular adenoma (FA) and follicular carcinoma (FC) [2]. Since FA and FC, particularly the microinvasive type, exhibit similar cellular atypia, the key to their diagnosis is based on the presence/absence of capsular invasion.
Tumors are composed of parenchymal tissue (the tumor cells themselves) and the stroma, which contains the extracellular matrix (ECM) and myofibroblasts (cancer-associated fibroblasts: CAF). The stroma is not only a supportive tissue, it also regulates the migration, differentiation, and/or growth of tumor cells in the tumor microenvironment [3]. Periostin (PON) is a noncollagenous ECM molecule that is deposited on the periosteum and periodontal ligaments [4,5]. PON was recently shown to be expressed in several cancers, including oral and breast cancers, and plays direct roles in the invasion and migration of tumor cells [6][7][8][9]. Moreover, PON-positive cancers have poor prognoses. We herein examined the expression and localization of PON in thyroid nodules.

Materials and methods
We randomly selected 105 cases of thyroid nodules from our institution's pathology files (study period: 2014-2018). They included 10 cases of adenomatous goiter (AG), 13 cases of microcarcinoma (MIC), 20 cases of FA, 18 cases of FC, 41 cases of PAC, 2 cases of poorly differentiated carcinoma (PDCa), and one case of undifferentiated carcinoma (UCa). As negative controls, background normal thyroid tissues were selected in matching cases. The clinicopathological features of the cases used in the present study were summarized in Supplemental Table 1.
We re-diagnosed each case based on hematoxylin and eosin (H&E) staining and selected specimens that exhibited typical histology. Immunohistochemical staining was performed on 4-μm-thick sections, which were cut from formalin-fixed and paraffin-embedded tissue. Immunostaining was performed according to the relevant manufacturer's instructions using a Leica BOND MAX automated immunostainer (Leica, Bannockburn, IL, USA) or Ventana Benchmark GX automatic immunostainer (Roche Tissue Diagnosis, Oro Valley, AZ, USA). The antibodies used in the present study are summarized in Table 1. Tumors were considered to be diffusely positive (++), positive (+), focally positive (F+), and negative when ≥50, 10-49, 1-9, and 0% of neoplastic cells were positive, respectively. In the immunohistochemical assessment of PON expression, stromal localization was recorded as S and cytoplasm localization as CY. We used ImageJ (National Institutes of Health, Bethesda, MD, USA) to estimate the percentage of Ki-67-positive tumor cells.

Histological findings
All MIC were histologically classified as tiny PAC (Fig. 1A). There were 0 cases of the encapsulated (cap)/ sclerosing (scl) subtype, 5 of the non-cap/scl subtype, 6 of the cap/non-scl subtype, and 2 of the non-cap/nonscl subtype. All PAC belonged to the well-differentiated type (Fig. 1B), and, according to their macroscopic findings, there were 23 cases of the localized/solid subtype, 7 of the localized/cystic subtype, and 11 of the mixed subtype. The present study did not include any cases of the diffuse scl subtype of PAC. FA showed dense follicle proliferation and thick fibrous capsules without invasion (Fig. 1C). On the other hand, 15 out of the 18 cases of FC harbored dense fibrous capsules with extracapsular invasion, which were subclassified into minimally invasive FC (MinI-FC) (Fig. 1D). The remaining 3 cases of FC did not possess fibrous capsules, and were classified as widely invasive FC (WI-FC). PDCa showed a solid and/or insular pattern. UCa was composed of densely packed proliferating spindle-shaped cells and polygonal cells, which exhibited marked cellular atypia, and a small focus of the follicular variant of PAC (Fig. 1E). AG did not possess fibrous capsules, but was well-defined. Some areas exhibited dense follicle proliferation, whereas others showed loosely distributed follicles with an edematous stroma (Fig. 1F).

Immunohistochemical analysis of PON expression
The immunohistochemical results for PON are summarized in Table 2.
The scl subtype of MIC showed moderate positivity for PON, whereas the non-scl subtype did not express PON. In the cap and non-cap subtypes of MIC, PON localized to the fibrous capsule-like stroma and hyalinous stroma, except in non-scl-type MIC ( Fig. 2A).
The localized cystic subtype of PAC showed weak positivity for PON on cyst walls, whereas moderate and  FA showed weak positivity for PON on the fibrous capsule (Fig. 2C), which was similar to that observed in MinI-FC. However, WI-FC showed stronger positivity for PON at the invasive tip than in the inner stroma (Fig. 2D).
Since PDCa exhibited medullary growth, PON localized to the scant stroma. UCa showed both stromal and cytoplasmic positivity for PON (Fig. 2E). AG showed very weak or no positivity for PON (Fig. 2F).

Immunohistochemical analysis of other markers
The immunohistochemical results obtained for other markers are summarized in Table 3.

Discussion
PON is a glycoprotein that consists of a 93.3-kDa homodimer and was initially described as osteoblast-specific factor 2 in 1993 [4,10]. Although PON was previously considered to contribute to the adhesion of preosteoblasts [4], it has since been shown to play important roles in the formation of collagen fibers, cell-cell adhesion, and wound repair [11][12][13][14]. Moreover, PON has been associated with invasion capacity and stromal formation in several cancer types [6-9, 15, 16]. CAF secrete epidermal growth factor and insulin-like growth factor-1, which promote tumor growth. Moreover, CAF secrete hepatocyte growth factor/scattering factor, which promote cell migration [3]. On the other hand, CAF are also involved in the construction and/or remodeling of the ECM; i.e., they play a role in dissolving type I, III, and V collagen, fibronectin, and laminin, and, thus, promote the migration and invasion of cancer cells [17]. PON has been implicated in non-small cell lung cancer, oral cancer, and liver cancer [6-9, 15, 16]. In numerous cancer types, cancer cells themselves have been reported to secrete PON; however, stromal CAF also secrete PON, which promotes the migration and/or invasion of cancer cells [18,19]. Ratajczak-Wielgomas et al. showed that benign lesions of the breast showed no stromal PON deposition, whereas PON was deposited around malignant mammary ducts in ductal carcinoma in situ, and was more widely deposited in the cancer stroma of invasive ductal carcinoma of the breast, particularly in cases showing high-grade histological atypia [19]. PON secreted by CAF is not only involved in tumor cell migration and invasion, but is also associated with tumor cell differentiation and high-grade malignancy via epithelialmesenchymal transition [19,20].
In the present study, we examined the expression and localization of PON in thyroid tumors. Cytoplasmic PON localization was not observed in our series, except in UCa. This may have been because the antibody used in our study recognized a different epitope to those used in previous studies. Alternatively, it may have been due to the slow growth of thyroid tumors (even malignant tumors), except for UCa.
Since AG is not associated with CAF recruitment and is a hyperplastic lesion, AG did not show any PON expression. On the other hand, PON expression was low on fibrous (pseudo-)capsules in tumors with fibrous capsules (i.e., cap-MIC, FA, and the localized/cystic type of PAC), whereas it was clearly expressed in the hyaline stroma of stroma-rich tumors. UCa, which is a rapidly growing tumor, showed weak positivity for PON in the stroma and cytoplasm of tumor cells. Based on these results, PON does not contribute to the growth capacity of thyroid tumors.
Two reverse transcription-polymerase chain reaction (RT-PCR)-based studies examined PON expression in PAC [21,22]. The findings obtained indicated that PON mRNA expression was higher in tumor tissues than in  [23], and variant I was associated with in vitro invasiveness and in vivo metastatic capacity. Similar relationships may exist in PAC.
In the present study, we examined thyroid tumors other than PAC, and PON deposition was detected on the thick fibrous capsules of FA and MinI-FC. However, MIC showed stronger PON deposition than FA and MinI-FC. These results suggest that PON plays a role in capsular formation and/or acts as barrier against tumor extension or invasion, whereas PON in MIC plays a role in invasiveness rather than barrier function.
We immunohistochemically examined the expression of HMBE-1 and Gal-3. Consistent with previous findings, most cases of PAC were positive for both markers, whereas approximately 50% of cases of FA and 70% cases of MinI-FC showed the partial expression of these Fig. 3 Immunohistochemical staining results for other thyroid tumor markers. In microcarcinoma, cancer cells exhibited strong positivity for galectin-3 (Gal-3) (A) and nuclear positivity for cyclin D1 (B). In papillary carcinoma, the cytoplasm of cancer cells was diffusely positive for Gal-3 (C). Undifferentiated carcinoma showed cytoplasmic immunopositivity for Gal-3 with a mosaic-like pattern (D). In papillary carcinoma, the luminal side of papillary structures was positive for Hector Battifora mesothelial 1 (HBME-1) (E), and tumor cells were diffusely positive for cytokeratin 19 (F). In undifferentiated carcinoma, cancer cells were diffusely positive for Ki-67 (G), whereas tumor cells in papillary carcinoma were sporadically positive for Ki-67 (H) PAC markers [24,25]. Immunohistochemical examinations of both HMBE-1 and Gal-3 expression are considered to be useful for the differential diagnosis of thyroid tumors. The high expression of cyclin D1 in MIC (tiny PAC type) was recently reported to positively correlate with extracapsular invasion [26], and the present study showed that all cases of MIC belonged to the tiny PAC type. Twelve (92%) out of the 13 MIC cases and 38 (93%) out of the 41 PAC cases were positive for cyclin D1; however, cyclin D1 positivity was not related to extracapsular invasion or lymph node metastasis.

Conclusions
In conclusion, the present results suggested that stromal PON deposition in PAC was related to stromal formation and invasion, whereas PON deposition on the fibrous capsules of FA or MinI-FC may function as a barrier against tumor extension. Although PON may play different roles in each histological type of thyroid tumors, the biological roles of PON in thyroidal tumors remain unknown.